Brain Development: Normal Processes and the Effects of Alcohol and Nicotine

BRAIN DEVELOPMEN T

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BRAIN DEVELOPMEN T NORMAL PROCESSE S AND THE EFFECT S O F ALCOHOL AND NICOTIN E

EDITED BY Michael W . Miller

OXPORD UNIVERSITY PRES S 2006

OXPORD UNIVERSITY PRES S

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Copyright © 200 6 by Oxford Universit y Press, Inc . Published b y Oxford University Press, Inc. 198 Madison Avenue, New York, New York 1001 6 www.oup.com Oxford i s a registered trademark of Oxford University Press All rights reserved. No par t of this publication may be reproduced , stored i n a retrieval system, o r transmitted, i n any form or by any means , electronic, mechanical, photocopying , recording , or otherwise, without the prio r permission of Oxford Universit y Press. Library of Congress Cataloging-in-Publication Data Brain development : normal processes and th e effect s o f alcohol an d nicotin e / edited b y Michael W. Miller. p. ; cm. Includes bibliographical references and index. ISBN-13 978-0-19-518313- 9

ISBN 0-19-518313-4

1. Developmenta l toxicology. 2 . Nenrotoxicology . 3 . Alcohol-Toxicology . 4 . Nicotin e —Toxicology. 5. Alcoholis m i n pregnancy—Pathophysiology. 6 . Pregnan t women—Tobacc o use —Physiological effect . [DNLM: 1 . Brain—dru g effects. 2 . Brain—growt h & development. 3 . Ethanol—pharmacology . 4 . Fetus—dru g effects . 5. Neurons-dru g effects. 6 . Nicotin e -pharmacology. WL 300 B8125 2006 ] I . Miller , Michael W. RA1224.45.B73 2006 616.8'6471-dc22 200501285 4

1 3 5 7 9 8 6 4 2 Printed i n the Unite d States of America on acid-fre e paper

Acknowledgments

As such, the present volume i s the produc t o f decades of work by teams o f neuroscientists and developmen tal biologists . I than k th e name d an d nameles s o f these dedicate d researcher s wh o hav e reache d m e through bot h ora l and/o r writte n communications . This volume build s o n the foundation of research described i n a boo k publishe d 1 3 year s ago , Development of the Central Nervous System, Effects of Alcohol and Opiates (Miller , 1992) . Since tha t boo k was published, ou r understandin g o f the mechanism s under lying norma l brai n developmen t an d th e effect s o f ethanol neurotoxicit y o n neura l ontogen y ha s grown exponentially. Interes t i n th e clinica l proble m o f nicotine exposure has also increased. Personally, m y researc h ha s bee n supporte d b y a host of postdoctoral trainees, graduate and undergraduate students, an d technicians . I am greatl y indebte d to them fo r working toward ou r commo n goa l o f un derstanding th e intricacie s o f brai n development . Some o f my current student s (Mari a Bruns and Juli e Siegenthaler) an d collaborator s (Sandr a Mooney )

contributed t o the writin g of this book. My career ha s been consistentl y an d substantivel y supporte d b y the federal government , notabl y the Nationa l Institute of Alcohol Abus e an d Alcoholis m an d th e Departmen t of Veterans Affairs. Without the suppor t of these agen cies I would no t hav e been abl e t o study normal an d abnormal brain development, no r woul d I have been able t o mee t th e peopl e wh o wrot e chapter s fo r this volume. These people hav e brought considerabl e expertise and kee n insight s to their contributions of outstanding chapters . The dua l sculptors, nature and nurture , have bee n essential factor s in m y development as a scientist. M y fascination wit h science—ho w thing s wor k an d ho w they fail—wa s fostere d b y m y parent s an d th e post Sputnik America n culture . I t i s m y grea t hop e tha t children of all ages, boys and girls , retain their wonder as to wh y things ar e a s they are . This sense o f marvel should b e celebrate d an d hel d i n aw e by society. My candle was stoked by high schoo l an d colleg e teacher s (including Willia m Harri s an d Willia m Tully , an d

vi ACKNOWLEDGMENT S Edward Hodgson an d Edwar d Maly, respectively), re- Finally , I would lik e to than k m y family , m y wif e search mentor s (includin g Pedr o Pasi k an d Ala n Pe - an d thre e children , fo r thei r continue d suppor t an d ters), and collaborator s (notably Richard Nowakowski goo d humor . The y ar e m y sustenance , grounding , and Rober t Rhoades). They encourage d m e t o thin k an d reaso n for being, broadly and not to be confined by dogma o r methods .

Contents

List of Abbreviations i x Contributors xii i 1. Model s o f Neurotoxicity Provide Uniqu e Insight into Normal Developmen t 3

5. Cel l Death 7 3 Stevens K Rehen Jerold J.M. Chun

Michael W. Miller

Part I. Norma l Developmen t 6. Intracellula r Pathways of Neuronal Death 9 1

2. Cel l Proliferatio n 9 Bernhard So ter Pradeep G . Bhide

Sandra M. Mooney George I. Henderson

3. Neurona l Migration 2 7 Huaiyu Hu

7. Developmenta l Disorder s an d Evolutionary Expectations: Mechanism s of Resilience 10 4

4. Neurona l Differentiation : From Axons to Synapses 4 5 C. David Mintz Iddii H. Bekirov Tonya R. Anderson Deanna L. Benson

Barbara L. Finlay Jeremy C. Yost Desmond T . Cheung

vii

viii CONTENT

S

Part IL Ethanol-Affecte d Developmen t 8. Prenata l Alcohol Exposure and Huma n Development 12 3 Claire D. Coles

9. Influenc e of Alcohol on the Structur e of the Developing Human Brai n 14 3 Susanna L, Fryer Christie L. McGee Andrea D. Spadoni Edward P. Riley

10. Prenata l Ethanol Exposur e and Feta l Programming: Implications for Endocrine and Immun e Developmen t an d Long-Term Healt h 15 3 Joanna H, Sliwowska Xingqi Zhang Joanne Weinberg

11. Earl y Exposure to Ethanol Affects th e Proliferation o f Neuronal Precursor s 18 2 Michael W. Miller 12. Effect s o f Ethanol o n the Regulatio n of Cell Cycle in Neural Ste m Cell s 19 9 W. Michael Zawada Mita Das

13. Mechanism s of Ethanol-Induce d Alterations in Neuronal Migration 21 6 Julie A. Siegenthaler Michael W. Miller

14. Effect s o f Ethanol on Mechanism s Regulating Neuronal Process Outgrowth 23 0 Tara A. Lindsley

15. Neurona l Surviva l Is Compromised b y Ethanol: Extracellula r Mediators 24 5 Michael W. Miller Maria B. Bruns Paula L. Hoffman

16. Intracellula r Events in Ethanol-Induce d Neuronal Death 26 7 Sandra M. Mooney Michael W . Miller George I. Henderson

17. Neura l Crest and Developmenta l Exposur e to Ethanol 27 9 Susan M. Smith Katherine A. Debelak-Kragtorp

18. Glia l Targets o f Developmental Exposur e to Ethanol 29 5 Consuelo Guerri Gemma Rubert Maria Pasqual

Part III. Nicotine-Affecte d Developmen t 19. Tobacco use During Pregnancy : Epidemiology an d Effect s o n Offsprin g 31 5 Jennifer A. Willford Nancy L . Da y Marie D. Cornelius

20. Prenata l Nicotin e Exposur e and Animal Behavior 32 9 Brenda M. Elliott Neil E. Grunberg

21. Neurona l Receptors for Nicotine: Functiona l Diversity and Developmental Change s 34 1 Huibert D. Mansvelder Loma W. Role

22. Neura l Precursor s as Preferential Targets for Dru g Abuse: Long Term Consequence s and Latent Susceptibilit y to Centra l Nervous System Disorders 36 3 Kurt F. Hauser Nazira El-Hage Shreya Buch Gregory N. Barnes Henrietta S . Bada James R. Pauly

23. Nicotini c Receptor Regulatio n of Developing Catecholamin e System s 38 1 Frances M. Leslie Layla Azam Kathy Goliardo Kathryn O'Leary Ryan Franke Shahrdad Lotfipour

Index 39 9

List of Abbreviations

ALC prenatall y exposed to alcohol, bot h wit h and without dysmorphic feature s

5-HT serotoni n 8-OH-DPAT 8-hydroxy-2-(di-n-propylamino)tetrali n

AMPA a-amino-B-hydroxy-S-methyM-isoxazole propionic acid

ABCG2 ATP-bindin g cassette protein otBgTx a-bungarotoxi n ACh acetylcholin e

AMPAR a-amino-3-hydroxy - 5-methyl-4-isoxazole propionic acid receptor

AChE acetylcholinesteras e

AP-1 activatin g protein-1

ACTH adrenocorticotropi c hormone

APAF apoptoti c protease activating factor

ADH alcoho l dehydrogenase

ARIA acetylcholin e receptor-inducin g protei n ARND alcohol-relate d neurodevelopmenta l disorder

ADHD attentio n deficit hyperactivity disorder ADNF activity-dependen t neurotrophic factor

ASD atria l septic defect

ADNP activity-dependen t neuroprotective protein

ASR acousti c startle reflex

ADP adenosin e diphosphate

Astn astrotacti n

ADX adrenalectom y

ATP adenosin e triphosphate

AHP adul t hippocampal progenito r

AVP arginin e vasopressin

AIDS acquire d immunodeficiency syndrome

BDNF brain-derive d neurotrophi c facto r

AI F apoptosi s inducing facto r

BEG bloo d ethano l concentratio n

ix

x LIS

T O F ABBREVIATION S

β-E P β-endorphi

n

DO I l-(2,5-dimethoxy-4-iodophenyl)-2 aminopropan e hydrochlorid e

-

bFG F basi c fibroblas t growth facto r (aka fibroblas t growth facto r 2 )

DO R δ-opioid

BH Bcl- 2 homolog y

DR G dorsa l roo t ganglia

BMP bon e morphogeni c protei n

DT I diffusio n tenso r imagin g

BSID Bayle y Scales o f Developmen t

EC 50 concentratio respons e

n effectin g a hal f maxima l

EC M extracellula

r matrix

BrdU bromodeoxyuridin [Ca ]i intracellula

e

r concentratio n o f Ca

2+

2+

receptor s

CAM cel l adhesio n molecul e

EG F epiderma

cAMP cycli c adenosin e monophosphat e

EGF r epiderma

CAP cel l adhesio n protei n

EG L externa

CBG corticosteron

EM electro

CC corpu

e bindin g globulin

s callosum

CC K cholecystokini

l growt h facto r l growth facto r recepto r

l granula r layer of the cerebellu m n microscop y

ER K extracellula r signal regulate d kinas e

n

ES embryoni

CD13 3 cel l determinan t 13 3 (hematopoieti c ste m cell marker , aka promini n cdk cyclindependen t kinas e Cd k cyclin-dependen

t kinase

c stem

ET S environmenta

l tobacc o smok e

FAD D Fas-associate

d deat h domai n

FAS feta l alcoho l syndrom e

cGM P cycli c guanosin e monophosphat e

FASD feta l alcoho l spectru m disorder s

ChAT cholin

FasL Fa

e acetyltransferas e

CK I cyclin-dependen CN S centra

t kinase inhibito r

l nervou s system

CO N A concanavali

nA

COR T corticosteron

e

CP cortica

s ligand

FCM D Fukuyama-typ dystroph y

FE E feta l ethanol-expose d FG F fibroblas t growth facto r FILI P filami n A-interactin g protei n

l plate

CRE B cycli c AMP respons e elemen t bindin g protein

fMR I functiona

CR H corticotropi

G gestationa

n releasin g hormon e

CRH-R 1 corticotropi recepto r CRM P collapsi CT compute

n releasin g hormon e typ e 1

r tomograph y

CYP2E 1 cytochrom CytO x cytochrom

e P450 2E 1 e C oxidas e

GABA γ-aminobutyri c aci d GD F growt h differentiatio n facto r y

G E ganglioni c eminenc e G F growt h fractio n GFA P glia l fibrillar y acidi c protei n GM C ganglio n mothe r cel l GnR H gonadotropin-releasin

Da b disable d d in colorecta l cance r

DHβ E dihydro-β-erythroidin DIABL O direc

l day

GD X gonadectom

a Verbal Learnin g Test- children' s

DA dopamin e DC C delete

l magneti c resonanc e imagin g

Gadd45 γ growt h arres t and DN A damage-inducibl e

n respons e mediato r protei n

CVLT- C Californi version

e congenita l muscular

e

t IAP bindin g protein

DI V day s in vitro DNA-PKc s DNA-dependen t protei n kinas e catalyti c subuni t

G R glucocorticoi

d recepto r

GR P glial-restricte GS H glutathion

d precursor

e

GTPas e guanosin HG F hepatocyt hn heteronuclea

g hormone

e 5'-triphosphatas e e growth facto r r

LIST OF ABBREVIATION S x i HNE 4-hydroxynonena l

MNTB media l nucleu s o f the trapezoi d body

hNSC huma n neura l stem cel l

MOR fl-opioi d receptor s

HPA hypothalamic-pituitary-adrena l

MR mineralocorticoi d recepto r

HPG hypothalamic-pituitary-gonada l

MRI magneti c resonance imaging

IAP inhibitor y apoptotic protei n

MZ margina l zon e

Ig immunoglobuli n IGF insulin-lik e growth factor IGF-lr insulin-lik e growth factor-1 receptor IGF-BP insulin-lik e growth factor bindin g protein IHZ intrahila r zone; or hippocampal field CA4 or the subgranular zone IL interleuki n IP intraperitonea l IP3 inosito l 1,4,5-triphosphat e IQ intelligenc e quotien t ISEL i n situ end-labelin g IZ intermediat e zon e KC1 potassiu m chlorid e Ki67 proliferation-associate d nuclear antige n

NAc nucleu s accumben s nACliR nicotini c acetylcholine receptor NAD nicotinamide-adenin e dinucleotid e NAP asn-ala-pro-val-ser-ile-pro-gl n NBL neuroblasti c layer nCAM neura l cel l adhesio n molecul e NE norepinephrin NGF nerv

e

e growth facto r

NgCAM neuron-gli a cell adhesio n molecul e NHEJ non-homologou s end-joinin g NMD neurona

l migration disorder

NMDA N-methyl-D-aspartat e NMDAR N-methyl-D-aspartat e recepto r NMJ neuromuscula r junction NO nitri c oxide

KOR K-opioi d receptor s

NOND naturall y occurring neuronal deat h

Large like-acetylglucosaminyltransferas e

NPC neura l progenitor cel l

LBW lo w birth weight

Nr-CAM NgCAM-relate d cell adhesio n molecul e

LC locu s coeruleus

NRT nicotin e replacemen t therap y

LC-NE locu s coeruleus-noradrenergic (sympathetic adrena l medullary )

NSC neura l ste m cel l NT neurotrophi n

LCN loca l circuit neuro n

NTS nucleus

LF leavin g fraction

NT-4 neurotrophi n 4

LHRH luteinizin g hormone releasin g hormone

OD ocula r dominance

tractus solitarius

LiglV ligasel V

ODC ornithin e decarboxylase

LPA lysophosphatidi c acid

OPPS Ottaw a Prospectiv e Prenata l Stud y

LPS lipopolysaccharid e

P postnata l day

LTP long-ter m potentiation

PA phosphatidi c aci d

LV laten t variable

PACAP pituitar y adenylyl cyclase activating polypeptide

mAChR muscarini c acetylcholine recepto r MAP microtubule-associate d protei n MAPK mitogen-activate d protein kinase

PAFAHIb platelet-activating facto r acetylhydrolas e isoform Ib , aka LISI

MDA malondialdehyd e

PARP poly-adenosin e diphosphate ribos e polymerase

MEB muscle-eye-brai n disease

PCD programme d cel l deat h

MHC majo r histocompatibility comple x

PDGF platelet-derive d growth facto r

MHPCD Materna l Healt h Practice s and Child Development

PET positro n emission tomograph y

PEE prenata l ethanol exposure

xii LIS T O F ABBREVIATION S PE t phosphatidy PF C prefronta

l ethanol

Smo Smoothene

l cortex

sMR I structura

PB К phosphatidylinosito

l З'-phosphokinas e

РК А protei n kinase A e

PL S partia l least square s analysis PM pia l membran e

SN substantia l nigr a

SNAP-2 5 synaptosom

l nervou s system

POMGnT l UDP-N-acetylglucosamin e protein-O mannos e ßl ,2-N-acetylglucosaminyltransferase POMC pro-opiomelanocorti n POMT1 protein-O-mannos e ßl,2-Nacetylglucosaminyltransferase

e associate d protein-2 5

SNR P stres s nonresponsiv e perio d SOD Superoxid

PN projectio n neuro n PN S periphera

l magnetic resonanc e imaging

SNARE S solubl e n-ethylmaleimide-sensitiv e fusion protein-attachmen t protei n receptor s

PK C protei n kinase С PLD phospholipas

d

e dismutas e

SPEC T singl e photo n emissio n compute d tomograph y SPP secondar y proliferativ e populatio n SV synapti c vesicle SynCAM synapti

c cel l adhesio n molecul e

SZ subventricula r zon e SZa anterio

r subventricula r zone

PPI prepuls e inhibitio n

T c tota l lengt h o f the cell cycl e

PSD post-synapti c densit y

TC R T

PSN principa l sensor y nucleu s of the trigemina l nerve

TER T telomeras e reverse transcriptas e

PSR phosphatidylserin e receptor РТ Е prenata

l tobacc o exposur e

PTV Piccolo-Bassoo

n transpor t vesicle

PVE pseudostratifie

d ventricula r epitheliu m

PVN paraventricula

r nucleu s

TGFßlr transformin g growt h facto r ßl receptor TGFßllr transformin g growt h factor ßll receptor TH tyrosin e hydroxylas e TNF tumo r necrosis facto r

TÚNEL termina l deoxynucleotidy l transferas e mediated deoxyuridine nick en d labeling

c aci d c acid recepto r

Rb retinoblastom

g growth facto r

Trk tyrosin e kinas e recepto r

Q probabilit y of cell cycl e exit RAR retinoi

TG F transformin

TRF telornerase-repeat-bindin g factor

PWM pokewee d mitoge n RA retinoi

cel l receptor s

UCS unconditione d stimulu s

a protei n

VB ventrobasa l nucleus of the thalamu s

RG radia l glia

VBM voxel-base d morphometry

RO S reactiv e oxygen specie s

VDCC voltage-dependen t Ca 24" channel

SC subcutaneou

VIP vasoactiv e intestinal polypeptide

SES socioeconomi

s c statu s

VLM ventrolatera l medulla

Shh soni c hedgeho g

VSD ventra l septi c defec t

SHR P stres s hyporesponsiv e perio d

VTA ventra l tegmental area

SKY spectra l karyotypin g

VZ ventricula r zone

SID S sudde n infan t deat h syndrom e

VLDLr ver y low density lipoprotei n recepto r

SIN- 1 3-morpholinosydnonimin

WCST Wisconsi n Card Sort Task

e

Smac secon d mitochondria-derive d activato r ofcaspase

WRAML Wid e Range Assessment of Memory and Learning

SMase sphingomyelinas

WWS Walker-Warbur g syndrom e

e

Contributors

TONYA R . ANDERSO N Mount Sinai School of Medicine New York NY

PRADEEP G . BHID E Massachusetts General Hospital Harvard Medical School Boston MA

LAYLA AZAM

MARLA B . BRUN S SI/NY- Upstate Medical University Syracuse NY

University of California College of Medicine Irvine CA HENRIETTA S . BADA University of Kentucky College of Medicine Lexington KY

SHREYA BUCH University of Kentucky College of Medicine Lexington KY

GREGORY N . BARNE S University of Kentucky College of Medicine Lexington KY

DESMOND T . CHEUN G Cornell University Ithaca NY

IDDIL H . BEKIROV Mount Sinai School of Medicine New York NY

JEROLD J.M. CHUN Scripps Research Institute La Jolla CA

DEANNA L. BENSO N Mount Sinai School of Medicine New York NY

CLAIRE D. COLE S

Emory University School of Medicine Atlanta GA xiii

xiv CONTRIBUTOR S MARIE D . CORNELIU S University of Pittsburgh School of Medicine Pittsburgh PA

PAULA L. HOFFMA N University of Colorado Health Sciences Center Denver CO

MITA DAS University of Colorado Health Sciences Center Denver CO

HUAIYU H u SUNY-Upstate Medical University Syracuse NY

NANCY L. DAY University of Pittsburgh School of Medicine Pittsburgh PA

FRANCES M . LESLI E University of California College of Medicine Irvine CA

KATHERINE A. DEBELAK-KRAGTOR P University of Wisconsin School of Medicine Madison WI

TARA A. LINDSLE Y Albany Medical School Albany NY

NAZIRA EL-HAGE University of Kentucky College of Medicine Lexington KY

SHAHRDAD LOTFIPOUR University of California College of Medicine Irvine CA

BRENDA M. ELLIOT T Uniformed Services University of the Health Sciences Bethesda MD

HUIBERT D . MANSVELDE R Vrije Universitat, Amsterdam, The Netherlands

BARBARA L. FINLA Y Cornell University Ithaca NY

CHRISTIE L. McGE E San Diego State University San Diego CA

RYAN FRANKE University of California College of Medicine Irvine CA SUSANNA L. FRYE R San Diego State University San Diego CA

MICHAEL W. MILLE R Department of Neuroscience and Physiology and Developmental Exposure Alcohol Research Center SUNY-Upstate Medical University Research Service Syracuse Veterans Affairs Medical Center Syracuse NY

KATHY GALLARD O University of California College of Medicine Irvine CA

G. DAVI D MINT Z Mount Sinai School of Medicine New York NY

Centro de investigacion Principe Felipe Valencia, Spain

CONSUELO GUERRI

SANDRA M. MOONEY SI/NY- Upstate Medical University Syracuse NY

NEIL E. GRUNBER G Uniformed Services University of the Health Sciences Bethesda MD

KATHRYN O'LEARY University of California College of Medicine Irvine CA

KURT F . HAUSE R University of Kentucky College of Medicine Lexington KY

MARIA PASCUA L Centro de investigacion Prìncipe Felipe Valencia, Spain

GEORGE I. HENDERSO N University of Texas Health Science Center San Antonio TX

JAMES R. PAUL Y University of Kentucky College of Medicine Lexington KY

CONTRIBUTORS x v STEVENS K . REHEN Scripps Research Institute La Jolla CA

ANDREA D . SPADON I San Diego State University San Diego CA

EDWARD P. RILE Y San Diego State University San Diego CA

BERNHARD SUTE R Massachusetts General Hospital Harvard Medical School Boston MA

LORNA W. ROL E Columbia University New York NY GEMMA RUBER T Centro de investigacion Principe Felipe Valencia, Spain

JOANNE WEINBER G University of British Columbia Vancouver BC JENNIFER A. WILLFORD University of Pittsburgh School of Medicine Pittsburgh PA

JULIE A. SlEGENTHALE R

SI/NY- Upstate Medical University Syracuse N Y

JEREMY C.YOS T Cornell University Ithaca NY

JOANNA H. SLIWOWSK A University of British Columbia Vancouver BC

XINGQI ZHAN G University of British Columbia Vancouver BC

SUSAN M. SMIT H University of Wisconsin School of Medicine Madison WI

W. MICHAE L ZAWADA University of Colorado Health Sciences Center Denver CO

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BRAIN DEVELOPMEN T

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1 Models of Neurotoxicity Provide Unique Insight into Normal Development Michael W . Miller

include changes cascading from a spontaneous muta tion i n th e genome . Externa l challenge s ca n b e sub tle (respons e t o a stress) or profound (change s cause d by exposur e t o a toxi n o r drug) . Alterations induce d by interna l an d externa l challenge s ca n b e superim posed an d change s ma y b e additiv e or synergistic . I n any case , a n understandin g o f th e response s o f th e nervous syste m provide s uniqu e insigh t int o it s nor mal developmen t an d it s plasticity . Doe s th e chal lenged nervou s syste m consistentl y respon d i n th e same fashion , o r doe s i t respon d usin g a variet y o f strategies? Tolstoy's comment on his own work was that Anna Karenina did not solve a single problem; rather, it presented the m all, beautifully. Environmental exposur e to substance s suc h a s ethano l an d nicotin e present s situations o f compellin g clinica l interest . Ethano l i s arguably the most full y studied teratogen . Unlike Tol stoy's self-critique , studie s o f ethano l toxicit y hav e not bee n restricte d t o phenomenologica l studies ; many mechanism s o f ethano l neurotoxicit y hav e

Happy familie s ar e al l alike ; ever y un happy family i s unhappy i n it s own way. Anna Karenina, Leo Tolstoy

The developin g nervou s system is comprised o f com plex set s o f element s tha t assemble int o a dynamic , interactive physical , chemical , an d electrica l mesh . In th e norma l animal , thi s assembl y proceeds i n a n orderly progression amountin g t o the su m o f additive (cell proliferation , migration , and neurit e outgrowth ) and subtractiv e processe s (prunin g of axons and den drites and neuronal death). Each o f these ontogeneti c processes involve s the timely , intricat e choreograph y of molecular an d cellula r players . That said, develop ment doe s not follow a rigid sequence . The developin g brai n ha s considerabl e flexibility and adaptability . Thi s adaptabilit y ca n b e overcom e by a multitud e o f challenges , an d th e variet y o f re sponses ca n be as diverse as the challenges . Th e chal lenges can be interna l or external. Internal alterations 3

4 INTRODUCTIO N been pursued . Indeed , wor k o n ethano l provide s a model o f ho w t o stud y the negativ e effect s o f othe r substances. Moreover , study of the effect s o f ethanol provides a uniqu e insigh t int o norma l development . The approac h of this book is to look at three contrast ing situations: normal development, an d th e effect s of two differen t type s of challenges. Examinatio n o f th e normal and abnormal situations informs eac h o f these situations. Thus, i t i s only throug h geneti c an d envi ronmental challenge s tha t a ful l appreciatio n o f th e complexity of brain development ca n be achieved.

NORMAL DEVELOPMEN T

Our understandin g o f norma l neura l developmen t has progressed by quanta during the las t dozen years . Not onl y hav e w e begu n t o understan d th e myria d genes tha t ar e expresse d i n th e developin g centra l nervous syste m (CNS) , bu t function s fo r th e tran scripts an d th e protein s have been ascribed . Two examples ar e rln an d dix, gene s tha t cod e fo r protein s important in stopping neuronal migration and i n promoting the migration of neocortical loca l circuit neurons from th e ganglion eminence . The compellin g abnorma l organizatio n o f th e brain an d th e associate d dysfunctio n o f th e reeler mouse hav e fascinate d developmenta l neurobiolo gists for more tha n five decades (e.g. , Falconer, 1951 ; Alter et al, 1968; Bruc k and Williams, 1970 ; Lamber t de Rouvroit and Goffinet , 1998) . Th e invers e organi zation o f th e reele r mous e ha s provide d uniqu e in sights int o mechanism s o f normal neurogenesi s an d the formatio n of neural networks . Yet it i s only since 1995 that the genetic basi s for this murine mutant has been discovered , the knockou t of the gen e rln (D'Arcangelo e t al. , 1995 ; Hirotsun e e t al. , 1995 ; Ogaw a et al. , 1995) . Th e functio n o f th e gen e product , reelin, continues to be debated. The gen e dix i s a ke y player i n cortica l develop ment (Mari n an d Rubenstein , 2003) . It i s critical for the generatio n o f loca l circui t neuron s i n th e gan glionic eminence. A dearth of local circuit neurons is evident i n dix knockou t mice . Thus , a ne w doo r t o understanding th e complexit y o f cortica l neurono genesis an d tangentia l pathway s o f neurona l migra tion has been opened . The sectio n on normal development describes our current understandin g o f cellula r an d molecula r events tha t defin e developmenta l phenomena . Fo r

convenience, neura l development ha s bee n divided into fou r phases : cel l proliferation , migration, differ entiation, and deat h (Chapter s 2, 3 , 4, an d 5 and 6 , respectively). Althoug h thes e ar e discret e phases , they are highly integrated. That is, the numbe r o f cycling cells ha s a direc t effec t o n th e wa y in whic h cells differentiate and o n the incidenc e o f cell death. Thus, these chapter s addres s a single developmental phase whil e placin g ontogeneti c event s i n th e con text o f th e continuu m o f neura l development . I n a provocative essa y (Chapter 7 ) at the en d o f this sec tion, basi c informatio n on ontogeneti c event s is discussed i n term s o f mammalian evolution , an d thei r ramifications o n th e respons e of the nervou s system are addressed.

ETHANOL-AFFECTED DEVELOPMEN T

The concep t that ethano l affect s th e developmen t o f the mammalia n (human ) brai n cam e t o th e for e i n 1973 wit h th e publicatio n o f paper s b y Jone s an d Smith (Jone s an d Smith , 1973 ; Jone s e t al , 1973) . These were not the first papers to identity fetal alcoho l effects. Thoug h les s celebrated , report s b y Lemoin e and colleagues (1968) and Ulleland (1972) had already described feature s o f feta l alcoho l syndrom e (FAS) . Although i t has been suggeste d tha t the first evidence linking alcoho l consumptio n durin g pregnanc y an d deleterious effect s o n offsprin g come s fro m Gree k and Roma n mytholog y an d fro m th e Bible , this no tion was debunked by Abel (1984). To the bes t of my knowledge, the first documentation o f someon e wit h FA S can b e attribute d t o th e artists Harvey Kurtzman i n 195 4 and Norma n Ming o in 1956 . The y dre w a perso n originall y know n as Melvin Coznowsk i o r Me l Haney , late r know n a s Alfred E . Neuman (also spelled Newman). 1 Mr. Neu man ha s al l o f the classi c pathomnemonic craniofa cial malformation s of FAS (thin uppe r lip , deficient , philtrum, narro w palpebrai fissures , flattene d bridge of th e nose , an d lo w se t ears) , alon g wit h acknowl edged menta l dysfunctio n and probabl e intrauterine growth retardation (a s evidenced b y microencephaly). Although verificatio n o f prenata l exposur e i s un known, such evidenc e i s not obligatory for a diagnosis of FAS (Stratton e t al. , 1996) . As is typical of accomplished artists , Kurtzman and Ming o were perceptive observers and mad e associations tha t wer e a s insightful a s thos e o f th e experience d pediatrician s wh o

NEUROTOXICITY AND NORMA L DEVELOPMENT 5 described FA S more tha n a doze n year s later . Kurtzman and Mingo did not associate the craniofacial malformations an d brai n dysfunctio n wit h prenata l exposure t o alcohol , bu t the y di d lin k th e diagnosti c features that characterize FAS. Ethanol ca n ac t through a number of mechanisms. This is not to say that ethanol is a "dirty drug". Indeed , evidence show s tha t i t affect s th e nervou s syste m i n very specifi c ways . Moreover , th e manne r i n whic h ethanol alter s developmen t instruct s u s abou t th e adaptability an d resilienc y of the developin g nervous system. Fro m a clinical perspective, a n understandin g of the consequence s o f early exposure to ethano l i s of paramount importance . Prenata l exposur e to alcoho l affects upward s o f 1 % o f al l liv e birth s an d i t i s th e prime cause of mental retardation in the Unite d States (Sokol e t al., 2003) . Furthermore, ou r appreciatio n o f ethanol-induced change s provide s a model o f and in sights int o th e etiolog y o f numerou s neurodevelop mental disorders, such as autism and schizophrenia . The firs t se t of chapters i n Par t II addresses global effects o f prenata l exposur e t o ethanol . Thes e in clude th e prevalenc e o f alcohol-induce d deficit s among human s (Chapte r 8) , th e structura l malfor mations cause d b y prenata l exposur e t o ethano l (Chapter 9) , an d th e effect s o f ethano l o n nervou s system-endocrine system interaction s (Chapte r 10) . Following this discussio n i s a serie s of chapters tha t focus o n th e fou r ontogeneti c phases : cel l prolifera tion (Chapter s 1 1 and 12) , migration (Chapte r 13) , differentiation (Chapte r 14) , and deat h (Chapter s 1 5 and 16) . The las t two chapters o f this sectio n exam ine th e effect s o f ethano l o n tw o population s o f neural-related cells : neura l cres t cell s (Chapte r 17 ) andglia (Chapte r 18) .

NICOTINE-AFFECTED DEVELOPMENT

The fina l sectio n o f the boo k examine s the respons e of th e nervou s syste m t o a nicotin e challenge . Whereas ethano l i s acte d upo n b y endogenou s enzymes—for example , catalas e an d alcoho l dehydrogenase —it does not ac t at a specific receptor . In contrast , nicotin e allegedl y act s thoug h a focused endogenous mechanis m a t receptor s fo r cholinergi c neurotransmission. An understanding o f the effect s of nicotine i s no t onl y importan t fo r appreciatio n o f a basic scienc e issue , i t i s o f critica l clinica l impor tance. A fetus i s exposed to nicotine thoug h increase d

blood concentration s in the pregnan t woman as nicotine freel y crosse s the placenta l "barrier, " and infant s are exposed to nicotine through secondhand smoke. The clinica l problem o f fetal nicotin e exposur e is discussed i n Chapte r 19 . Nicotine i s a rathe r ubiqui tous substanc e — for thos e wh o smok e an d fo r thos e who ar e expose d t o secondhan d smoke . Nicotin e ex posure i s often accompanie d b y exposure to other substances suc h a s alcohol . Thus , clinica l studie s hav e fostered th e nee d t o conduc t well-controlle d anima l studies to understand the behaviora l consequences of exposure t o nicotin e pe r s e (Chapte r 20) . Althoug h nicotine i s thought to act through specifi c cholinergic receptors, it has broader effects throug h catecholaminergic and othe r systems . These issues are examined i n Chapters 2 1 and 22 , respectively. Chapter 2 3 explores the effect s o f nicotine o n specific ontogenetic events . Separate studie s o f norma l developmen t an d o f development altere d b y ethano l o r nicotin e provide unique, thoug h limited , insigh t int o brai n develop ment. Eac h lin e o f inquiry opens window s of understanding tha t otherwis e migh t b e inaccessibl e fro m other vantag e points . B y consolidating th e informa tion gathered from th e three approaches, however, we can gai n a fuller appreciatio n (a ) of the complexit y of neural development , an d (b ) o f how i t can g o wrong during a disease process. Abbreviations CNS centra l nervou s system FAS feta l alcoho l syndrom e Note 1. I t wa s alleged that Harve y Kurtzma n an d Norman Mingo were no t th e first to dra w a fac e resemblin g Alfred E . Neuman . Indeed , two lawsuits (base d on copyrights file d i n 191 4 an d 1936 ) atteste d tha t Kurtzman and Mingo were not the originators of the caricature no w referre d t o a s Alfred E . Neuman . It was used in advertisements for dental practices (see a copy of the Manitob a Free Press in 192 8 [http://www .imagehosting.us/index.php?action = show&ident = 728813]) and a n imag e was even used by the Nazi s as par t o f their anti-Semiti c campaig n i n th e 1930 s (Djerassi, 1988 ; see http://garfield.library.upenn.edu/ essaysM2pl61yl989.pdf). [n.b. Th e nam e Alfred E . Neuman an d th e imag e o f the gap-toothe d person were first joined i n 1956. ] The principa l (winning ) defense was that earlier artists had themselves copied

6 INTRODUCTIO N a likenes s produced befor e 1914 , notabl y "Mickey Dugan, The Yello w Kid" drawn by Richard Felto n Outcault i n th e lat e nineteent h century , an d vari ous othe r cartoon s and advertisement s (see a cop y of th e Winnipe g Tribun e i n 190 9 [http://www r .imagehosting.iis/index.phpFaction = show&ident = 728816]). Interestingly , mos t o f th e earlie r versions depict a norma l uppe r lip , a distinc t philtrum , an d normal-appearing eyes. Therefore, it does appear that Kurtzman an d Ming o were the first to associate th e three characteristi c features o f FAS : facia l dysrnor phology, mental dysfunction , and microencephaly. References Abel E L (1984 ) Fetal Alcohol Syndrome an d Fetal Alcohol Effects. Plenum , Ne w York . (1995) An update on incidenc e of FAS: FAS is not an equa l opportunit y birt h defect . Neurotoxico l Teratol 17:437-443 . Alter M, Lieb o J , Desnick SO , Stromme r B (1968) Th e behavior of the reele r neurologica l mutan t mouse . Neurology 18:289 . Bruck RM , Williams TH (1970 ) Light and electro n microscopic stud y of the hippocampa l regio n o f normal and reele r mice. J Anat 106:170-171. D'Arcangelo G , Mia o GG, Chen SC , Soare s HD, Mor gan JI, Curran T (1995 ) A protein relate d to extracellular matri x proteins delete d i n th e mous e mutan t reeler. Nature 374:719-723. Djerassi C (1988 ) Th e ques t fo r Alfre d E . Neuman . Grand Stree t 8:167-174. Falconer D S (1951 ) Tw o ne w mutations , trembler an d reeler with neurological action i n the hous e mouse. J Genet 50:192-201 .

Hirotsune S , Takahara T, Sasak i N, Hiros e K, Yoshiki A, Ohashi T , Kusakab e M , Murakam i Y, Muramatsu M, Watanabe S , Nakao K, Katsuki M, Hayashizakiy (1995) Th e reeler gen e encode s a protei n wit h a n EGF-like moti f expresse d b y pioneer neurons . Na t Genet 10:77-83. Jones KL , Smit h D W (1973 ) Recognitio n o f th e feta l alcohol syndrom e i n earl y infancy . Lance t 2:999 1001. Jones KL , Smit h DW , Ullelan d CN , Streissgut h A P (1973) Patter n o f malformatio n i n offsprin g o f chronic alcoholic mothers . Lance t 1:1267—1271 . Lambert de Rouvrai t C, Goffine t A M (1998) The reele r mouse a s a model o f brain development. Ad v Anat EmbryolCellBioll50:l-106. Lemoine P , Haroussea u H , Borteyr u J-P , Menue t J- C (1968) Le s enfant s de parent s alcooliques : anom alies observées à propos de 12 7 cas. Ouest Me d 21 : 476-482. Marin O , Rubenstei n J L (2003 ) Cell migratio n i n th é forebrain. Annu Rev Neurosci 26:441-483. Ogawa M , Miyat a T , Nakajim a K , Yagy u K , Seik e M , Ikenaka K , Yamamoto H , Mikoshib a K (1995) Th e reeler gene-associated antigen o n Cajal-Retzius neu rons is a crucial molecule for laminar organization of cortical neurons . Neuro n 14:899-912 . Sokol RJ , Delaney-Blac k V , Nordstro m B (2003 ) Feta l alcohol spectru m disorder . JAM A 290:2996 2999. Stratton K, Howe C, Battagli a F ( 1996) Fetal Alcohol Syndrome: Diagnosis, Epidemiology, Prevention and Treatment. Nationa l Academy Science Press , Washington, DC . Ulleland CN (1972 ) The offsprin g o f alcoholic mothers. Ann N Y Acad Sci 197:167-169.

I NORMAL DEVELOPMENT

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2

Cell Proliferatio n Bernhard Suter Pradeep G. Bhide

Cell proliferatio n i s the earlies t step in the protracted process of development o f the mammalia n brain , and the pac e o f cel l proliferatio n and th e numbe r an d types of cells produced ca n be modulated b y a variety of genetic an d environmenta l factors . The dynamic s of the proliferatio n o f precursor cell s i n th e develop ing mammalia n brai n ar e shape d b y various factors. This chapte r describe s th e spatiotempora l feature s of cell proliferatio n and effect s o f neurotransmitters, major constituent s o f th e chemica l environmen t o f the developin g brai n tha t modulat e th e proces s o f precursor cell proliferation. It focuses on three neurotransmitters tha t ar e among th e mos t abundan t one s in th e developin g brain : dopamine , y-aminobutyri c acid (GABA) , an d glutamate . Th e goa l i s to presen t an overview of the organizatio n and activit y of precursor cell populations and discuss the potential fo r modulation of precursor cell activity by neurotransmitters. The discussio n i s limited t o cel l proliferatio n i n tw o exemplary structure s i n th e centra l nervou s syste m (CNS): the cerebral cortex and neostriatum.

ORGANIZATION O F PRECURSOR CELL POPULATIONS I N THE EMBRYONIC TELENCEPHALON

Cells of the nervou s system arise from precurso r cell s lining th e inne r o r ventricula r surface o f the neura l tube. Th e precurso r cell s ar e organized a s a pseudos tratified columna r epithelium. The epitheliu m is commonly referre d t o a s th e pseudostmtified ventricular epithelium (PVE) , a s i t border s th e ventricula r cavi ties of the embryoni c nervous system (Boulder Committee, 1970 ; Takahash i e t al. , 1993) . Th e regio n of the neura l tub e o f the feta l nervou s system that con tains th e PV E i s called th e ventricular zone (VZ) , a histologically define d regio n surroundin g th e ventri cles. Most cells in the PVE are bipolar and span virtually the entir e distanc e betwee n th e pia i (outer ) an d the ventricular (inner) surfaces (Fig . 2-1A), especially during earl y neura l tub e stages . A small numbe r o f cells ar e roun d an d contac t only the ventricula r surface; most of these cells are mitotically active. 9

FIGURE 2- 1 Diagra m o f th e architectoni c an d cytokineti c compartment s i n the developin g mammalia n brain . The ventricula r surface is to the lef t an d th e piai surface t o the right . A. Four cytoarchitectonic compartments ca n be recog nized o n th e basi s o f cytologica l criteri a (Boulde r Committee, 1970 ; Smart , 1976): the ventricula r zone (VZ , o r the ependyma l layer), subventricular zone (SZ, o r th e subependyma l layer) , the intermediat e zon e (IZ , o r th e externa l sagittal stratum), and three differentiatin g fields—a subplate (SP), cortical plate (CP), and margina l zone (MZ). Cells in the VZ are organized a s a pseudostratified columna r epithelium , wherea s the S Z and SP/CP/M Z contain randoml y oriented, nonepithelial cells. The I Z consists predominantly of columnar arrays of bipolar cells and growin g axons (black lines). The border s of the S Z and I Z are ambiguous. Th e dashe d lines indicate the approximate borders between th e different compartments . B . Precursor cells can b e classifie d int o two groups on the basi s of their interkineti c nuclea r migratory pattern: a pseudostratified ven tricular epithelium (PVE ) and a secondary proliferati ve population (SPP) . PVE cells undergo interkineti c nuclea r migratio n (blac k arrows) such tha t nuclei of cells in M phas e of the cel l cycl e are located a t the ventricula r surface an d th e nuclei o f cells i n S phas e ar e farthes t fro m th e ventricula r surface. Nucle i o f cells i n Gl an d G 2 phase s ar e at intermediate distances . SP P cells do not un dergo interkinetic nuclear migration, and the nucle i of cells in the differen t cel l cycle phases are scattered throughou t the VZ and I Z (soli d blac k arrows). PVE cells that leave the cel l cycl e exit the VZ, migrate throug h th e S Z and I Z (broken, curved arrows) , and settl e i n the CP/MZ. SPP cells that exit the cel l cycl e also migrate to the CP/MZ. C. The spatia l distribution of PVE, SPP , and CP/MZ cells i s illustrated to sho w the overla p among th e proliferativ e and postmitoti c populations. Th e PV E nea r the ventricula r surface is a "pure" population; how ever, the S-phas e zone (wher e nuclei of cells undergoing S phase are located) of the PV E overlap s with the SPP . Th e CP/M Z i s a populatio n o f predominantly postmitotic cell s near the pia i surface (right-hand side of the figure); however, in the SZ/IZ area, some postmitotic cell s mi x with precursor cells of the SPP . Thus, the architectonic and cytokinetic compartments show considerable overlap in the developing nervous system. (Source: Modifie d from Bhide , 1996) .

10

CELL PROLIFERATION 1 1 At late r stage s o f feta l development , variou s re gions interpose betwee n th e VZ and th e piai surface . Thus, additiona l architectoni c subdivision s an d a n additional precurso r populatio n emerge . A n earl y population o f precurso r cell s tha t emerge s i s calle d the secondary? proliferative population (SPP) . The ar chitectonic subdivision in which the SP P comes to lie is called the subventricular zone (SZ ) (Boulde r Com mittee, 1970) . Around the sam e time , a preplat e ap pears betwee n th e S Z an d th e pia i surfac e (Boulde r Committee, 1970 ; Marin-Padilla, 1983) . Shortly thereafter, th e preplat e i s split int o a marginal zon e (MZ ) and subplat e b y the cortica l plat e (CP) . Thes e thre e zones (the subplate, CP, and MZ) ar e fields that con sist largel y of postmitotic o r differentiatin g cell s pro duced b y the PV E and SP P precursors. In some parts of the nervou s system such as the cerebra l cortex, another architectoni c subdivisio n called th e intermediate zone (IZ ) develops . Th e I Z lie s betwee n th e S Z and th e M Z an d contain s growin g axons, migrating postmitotic neurons , an d cell s i n glia l lineages. Not e that the VZ, SZ, CP, and MZ are architectonic subdi visions o f th e nervou s system whereas th e PV E an d SPP are cytokinetic subdivisions of the precursor populations. Th e architectoni c an d cytokineti c subdivi sions overlap extensively, as shown i n Figure 2-1C. The PV E i s a n interactiv e communit y o f cells , coupled t o on e anothe r vi a intercellula r junction s such as gap junctions (LoTurco and Kriegstein , 1991 ; Bittman e t al, 1997) . PV E cell s shar e biochemica l characteristics with radial glia (Hockfield and McKay, 1985; Mille r an d Robertson , 1993 ; Malatest a e t al , 2000, 2003; Noctor et al, 2001; Weissman et al, 2003). Until recently, radia l gli a wer e considere d onl y t o serve th e dua l function s o f guiding newl y generate d neurons t o thei r fina l destination s an d actin g a s a transitional stage i n astrocytic ontogeny (Rakic , 1972; Voigt, 1989 ; Takahash i e t al. , 1990 ; Misso n e t al , 1991; Mille r and Robertson , 1993) . The PV E cell s underg o interkineti c nuclea r mi gration, a hallmar k o f pseudostratifie d epitheli a i n general (Sidma n e t al. , 1959 ; Fujita , 1960) . Thus , nuclei of the PV E cells are distributed within the VZ in accordanc e with the cel l cycl e phase (Fig . 2-1B). Nuclei undergoing mitoses are located a t the ventricular border, whereas the nucle i in the DN A synthesis or S-phas e ar e i n abventricula r locations . Nucle i o f cells passing through Gl an d G2 ar e located at intermediate distance s between th e tw o poles. Th e inter kinetic nuclea r migrator y pattern distinguishe s th e

PVE from th e SP P (His , 1887; Sauer , 1936 ; Boulde r Committee, 1970 ; Takahash i e t al, 1993) . SPP cells are not organized into an epitheliu m an d d o not display interkineti c nuclea r migratio n (Raki c e t al. , 1974). The SP P is a major componen t of the precur sor cel l poo l i n som e part s o f th e nervou s system , such a s the neostriatu m and th e cerebra l cortex , an d not i n othe r parts , suc h a s the hippocampu s o r th e hypothalamus. The PV E i s a "pure" populatio n o f precursor cell s in th e sens e tha t daughte r cell s produce d b y i t an d that embark upon the path o f differentiation int o neu rons o r gli a exi t th e PV E rapidl y (Miller , 1999) . I n other words , a snapshot o f the VZ , whic h house s th e PVE, reveal s proliferatin g precurso r cell s an d virtu ally n o differentiatin g neuron s o r gli a (Fig . 2-1C) . The SZ , b y contrast , whic h include s th e SPP , i s a mixture o f proliferating SPP cell s an d differentiating daughter cell s generated fro m bot h th e PV E an d th e SPP. Thes e difference s i n th e cellula r environment s may bestow unique physiological features on the PVE and SP P precursors and their differential response s t o environmental substances such a s ethanol (see Chapter 11) . The intimat e cell-cell contact amon g precur sor cell s i n th e V Z ma y facilitat e contact-mediate d signaling betwee n proliferatin g cells , whic h i s les s likely t o occu r i n th e SZ , althoug h contac t amon g precursor cell s an d postmitoti c cell s ma y b e mor e likely in the SZ . Close proximity of the SP P to growing axons in th e I Z (Fig . 2-1A), i n som e part s of the nervous syste m suc h a s th e cerebra l wall , increase s the molecula r diversit y of the environmen t further . Neurons and glia arise from bot h the PV E and th e SPP. Contributions o f the PV E and the SP P to the total number s of cells i n differen t part s of the brai n vary significantly. Fo r example , i t ha s bee n suggeste d tha t the PV E an d SP P generat e neuron s primaril y in th e deep and superficial laminae o f neocortex, respectively (Miller, 1989 ; Miller and Nowakowski, 1991) (Chapte r 11). In the neostriatum, the SP P appears to be the ma jor contributor of neurons and glia, whereas in the hip pocampus proper , th e PV E generate s al l cel l types . The PV E i n the hippocampa l formatio n is the sourc e of pyramidal neurons of the hippocampu s and granule neurons o f the dentat e gyrus , earl y in the feta l perio d (Angevine, 1965 ; Stanfield an d Cowan , 1979 ; Altman and Bayer , 1990) . I n th e dentat e gyru s cells derive d from th e PV E earl y i n th e embryoni c period settle a t the border between th e granule cell layer and the hilar region an d continu e t o proliferate , formin g a secon d

12 NORMA L DEVELOPMENT and apparentl y permanen t precurso r poo l calle d th e subgranular, or intrahilar zone (IHZ) (Angevine, 1965 ; Stanfield an d Cowan , 1979 ; Altman and Bayer , 1990) . The IH Z i s the sourc e o f the majorit y o f granule neu rons of the dentat e gyru s later in development, including adult life (Angevine , 1965 ; Bayer , 1982 ; Camero n

et al, 1993 ; Gag e et al, 1998 ; va n Praag e t al, 2002 ; Kempermann et al., 2004). Although th e architectoni c an d cytokineti c subdivisions mentioned abov e appea r i n nearl y all parts o f the developin g telencephalon , considerabl e regiona l variation occur s i n term s o f the siz e o f the differen t

FIGURE 2- 2 Regiona l difference s i n the siz e of the cytoarchitectoni c compart ments i n th e developin g brain . A histologica l sectio n i n th e corona l plan e though th e hea d o f an 11-day-ol d mouse fetus i s shown. The fetu s was exposed to th e S-phas e marke r bromodeoxyuridine (BrdU ) fo r 6 hours ; Brd U wa s injected int o th e pregnan t da m an d th e fetu s wa s harvested 6 hour s later . Th e section wa s processe d immunohistochemicall y t o revea l th e distributio n o f BrdU i n proliferatin g cells. BrdU-labele d cell s appea r dar k and surroun d th e ventricular cavities of the brai n (empty space a t the cente r o f the micrograph) . The compartment s o f the telencephalo n visibl e a t thi s corona l leve l ar e th e hippocampus (HIP) , cerebra l neocortica l wal l (CX) , latera l ganglioni c emi nence (LGE) , an d medial ganglionic eminence (MGE) . O n the left-hand side , the broken lin e marks the approximate boundar y between th e ventricular zon e (VZ) and the subventricular zone (SZ) , and the soli d lin e marks the boundar y between th e S Z and th e intermediat e zon e (IZ) . The broke n lin e i s drawn on the basi s o f difference s i n th e packin g densit y an d dispositio n o f th e BrdU labeled cell s (highe r densit y i n th e VZ , aligne d i n compariso n t o th e SZ , which ha s a lower density of BrdU-labeled cell s that are randomly distributed) . The soli d line i s at the boundar y between BrdU-labele d (precursor ) and unla beled (nonproliferatin g o r differentiating) cells . A relatively large VZ, SZ , an d IZ area i s in th e ganglioni c regio n (LG E an d MGE ) compare d t o that i n th e dorsal C X an d hippocampa l anlag e (HIP) , eve n a t thi s earl y stag e o f brai n development. Th e higher-magnificatio n image s (right ) sho w th e histolog ical divisions of VZ, SZ, and PPZ in the differen t telencepahli c compartments . The primordi a o f eyes (EYE ) are also visible in thi s section. (Source: Modifie d from Shet h and Bhide, 1997 )

CELL PROLIFERATIO N 1 3 subdivisions an d th e tim e o f emergence o f each sub division i n th e cours e o f embryonic development . I n fact, these regiona l difference s distinguish th e precur sors o f the differen t telencephali c compartments . I n the telencephalon of the developin g mous e brain, regional variatio n is evident a s earl y as gestational da y (G) 1 1 (the day of conception i s designated a s GO). In coronal section s o f 11-day-ol d feta l mous e brain , a sizeable SZ can be seen i n the lateral ganglionic emi nence (GE ) an d media l GE . Thes e specialize d seg ments o f the S Z contain precursor s of neurons i n th e basal ganglia and a source o f neocortical G AB A neu rons (Fig . 2-2) . Th e murin e cerebra l cortex , hip pocampus, an d hypothalami c region s d o no t hav e a SZ at G11. In fact, an SZ does not develop i n the hippocampal and hypothalamic regions , although it does develop i n th e cerebra l corte x abou t 2- 3 day s later (Miller, 1999) . The V Z i s the earlies t structure i n the developin g nervous system , thu s i t contain s th e earlies t formin g precursor cel l populatio n (i.e. , the PVE) . Th e V Z is present i n al l part s o f th e Gi l telencephalo n (Fig . 2-2). A distinct and sizeabl e MZ , consisting o f newly generated differentiatin g cells, is evident i n the latera l and media l GEs by Gl 1, whereas the othe r region s of the telencephalon hav e a relatively smaller MZ at this age. Thus, the siz e and distributio n o f both proliferative and postmitotic subdivisions of the telencephalon show considerabl e variatio n i n th e feta l forebrain . The emergenc e o f heterogeneit y withi n th e telen cepahlic anlage reflect s influence s of transcription factors (Puelle s and Rubenstein , 1993 ; Rubenstein e t al., 1998) and/o r neurotransmitter s (Ohtan i e t al. , 2003 ) that distribute in a regionally specific manner . The re gional variation also foreshadows the structura l differ ences tha t emerg e i n thes e region s a s developmen t proceeds.

MOLECULAR REGULATIO N OF CELL PROLIFERATION

Cell proliferatio n in th e nervou s syste m i s a n intri cately regulated process . The regulatio n occur s by the concerted action s of a variety of genetic an d environ mental factor s a t th e leve l o f the cyclin g cell s i n th e PVE an d SPP . Ther e ar e a t leas t thre e interrelate d tiers o f regulation: (1 ) th e tota l numbe r o f cells pro duced b y the PVE and SPP , (2 ) the emergenc e o f specific phenotype s amon g th e daughte r cells , an d (3 )

FIGURE 2- 3 Rol e played b y the cyclin-dependen t kinase (Cdk ) inhibito r p27 KlP1 i n the regulatio n o f cell cycle a t th e Gl-to-S-phas e transition . Th e progres sion of the cel l cycle can be regulated at the transition from th e G 2 t o M phas e o r from th e G l t o S phase . Genetic an d environmenta l factor s tha t modulat e progression o f cell cycl e i n th e developin g brai n ap pear t o influenc e th e Gl-to-S-phas e transition . Thi s transition i s regulate d b y Cdk s (Cdk2 , Cdk4 , an d Cdk6) an d their cyclin partners (cyclins A, E, and D) . Association o f Cdks an d thei r cycli n partner s inacti vates the retinoblastom a protein (Rb ) by phosphorylation, which i n turn promote s th e transitio n fro m G l to S phase b y releasing th e transcriptio n facto r E2F . p27KlP1 inhibit s th e activit y of the Cdk-cycli n com plexes an d therefor e inhibit s th e transitio n fro m G l to S phase. Thus , geneti c an d environmenta l factor s that alte r th e expressio n an d activit y of p27 KlP1 ca n modulate cel l proliferation in the developin g brain.

the temporal sequenc e of cell productio n an d specification tha t i s unique t o eac h regio n o f the brain . A n understanding of the molecula r mechanism s that regulate these processe s i s important no t onl y for appreciating cytogenesi s durin g norma l developmen t bu t also fo r understanding (a ) the wa y in which cel l pro duction ca n b e induce d i n specifi c region s o f th e adult brain t o repai r injur y b y trauma o r diseas e an d (b) th e mechanism s b y which neoplasms i n the CN S originate. Genetic an d environmenta l factor s regulat e cel l proliferation b y actin g upo n th e molecula r machin ery that regulate s the cell cycle . This machinery con sists of protein kinase s and thei r regulator y subunits , known a s cyclin-dependen t kinase s (Cdks ) an d cy clins, respectively (Fig. 2-3). Essentially , Cdk activity

14 NORMA L DEVELOPMEN T depends o n the synthesis and destructio n o f cyclins as well a s on th e functio n o f Cdk-activating kinase s and Cdk inhibitor s (LaBaer et al. , 1997 ; Morgan , 1997 ; Cheng et al, 1999 ; Sher r and Roberts , 1999 ; Murray, 2004). Eac h Cd k ha s a preferre d cycli n partner(s) . For example , Cdk 2 associate s wit h cyclin s A and E , whereas Cdk 4 an d Cdk o associat e wit h D-typ e cy clins. Associatio n of Cdk s wit h cyclin s results i n th e regulation o f the cel l cycle at two principal transition points: th e transition s fro m th e G l t o S phase , an d from G 2 t o M (Murra y and Hunt , 1993 ) (Fig . 2-3) . Cdk2, Cdk4 , an d Cdk 6 ar e principall y involved i n the regulatio n of the transitio n from G l t o S, whereas Cdkl regulate s the G2- M transition . The transitio n from G l t o S phas e i s associate d wit h additiona l mechanisms o f regulatio n involvin g th e retinoblas toma protei n (Rb ) an d th e transcriptio n facto r E2F . Association o f Cdk s wit h cyclin s inactivate s R b b y phosphorylation, which i n tur n result s in th e releas e of E2F . E2 F promotes th e transitio n fro m G l t o S and DNA synthesis (Dyson, 1998 ; Frolov e t al, 2001 ). Control ove r cel l cycl e progressio n a t th e Gl-to S-phase transitio n i s exercise d b y Cd k inhibitors , which inhibi t th e activit y of th e Cdk-cycli n com plexes (Ferguso n e t al. , 2000) . Inhibitio n o f Cdk ac tivity b y specifi c Cd k inhibitor s suc h a s p27 KiP] o r p21Wafl suppresse s the entr y into the S phase (Zind y et al, 1997 , 1999 ; Delall e e t al. , 1999 ; Siegenthale r and Miller , 2005) (Fig . 2-3). Muc h o f the regulation of cytogenesi s i n th e developin g brai n i s thought t o occur a t th e Gl/ S transitio n (Cavines s e t al, 1996 ; Ross, 1996) . Transcriptio n factors , growt h factors , hormones, an d neurotransmitter s ar e amon g th e most commo n regulator s o f cel l proliferatio n i n the developin g brain . They are believe d t o act upo n the Gl/ S transitio n poin t t o increase o r decrease th e output of neurons or glia (Cavines s et al., 1996 ; Ross , 1996). Much o f ou r understandin g of cel l cycl e regula tion derive s from i n vitro studies. That said, the PV E and SP P cell s i n th e intac t mammalia n brai n diffe r from th e precurso r cells maintained i n a Petri dish i n one importan t aspect. PV E an d SP P cell s i n th e in tact brain serve a histogenetic agenda, which warrants generation o f a specific numbe r o f specific cel l type s in a spatiotemporal gradien t over a restricted perio d of time. Therefore, t o understand mechanism s tha t reg ulate CNS histogenesi s we need t o analyze cell prolif eration i n population s o f PV E o r SP P cell s i n th e intact brain . A challeng e face d b y developmenta l

neurobiologists i s to assimilat e mechanisti c insights , gained b y analysis of cell cycl e regulator y machiner y from studie s o f dissociated cells , int o analyse s of cel l cycle kinetic s an d cel l outpu t fro m population s o f PVE o r SP P cells i n th e intac t brain . Th e followin g section describe s recen t progress i n efforts alon g those lines. Complementary insigh t into these issue s is provided b y studyin g th e proliferativ e response i n th e developing brai n followin g ethano l exposur e (se e Chapters 1 1 and 12) . Much of our knowledg e o f th e kinetics of PVE and SP P cell cycle in the intac t brain is derive d fro m analyse s o f neocortica l PV E cells . Therefore, th e followin g section focuse s mainl y o n cell cycl e kinetics of neocortical PVE .

CELL CYCLE KINETICS

AND DYNAMIC S OF CELL PROLIFERATION I N THE NEOCORTE X

In an y tissue or organ system, the precurso r cell popu lation initiall y expands by producing large numbers of new precurso r cells . Th e precurso r cel l populatio n subsequently deplete s itsel f through (a ) the productio n of large number s o f daughter cells , which exi t the cel l cycle, differentiate , an d acquir e matur e phenotypes , and (b ) th e deat h o f cyclin g cell s (se e Chapte r 5) . This type o f biphasic growt h pattern ensure s the pro duction o f a larg e numbe r o f cells withi n a specifie d time interval . Th e neocortica l PV E follow s thi s growth pattern. It undergoes two phases of growth: an initial phas e o f expansion durin g which mos t daugh ter cell s retur n t o th e precurso r pool , an d a late r phase o f depletion whe n mos t o f the daughte r cell s differentiate int o neuron s o r glia (Smart , 1976 ; Cavi ness e t al, 1995 ; Mille r an d Kuhn , 1995 ; Takahash i et al, 1996 , 1997) . Interestingly , during the phas e of expansion, th e PV E grow s i n th e tangentia l dimen sion, and thi s growth result s in expansion o f the PV E such tha t i t cover s th e underlyin g subcortica l struc tures. There i s little expansio n o f the PV E i n th e ra dial dimension ; growt h i n th e radia l dimensio n i s seemingly reserve d fo r late r developmenta l period s when newbor n neuron s an d gli a ar e release d fro m a depleting PV E (Miller, 1989) . The us e o f th e S-phas e marke r [ 3H]thymidine (pH]dT) revolutionize d th e analysi s of neuroepithelial cel l cycl e kinetic s b y permitting identificatio n of nuclei i n S phase an d tracking the nuclei labele d i n S

CELL PROLIFERATIO N 1 5 phase a s they passed throug h G2 , M , and Gl phase s of the cel l cycl e (Sidma n e t al. , 1959 ; Angevine and Sidman, 1961 ; Fujit a an d Miyake , 1962 ; Sidman , 1970). I t becam e possibl e t o estimat e cel l cycl e pa rameters an d th e tim e o f generation o f different cel l types throug h cumulativ e o r puls e labelin g wit h [3H]dT. Recen t developmen t o f nonradioactiv e S-phase marker s bromodeoxyuridin e (BrdU ) an d iododeoxyuridine furthe r enhance d th e versatilit y of these method s (Mille r and Nowakowski , 1988, 1991 ; Takahashi e t al , 1992 ; Haye s an d Nowakowski , 2000; Tarui et al., 2005). S-phase labelin g methods hav e been exploited, often combinin g th e radioactiv e an d nonradioactiv e markers t o perfor m sophisticate d analyse s of cell cy cle kinetic s an d cel l outpu t function s o f PV E an d SPP cells in the embryoni c murine neocortex (Miller and Nowakowski , 1991; Cavines s e t al, 1995 ; Haye s and Nowakowski , 2000 ; Takahash i e t al , 2002 ; Siegenthaler an d Miller , 2005 ; inter alia) . This work shows that the neocortica l PVE cells in fetal mic e executed 1 1 cell cycle s throughout th e cours e of generation of neocortical neuron s ove r the week from Gi l to G17. During the 1 1 cell cycles, the length of the cel l cycle increase s (from abou t 8 to 1 8 hours) (Fig. 2-4) , the increas e bein g du e t o th e lengthenin g o f G l (from abou t 3 t o 1 2 hours). This patter n i s also de tected i n ra t neocortica l proliferativ e zone s (Mille r and Kuhn, 1995). The probabilit y that a new daughter cel l exit s th e cell cycl e (calle d Q ) increase d durin g th e perio d of cortical neuronogenesi s (Miller , 1993 , 1999 ; Mille r and Kuhn , 1995 ; Takahashi e t al, 1999) . I t was near zero fo r cel l cycl e 1 (o n Gi l fo r th e mouse ) an d nearly 100 % for cell cycle 1 1 (G16-17). Eac h cell cycle i s associated wit h specific values of Q an d cel l cycle length (i.e. , the lengt h o f the Gl-phase) , the tw o parameters that chang e (increase ) systematically over the cours e o f the 1 1 cel l cycle s (Fig . 2-4) . Th e Q value reache s th e halfwa y mar k durin g th e sevent h and eight h cel l cycles , o n approximatel y G1 4 (Fig . 2-4). Thi s probability value marks a significant mile stone i n the lif e histor y of neocortical PVE , a s it indicates th e switc h fro m th e expansiv e to th e depletiv e phase of growth and fro m tangentia l to radial expansion o f the neocortex . Furthermore , i t mark s a sig nificant mileston e i n neocortica l neuronogenesis : generation of neurons for the infragranula r layers (layers V an d VI ) cease s an d tha t fo r the granula r layer (layer IV ) and supragranula r layer s (layer s I I an d III )

begins (Fig . 2-4) . Thus , quantitative estimate s o f the values o f Q a t eac h intege r cell cycl e hav e enable d the linkag e of Q to laminar destination of the daugh ter cells . In other words , a novel concept emerges, i n which th e mechanism s regulatin g Q ar e linke d t o mechanisms regulatin g cel l fat e o r cel l clas s (Taru i et al, 2005). Neuronogenesis acros s th e cortica l mantl e doe s not procee d simultaneously ; i t abides b y various spatiotemporal gradients (Smart, 1961; Smar t and McSh erry, 1982 ; Miller , 1988a ; Baye r and Altman , 1991) . The transvers e gradients reflec t th e gradient s in cel l cycle kinetics, especially the valu e o f Q, i n th e PV E (Caviness et al., 2000, 2003; Tarui et al, 2005). Thus, PVE cell s are distribute d along a spatia l continuu m of cell cycl e parameters an d Q . PV E cell s located a t the rostrolatera l locatio n ar e mor e advance d tha n those a t the caudomedia l location with respect to cell cycle length and values of Q. That is, rostrolateral precursor cell s hav e longe r cel l cycl e time s an d highe r values of Q than those o f caudomedial precurso r cell s on any given day during the interva l Gil t o G17. For example, a given location i n the neocortical PV E contains precursor cell s passin g through th e entir e rang e of cell cycle times and Q values over the interva l Gil to G17. At any given time , th e neuroepitheliu m con tains a rang e o f cell cycl e time s an d Q values . Cells destined t o multiple layer s in multiple neocortica l ar eas aris e contemporaneousl y a t multipl e location s within the neuroepithelium . Apart fro m th e tangentia l neurogeneti c gradien t discussed above, neocortical neuronogenesis proceeds along a radial dimension, a characteristic commo n t o laminar structures throughout th e nervou s system. At any give n locatio n o f the neocortica l anlagen , neu rons are generated i n an inside-ou t pattern (Angevin e and Sidman , 1961 ; Berr y et al , 1964 ; Berr y and Rogers, 1965 ; Caviness , 1982 ; Miller , 1985) . Deep layer neuron s ar e generate d earlie r than th e superfi cial laye r neurons . Usin g a doubl e S-phas e labelin g method, Takahashi an d colleague s (1999 ) correlate d the lamina r position of a neocortica l neuron wit h its "birth hour. " Throug h th e us e o f this method , the y were able to correlate the time of generation and laminar destination of neocortical neurons with considerably higher spatiotempora l resolutio n tha n ha d bee n possible previously. A mode l correlatin g th e probabilit y o f cell cycl e exit (which is equivalent t o Q) with th e probabilit y of the destinatio n o f a neuro n i n a specifi c neocortica l

16 NORMA L DEVELOPMEN T

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FIGURE 2- 4 Spatiotempora l gradient s o f cell proliferatio n and neurogenesi s i n th e cerebra l cortex . Neurogenesis in th e cerebra l cortex progresses along tangential (A ) and radia l (B) gradients. A. Neo cortical neurogenesi s begin s at the rostroventrolatera l regio n o n gestationa l da y (G) 1 1 i n mic e an d proceeds towar d the caudodorsomedial regio n in a systematic gradient (curved arrow). This transverse neurogenetic gradien t translates into a correspondin g gradient in cel l cycle lengt h o f the precurso r population an d th e probabilit y of cell cycl e exi t (Q) for the daughte r cells . Thus, precursor cell s i n the pseudostratifie d ventricula r epithelium (PVE ) i n th e rostroventrolatera l region have longer cell cycle time and highe r values of Q compare d t o their counterparts in the caudodorsomedia l region. B. Neurogenesis als o proceeds alon g a radial gradient. Neurons o f the dee p layers are produced ear lier than those in the superficia l layers . Each neocortica l PVE cell in mice, on average, undergoes 1 1 cell cycles over a 7-day period fro m Gi l t o G17. Cell cycle length increase s with each cel l cycle and each cycl e is associated wit h a unique value of Q. Thus, the cel l cycle length increase s from abou t 8 to 1 8 hours over the cours e o f 1 1 cell cycles . The valu e of Q increase s from abou t 0. 1 t o 0.8 , ap proaching the maximal value of 1.0, during this interval and reaches th e halfwa y mar k (0.5) betwee n cell cycle s 7 and 8 on G1 4 (broke n line an d asterisk) . Each cel l cycl e produce s cell s destine d fo r specific neocortica l layer(s). Thus, cell cycle number, cell cycle length, Q , an d laye r destination are linked to one anothe r durin g normal development . (Source: Base d on data o f Takahashi et al., 1999 ; Tarili et al., 2005)

layer wa s the n develope d b y Takahash i an d col leagues (1999) . Theoretically , neuron s produce d a t any time point during the neurogenetic sequenc e ca n have som e probabilit y of occupying an y neocortica l layer. I n reality , however , durin g norma l develop ment neuron s generate d durin g cel l cycl e 1 hav e

nearly a 100 % probabilit y of occupying laye r VI an d 0% probability of occupying layer II—III, and neuron s generated durin g cel l cycl e 1 1 (th e fina l cel l cycle ) have a near 100 % probability of occupying laye r II—III and 0 % probabilit y of occupyin g laye r VI . Sinc e a given cell cycle number is associated wit h given values

CELL PROLIFERATION 1 7 of Q, anothe r wa y of interpreting these finding s i s that the probability that a cell arising from a given cell cycle will occupy a specific laye r is tightly linked t o the val ues of Q. Lower Q values predict destination to deeper layers and higher values predict destination t o more superficial layers . What factors set the probabilit y values? Overexpression o f the cd k inhibito r p27 kiP* i n th e PVE increases Q, and that the neurons produced during the cell cycles with the experimentally elevated Q values assum e superficia l lamina r position s tha t would be appropriate for the higher valu e of Q (Taru i et al. , 2005) . Therefore , Q an d lamina r destinatio n appear t o b e highl y correlated . Also , thes e finding s suggest that p27 klP* expression and activit y play a role in settin g the value s of Q. Recen t dat a sho w that an other cdk inhibitor , p21, i s also involved i n cell cycl e exit (Siegenthale r an d Miller , 2005) . Furthermore, a variety of genetic an d environmenta l factors , such as transcription factors , growt h factors , an d neurotrans mitters, influenc e intracellula r p27 kiPJ an d p21 wofl content. Th e combine d action s o f these factor s ma y regulate Q and , therefore , neocortica l neurona l fat e during normal development . The discussio n abov e pertains directl y to the regu lation o f cel l proliferatio n i n th e neocortica l PVE . Neocortical neuron s aris e not only from th e neocorti cal PVE but also from the proliferative cells in the GE . Neocortical PV E i s the sourc e o f neocortica l (gluta matergic) projection neurons (PNs) , which constitut e 70%-90% o f al l neocortica l neuron s (Peter s e t al , 1985; Re n e t al, 1992 ; Beaulieu , 1993) . I n rodents , the majorit y o f GABAergi c loca l circui t neuron s (LCNs) o f th e cerebra l cortex , whic h constitut e 10%-30% o f all neocortica l neurons , i s produced i n the media l an d cauda l GE s o f th e basa l forebrai n (Anderson e t al, 1997a ; Tamamaki e t al, 1997 ; Lavdas et al, 1999 ; Powel l e t al., 2001; Nery et al., 2002; Ang et al, 2003). These LCNs arrive in the cerebra l cortex via tangential migrator y pathways. In primates , however, i t appear s tha t mos t GABAergi c LCN s o f the cerebra l corte x derive from th e neocortica l PV E and only a minority come from th e GÈ (Letinic et al., 2002). Although neocortica l PN s an d LCN s ar e pro duced i n different locations , bot h types of neurons located i n th e sam e neocortica l laye r appea r t o b e produced o n the same day , at least i n rodents (Miller , 1985, 1988b ; 1999 ; Fairé n e t al , 1986 ; Goba s an d Fairén 1988 ; Valcanis and Tan , 2003). Neocortical gli a ar e generate d i n th e SP P (Smart, 1961 ; Smar t an d LeBlond , 1961 ; L e Vine

and Goldman , 1988a , 1988b ; Levison and Goldman , 1993). One notabl e exceptio n to this pattern is that of radial glia , which commonl y hav e cel l bodie s i n th e PVE, an d may undergo transformation into astrocytes near th e en d o f th e neuronogeneti c epoc h (Voigt , 1989; Takahash i e t al , 1990 ; Misso n e t al , 1991 ; Miller an d Robertson , 1993) . A distinguishable SP P (SZ) appear s i n th e neocortica l proliferativ e regio n soon afte r th e first cortical neuron s ar e generated, b y G13 i n th e mous e (Miller , 1989 ; Takahash i e t al , 1995). At this time, th e siz e of the SP P is <10% of the total neocortica l precurso r poo l (th e PV E bein g th e dominant population) , bu t th e SP P size increase s t o nearly 50 % until abou t G16 . Thus, a t late r stage s of gestation, a s th e PV E wane s th e SP P become s th e dominant population . Th e cel l cycl e lengt h o f th e SPP doe s no t appea r t o chang e systematicall y wit h age an d remain s relativel y constan t a t aroun d 1 5 hours, durin g th e interva l G1 3 t o G1 6 (Takahash i et al., 1995) . The SP P may also contain a population of precursor cell s that aris e from th e PV E an d "wait " in th e S Z for varying lengths o f time (u p to 48 hours) prior t o producin g daughte r cell s tha t differentiat e into neuron s (Baye r and Altman, 1991 ; Miller, 1999 ; Noctoretal, 2004) .

CELL CYCLE KINETIC S AND NEUROGENESI S IN THE NEOSTRIATUM

Unlike the cerebra l cortex , th e neostriatu m i s a non laminar structure . I t consist s o f the caudat e nucleu s and th e putame n an d i t i s th e inpu t cente r o f th e basal ganglia . I n histologica l section s o f th e roden t brain i t i s difficul t t o distinguis h th e boundar y be tween th e caudate nucleu s an d the putamen, makin g the ter m neostriatum o r caudoputamen particularl y useful. Nearl y 90 % o f neostriata l neuron s ar e GABAergic projectio n neuron s an d th e remainin g cells ar e cholinergi c LCN s (Gerfe n an d Wilson , 1996). Neostriata l projectio n neuron s ar e generate d in th e latera l GE (Hewitt , 1958 ; Smart , 1976 ; Smar t and Sturrock , 1979 ; Lammer s e t al, 1980 ; Fentres s et al., 1981) , and the LCN s are generated i n the medial G E o r th e preoptic/anterio r entopeduncula r ar eas (Marin et al, 2000). The latera l G E consist s of a larger SP P tha n tha t o f the neocortica l neuroepithe lium. Th e SP P i n th e latera l G E i s establishe d a s early a s Gil i n th e mous e (Bhide , 1996 ; Shet h an d

18 NORMA L DEVELOPMENT Bhide, 1997 ) (Fig . 2-2) . Therefore , th e SP P i s likely to b e a majo r sourc e o f neurons o f the neostriatum . This findin g concur s wit h tha t i n th e neocorte x (se e Chapter 11) . Studie s o n th e Distalless-relate d tran scription factor s dlxl, dlx2, an d dlxS, whic h regulate cell proliferatio n and migratio n i n th e GE , confir m the origi n of striatal projection neuron s from th e SP P of th e G E (Anderso n e t al, 1997b ; Stenma n e t al. , 2003). Furthermore , analyse s o f transgeni c mic e show that the radia l glia i n the PV E o f the latera l an d medial GE s giv e ris e t o nearl y all o f th e gli a i n th e neostriatum and onl y to a subset of LCNs (parvalbumin an d Er81-positiv e LCNs). Two studies using S-phase labelin g methods com parable t o thos e use d fo r th e neocortica l PV E esti mated th e cel l cycl e parameters and cel l outpu t fro m the PV E o f the latera l G E (Bhide , 1996 ; Shet h an d Bhide, 1997) . The cel l cycl e wa s longer an d th e val ues o f Q wer e greate r i n th e latera l G E o f mice o n Gil an d G12 compared t o the corresponding parameters i n the neocortica l PVE . Estimatio n o f cell cycl e kinetics of the PV E o f the latera l GE a t later stages of development i s complicated b y th e fac t tha t durin g the late r developmenta l stage s thi s regio n acquire s migrating cell s produce d i n the adjacen t media l G E (Anderson et al., 2001). The strikin g structura l and functiona l complexity of th e neostriatu m (Graybiel , 1990 ; Gerfen , 1992 ) has le d t o a numbe r o f investigation s of the tim e o f generation o f neostriata l neurons . Earl y studie s fo cused o n the tim e of generation o f neurons destine d for th e patc h (striosome ) an d matri x compartments . These can b e identified on the basi s of distribution of opioid receptor s o r neuropeptide conten t of the neu rons. Neuron s destine d fo r th e patc h compartmen t are generate d earlie r than thos e destine d fo r the ma trix i n rodents , carnivores , and primate s (Bran d an d Rakic, 1979 ; Graybie l an d Hickey , 1982 ; Fishel l an d van der Kooy, 1987 ; Fishel l e t al, 1990; Son g and Harlan, 1994) . Other studies, which examined the time of generation o f neostriata l neuron s regardles s o f loca tion o f a neuron withi n the patc h o r matrix compartments, foun d some overla p in th e tim e o f generation of PN s an d LCN s (Bran d an d Rakic , 1979 ; Fernan dez et al, 1979; Bayer , 1984). In rodents, striatal neu rons containing different type s of neurotransmitters or neuropeptides ar e generate d a t distinc t period s o f neurogenesis (Semb a e t al. , 1988 ; Sadiko t an d Sas seville, 1997) . Neostriata l neurogenesi s als o follow s spatiotemporal gradient s (Fernande z e t al. , 1979 ;

Marchand an d L a joie, 1986 ; Bayer and Altman, 1987 ) that ar e les s systematic than th e transvers e and radial gradients definin g neocortica l neuronogenesis . I n fact, gradient s o f neuronogenesi s ar e difficul t t o de fine in nonlaminar structures suc h a s the neostriatu m (Bayer and Altman, 1987) .

NEUROTRANSMITTERS AN D

CELL PROLIFERATION

Cell proliferatio n i s shape d b y a variet y of environ mental factor s suc h a s growth factor s an d neurotrans mitters. Growth factors , as the nam e indicates, tend t o be growth-promotin g agent s an d thei r influence s o n cell proliferatio n are no t unexpected . Som e growt h factors, such as transforming growth factor (3 , however, have a negative effec t o n cel l proliferation (see Chapter 11) . Neurotransmitter s hav e well-characterize d functions a t the synaps e i n the matur e brain. Neuro transmitters and thei r receptors appear in the nervous system earl y i n th e feta l period , befor e synapti c contacts develop . Thus, it can be inferred that neuro transmitters influenc e developmental processes . Non vesicular transpor t an d releas e o f neurotransmitter s (Démarque e t al. , 2002 ) an d paracrin e mechanism s for neurotransmitte r receptor activation (Owen s et al., 1999) hav e bee n propose d i n th e feta l brain . Suc h mechanisms ar e distinc t fro m thos e use d b y neuro transmitters i n th e matur e brain , a s are the composi tion an d activit y o f neurotransmitte r receptors . Fo r example, GAB A receptors i n th e feta l brai n diffe r i n their subunit composition, affinity , sensitivity , and sig nal transductio n mechanism s relativ e to thei r coun terparts i n th e matur e brai n (Owen s an d Kriegstein , 2002). Thus, a rol e fo r neurotransmitters in th e feta l brain tha t ma y b e separat e fro m thei r rol e a t th e synapse i n th e matur e brai n appear s likel y (Lander , 1988; Levit t et al, 1997 ; Nguye n e t al, 2001; Owen s and Kriegstein , 2002; Ohtani e t al, 2003). The influ ence of dopamine, GABA, and glutamate o n cell pro liferation an d neurogenesi s i n th e neocorte x an d th e neostriatum is discussed below . Dopamine Dopamine appear s in the developin g mouse brai n as early as G13 (Ohtan i e t al., 2003) . The cerebra l cor tex an d th e neostriatu m expres s amon g th e highes t amounts o f dopamin e receptor s i n th e feta l brai n

CELL PROLIFERATIO N 1 9 (Sales e t al , 1989 ; Guennou n an d Bloch , 1993 ; Schambra e t al, 1994 ; Bakowsk a and Morrell , 1995 ; Jung and Bennett , 1996 ; Dia z e t al., 1997 ; Shearma n et al., 1997 ; Ohtani et al., 2003). Dopaminergic axons originating in the midbrai n penetrat e th e VZ and S Z of th e prefronta l cortex an d latera l G E (precurso r of the neostriatum ) o f the mous e brai n a s early as G13, and growt h cone s of these axons are i n close proxim ity t o PV E an d SP P cell s i n thes e region s (Ohtan i et al., 2003; Popolo e t al., 2004) . Dopamine recepto r activatio n influences cell pro liferation i n th e latera l GE an d th e dorsomedia l pre frontal corte x (PFC) . I n th e latera l GE , Dl-lik e receptor activatio n reduce s entr y int o S (i.e. , an an timitogenic effect ) an d D2-lik e receptor activatio n in creases th e Gl/ S transitio n (i.e. , a mitogeni c effect ) (Zhang and Lidow, 2002 ; Ohtani et al, 2003 ; Popol o et al, 2004 ; Zhang et al, 2005) . Dopamine itself produces Dl-lik e effects , presumabl y becaus e o f th e

preponderance o f Dl-lik e bindin g site s i n th e feta l brain (Ohtan i e t al, 2003) . Dopamine recepto r acti vation influence s cel l proliferatio n i n th e neuroep ithelium o f the PF C i n a manner comparabl e t o tha t in the lateral GE . Activating dopamine receptor s differentiall y affect s precursor population s i n th e V Z an d S Z (Fig . 2-5) . Precursor cell s i n th e V Z respon d predominantl y t o Dl-like receptor activation , whereas the precursors in the S Z respond t o D2-like recepto r activatio n (Ohtani et al, 2003 ; Popol o e t al, 2004) . VZ cells i n the lat eral ganglionic region ar e more responsiv e to Dl-lik e receptor activatio n tha n thei r counterpart s i n th e PFC; a large r dos e o f th e Dl-lik e recepto r agonis t SKF 8129 7 is required t o elici t a significant respons e from th e PF C (Popol o e t al , 2004) . Furthermore , D2-like recepto r activatio n di d no t produc e change s in th e proliferatio n o f precursor cel l i n the neocorti cal VZ, althoug h i t produced effect s i n the V Z o f the

FIGURE 2- 5 Schemati c representatio n o f distributio n o f Dl-lik e an d D2-lik e receptors (R) . Dl-R and D2- R i n the ventricular an d subventricula r zone s (V Z and SZ , respectively ) of the telencepahli c neuroepitheliu m (A ) and th e effect s of dopamine recepto r activatio n on cel l proliferatio n (B ) are shown. A . On th e basis o f dat a o n incorporatio n o f th e S-phas e marke r bromodeoxyuridin e (BrdU), precurso r cell s i n th e V Z appea r t o respon d t o Dl- R activatio n t o a greater extent than D2- R activation , whereas the precursor cells in the S Z have an opposite respons e (Ohtan i e t al, 2003 ; Popolo e t al, 2004) . B. Activation of Dl-R decrease s th e Brd U labeling index , which i s a reflection o f a decrease i n the relativ e proportions of proliferating cells entering S phase. Activation of D2R produces th e opposit e effect (i.e. , increase s Brd U labelin g index) . Dopamine produces effect s simila r to thos e produce d b y Dl-R activation . (Source: Modi fied from wor k by Popolo e t al, 2004) .

20 NORMA L DEVELOPMEN T lateral ganglioni c region. Thus , significan t regiona l variation exist s in th e responsivenes s of progenitors i n the neuroepithelia l domain s of the dorsa l and ventra l forebrain of the mouse fetus. This pattern likel y reflects variation i n th e relativ e abundanc e o f dopamin e re ceptor bindin g site s or differences i n signa l transduction mechanisms . The Dl-lik e famil y o f dopamin e receptor s i s coupled to G proteins, which activate adenylate cyclase and increase intracellula r cyclic adenylate monophosphat e (cAMP) concentration s (Monsm a e t al. , 1990) . In creased intracellula r cAM P conten t leads t o increase s in the amount s of the Cd k inhibito r p27Kl P ] , which in turn inhibit s entry int o S (Kat o e t al. , 1994 ; L u an d DiCicco-Bloom, 1997) . Thus , th e antimitogeni c ef fects o f Dl-lik e recepto r activatio n ma y b e mediate d via th e cAMP-p27 KlP1 pathway. Other report s suggest that th e effect s o f Dl-lik e recepto r activatio n o n neocortical cel l proliferation is mediated vi a a cAMP independent pathway involving Raf-1, a component o f the recepto r tyrosin e kinase-mitogen-activate d kinas e pathway (Zhang et al., 2005). Therefore, dopamin e re ceptor activation may activate multiple second messen ger systems in neuroepithelial cells . GABA GABA receptors appear early in the neuroepitheliu m and M Z o f the feta l brai n (Huntle y et al, 1988 ; Lo Turco and Kriegstein , 1991 ; LoTurco e t al., 1995 ; Behar e t al., 1998 ; Hayda r et al., 2000). GABA receptors in th e feta l brai n ar e excitator y an d influenc e cell proliferation i n the VZ and SZ . Early studies indicate that GABA A recepto r activatio n decrease d entr y int o S (antiproliferative ) i n neocortica l precurso r cell s (LoTurco e t al., 1995) . These effects appea r to be me diated b y endogenously released GABA , as inhibition of the GABA A receptor s increase d Gl - to S entr y i n expiants o f neocortica l tissu e take n fro m ra t fetuse s (LoTurco e t al, 1995) . The effect s o f GABA A recep tor inhibition were developmentally regulate d i n tha t the effect s wer e pronounce d a t G19 , bu t no t a t G14-G16 (LoTurc o e t al , 1995) . Presumably , thi s finding reflects maturational changes i n the activity of the receptors . Th e effect s o f GABA A recepto r activa tion, lik e th e effect s o f dopamine recepto r activation , differ betwee n th e VZ and the SZ . GABAA receptor activation increase s Gl - t o S entr y i n th e V Z an d de creases i t i n th e S Z (Hayda r e t al , 2000) . Th e

intracellular signalin g cascad e tha t mediate s th e ef fects o f GAB A recepto r activatio n o n th e cel l cycl e machinery is not fully understood . The effect s ma y be secondary t o suppressio n o f the actio n o f mitogeni c factors suc h a s basic fibroblast growth factor (Antono poulosetal, 1997) . Glutamate Glutamate recepto r activatio n influences the prolifera tion o f precursor cell s i n th e cerebra l corte x an d th e neostriatum. Activatio n o f bot h N-methyl-D-aspartat e (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazol e propionate (AMPA)/kainat e receptors influence s Glto-S entry in the precursor cells . Early studies indicate d that activatio n of AMPA/kainate receptors b y endoge nous glutamate decrease d entr y into S in the neocorti cal V Z (LoTurc o e t al. , 1995 ; Hayda r e t al , 2000) . Activation o f AMPA/kainit e receptors , however , pro duced th e opposit e effect s i n the S Z by increasing en try int o S (Hayda r e t al, 2000) . Although glutamat e receptor activatio n als o influence d proliferatio n o f neostriatal precursors , th e effect s wer e mediate d vi a the NMD A recepto r famil y rathe r tha n throug h th e AMPA/kainate receptor s (Lu k e t al. , 2003 ; Lu k an d Sadikot, 2004) . NMD A recepto r activatio n promote d entry into S among neostriata l precurso r cell s (Sadiko t et al, 1998 ; Luk et al., 2003; Luk and Sadikot , 2004).

CONCLUSIONS

The discussio n abov e presents a n overvie w of the ef fects o f neurotransmitters on th e proces s of cell prolif eration. A summary of the effect s o f dopamine, GABA , and glutamat e o n cel l proliferatio n in th e neocorte x and neostriatu m o f the developin g brain i s presented in Table 2-1 . Thes e data sho w that imbalanc e i n the neurochemical environmen t o f the developin g brai n can alte r th e proces s o f generatio n o f neuron s an d glia. Although th e directio n of the effect s ma y vary depending o n th e recepto r typ e activate d an d th e pre cursor populatio n involve d (i.e. , increas e o r decreas e in proliferation), it is reasonable t o assume tha t expo sure o f proliferatin g precurso r cell s t o therapeuti c or illici t drug s tha t targe t an d produc e imbalance s in th e neurotransmitte r system s ca n produc e signifi cant change s i n th e numbe r an d phenotyp e o f cell s generated.

CELL PROLIFERATION 2 1 TABLE 2- 1 Summar y of effects o f dopamine, GABA, and glutamat e receptor activation on S-phas e labeling indices in neocortical and neostriata l precursor populations of the feta l brain Dopamine

Neocortex

Glutamate

Receptor Subtype

Precursor Population

Dl

D2

GABAA

NMDA

AMPA/Kainate

VZ

Decrease*

No effect 1

No effect -

sz

Decrease1

Increase *

Increase** Decrease ft Decrease**

Increase** Decrease**-"'** Decrease** No effect * *

Decrease f f Decrease n

Increase fî Increase u

Not known Not known

Increase1 f No effec t

VZ

Neostriatum

GABA

sz

No effect * *

Noeffect u No effect 1 '

*Zhang and Lidow (2002); Popolo et al. (2004). f

Popolo et al. (2004).

'Ohtanietal. (2003). **Haydar et al. (2000); Owens and Kriegstei n (2002) . M

LoTurcoetal. (1995).

-Sadikot et al. (1998); Luk et al. (2003); Luk and Sadiko t (2004).

Abbreviations AMPA a-amino-34iydroxy-5-methyl-4-isoxazol e propionate

SZ subventricula r zone VZ ventricula r zon e

BrdU Bromodeoxyuridin e

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3

Neuronal Migration Huaiyu Hu

During developmen t o f the mammalia n centra l ner vous syste m (CNS) , neuron s ar e commonl y gener ated at sites far from thei r final location (se e Chapters 2, 11 , and 12) . They migrate to a n anlag e before differentiating int o thei r fina l morpholog y an d makin g synaptic connection s (se e Chapte r 4) . Disturbance s of neurona l migratio n underli e brai n disorder s suc h as menta l retardatio n an d epileps y (Dobyn s an d Truwit, 1995 ; Raymon d e t al. , 1995 ; Dobyn s e t al, 1996). Neuronal migratio n i s widespread throughout th e developing periphera l nervou s system. Fo r example , neurons usin g luteinizin g hormon e releasin g hor mone (LHRH ) are generated i n the developin g olfac tory placode , a transien t structur e outsid e th e brain . They migrat e lon g distance s t o thei r fina l location s in th e media l basa l forebrai n an d hypothalamus . I n contrast, som e neuron s migrat e fro m th e CNS . A prime exampl e i s neural cres t cells . Thes e cell s ar e derived fro m th e dorsa l neura l tub e an d giv e ris e t o

the neurons an d glia of the periphera l nervou s system as wel l a s non-neural cells , fo r example , facia l skele ton, segment s o f the hear t an d great arteries, and pig ment cells in the ski n (see Chapter 17) . Most studie s o f neuronal migratio n hav e focuse d on neocortex . This is (a) because defect s in neuronal migration causing clinical disorders are commonly associated wit h corte x an d (b ) becaus e corte x ha s a highly defined structural an d functiona l organizatio n that derive s chiefly fro m a n orderl y neuronal migra tion. Cortica l neuron s ar e generated i n the neuroep ithelium surroundin g the latera l ventricles (Chapter s 2, 11 , and 12) . The rule s defining neuronal migratio n appear to be similar among mammalia n species. This present chapter focuse s on rodents . Neuronal migratio n involve s fou r coordinate d steps outlined below. 1. Th e proces s begins as cells pass through mitosis and permanentl y exi t fro m th e cel l cycl e (se e

27

28 NORMA L DEVELOPMENT Chapters 2 and 11) . I n th e rodent , thi s occur s during th e secon d hal f o f gestation (Angevin e and Sidman , 1961 ; Lun d an d Mustari , 1977 ; Gardette e t al, 1982; Miller, 1985) . 2. Postmitoti c neuron s prepar e t o migrate . The y remain i n th e proliferativ e zone s for up t o 18 26 hours , th e amoun t o f time increasin g wit h the age of the fetu s (Miller, 1999) . Durin g thi s time youn g neuron s exten d thei r leadin g pro cesses whic h boas t pioneerin g filopodi a an d laniellipodia tha t explor e th e environmen t an d define th e directio n o f movemen t (Rakic , 1972). They may move laterally within the pro liferative zones (Nocto r e t al., 2004) . 3. Th e neuron s actively move fro m th e prolifera tive zone s throug h th e intermediat e zon e and ente r th e immatur e gra y matter. Thi s ste p involves th e choreographe d movemen t o f the nucleu s toward the leadin g process and th e retraction o f th e trailin g proces s (Hatte n an d Mason, 1990 ; Raki c et al, 1996) . Neurona l migration i n th e neocorte x o f mic e an d rat s i s completed b y the end of the first postnatal wee k (Berry and Rogers , 1965 ; Miller, 1999) . 4. Whe n a neuron reaches it s final destination, its nucleus cease s th e forwar d movement . A t this site , th e neuro n differentiate s and elabo rates a characteristic morphology (Miller, 1981 , 1986). The migratio n o f neocortical neuron s ca n follo w a radial o r tangentia l pat h (Rakic , 1990; Miller , 1992 ; Nadarajah an d Parnavelas , 2002) (Fig. 3-1) . Th e former describes a process whereby neurons migrate in a direction perpendicula r t o th e surfac e o f th e latera l

FIGURE 3- 1 Multipl e migratio n route s o f cerebra l cortical neurons . Cerebra l cortica l projectio n neu rons ar e generate d i n th e ventricula r zon e (VZ ) and subventricula r zon e (SZ ) o f the developin g ce rebral wall . Th e migratio n rout e o f thes e neuron s is radia l (1) . Loca l circui t neuron s ar e generate d in th e media l ganglioni c eminenc e (GE) . The y undertake severa l migration routes (2 , 3 , and 4 ) that involve tangentia l migratio n an d radia l migration . CP, cortical plate ; IZ, intermediat e zone ; MZ, mar ginal zone .

ventricles (pathwa y 1) , wherea s th e latte r refer s t o neurons as they migrate along a pathway that parallels the surfac e o f th e latera l ventricl e (pathway s 2-4) . This chapter review s the basic principles o f radial and tangential migration , molecula r event s underlyin g normal neurona l migration , an d som e molecula r de fects tha t distur b neuronal migratio n to caus e neuro logical disorder s (Table 3-1) .

TABLE 3- 1 Genetic s of neuronal migration disorders in human s Protein and Function

Gene

Structural Phenotype

Filamin A, actin binding protein

FLNA

Peri ventricular heterotopia

Doublecortin, microtubule-associated protei n

Dcx

Double cortcortex syndrome s Subcortical ban d heterotopi a

LISl/PAFAHIb, interacts with tubulin, dynein, NUDEL, mNUDE

PAFAHIB

Type I lissencephaly

Reelin, ECM protei n

Rein

Lissencephaly

POMT1, presumed glycosyltransferas e

POMT1

Walker- Warburg syndrome Polymicrogyria

POMT2, presumed glycosyltransferase

POMT2

Walker- Warburg syndrome Polymicrogyria

POMGnTl, glycosyltransferas e

POMGnTl

Muscle-eye-brain disease Polymicrogyria

Eukutin, presumed glycosyltransferase

FCMD

Fukuyama congenital muscula r dystroph y Polymicrogyria

Large, presumed glycosyltransferas e

Large

MDC1D, Polymicrogyria

NEURONAL MIGRATION 2 9 PATHWAYS O F MIGRATIO N

Radial Migratio n Most cortica l neurons , notabl y projectio n neurons , are derive d fro m progenitor s i n th e ventricula r zon e (VZ) an d subventricula r zon e (SZ) . These zone s are located alon g the interna l aspec t o f the cerebra l wall . Most V Z an d S Z cell s migrat e t o corte x via a radial pathway. Radia l migration appear s t o involv e at leas t two mechanisms. The firs t cohor t o f neurons tha t move s ou t o f th e germinal are a forms th e preplat e (Marin-Padilla , 1978 ; Allendorfer and Shatz, 1994). Durin g the earliest stages of cortical development, neuron s take position s in th e preplate vi a somatic translocatio n (Britti s e t al. , 1995 ; Miyata e t al, 2001; Nadaraja h e t al, 2001; Nadaraja h and Parnavelas , 2002). Each o f these neuron s send s a long process toward the pia i surface a s it leaves the VZ . Subsequently, a migrating neuron release s it s ventricular attachments, but maintains its piai contacts. The second , an d large r cohor t o f migrating neu rons split s the preplat e int o tw o layers : th e margina l zone (MZ ) and the subplate (SP) , the anlage of layers I an d VIb , respectively. These newl y arrived neurons take a position betwee n th e MZ an d S P and form the cortical plate (CP). The C P i s laid down by an insideout sequenc e i n whic h early-generate d neuron s tak e a positio n i n th e deepe r C P an d wave s o f later generated neuron s migrat e t o positio n i n succeed ingly superficia l position s (Angevin e an d Sidman , 1961; Rakic , 1974 ; Miller , 1985 ; McConnell, 1995) . The C P differentiate s int o layers Il-VIa. Neurons mov e int o th e C P migrat e radiall y via a different mod e o f migratio n tha n tha t use d b y neu rons forming the preplate . Th e CP-destine d neuron s use radia l glia a s a physica l substrat e fo r their migration (Rakic, 1972,1990; Hatten, 1999) (Fig . 3-2). Dur ing migration , eac h neuro n ha s a shor t leadin g process tha t i s not attache d t o th e pia i surfac e and a trailing proces s tha t eventuall y lose s it s contact wit h the ventricular surface. Radial glial guidance provides a mechanism fo r later-generated neuron s to find positions beyond the postmigrator y neurons, hence estab lishing the inside-ou t patter n characteristi c of the CP . This migratio n is relatively fast. Apparently, regardless of when th e neuron s ar e generated, the y migrat e at a rate o f ~120|im pe r da y (Miller, 1999) . Simila r rate s are eviden t i n th e radia l migratio n o f hippocampa l neurons from th e VZ (Nowakowski and Rakic , 1981).

FIGURE 3- 2 Radia l migration durin g development o f the cerebra l cortex. Neurons migrate along the radial glia (RG ) t o the cortica l plate (CP ) wher e later-born neurons migrat e pas t th e early-bor n neurons . Reeli n (illustrated a s dots) secreted b y Cajal-Retzius neuron s (CR) i n th e margina l zon e (MZ ) i s required fo r th e inside-out patter n o f lamination . Withi n th e MZ , radial glia l endfee t (EF ) for m th e so-calle d glial limiting membrane. Togethe r wit h some Cajal-Retziu s cells, thes e endfee t appos e th e brai n surfac e base ment membrane. SP , subplate.

Radial gli a spa n th e ful l dept h o f the developin g cerebral wall . In th e intermediat e zon e (IZ) , the ra dial glia l fiber s ar e groupe d i n fascicle s (Gressen s and Evrard , 1993). I n the mouse , thes e cell s ar e evident durin g th e perio d o f neurogenesis an d beyon d the cessatio n o f neurona l migratio n i n neocorte x (Miller an d Robertson , 1993) . After neurona l migra tion i s complete , radia l gli a appea r t o transfor m t o astrocytes (Schmeche l an d Rakic , 1979 ; Misso n et al , 1988 ; Voigt , 1989 ; Mille r an d Robertson , 1993). Fo r many years, the identit y of radial glia ha s been considere d par t o f th e astrocyt e lineage . O n the basi s o f recent data , however , i t appears tha t radial gli a ar e no t gli a —rather, the y ar e neura l ste m cells (Malatest a e t al. , 2000 ; Miyat a e t al. , 2001 ; Noctor e t al , 2001) . The y expres s marker s for cytoskeletal protein s fo r immature (stem ) cells , fo r example, nesti n an d RC-2 , an d the y ca n generat e neurons an d glia .

30 NORMA L DEVELOPMEN T Tangential Migratio n Analyses o f forebrain neuron s with retrovira l markers show tha t a neurona l clon e derive d fro m a singl e founder i s dispersed fa r wider than woul d hav e heen predicted i f neuron s onl y migrate d radiall y (Luski n et al, 1988; Walsh and Cepko, 1988) . Although mos t neurons ar e tightl y distribute d i n radia l column s of -200 |Llm, a notabl e numbe r ar e locate d a s muc h as 2 mm fro m thi s column. I n these clones , cells tend to b e projectio n neuron s o r loca l circui t neuron s (LCNs) (Luski n et al., 1988 , 1993 ; Parnavelas e t al, 1991). Th e distributio n o f th e retrovirall y labele d cells i s equally broad fo r clones o f projection and lo cal circuit neurons. This dispersio n principally results from th e tangential movemen t o f cells within the proliferative zones before they ascend radially. On th e basi s of findings from trac t tracing studies, it appear s tha t neuron s generate d i n th e ganglioni c eminence (GE ) migrat e t o neocorte x (d e Carlo s et al., 1996) . Molecular an d anatomica l studie s show that thes e neuron s ar e LCN s (e.g. , Anderso n e t al. , 1997; Powel l e t al , 2001 ; Marin an d Rubenstein , 2003). Thes e LCN s migrat e throug h th e SZ , IZ , or MZ (Fig . 3-1) (Tamanak i e t al, 1997 ; Lavdas et al , 1999; Anderso n e t al , 2001 ; An g et al , 2003) . Accordingly, th e neuron s loo p aroun d th e latera l G È and cours e alon g th e VZ-SZ interface , in the I Z external t o th e SZ , o r throug h th e MZ . Afte r findin g their tangential position, the neuron s associate with a radial gli a an d migrat e radiall y t o thei r appropriat e laminar residence. Thi s is supported b y data showin g that LCNs migrate to cortex via an inside-ou t patter n (Miller, 1985 , 1986, 1992; Fairé n étal, 1986 ; Gobas and Fairén , 1988) . A n exceptio n i s a subpopulatio n of LCN s tha t expres s vasoactiv e intestina l polypep tide (Miller , 1992) . Fo r thes e neurons , ther e i s n o birthdate- or lamina-specific pattern o f the migration. The tangentia l pathway s ar e importan t becaus e many neocortica l LCN s migrat e vi a on e o f thes e pathways; dix knockout mice (mic e i n which n o neurons ar e generate d i n th e GE ) hav e les s tha n onequarter o f th e LCN s eviden t i n wild-typ e mic e (Anderson e t al , 1997) . Thi s findin g concur s wit h those fro m retrovira l tracin g studie s showin g tha t clones commonly consist of either projection neurons or LCN s (Luski n et al, 1988 , 1993; Parnavela s et al , 1991). Recen t evidenc e (An g et al , 2003 ; Nocto r et al , 2004 ) show s that LCN s derive d fro m th e G E that migrat e at the VZ-SZ interfac e can di p int o th e

VZ and re-ente r th e cell cycl e before they begin thei r radial migration . This observatio n contribute s t o th e finding that cells remain in the proliferative zones before the y commenc e thei r radia l migratio n (Miller , 1999). Nevertheless , th e tangentia l migratio n is suffi ciently fas t tha t LCN s generate d i n th e G E an d co hort projectio n neuron s generate d concurrentl y i n the V Z an d S Z migrat e t o cortex i n tande m (Miller , 1985, 1986 , 1988, 1997). Neurons migratin g tangentiall y rel y o n divers e substrates. LHR H expressin g neurons migrat e o n axons a s they mov e t o thei r targe t (Schwanzel-Fukud a and Pfaff , 1990) . Othe r migratin g neuron s migrat e over "resident " cells . Thes e includ e olfactor y LCN s migrating throug h th e anterio r subventriciila r zon e (SZa) o n thei r wa y t o th e olfactor y bul b (Luskin , 1993; Loi s e t al.,1996). The substrat e for the tangen tial migratio n of neurons from th e media l GE t o neocortex i s unknown; there i s no axona l or glia l syste m for guidance . Thus , thes e neuron s presumabl y mi grate ove r cell s i n th e variou s compartments o f th e developing cerebral wall (Anderson et al, 1997) .

FACTORS REGULATIN G

NEURONAL MIGRATION

Soluble Stimulatin g Factor s Soluble factors can modulate radial migration. Migrating cortical neuron s express TrkB, the high-affinit y re ceptor fo r brain-derived neurotrophi c facto r (BDNF ) and neurotrophin- 4 (NT-4 ) (Beha r et al, 1997) . Thi s suggests a role of BDNF and NT- 4 in their migration. Indeed, BDN F an d NT- 4 promote th e migratio n o f cortical neuron s i n vitro. Consistent wit h culture stud ies, BDNF or NT-4 infusion int o th e latera l ventricl e or applicatio n i n cultur e slice s induce s heterotopias , which result from th e overmigration of neurons (Brunstrom e t al, 1997) . Epiderma l growt h facto r recepto r (EGFr) an d it s ligands, includin g epiderma l growt h factor an d transformin g growth facto r (TGF ) a, ar e expressed i n th e developin g cor fex (Kornblu m e t al , 1997). Mic e lackin g EGF r exhibi t mor e cell s i n th e proliferative zones , a finding suggestin g inhibite d mi gration, wherea s hig h concentration s o f EGFr i n th e embryonic cortex enhance radia l migration (Threadgill et al, 1995 , Carie et al, 2001) . Tangential migratio n is stimulated by growth factors. One suc h ligan d i s hepatocyte growth factor (HGF). It

NEURONAL MIGRATIO N 3 1 is expresse d in th e teleneephalo n durin g LCN migra tion from the medial G E t o the pallium (Powel l et al., 2001). HG F increase s the numbe r o f cells migratin g away from th e subpallium , and neutralizatio n o f HG F by blocking antibodie s inhibit s this migration. In addi tion, BDNF an d NT-4 strongly stimulate the migratio n of LCNs that use y-aminobutyric acid (GABA) as a neurotransmitter (Polleux et al, 2002). TGFp promotes neuronal migration. TGFpl and 2 are expressed by cells in the developing cortex; TGFpl, by neurons ; an d TGFp2 , b y radia l glia , particularly their inner segments (Miller, 2003). Elements in developing corte x als o expres s TGF p receptors—TGFpI r predominantly by radial glia and TGFpIIr by neurons. Thus, developin g corte x contains th e component s needed fo r TGFp to mediate neuron—gli a interaction s through bidirectional paracrine mechanism. Also, it appears that TGFp is involved in early stages of neuronal migration (stage s 1 , 2 , an d 3) . These conclusion s are supported b y finding s fro m complementar y i n vitro studies. TGFpl inhibit s the proliferation of B104 neuroblastoma cell s (Lu o and Miller , 1999 ) an d culture d cortical neurons (Mille r an d Luo , 2002a) . In addition, TGFpl move s VZ cells i n slices of cerebral corte x out of th e cyclin g populatio n an d accelerate s th e rat e o f neuronal migratio n b y 70% (Siegenthaler an d Miller , 2005). Th e latte r effec t i s concentration-dependent . Migration i s maximall y potentiate d b y exposur e t o TGFpl a t a concentration of 10 ng/ml. In contrast, exposure to a higher o r lower concentration ha s little effect or it can even retard neuronal migration. Besides growt h factors , neurotransmitter s ar e in volved i n modulatin g neurona l migration . GAB A i s present i n th e developin g corte x (Rickman n e t al. , 1977; Miller , 1986) . I t promote s cortica l neuron s t o migrate throug h GAB A receptors (Beha r e t al. , 1996 , 2001). It appears that GABA A/C receptor s ar e involved in migratio n fro m th e proliferativ e zones t o th e I Z whereas GABA B receptor s ar e involve d i n migratio n from th e I Z t o th e CP . Anothe r neurotransrnitter , N-methyl-D-aspartate (NMDA), is also shown to modulate radial migration (Behar et al., 1999). Pharmacolog ical blockad e o f NMD A recepto r activit y decrease s migration whereas enhancemen t o f NMDA recepto r activity increases the rate of cell movement. Cell Adhesion Molecules Interactions betwee n migratin g neuron s an d thei r substrates ar e mediate d b y cel l adhesio n molecules .

Astrotactin (Astn ) 1 is a glycoprotein expressed by th e migrating neuron s (Fishel l an d Hatten , 1991 ; Zhen g et al., 1996) . Antibodies that block the functions of astrotactin abolis h th e adhesio n betwee n neuron s an d glia, thu s reducin g th e rat e o f migratio n (Stit t an d Hatten, 1990) . I n vivo , mice deficien t i n astrotacti n display slowed radial migration (Adams et al., 2002). Integrals are cell surface receptors tha t hav e been shown to regulate the migratio n of many cell types including neurons . The y mediat e essentia l cell-cel l and cell-extracellula r matri x interactions required for cell migration . Blocking of integrili oc ^ by specific antibodies reduce s th e rat e o f migration an d result s i n detachment of neurons fro m radia l glial fibers (Anton et al., 1999 ; Dulabo n e t al, 2000). In mic e deficien t in integrin OC 3, some neuron s appear to stop migrating prematurely an d fai l t o migrat e t o thei r appropriat e layer. Similarly , antibodie s agains t oc v integri n als o perturb neuron-glia interaction s and ma y affect neu ronal migration as well. The neural cell adhesion molecule (nCAM ) i s a member of the immunoglobuli n superfamil y of cel l adhesion molecule s though t t o pla y a rol e i n chai n migration. I t ha s a n unusua l carbohydrat e modifi cation calle d polysialic acid, a linear polymer of sialic acid (Finn e e t al , 1983 ; Rutishauser , 1996) . I n nCAM knockou t mice , man y olfactor y LCN precur sors are retarded i n the SZ a and fai l t o migrate to th e olfactory bul b (Tomasiewic z et al. , 1993 ; Grenie r et al. , 1994) . Interestingly , removal o f the polysiali c acid moiet y of nCAM b y a specifi c enzyme i n wild type animal s completel y mimicke d thi s knockou t phenotype (Ono e t al., 1994) . Cell migration in vitro is severel y abolishe d i n collage n culture s i n whic h polysialic acid is removed (H u et al., 1996) . Thus, the carbohydrate modificatio n of nCAM, polysialic acid , plays an importan t rol e i n th e migratio n o f olfactory LCNs. TGFpl induce s nCA M an d integri n expression. Studies o f cortica l slice s sho w tha t thes e effect s ar e concentration-dependent; nCA M an d integri n ex pression increas e wit h increase d TGFp l concen tration (Siegenthale r an d Miller , 2004) . Likewise , TGFpl potentiate s nCA M expressio n b y culture d cortical astrocyte s (Lu o an d Miller , 1999) , neurons , and neuroblastom a cell s (Mille r an d Luo , 2002b) . In ligh t o f the effec t o f TGFpl o n th e rat e o f neu ronal migratio n (se e above), i t appears that increase d nCAM an d integri n expression is facilitatory, but only to a certain point. I f the extracellula r space become s

32 NORMA L DEVELOPMEN T too rich in cell adhesion proteins, neuronal migration is impeded , possibl y because th e microenvironmen t becomes too sticky. Extracellular Matrix Molecule Reelin Modern mous e genetic s hav e provide d som e muta tions wit h neurona l migratio n disorder s (Tabl e 3-2) . The reeler mouse ha s receive d greate r attentio n fro m developmental neurobiologist s tha n ha s an y othe r mouse mutants . Thi s mous e exhibit s characteristi c lamination defect s of cerebral cortex , the hippocam pus, an d th e cerebellu m (Raki c and Caviness , 1995 ; Lambert de Rouvroit and Goffinet , 1998) . I n the mu tants, neocortica l neuron s ar e generate d i n th e V Z and S Z as in th e wild-typ e animals and initiall y their migration seems normal. A major differenc e i s that th e preplate i s never split into the M Z an d th e SP ; therefore, th e C P form s unde r th e so-calle d superplate . As the migratin g neurons approac h th e CP , neuron s i n the reeler mic e fai l t o for m norma l architectoni c or ganizations. Instea d o f forming b y a n inside-ou t pattern o f neuronogenesis , th e birthdat e o f neuron s i n the mutant cortex is reversed. Early-generated neurons are locate d superficiall y and late-generate d neuron s are distributed in deep cortex. Remarkably, despite the

positioning defects, neuron s do make correct connec tions, although the axonal pathways are distorted. The clonin g o f th e defectiv e gen e relin ha s in creased interes t i n thi s mutatio n (D'Arcangel o e t al. , 1995). Reelin is expressed by Cajal-Retzius neuron s i n the M Z o f the developin g cerebral wal l (D'Arcangel o et al., 1997) . How reelin function s in neuronal migra tion i s an are a o f intense research ; n o consensu s ha s been reached. Mos t results support the idea that reeli n controls laminatio n b y acting a s a sto p signa l fo r mi grating neuron s (Dulabo n e t al. , 2000) . Accordingly , neurons migrate past the previously deposited neurons in the lower CP wher e reelin is not expressed. The mi grating neurons proceed t o the superficia l edg e o f the CP borderin g th e M Z where reeli n i s expressed (Cur ran and D'Arcangelo , 1998) . Thi s finding implies tha t reelin tells the neuron s to stop migrating. Some o f the migration defects characteristic o f the reeler mice, such as th e lac k o f splittin g o f th e preplate , i s rescue d i n transgenic mic e i n which reeli n expressio n i s targeted in th e VZ and SZ . Thus, the role of reelin in developing cortex is more complicated tha n simply providing a migratory stop signal (Magdaleno et al., 2002). Reelin binds to two receptors expressed by migrating neurons , Apo E recepto r 2 an d ver y low-density lipoprotein recepto r (VLDLr ) (D'Arcangel o e t al. ,

TABLE 3- 2 Mous e mutations that affect neurona l migration Protein and Function

Gene

Structural Phenotype

Reelin, ECM protei n

rein

Astrotactin 1 , neuron-glia cell adhesion molecul e Integrin a^ subunit, cell surface receptor bind s to reelin Integrin oc 6 subunit, cell surface receptor bind s to laminin

astn

reeler mouse, inverted cortical layering , absence of preplate spli t Slowed radial migration Abnormal laminar positioning of projection neurons Basement membrane breac h Cortical layering perturbation

Integrin (3j , cell surface receptor interacts with reelin, laminin Laminin Y J subunit, ECM protei n

itgbl

Very low-density lipoprotei n receptor , reeli n recepto r Low-density lipoprotein receptor-related protein, reeli n receptor also known as ApoE receptor 2 Disabled homolog 1 , interacts with VLDLr/Lrp8 Cyclin-dependent kinase 5, phosphorylates Dabi and NUDE L

vldlr

LISl/PAFAHIb, binds to dynein, microtubule

PAFAHIb

Doublecortin, microtubule-associated protei n

dcx

itga3 itgao

lame I

Irp8 dabl cdkS

Basement membrane breach Cortical layerin g perturbation Basement membrane breac h Cortical layering perturbatio n Reeler phenotype in Vldlr/LrpS doubl e knockout Reeler phenotype in Vldlr /Lrp8 doubl e knockout Reeler phenotype, mutated in yotari and scrambler Inverted cortical layering Null is lethal, hypomorphic mutations caus e migration defec t No migration phenotyp e

NEURONAL MIGRATION 3 3 1999; Hiesberger et al, 1999) . Mice deficient i n both of these receptor s exhibi t phenotypes identical to that of the reeler mouse (Trommsdorf f e t al., 1999) . There may b e othe r receptor s involve d in transducin g the reelin signal . Reeli n als o bind s t o integri n oc 3pj an d cadherin-related neurona l receptor s (Senzak i e t al. , 1999; Dulabo n e t al , 2000) . Apparently , reelin integrin bindin g i s no t require d fo r reeli n functio n because integrin pj knockout mouse does not exhibit a ree/er-like phenotype. Downstream reelin-induce d signalin g involves the phosphorylation o f Drosophila disable d homologu e (Dab) 1 (Howell et al., 1999). Mice with mutations i n the gene s dabl, scrambler, and yotari exhibi t phenotypes similar to that of reeler mice. Dabi bind s to th e intracellular domain s o f lipoprotein receptor s an d i s tyrosine phosphorylate d upo n ligand-recepto r bind ing (Trommsdorf f et al, 1998 ; Keshvar a et al., 2001) . Surprisingly, reeli n ha s bee n show n t o posses s a serine proteas e activit y and can diges t extracellula r matrix molecules (Quattrocchi et al, 2002). Whether its enzymatic activit y regulate s neurona l migratio n re mains to be evaluated. The mous e knockou t of cyclin-dependent kinase (Cdk) 5 (a serine/threonine kinase ) o r o f its activator protein, p35 , exhibit s a reeler-like cortica l migratio n phenotype (Gilmor e e t al. , 1998 ; Kwo n an d Tsai , 1998). A major differenc e is that th e preplat e i s split into the MZ and the S P in these mutan t mice. Thus, Cdk5 and p3 5 ma y function in a different pathwa y to control neurona l migratio n fro m reelin . Cdk 5 ca n phosphorylate Dabi independen t o f reelin binding to its receptor s (Keshvar a et al. , 2002) . There ma y b e crosstalk betwee n th e reeli n signalin g pathwa y an d the Cd k 5 pathway.

MIGRATION DEFECTS

X-linked Periventricular Heterotopias: Failure to Initiate Migratio n X-linked periventricula r heterotopia s ar e nodule s o f neurons linin g the V Z o r SZ . Presumabl y there i s a failure o f neuron s t o migrat e ou t o f proliferat i ve zones. The geneti c defect in X-linked periventricular heterotopia i s mutations i n FLNA , a n X-linke d gen e encoding filami n A (Fox et al. , 1998) . Filami n A is a large actin-bindin g phosphoprotein wit h a molecular

weight of 280 kDa. I t i s necessary for the locomotio n of several cell types and i s expressed by cells in all layers o f th e developin g cerebra l cortex . Hemizygou s males with null mutation s di e during the embryoni c period. Heterozygou s female s have epileps y that ca n be accompanie d b y othe r manifestation s suc h a s patent ductus arteriosus. I t i s believe d tha t rando m X-chromosome inactivatio n result s i n inactivatio n o f FLNA expressio n in strande d neurons . Recently , affected male s with likely partial loss-of-function muta tions i n FLNA (e.g. , amino aci d 65 6 Leu t o Phe an d amino aci d 230 5 Tyr t o sto p codon ) hav e bee n re ported (Shee n e t al., 2001 ; Moro e t al, 2002) . Interestingly, male s wit h thes e mutation s hav e neuron s that eithe r migrat e normally or exhibit complete mi gratory arrest . Thi s dichotom y suggest s tha t othe r functionally relate d gene s can compensat e fo r filamin A deficiency . Indeed , a structurall y relate d gene , FLNB, i s als o expresse d i n th e developin g cerebra l cortical wall, and both proteins can form heterodimer s (Sheen et al., 2002). The mechanis m throug h which filamin A regulates the initiatio n of migration is unclear. Likely it involves the abilit y o f filami n A t o cross-lin k F-acti n int o isotropie, orthogonal arrays (Stossel et al., 2001). Crosslinking of F-actin increase s the viscosit y and stiffnes s of actin an d ma y b e involve d in th e initiatio n of migration. I n the V Z an d SZ , FLNA i s expressed by all cells —mitotic and postmitoti c cells. If filamin A regulates the initiatio n of neuronal migration, why do only postmitotic neuron s migrat e ou t o f the V Z an d S Z when all cells express FLNA? A potential mechanism involves filamin A-interacting protei n (FILIP) . FILIP is expressed in the VZ and SZ , but no t in postmitoti c migrating neuron s (Nagan o e t al. , 2002) . FILIP-fil amin A binding induces the degradatio n o f filamin A. Thus, the loss of FILIP expressio n in postmitotic neu rons may enabl e filami n A to contro l th e star t of migration. Double Cortex Syndrom e an d Type I Lissencephaly: Prematur e Cessatio n of Neuronal Migratio n Double cortex describe s a conditio n i n whic h a subcortical-band heterotopi a form s i n the subcortica l IZ, th e anlag e o f the whit e matter . Mutation s i n a n X-linked gene, doublecortin (dcx)y ar e a genetic caus e of the disorder (des Portes et al, 1998 ; Gleeso n e t al, 1998). Doublecortin i s a 40 kDa protein , expressed by

34 NORMA L DEVELOPMENT migrating an d man y differentiatin g neurons . I t i s a microtubule-associated protei n wit h n o know n ho mology wit h othe r microtubule-associate d protein s (Francis et al., 1999; Gleeso n e t al., 1999 ; Hores h et al., 1999). Doublecorti n stimulate s microtubul e polymer ization b y binding t o 1 3 protofilament microtubule s (Moores e t al., 2004). I t is principally localize d i n th e leading processe s o f migratin g neuron s (Friocour t et al., 2003). Thus, doublecortin ma y promote move ment by inducing microtubule polymerizatio n i n th e leading processes of migrating neurons. Classical lissencephal y (type I ) i s a brai n malfor mation characterize d b y absent o r reduce d gyratio n and a cortex that is thicker and ha s a rudimentary four layers. I t is associated with severe mental retardation , epilepsy, an d cerebra l palsy . Mos t case s ar e du e t o mutations o f lisi, encoding LIS I o r platelet-activatin g factor acetylhydrolas e isofor m I b (PAFAHIb , a non catalytic subunit o f the enzyme ) (Reine r e t al., 1993 ; Hattori et al., 1994). Besides being part of an enzyme , LISl/PAFAHIb ha s a nonenzymati c function . Th e homolog i n fungu s Aspergillus i s NudF, a n essentia l part of a signaling pathway that regulate s nuclea r mi gration (Wynshaw-Bori s an d Gambello , 2001) . Evi dence indicate s that LISl/PAFAHIb regulates nuclear movement b y an evolutionary-conserve d mechanis m similar to that of the fungus . It binds to and regulate s the functio n an d distributio n of dynein (mammalia n NudA homolog) , whic h function s a s a minu s end directed microtubule-associate d moto r protein . Thus , LISl/PAFAHIb ma y b e par t o f the protei n comple x that exert s force s o n th e microtubule s surroundin g the nucleu s i n migratin g neuron s an d pullin g th e nucleus into the leading process (Tanaka et al., 2004). A rol e fo r LISl/PAFAHI b i n nuclea r translocatio n i s also supported b y its binding t o two other dynei n in teracting proteins , NUDE L an d mNUD E (mam malian Nud E homologs ) (Fen g e t al. , 2000 ; Lian g et al, 2004). NUDEL and mNUDE colocalize a t the centromere tha t migrate s ahea d o f the nucleus . Th e distance betwee n th e centromer e an d th e nucleu s is enlarge d i n LISl/PAFAHIb-deficien t neuron s (Tanaka e t al, 2004) . Thus , LISl/PAFAHI b play s an important rol e i n nuclea r translocatio n durin g neu ronal migration. Type II Lissencephaly: Overmigration Several form s o f congenita l muscula r dystrophies — Walker-Warburg syndrome (WWS) , muscle-eye-brai n

disease (MEB) , an d Fukuyama-typ e congenita l muscular dystroph y (FCMD ) —are associate d wit h cortical dysplasia . These ar e autosoma l recessiv e dis orders characterize d b y congenita l muscula r dystro phy, ocula r abnormalities , an d cortica l dysplasi a (Santavuori e t al. , 1989) . Magneti c resonanc e imag ing of MEB patient s reveal s hydrocephalus associate d with polymicrogyra o r pachygyra caused b y ectopie lo cation o f neura l tissue s i n th e leptomeninges , als o known a s cobbleston e complexe s o r typ e I I lissencephaly (Valann e et al. , 1994 ; Corman d e t al. , 2001). Presumably , thes e abnormalitie s result whe n the neuron s migrat e to o far, passing through th e MZ , the glial-limiting membrane, th e piai basement mem brane (PM) , and th e pia (Ross and Walsh, 2001). Each o f th e congenita l muscula r dystrophie s ha s been associate d wit h mutatio n o f a spécifi e gene . In WWS , mutation s ar e i n th e gene s codin g fo r protein-O-mannosyl transferase s (POMT 1 an d POMT2) (Beltran-Valer o e t al, 2002 ; va n Reeuwij k et al. , 2005) . Th e defectiv e gen e fo r ME B map s t o chromosome lp32-3 4 (Corman d e t al. , 1999) , an d the mutated gen e is POMGnTl. Peopl e wit h FCMD have a retrotransposo n insertio n int o the 3 ' untrans lated regio n o f FCMD . Thi s insertio n result s i n a dramatic reductio n i n FCMD expression . The myodystroph y (myd) mous e ha s muscle , eye , and neurona l migratio n defects in the brai n similar to those i n humans wit h congenital muscula r dystrophie s (Holzfeind e t al., 2002) . This myd mous e ha s muscle , eye, an d brai n defect s associate d wit h overmigratio n (Holzfeind e t al, 2002). In the myd mouse, the geneti c defect i s a functional deletio n i n th e Large gene (like acetylglucosaminyltransferase) (Grewa l e t al. , 2001) . The my d mutatio n i s now designated Large myd. I n hu mans, mutation s i n Large gen e cause congenita l mus cular dystroph y MDC1 D (Longman , e t al , 2003 ) Thus, four ne w genes, POMT1, POMGnTl, FCMD, and Large, have bee n associate d wit h congenital mus cular dystrophies , an d eac h cause s simila r overmigra tion defects when mutated . The functio n of POMGnTl ha s been describe d in vitro. POMGnTl encode s a n enzym e involve d i n O-mannosyl glycosylatio n o f protein s calle d UDP N-acetylglucosamine: protein-O-mannos e p-l,2-N acetylglucosaminyl transferas e (POMGnTl), Th e enzyme catalyze s the following reaction: UDP-GlcNAc + Man-R -> GlcNAc(31-2Man-R + UDP

NEURONAL MIGRATION 3 5 where R is a protein an d Ma n i s anchored t o Ser/Thr residues (Takahashi et al., 2001; Yoshida et al, 2001). The function s o f POMTT, fukutin , an d Large , gen e products o f POMTI , FCMD , an d Large, hav e no t been determined . Bioinformatic s studies , however , show tha t eac h protei n ha s a glycosyltransferase-lik e domain (Aravin d an d Koonin , 1999 ; Jurad o e t al. , 1999; Peyrar d e t al. , 1999 ; Beltran-Valer o e t al , 2002). The implicatio n is that the dystrophies and migration defects result from abnorma l protein glycosyla tion. How can protei n glycosylatio n enzyme s regulate neuronal migration? Conceivably, migration involves glycosylation substrate proteins. A candidate substrate is a-dystroglycan , a heavily glycosylated membrane protein . The mucin like domain o f a-dystroglycan is heavily substituted b y O-linked mannosy l glycan s tha t contai n th e linkag e catalyzed b y POMGnTl (Chib a e t al, 1997) . Th e O-mannosyl glycans may play important role s in me diating a-dystroglyca n interaction s with lamini n (Er vasti and Campbell , 1993 ; Smalheiser , 1993), a majo r component o f th e basemen t membrane . Further , a-dystroglycan i n som e o f these disease s i s underglycosylated and bindin g to laminin is reduced (Hayash i et aì., 2001; Holzfeind e t al, 2002; Kano et al, 2002 ; Michele et al., 2002). These results suggest a connection between hypoglycosylatio n of a-dystroglycan an d neuronal migratio n defect s i n th e brain . A n impor tant rol e for a-dystroglycan i n this process is also supported b y finding s o f neurona l migratio n defect s i n the brain s of mice i n which dystroglyean is conditionally knocked ou t (Michel e e t al., 2002 ; Moore e t al. , 2002). Mechanisms o f overmigration may involve abrogation o f th e PM . Th e P M i s compose d mainl y o f laminin, collage n IV , nidogen , an d perlecan , al l o f which regulat e cel l proliferation , migration , an d dif ferentiation b y interactin g wit h mainl y tw o cel l sur face receptors : integrilis and a-dystroglycan. Th e PM , to whic h radia l glia l endfee t ar e attached , i s locate d between th e pi a mater an d th e MZ . Electro n micro scopic analyse s show breache s i n th e P M a t site s of ectopie neura l cluster s o f patient s wit h FCM D (Nakano e t al. , 1996 ; Ishi i e t al , 1997 ; Sait o e t al. , 1999). Laminin i s reduced o r abnormall y distribute d in th e brai n surfac e P M o f several mutan t mice wit h overmigration, includin g Lmxl a (Dreher) mic e (Sekiguchi e t al, 1994 ^ Costa e t al, 2001 ) and mic e lacking rnyristolate d alanine-rich C kinas e substrat e (Blackshear e t al , 1997) , integri n a 6 (Georges -

Labouesse e t al, 1998) , an d integri n p ] (Graus-Port a et al , 2001) . Interestingly , integrili (3 j nul l neuron s migrate to appropriate positions in the cerebral cortex in chimeri c mic e (Fassle r and Meyer , 1995) , imply ing tha t overmigratio n i s no t cause d b y a n intrinsi c defect o f the migratin g neuron s but b y a defective environment i n thi s mutant . Whethe r breache s i n th e PM ar e th e caus e o r th e resul t o f overmigratio n re mains to be clarified.

DIRECTIONAL GUIDANCE OF NEURONAL MIGRATIO N BY DIFFUSABLE FACTORS

LCNs o f th e olfactor y bul b (granul e cell s an d periglomerular cells ) ar e mainl y generate d postna tally, during th e first 2 to 3 weeks after birt h (Altma n and Das , 1966 ; Hinds , 1968) , althoug h som e neu ronogenesis continue s i n th e adul t (Corott o e t al , 1993; Loi s an d Alvarey-Buvlla , 1994) . Retroviral labeling studie s demonstrat e tha t mos t o f the LCN s are generate d i n th e S Z nea r th e anterio r forebrai n (SZa) an d migrat e t o th e olfactor y bulb throug h a n SZ pathwa y (Fig . 3-3 ) (Luskin , 1993 ; Zigova e t al , 1996). Apparently , olfactor y LCNs do not migrate o n radially oriente d glia l processes, a s the orientatio n o f the radia l glia l fiber s i s orthogona l t o th e migratio n trajectory (Kish i e t al, 1990) . Further , th e migratio n pathway i n th e S Z i s also devoi d o f axon projections (Kishi, 1987) . How d o olfactor y LC N migrate ? Immunohisto chemical studie s o f polysialic aci d an d Tu J 1 expression i n th e adul t S Z pathwa y sho w tha t migratin g cells tend to travel in chains o r streams o f cells (Rousselot e t al, 1995 ; Doetsc h an d Alvarez-Buylla , 1996 ; Jankovski and Sotelo, 1996 ; Loi s et al, 1996 ; Doetsc h et al , 1997 ; Garcia-Verdug o e t al , 1998) . Althoug h these chain s o f migrating cells cannot b e observe d in newborn animal s because man y cells are migrating at the sam e time , suc h chain s are often observed i n cul tures of SZ cell s plate d on collage n gel s (H u et al, 1996) o r Matrigel (Wichterle et al, 1997) . Therefore , chain migratio n of olfactory LCN precursor s seems to be mechanisticall y distinc t fro m radia l migration relying on radial glia. In vivo studies show that olfactory LCN precursor s migrate fro m th e SZ a to the olfactor y bulb in a unidirectional manner (Luskin , 1993; H u and Rutishauser, 1996). Thi s findin g implie s a n activ e guidanc e

36 NORMA L DEVELOPMENT

FIGURE 3- 3 Tangentia l migratio n o f olfactor y loca l circuit neuron s (LCNs) . A parasagitta l sectio n through th e middl e o f the olfactor y bul b (OB ) of the forebrain o f the mous e i s shown. Olfactory LCNs ar e generated i n the anterio r subventricular zone (SVZa ; shown betwee n arrows) . The y migrat e t o th e O B within the SVZa (show n in dotted areas) . After arriving in th e bulb , they migrate i n a radial fashion to reac h their fina l location s i n th e granul e cel l laye r an d glomerular layer . Th e septu m i s locate d media l t o this section . CC , corpu s callosum ; CP , caudoputa men; CTX , cerebra l cortex ; fmi , forceps mino r cor pus callosum .

mechanism. Th e olfactor y bul b ma y secret e a chemoattractant(s) (Li u an d Rao , 2003) . Further more, th e cauda l part s o f th e septu m an d th e choroid plexus , which li e behind th e migratio n pathway, secret e a chernorepulsiv e factor(s ) (H u an d Rutishauser, 1996) . Th e chernorepulsiv e activit y secreted fro m th e septum is a member of the Sli t family of protein s (Siiti , SlitZ , an d Slit3 ) (Hu , 1999 ; W u et al. , 1999) . Slit , first discovered i n Drosophila, an d also found in mammals, regulates the growth of axons crossing a t th e midlin e (Ba-Charve t e t al. , 1999 ; L i et al. , 1999 ) b y interactin g with roundabou t (Robo) receptors (Kidd et al, 1998) . Robo receptors are members of the immunoglobuli n superfamily of transmembrane molecule s (Zalle n e t al. , 1998 ; Kid d e t al. , 1999). Th e Slit/Rob o repulsiv e signa l i s mediated i n part by the protein s enable d an d abelson , Rh o famil y guanidine triphospotase s (GTPases) , an d Slit-Rob o GTPase activatin g proteins (srGAPs ) (Bashaw et al. , 2000; Wong et al, 2001). Soluble factor s ma y als o b e involve d i n activ e guidance i n th e migratio n o f cortical neurons . Thi s conclusion i s supported b y studies of LCN migratio n from th e GE to the cerebral cortex . In vitro, these migrating neurons are repelle d b y the Sli t protein s that

are expresse d b y th e G E (Zh u e t al. , 1999 ) an d at tracted by substances released by the developing cortex (Marin e t al. , 2003 ; Wichterl e e t al , 2003) . Neuropilins are transmembrane receptor s that mediate th e repulsive guidanc e activitie s o f class 3 semaphorins. Neuropilin 1 and 2 ar e expresse d by the LCN s tha t migrate from th e G E t o cerebral cortex (Marin et al., 2001). Neuropili n knockou t mic e hav e decrease d numbers of LCNs in neocortex. Successful navigatio n o f neuron s involve s mor e than attractio n an d repulsion. While e n route within the SZ a pathway , olfactor y LCN s d o no t deviat e from thei r rout e int o surroundin g region s (Luskin , 1993). Wha t confine s the cell s to th e SZ a pathway? The S Z migration pathway in adult mice i s rich in astrocytes, tenascin , and chondroiti n sulfat e proteogly can (Thoma s et al., 1996). Thus, it is possible that th e migrating cell s migh t hav e stronge r affinit y fo r th e SZ, becaus e th e S Z contain s differen t extracellula r matrix molecule s an d cel l type s fro m thos e i n sur rounding regions.

SUMMARY AND CONCLUSION S

Neuronal migration is a complex phenomenon regu lated b y man y factors . I n vitr o perturbatio n studie s and molecula r studie s i n bot h th e mous e an d hu mans identif y som e ke y molecules that are involved. Examples includ e factor s extrinsic an d intrinsi c to a migrating neuron . Thes e extrinsi c factors , suc h a s soluble an d extracellula r matri x molecule s (neu rotrophins, reelin , an d Slit) , an d intrinsi c factors, such a s cytoskeleta l proteins (filamin , doublecortin , and PAFAHIb) , work in concert. They likely interact with cel l surfac e receptor s an d signalin g pathways. Some o f th e mechanism s ar e jus t beginnin g t o emerge. I n addition , ther e ar e critica l interaction s between geneti c an d environmenta l o r epigeneti c factors. Abbreviations Astn astrotacti n BDNF brain-derive d neurotrophic facto r Cdk cyc l in-dependent kinase CP cortica l plate Dab disable d EGFr epiderma l growth factor recepto r

NEURONAL MIGRATIO N 3 FCMD Fukuyama-typ e congenita l muscula r dys trophy FILIP filami n A-interactin g protein GABA y-aminobutyri c acid GÈ gangliom a eminence GTPase guanidin e triphosphotase HGF hepatocyt

e growth facto r

IZ intermediat e zone Large like-acetylglucosaminyltransferas e LCN local

circuit neuron

LHRH luteinizin g hormone releasing hormone MEB muscle-eye-brai n disease MZ margina l zone nCAM neura l cell adhesion molecule NMDA N-methyl-D-aspartat e NT-4 neurotrophin- 4 PAFAHIb platelet-activatin g facto r acetylhydro lase isoform Ib , a.k.a. LISI PM pia i basement membrane POMGnTl UDP-N-acetylglucosamin e protein O-mannose ßl,2-N-acetylglucos a minyltransferase POMT1 protein-O-mannosy l transferas e SZ subventricula r zone SZa anterio r subventricular zone TGF transformin

g growth facto r

VZ ventricula r zone VLDLr ver y low-density lipoprotein receptor WWS Walker-Warbur g syndrome

ACKNOWLEDGMENTS Th e autho r thank s Dr . Eri c Olson fo r critica l readin g o f th e manuscript . Result s from th e author' s laborator y wer e supporte d b y grant s NS038877 an d HD04401 1 fro m th e Nationa l Institute s of Health. References Adams NC , Tomoda T, Cooper M, Dietz G, Hatten ME (2002) Mic e that lac k astrotacti n hav e slowe d neu ronal migration . Development 129:965-972 . Allendoerfer KL , Shatz C J (1994 ) Th e subplate , a tran sient neocortica l structure : it s role i n th e develop ment of connections between thalamus and cortex . Annu Rev Neurosci 17:185-218 .

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4

Neuronal Differentiation : From Axons to Synapses C. David Mintz Iddii H. Bekirov Tonya R . Anderson Deanna L . Benson

are usually shorter, taper with distance from th e soma and generall y branch a t acute angles. Earl y in devel opment, neurite s ar e morphologicall y indistinguish able from on e another. Over the cours e of maturation they assume the characteristic s of axons and dendrites in a process known as polarization.

A newl y born neuro n generate s a singl e axo n an d a somatodendritic domain. As it matures, i t engages, by way o f synapses, a select populatio n o f the 2 0 billion other differentiatin g neuron s i n th e huma n centra l nervous syste m (CNS) . Amazingly , the connection s that ultimately form are stereotyped and , for the mos t part, faithfull y recapitulate d acros s individuals . Thi s chapter outlines major event s in differentiation, high lighting the key cellular events and molecular mecha nisms governing the process .

Stages of Neurite Maturatio n Time-lapse imagin g of individual neurons growin g in dissociated culture s reveals a stereotyped sequence of stages in neurite development, diagramme d in Figur e 4-1 (Dott i e t al., 1988) . I n Stag e 1 , the neuro n exist s as a round som a with lamellipodia an d filopodia , bu t without neurites . Immatur e neurites , whic h canno t be identified as either axons or dendrites, emerge dur ing Stag e 2 . I n Stag e 3 , a singl e proces s undergoe s rapid growth and becomes the axon . The shorte r processes develop int o dendrites i n Stage 4. During Stag e 5, there i s further elaboration an d maturatio n o f dendrites and axon s as synapses form. Though it is not yet

ACQUISITION O F NEURONAL POLARITY

The neuroanatomis t Jan Purkinje was the first to note that ther e ar e tw o morphologicall y distinc t type s of nerve fiber s i n th e adul t nervou s system, axons an d dendrites (Shepherd , 1991) . Axons traverse long distances, maintai n a constan t caliber , an d commonl y branch a t righ t angles . I n contrast , dendrites , which 45

46 NORMA L DEVELOPMEN T

FIGURE 4-1 Stage s of neurite development Th e Stag e 1 neuron lacks neurites entirely. In Stag e 2 , immature neurite s emerg e tha t are capabl e o f becomin g either axon s or dendrites . When on e o f the immatur e neurite s outgrows the others i t becomes th e axo n and th e neuro n reache s Stag e 3 . The remainin g immature processe s becom e dendrite s an d elaborat e an d branc h i n Stag e 4 . Synaptogenesis an d spinogensi s occur i n Stag e 5 , which i s not show n i n thi s diagram.

proven definitively , it i s generall y believe d tha t th e stages o f neurit e developmen t observe d i n cultur e also occu r i n situ . Nearl y all dat a tha t addres s ques tions about neuronal polarity are derived from experi ments i n whic h neuron s ar e grow n i n dissociate d cultures where individual processes can b e identified and followed over time. Polarization is Initiated b y Axon Specificatio n All earl y neurites o n a particular neuro n ar e capabl e of becomin g axon s or dendrites . On e ultimatel y acquires axona l characteristic s an d th e remainde r as sume a dendriti c fate . I n a Stag e 2 neuron , eac h process undergoe s cycle s o f extension an d retractio n until on e neurit e outgrow s th e other s b y a critica l length—lOjLim i n hippocampa l neurons—an d be comes th e axo n (Gosli n an d Banker , 1989) . I f a recently formed axo n is transected to a length compa rable to that of the other neurites, one of the other minor processe s typicall y become s th e axo n an d th e stump of the original axon adopts dendritic character istics (Dott i an d Banker , 1987) . Thus , ever y Stag e 2 proces s ha s the potentia l t o becom e th e axon , bu t the presenc e o f a specifie d axo n activel y promotes a

dendritic fat e fo r the remainin g neurites . Thi s mode l is supporte d b y dat a showin g tha t dendriti c matura tion ca n occu r onl y after axo n specification (Cacere s et al, 1991) . As one neurit e outgrows the other s b y virtue of interactions wit h loca l environmenta l factors , i t give s rise t o a positiv e feedbac k loo p tha t create s tw o dis tinct compartments (Crai g and Banker , 1994) . As development continues , som e protein s an d organelle s become segregate d int o on e o f th e tw o compart ments, an d th e differen t complement s o f proteins i n mature axon s an d dendrite s underli e th e functiona l and morphologica l difference s betwee n the tw o types of neurites . I n recen t years , some progres s ha s bee n made in elucidating the molecula r basis of axon specification an d subsequen t maturatio n events , bu t a complete mode l ha s not yet been formulated. Role of the Cytoskeleton in the Establishment of Polarity A considerabl e bod y o f evidenc e support s th e hy pothesis that microtubule s are involved in axon specification. Quantitativ e analyse s o f seria l electro n microscopy (EM ) reconstruction s o f minor processe s and axon s in Stag e 2 and Stag e 3 neurons revea l tha t

NEURONAL DIFFERENTIATION: FROM AXONS TO SYNAPSE S 4 7

axon specification coincides with a dramatic increase in microtubule number (Yu and Baas, 1994). This increase raises the possibilit y that selective transport of microtubules t o a singl e neurit e produce s rapi d growth and subsequen t acquisitio n o f axonal charac teristics (Baas, 1999) . The efficienc y o f microtubule assembl y and th e stability of microtubules once formed are inextricably linked t o th e loca l compositio n o f mierotubule associated proteins (MAPs). Data from culture d neu rons indicat e tha t MAP s suc h a s Tau an d MAPlb , which are enriched i n axons, may also play a role i n axon specification (Caceres and Kosik , 1990 ; DiTell a et al, 1996). Mice deficient in both Tau and MAPlb, however, have only modest defect s in axo n development (Takei et al., 2000), a finding suggesting that the dependence on MAPs is exaggerated in culture or compensated for over the long term in situ. At the very least, MAPs stabilize microtubules, and this stabilization contributes t o rapi d microtubule-dependen t neurit e out growth. Other proteins that regulate microtubules may play a more crucial role in axon specification. For example, collapsi n respons e mediator protein (CRMP ) 2 i s enriched i n th e dista l regio n o f Stag e 3 axons where i t apparently facilitates microtubule assembl y (Fukata e t al. , 2002a) . Supportin g thi s possibility , a dominant negativ e CRMP- 2 lackin g th e tubuli n binding sit e prevent s axon specificatio n in culture d neurons (Inagak i e t al. , 2001) . Also , recen t studie s have demonstrate d tha t glycogen synthas e kinase 3 $ and other signaling molecules directly upstream fro m CRMP-2 ca n influenc e axon formation (Jiang et al., 2005; Yoshimura et al, 2005). It must be noted, however, that CRMP- 2 an d th e axona l MAP s are multi functional proteins , makin g the precis e significanc e of microtubules in the establishmen t of neuronal polarity unclear. The acti n cytoskeleto n ha s bee n implicate d i n axon specification . Local applicatio n o f cytochalasin D, an actin-depolymerizing agent, to the growth cone of a single neurite o f a Stage 2 neuron increase s th e likelihood tha t thi s neurit e wil l becom e th e axon . Bath application of the sam e reagent results in an increase in neurons bearing multiple axons (Bradke and Dotti, 1999). It should be noted, however, that a subsequent study argues that this effect i s actually the re sult o f faste r growt h o f al l neurites , rathe r tha n aberrant polarization (Ruthel and Hollenbeck, 2000). In Aplysia growt h cones, actin depolymerization ca n trigger increase d microtubul e elongatio n (Forsche r

and Smith, 1988). Hence, it is likely that axon specification depends on the coordinated regulation of both actin depolymerization and microtubule assembly. Signaling Pathways That Control Polarization The emergenc e of polarity requires coordination between Stage 2 neurites, suggesting the existence of intracellular signalin g pathways . Th e Rh o famil y o f small GTPases , whic h include s Rho , Rac , an d Cdc42, are candidates to control polarization, as they can regulat e bot h acti n an d microtubule s (Burridg e and Wennerberg , 2004 ; Gunderse n e t al. , 2004) . Broad-based inhibitio n o f th e entir e Rh o famil y i n Stage 2 neurons results in the emergence of multiple axons (Bradke and Dotti, 1999). Additional work supports the notio n tha t Rho, Rac, and Cdc42 ca n each play distinc t roles . Expressio n o f a Ra c dominant negative mutant in Drosophila reduces axon outgrowth (Luo e t al. , 1994) , wherea s expression o f a constitu tively activ e Cdc42 mutan t i n culture d hippocampa l neurons induce s multipl e axon s (Schwambor n an d Puschel, 2004) . In contrast, Rho can induce the col lapse of the dista l ends o f neurites via phosphyorlyation of CRMP-2 (Arimur a et al, 2000). According to one model , the spee d of outgrowth of any given neurite i s determine d b y a balanc e betwee n retractio n and collaps e promote d b y Rh o an d outgrowt h pro moted by Rac and Cdc42 (Fukata et al, 2002b). This hypothesis, whic h ha s no t bee n full y validate d i n mammalian neurons, has the potential t o explain differential growth of axons and dendrites in terms of local Rho family activity. There ar e man y parallels betwee n neurona l an d non-neuronal cel l polarizatio n (Rodriguez-Boula n and Powell , 1992) , an d ther e i s evidence that some signaling pathway s related t o polarizatio n ar e con served amon g different cel l types. The Pa r complex, which consist s of Par3, Parò, and atypica l phosphokinase C , control s th e establishmen t o f polarit y i n a broad range of species and cel l types via regulation of actin an d microtubule s (Wodarz , 2002). Recent data suggest that the Pa r complex may control axon specification upstream from th e Rho family. Par3 and Parò are polarize d to th e axon s o f hippocampal neurons , and overexpressio n of ectopie Pa r prevents polarization (Sh i e t al. , 2004) . Segregatio n o f Pa r comple x members to a single neurite, which can occur as early as Stag e 2 , i s precede d b y polarizatio n o f Ra p IB,

48 NORMA L DEVELOPMENT a smal l GTPas e tha t i s a Ra s famil y membe r (Schwamborn and Puschel, 2004) . The dat a indicate that Ra p IB i s upstrea m o f bot h th e Pa r comple x and Cdc4 2 wit h respec t t o axo n specificatio n (Schwamborn an d Puschel , 2004) . I t i s thought tha t extracellular factors, a s yet unidentified, might medi ate th e effect s o f the loca l environmen t o n polariza tion b y acting upstrea m fro m intracellula r signaling molecules such as Rho, Par, and Ra p IB. Polarized Distribution of Proteins an d Organelles The difference s i n morphology and functio n that distinguish axons and dendrites reflect different comple ments o f protein s an d organelle s i n eac h typ e o f neurite. In the adult brain, certain proteins have a polarized distribution , meanin g tha t the y ar e concen trated or even exclusively present in either dendrites or axons. This asymmetr y must be created durin g development b y compartment-specifi c traffickin g an d re tention o f thes e proteins . Th e Stag e 2 neurit e tha t becomes th e axo n in Stag e 3 typically receives more organelles, cytosoli c proteins, and Golgi-derive d vesi cles than the processes that become dendrites (Bradke and Dotti , 1997) . Intac t traffickin g i s required for polarization, as it is possible to prevent axon specification by blockin g th e traffickin g o f Golgi-derive d vesicle s with brefeldin A (Jareb and Banker , 1997). The polar ized distribution of some proteins may either promote the emergin g difference s betwee n developin g axon s and dendrite s or simply reflect thos e differences . Th e details o f how eac h polarize d protei n reache s it s appropriate destination in one compartment bu t not the other remain largely speculative. The polarizatio n o f protein s ma y resul t fro m re tention specifi c t o the appropriat e compartment. Experiments with optical tweezers, which allow traction force t o b e applie d t o specifi c proteins, sho w that a barrier t o th e diffusio n o f som e axon-specifi c mem brane protein s exist s a t th e axo n initia l segmen t (Winckler et al, 1999; Fâche et al, 2004). The barrier appears to be an accumulation of actin-tethered mem brane proteins . Analysis of phospholipid diffusio n i n the membran e suggest s that the diffusio n barrie r does not exis t prio r t o axo n specificatio n (Nakad a e t al. , 2003), but it may play an important role in subsequent maturation. The exten t to which th e diffusio n barrie r is a cause or consequent of the establishmen t of neuronal polarity remains unknown.

NEURITE MOTILITY

The neuroanatomis t Santiag o Ramo n y Caja l (1894 ) observed tha t th e dista l exten t o f a growin g neurit e "end[s] in a spherical conical swelling . .. with a large number o f thick protrusion s and lamella r processes, " which he speculated wer e like "an amoebic mas s that acts as a battering ram to spread the elements alon g its path." H e name d thi s morphologica l specializatio n the growth cone, and decade s of subsequent researc h have supporte d hi s inferenc e tha t growt h cone s ar e highly motile structures required for the elongatio n o f both axon s and dendrites . Muc h i s known about th e structure and functio n of growth cones . Th e motilit y of growth cone s depend s o n acti n an d microtubule s and th e protein s tha t regulat e them . Additionally , growth cone s ar e th e sensor y organs for extracellular cues that guide axons to engage in the directed growth required for the establishmen t of correct connectivity. Though muc h wor k remain s t o b e done , a n under standing o f ho w growt h cone s functio n i n neurit e growth i s emerging. Growth Cone Structur e Although neuronal growth cones vary considerably in shape an d size , the y generall y share som e commo n structural features. Most investigators recognize thre e morphological zone s know n a s th e central , transi tional, an d periphera l domains , which ar e indicate d in Figur e 4- 2 (Forsche r and Smith , 1988 ; Bridgma n and Dailey , 1989) . Th e relativel y thic k centra l do main abut s th e neurit e an d i s enriched wit h micro tubules. The transitiona l domain, which lies between the centra l an d periphera l domains , contain s mesh work, arcs , and radia l ridge s o f F-actin. Th e periph eral domai n consist s o f th e lamellipodium , a wide , flat region, an d filopodia, which ar e fin e membran e protrusions tha t exten d distall y fro m th e lamel lipodium. The periphera l domain i s greatly enriched with F-actin , organized a s meshwork and arc s in th e lamellipodia. Filopodia contai n bundles o f filaments with th e barbe d end s pointing distally. Compared t o the centra l domain , th e periphera l domai n ha s a lower densit y o f microtubule s an d the y ar e notabl y more dynami c (Schaefe r e t al, , 2002) . Th e leadin g edge o f migratin g fibroblast s wa s thought t o hav e a structure similar to that of the neurona l growth cone . Recent findings, however, show that th e cytoskeletal configuration an d mechanic s o f motility in these tw o

NEURONAL DIFFERENTIATION : FRO M AXONS TO SYNAPSE S 4 9

FIGURE 4- 2 Structur e o f the growt h cone . Th e mor phological zone s o f a growt h con e ar e show n usin g a differential interferenc e contras t micrograp h o f a dendritic growth cone from a cultured ra t neocortical neu ron. Not e th e thic k centra l zon e (C) , separate d fro m the fla t periphera l zon e (P ) b y th e transitiona l zon e (T). Also , i n th e periphera l zone , th e lamellipodiu m and severa l filopodia are indicated by dashed lines.

structures diffe r considerabl y (Strasse r e t al. , 2004) . Thus, the presen t discussio n of growth con e motilit y is confined to models from experiment s don e o n neuronal growth cones . Growth Cone Motility The exac t relationship between filopodial, lamellipodial, an d neurit e motilit y i s no t completel y under stood. Filopodi a sampl e th e environmen t fo r cues t o determine th e directio n o f growth (O'Conno r e t al. , 1990), and thu s filopodial motility tends to reflect this scanning patter n rathe r tha n demonstratin g a tigh t link wit h neurit e growth . Th e movement s o f lamellipodia ar e better correlate d wit h neurite growt h tha n those o f filopodia , bu t lamellipodi a commonl y en gage i n extensio n an d retractio n tha t d o no t directl y correlate wit h alterations i n the trajector y of the grow ing neurite. Growth con e motilit y requires dynamic regulatio n of th e cytoskeleton . Th e treadmil l model , whic h i s based on observation s in filopodia, focuses o n the rol e of acti n (Fig . 4-3 ) (Li n e t al , 1994) . Filament s o f actin ar e simultaneously polymerized at the dista l tips of filopodi a an d retracte d proximall y b y myosi n

FIGURE 4- 3 Th e acti n treadmil l mode l o f motility. The tw o principle features of the treadmil l mode l ar e the retrograd e flo w o f actin, whic h i s due t o myosi n motors, and th e polymerization of actin at the leading edge o f the filopodi a (o n right) . I f polymerization i s faster tha t retrograd e flow , ther e i s forwar d move ment; i f it is slower, there i s retraction. Note tha t acti n is coupled t o the substrat e by linker proteins that bin d transmembrane CAMs .

motors (Li n e t al., 1996) . I f the rat e o f actin polymer ization a t th e leadin g edg e i s greater tha n th e rat e of actin retrograd e flow , th e filopodi a advances . I f polymerization is slower than retrograd e flow, the filopodia retracts. If polymerization and retrograd e flow are bal anced, ther e i s no ne t chang e i n th e positio n o f th e filopodia. The substrat e on which a neurite grow s can interact wit h transmembran e adhesio n molecules , which ar e attache d t o acti n indirectl y via linker pro teins. Th e couplin g o f the substrat e t o th e acti n cy toskeleton (a ) allow s fo r th e generatio n o f tensio n when force s are applie d t o actin an d (b ) accounts for the influenc e o f substrat e o n growt h con e motilit y (Suter an d Forscher , 1998) . Thi s mode l i s not com plete, a s axon s treate d wit h acti n depolymerizin g agents stil l demonstrat e ne t growt h (Bentle y an d Toroian-Raymond, 1986) , an d applicatio n o f rnicro tubule destabilizing reagents can preven t neurit e out growth (Bambur g et al., 1986) . I t should be noted tha t current model s o f growt h con e motilit y ar e largel y based o n experiment s i n unpolarized Aplysia neuron s or in spinal or sensory neurons isolate d from frog s an d chicks. Som e mechanism s o f growt h con e motilit y may differ amon g species , cell types, and neurit e type , but th e regulator y molecule s ar e likel y t o b e conserved. A larg e an d divers e grou p o f proteins tha t modu late cytoskeleta l dynamic s throug h intracellula r signaling o r direc t actio n i s involve d i n regulatin g growth con e motility . Th e Rh o famil y o f proteins ,

50 NORMA L DEVELOPMEN T which ca n influenc e bot h th e acti n an d microtubul e cytoskeleton, play s a n importan t rol e i n regulatin g growth con e motility . Th e contro l o f actin polymer ization a t the leadin g edge o f growth cones i s an are a of activ e study . Member s o f th e Ena/Vas p family , which preven t barbe d acti n capping , ca n regulate th e behavior o f filopodi a (Lebran d e t al. , 2004) . Othe r molecules tha t ma y pla y role s i n acti n dynamic s a t the leadin g edg e includ e profilin , fascili , an d acti n depolymerizing factor/cofili n (Will s e t al. , 1999 ; Meberg an d Hamburg , 2000 ; Coha n e t al , 2001) . Further wor k i s require d t o construc t a cohesiv e model o f ho w thes e an d othe r protein s regulat e cy toskeletal dynamics at the leadin g edge o f the growt h cone. Directed Growth Growth cone s ar e generall y considere d t o mediat e the respons e o f growin g axon s an d dendrite s t o th e myriad of guidance cue s that direct the establishmen t of appropriat e connectivity. It i s possible t o alte r th e trajectory o f neurite growt h b y applying a gradient of a chemotropi c molecul e t o a growt h con e i n vitr o (Zheng e t al. , 1994) . I n fact , thi s assa y i s now com monly use d t o tes t whether a protei n ca n serv e a s a guidance cue. Furthermore, many receptors for known guidance cues are expressed in the plasma membran e of growth cones. I t i s believed that gradient s of guidance cue s acros s the surfac e o f the growt h con e pro duce asymmetri c effect s o n receptor s tha t mediat e growth cone steerin g (Goodman, 1996) . The respons e o f growt h cone s t o guidanc e cue s depends o n bot h th e acti n an d microtubul e compo nents o f the cytoskeleto n an d th e effector s tha t regu late them downstrea m fro m guidanc e receptors . I t has been propose d that growth cone turnin g occurs when actin i s polymerize d asymmetricall y withi n th e growth cone an d whe n there i s a subsequent increase in microtubul e entr y int o an d polymerizatio n in th e actin ric h zon e (Den t and Gertler , 2003) . Consistent with this model, disruptions of regulators of either th e actin o r th e microtubul e cytoskeleto n ca n resul t i n defective axo n guidance. Example s include th e acti n interactors Arp2/3 and Ena/Vas p (Lanie r e t al. , 1999 ; Lee e t al, 2004 ; Strasse r e t al, 2004 ) a s well a s the microtubule interactors , plu s end-trackin g protein s (Lanier et al, 1999 ; Le e et al, 2004). The precis e significance o f these molecules in directed growth, however, remains a subject of active investigation.

Several molecule s tha t ca n signa l betwee n guid ance receptor s an d th e cytoskeleto n i n growt h cone s have been identified . Rho family members, which ca n regulate bot h acti n an d microtubules , hav e bee n im plicated i n signalin g downstrea m fro m guidanc e receptors (Gua n an d Rao , 2003) . Also, relativ e con centrations o f cycli c nucleotide s (cycli c adenosin e monophosphate [cAMP ] an d cycli c guanosin e monophosphate [cGMP] ) can modulate the respons e of growth cones t o guidance cues . Fo r instance, a gradient o f brain-derived neurotrophi c facto r (BDNF), a factor tha t i s usuall y attractiv e fo r Xenopus growt h cones, becomes repulsiv e if the growt h cone is treated with a cAM P inhibito r (Song e t al, 1997) . Anothe r potential signa l downstream from guidanc e receptor s is Ca 2+. Liv e imagin g o f growt h cone s i n cultur e shows tha t transien t increases in Ca 2+ o n on e sid e of the growt h con e correlat e wit h turnin g towar d th e contralateral sid e (Gomez e t al. , 2001) . Interestingly, larger Ca z+ transients can resul t in ipsilateral turning. This paradox i s explained b y data indicatin g tha t low concentrations o f Ca 2+ ac t primaril y throug h cal cineurin phosphatase, whereas higher concentration s act vi a calcium-calmodulin-dependen t protei n ki nase I I (We n e t al. , 2004) . Th e recen t findin g tha t Ca2+ concentrations i n the growth cone can be mod ulated by cAMP and cGMP control of calcium chan nels suggests that Ca 2+ acts on growth cone guidanc e downstream fro m th e cycli c nucleotide s (Nishiyam a et al., 2003). Though data on interactions between re ceptors, signals , effectors , an d th e cytoskeleto n ar e emerging, muc h remain s t o b e don e befor e a com plete mode l o f how a specific guidanc e cu e result s in a change i n growth cone motilit y can be constructed .

AXON GUIDANC E In th e lat e nineteent h century , Ramo n y Caja l pro posed tha t axon s coul d b e guide d chemotropically , but th e ide a fel l ou t of fashion and was untested unti l it was revisited by Roger Sperr y in th e 1940 s (Sperry, 1963). I n a serie s of classi c experiments, Sperr y severed a frog opti c nerve, rotated the ey e 180°, and the n allowed connections t o form again . These frogs mad e 180° error s i n their attempt s t o catch flies. In contra diction t o the popula r view, including that of his the sis advisor , Sperr y conclude d tha t th e regenerate d axons di d no t innervat e ne w targe t region s t o com pensate fo r th e ey e rotation , bu t projecte d t o thei r

NEURONAL DIFFERENTIATION : FRO M AXON S TO SYNAPSE S 5 1 original sites in the tectum . The outcome s o f this and other relate d experiment s le d Sperr y t o propos e tha t extending axon s ar e guide d b y "cytochemica l tags. " He furthe r propose d tha t neura l connectivity i s specified wit h onl y a fe w such tag s o r cue s distribute d in orthogonal gradient s (Sperry , 1963) . Severa l of thes e cues have no w been identified , validatin g these principles and indicatin g that axo n guidanc e i s best con sidered in their context . Guidance cue s ca n b e secrete d fro m a restricted source to form long-range, diffusible gradient s that act as molecula r beacons , wherea s membrane-attache d cues ca n for m short-range , standin g gradient s (se e Directed Growth , above) . Concurrently , adhesio n molecules expresse d alon g th e surfac e o f axon s ca n interact wit h othe r axon s o r particula r substrate s t o promote axo n fasciculation , extension , turning , o r stopping. Most cue s have differen t function s at differ ent stage s o f developmen t o r i n differen t pathways , and man y hav e multipl e receptor s an d co-receptor s that ca n mediat e differen t responses . Thi s hig h de gree of complexity make s it difficult t o sort out whic h interactions ar e importan t fo r th e generatio n o f particular connections , bu t i t supports Sperry' s ide a tha t a smal l numbe r o f guidanc e cue s ca n generat e th e diverse response s require d fo r high-fidelit y network development. I n ligh t o f thi s diversity , exemplar y cases o f axo n guidanc e hav e bee n selecte d t o illus trate how the challenge s o f directed growth and specificity are met. Many individua l growt h cone s appea r t o expres s all o f the receptor s require d a t th e appropriat e tim e and place t o respond t o the cue s necessar y for their final targeting. Some pathway s capitalize on the effort s of a fe w pionee r neurons . Pioneer s ar e bes t under stood in invertebrates, where it is well established that they differentiat e an d mak e connection s earl y in de velopment when distances are small. The connection s provide a pat h fo r axon s extendin g fro m later-bor n neurons. I n mammalian cerebra l cortex , th e develop ing cortical plate i s sandwiched between transien t layers called the marginal zone and subplate that contai n the earliest born neurons (Marin-Padilla, 1971) . Populations of subplate neurons send axons to the thalamus before th e laye r V and VI neurons that form th e adult projections are even born (McConnel l e t al., 1989) . In a very different way , Cajal-Retzius neurons i n the mar ginal zone pioneer local intracortical connections an d may b e importan t fo r generating a columna r organi zation o f cortical networks (Sarnat and Flores-Sarnat ,

2002). I n thi s way , pioneer neuron s produc e frame works that , fo r particula r networks , ar e critica l fo r specifying pathways. Midline Crossing in Vertebrate Ventral Spinal Cor d After closel y examinin g the appearanc e an d trajecto ries of Golgi-impregnated neuron s in the spina l cord , Ramon y Caja l hypothesize d tha t group s o f dorsally disposed neuron s giv e rise to axon s tha t ar e attracte d to th e ventra l midline . Th e proo f cam e nearl y 10 0 years later , whe n dissecte d midlin e expiant s wer e demonstrated t o b e attractiv e to commissurall y pro jecting axons (Tessier-Lavigne et al., 1988 ) (Fig . 4-4) . This system was used a s a bioassay to isolat e netrin-1 and -2 , whic h ar e solubl e cue s tha t ac t a s attractive proteins for commissural axons. Subsequent wor k ha s demonstrated tha t netrin' s attractiv e actio n i s medi ated b y binding t o it s receptor, delete d i n colorecta l cancer (DCC ) (Kenned y et al., 1994 ; Serafin i e t al, 1994; Keino-Mas u et al., 1996 ; Fazel i et al, 1997) . Interestingly, mutant mic e hypomorphi c fo r the netrin 1 allele or lacking DCC stil l show some commissura l axon crossing (Serafini e t al., 1996 ; Fazel i et al., 1997) . This findin g indicate s that netri n doe s no t ac t alone . Sonic hedgeho g (Shh) , a morphoge n secreted b y the floor plate , i s better know n fo r it s role i n neura l pro genitor specificatio n (Charron e t al., 2003; Yoshikawa and Thomas, 2004). Shh ca n also attract or influence the directio n o f polarizatio n o f commissura l axon s through it s receptor smoothened (Smo) . Assisting the actions o f both Sh h an d netrin , heterodimers of bone morphogenic protein 7 (BMP7) and growt h differenti ation facto r 7 (GDF7) secreted fro m dorsa l roof plat e cells repel th e incipien t commissural axons away fro m the dorsa l midlin e (Augsburge r et al. , 1999 ; Butle r and Dodd , 2003). Once attracte d t o the floor plate, commissural axons cross th e midlin e (Seege r e t al., 1993) . Othe r axons turn bac k and for m ipsilatera l longitudinal tracts. Crossing i s regulate d i n par t b y th e signalin g o f chemorepellants of the Sli t family which ar e secrete d by midlin e gli a an d bin d Rob o famil y receptor s o n commissurally projectin g growt h cone s (Rothber g et al , 1988 , 1990 ; Eros e e t al. , 1999 ; Lon g e t al , 2004). Unlike other Robo famil y members , th e recep tor Rig- 1 appear s t o attenuat e Sli t sensitivity , since commissural axon s neve r cros s th e midlin e i n Rig-1 deficient mic e (Sabatie r e t al. , 2004) . I n wild-typ e

52 NORMA L DEVELOPMEN T

FIGURE 4- 4 Midlin e guidance . Schemati c diagram s o f developin g mam malian spinal cord depic t the trajectorie s o f eommissurally projecting neurons relative to the roo f plate an d floor plate i n cross-section (A ) or molecular gradients i n longitudinal preparatio n through th e ventra l rnidline (B) . Pre-crossing axons are repelled fro m dorsa l midline b y BMP? in the roo f plate (no t shown) and attracte d ventrall y by netrin an d Sh h gradient s emanatin g from th e floor plate (shade d circles) . Post-crossing axons become sensitive to Slit - and Sema phorin 3 B an d F-mediate d repulsio n an d d o no t retur n t o th e floo r plate . (Source: Modifie d from William s et al, 2004)

animals, Rig- 1 concentration s decreas e an d Robo- 1 and - 2 amount s increas e soo n afte r crossing , whic h may serv e to expe l axon s from th e midlin e (Sabatie r et al. , 2004) . Thi s expulsio n i s probabl y aide d b y Robo-Ts abilit y t o silenc e DCC-mediate d attractio n to netrin b y binding directly to DCC i n the presenc e of Slit (Stein an d Tessier-Lavigne , 2001 ) or by growth cone desensitizatio n t o th e hig h concentration s o f netrin at the midline (Pipe r et al., 2005). Sema-3B, an additional solubl e repellan t secrete d b y th e floo r plate, prevent s axon s fro m returnin g t o th e midlin e via interactions with it s receptor neuropilin- 2 (Zou e t al., 2000) . Afte r crossing , th e positio n o f longitudi nally runnin g tract s appear s t o b e regulate d b y th e midline Sli t gradien t interactin g wit h Robo- 1 o r - 2 (Rajagopalan et al. , 2000 ; Simpso n et al. , 2000) . Concurrently, Wnt-4 , actin g throug h it s recepto r frizzled3, an d Shh , actin g throug h hedgeho g inter acting protein , hel p orien t axon s anteriorl y i n th e longitudinal axi s (Lyuksyutov a et al. , 2003 ; Bourikas er al., 2005) , but th e interactio n betwee n thes e cue s has ye t t o b e investigated . Collectively , thes e dat a support th e idea s tha t certai n guidanc e cue s ca n

trump th e other s an d sugges t that cue s ar e processe d hierarchically rather than bein g integrated. Generating Retinotectal Maps Once axon s reach thei r target region , they terminat e in highly organized pattern s tha t reflect their points of origin. Th e projection s extendin g fro m retina l gan glion cell s t o the tectu m i n fish and frog s an d t o th e superior colliculus in rodents has served as the canon ical syste m fo r studyin g the developmen t o f pattern ing. I n mos t mammals , a ligh t show n i n th e fa r lef t visual field projects o n th e ventronasa l part of the lef t retina, whic h i n tur n project s contralaterall y t o th e caudomedial regio n o f th e superio r colliculu s (Fig . 4-5). I f a n experimente r systematicall y move s a recording electrode rostrall y through th e superio r colliculus, th e stimulatin g ligh t sourc e need s t o b e moved i n a n orderl y progression fro m lef t t o righ t to activate th e neurons . Thus , th e superio r colliculu s contains topographic map s of the visual field. A larg e bod y o f dat a suppor t th e correlatio n o f ephrin gradient s i n the retin a an d superio r colliculus

NEURONAL DIFFERENTIATION : FROM AXONS T O SYNAPSE S 5 3 interactions (McLaughlin et al., 2003; van Horck et al., 2004). EphA receptor protein kinases are expressed in high i n temporal an d low in nasal retina. EphrinA ligands ar e expresse d mostl y in hig h cauda l t o lo w rostral gradient s in th e superio r colliculus. Th e ligand s guide the formatio n of retinotopic map s oriented i n a rostral-to-caudal progressio n b y repulsiv e eyes . Mice having targeted disruption s in both ephrinA 2 and A5 genes sho w a nea r complet e los s o f rostral-to-caudal patterning in the superio r colliculus (Feldheim e t al., 2000). Introducin g ne w gradient s b y overexpressing additional Eph A receptor s i n subpopulation s o f reti nal ganglio n cell s results in the creatio n o f two maps along th e rostral-to-cauda l axi s (Brow n et al. , 2000) . Orthogonal gradient s o f EphB Z i n th e retin a an d ephrin-Bl an d 2 in the tectu m labe l the dorsoventral axis (Braiste d et al. , 1997) . Member s o f the ephrin B subfamily appea r t o operat e principall y via attractiv e rather tha n repulsiv e interaction s (Hindge s e t al. ,

2002; Man n e t al. , 2002) . Th e mechanism s guidin g retinal ganglio n cel l axon s appea r t o follo w Sperry' s chemoaffinity mode l of targeting by orthogonal stand ing gradient s (Sperry, 1963) . I n a n importan t exten sion o n thi s model, recen t wor k indicate s that at low concentrations, EphA-ephrin A interaction s ar e at tractive fo r axo n extension , wherea s hig h concentra tions elici t a repulsiv e response . Thu s axon s wil l readily exten d int o th e superio r colliculus until the y reach a point where positiv e and negativ e influences are balanced (Hanse n et al, 2004). It is noteworthy that in mice lacking ephrinA2 an d A5, retinal ganglio n cel l axon s still target the superio r colliculus, an d thei r axona l arbors are evenl y distributed within the entir e space allotted (Feldhei m e t al., 2000). Th e mechanis m determinin g this interaxonal spacing remains unknown. Furthermore, i n the sam e mice, ther e i s evidenc e i n th e clusterin g o f opti c nerve terminal s tha t som e nearest-neighbo r relation ships, an d thu s som e topographi c ordering , i s maintained over small distances. Such clusterin g has been proposed t o be th e resul t o f correlated activit y (Feldheim e t al, 2000 ) and support s th e ide a tha t activity plays a majo r rol e i n th e prunin g an d shapin g o f the final map s tha t ar e generate d b y standin g Ep h an d ephrin gradients. Cues Are Bifunctional

FIGURE 4-5 Th e retinocollicula r map. Schemati c diagram depict s orthogona l gradient s o f Eph s an d ephrins tha t mediat e retinotopi c mappin g togethe r with the trajector y of a single projection i n the roden t projection fro m retin a t o superio r colliculus . Ligh t from a point in the fa r left visual field strikes the nasa l retina o f the lef t ey e (circle) in it s monocular region . Ganglion cell s i n thi s zon e projec t (dar k axon ) t o caudal-medial point s on the righ t superior colliculus (oval). Eph A tyrosin e kinase s bind an d ar e repelle d from hig h concentration s o f ephrinA ligands, whereas EphB tyrosin e kinases bind an d ar e attracted t o hig h concentrations of EphB ligand.

Netrins ar e generall y attractiv e an d EphA-ephrin A interactions, repulsive , but nearl y all guidanc e cue s can generat e diametricall y opposed response s in particular circumstance s (Fig . 4-6) . Differen t response s are sometimes th e functio n of different cel l types. For example, netrin-1 , secreted b y the ventra l floor plate, repels moto r axon s extending fro m th e trochlea r nu cleus such tha t they exit the dorsa l aspect of the brain at th e midbrain-hindbrai n border . Thi s stand s i n stark contras t to it s attraction for spina l commissura l axons (Colamarino an d Tessier-Lavigne, 1995) . More commonly, changes between attractio n and repulsion occur a t differen t stage s o f outgrowth an d appea r t o be employe d withi n individua l growt h cones . Suc h dynamic signalin g appear s t o b e require d fo r steady axon extensio n throug h transitio n zones, suc h a s th e ventral midline, and to titrate growth cone responsiveness withi n a targe t t o achiev e a smoot h representa tional map . Bifunctional response s resul t fro m differentia l activation o f downstrea m signalin g pathway s tha t

54 NORMA L DEVELOPMEN T

FIGURE 4- 6 Guidanc e cues . Example s of long- and short-rang e axon guidanc e proteins used i n vertebrates. Molecules are depicted acros s from on e anothe r a s they would be i n situ. Members o f the ephri n family generate topographic maps , such as that found in th e retinotectal projection . Integrins , an d immunoglobuli n (Ig ) superfamil y member s o f the calcium-dependen t (classi c cadherins) an d calcium-independen t classe s (e.g. , Ng CAM) mediat e short-rang e guidanc e throug h contact-dependen t mechanisms . Thre e major familie s o f long-range axo n guidanc e ar e indicate d here : netri n an d it s receptor , DCC; Sli t and it s receptor, Robo; an d clas s 3 Semaphorins and it s co-receptor complex , neuropilins an d plexins . (Source: Modifie d fro m Benso n e t al. , 2001 , an d Y u an d Bargmann, 2001)

ultimately regulat e acti n dynamic s (fo r review , se e Pasterkanip and Kolodkin , 2003). For instance, attractive growt h con e turnin g o f Xenopus spina l moto r growth cones to gradients of netrin-1 can be converte d to repulsio n upo n associatio n o f the cytoplasmi c do mains of the netri n DCC recepto r with a co-receptor, UNC5 (Hon g e t al , 1999) . Additionally , changing amounts o f cycli c nucleotide s ca n chang e growt h

cone response s t o BDNF , netrin-1 , an d Sema3 D (Ming et al, 1997 ; Son g e t al, 1998) , results suggesting that previous experience o r environment ca n alter responsiveness. I n suppor t o f this possibilty, Xenopus retinal ganglion cell axons grown on laminin have decreased ratios of cAMP/cGMP relative to those grow n on fibronectin, and ar e repulsed rathe r than attracte d by netri n (Hopke r e t al. , 1999) . Thes e example s

NEURONAL DIFFERENTIATION : FRO M AXONS TO SYNAPSE S 5 5 (a) suggest that the respons e to a cue ca n be modified by bot h outside-i n (recepto r t o signalin g pathway) and inside-ou t (signalin g molecule to altered stat e of receptor responsiveness ) mechanisms and (b ) support the ide a tha t growt h cone s hav e multipl e mean s by which to finely tune their responses . Adhesion and Guidance Growth cones explore their environment by regulating their attachment to other neurons, glia, or components of the extracellular matrix (ECM). Contact-dependent interactions betwee n cel l adhesio n molecule s (CAMs) o f th e immunoglobuli n superfamily , cad herin superfamily , integrins, an d th e EC M generat e traction sufficien t fo r growth cone extension . Interactions between CAMs and ECMs that are inhibitory to growth creat e boundarie s (Sno w an d Letourneau , 1992). It has long been suspecte d that standing gradients o f ECM s ma y als o b e abl e t o orien t o r stee r growth cones , bu t th e dat a directl y supportin g thi s role a s a guidanc e cu e ar e quit e recen t becaus e o f the difficult y o f generatin g continuous , substrate bound gradient s (Adam s et al. , 2005) . Cell-cel l an d cell-substrate interaction s can promote axon bundling (fasciculation) an d defasciculation , thereb y makin g the proces s o f guidance mor e efficient . O n th e othe r hand, there i s little evidenc e indicatin g that selective fasciculation serve s as a critica l guidanc e cu e (Ben son et al, 2001). Contact-dependent interaction s ca n sto p axo n extension when i t has reached it s final targe t region, preceding th e formatio n o f termina l branche s an d synapses. Axons usually terminate withi n small neural groups or a restricted number o f layers, greatly reducing th e numbe r o f potentia l individua l neura l targets. Homophilic N-cadherin adhesion appears to prevent thalamocortica l an d retinocollicula r axon s from extendin g pas t thei r targe t layer s (Inou e an d Sanes, 1997 ; Poskanzer et al., 2003) and can apically restrict mossy-fibe r terminal field s i n hippocampu s (Bekirov e t al. , 2002) . Similarly , attractive interactions betwee n axonin-1/transien t axona l glycopro tein 1 an d neuron-gli a CA M (Ng-CAM) , an d between Fll/contacti n an d Ng-CAM-relate d CA M (Nr-CAM) contribut e t o lamina-specifi c termina tions o f nociceptiv e an d proprioceptiv e fibers , re spectively, i n chic k spinal cord (Perri n et al., 2001). Thus, CAM s appea r t o encode lamina r recognitio n cues, bu t the y ma y als o provid e a sto p signal ,

preventing axon s fro m extendin g further . Whethe r recognition an d stoppin g ar e mediate d concomi tantly by a particular cue o r combination o f cues has yet to be investigated. Few experiments have addressed directly how adhesive cue s becom e integrate d wit h othe r guidanc e cues. Th e emergin g pictur e promise s a complicate d answer. The CAM L I can suppor t rapid axon extension, but in a cis complex with the receptor neuropilin1, i t als o appear s t o b e require d fo r repulsio n o f cortical axon s by the solubl e ligand Sema3 A (Castel lani et al., 2000). Additionally, soluble L I (whic h can be generate d b y metalloproteas e cleavage ) can convert a repulsive Sema3A response to attraction (Castellani et al., 2000) in a manner reminiscent o f laminin's ability to convert an attractiv e netrin response to on e of repulsio n (se e Cue s Ar e Bifunctional , above ) (Hopkeretal., 1999) . Interactions betwee n substrat e an d solubl e cue s may underli e ho w commissura l axon s ca n travers e the neutra l midlin e an d exten d laterall y toward longitudinal tracts . Rob o activatio n by Sli t ca n elimi nate N-cadherin-base d adhesio n tha t normall y promotes rapi d extensio n (Rhe e e t al. , 2002) . This activation correlates with the formatio n of a complex between Robo , N-cadherin , an d Abelso n kinas e an d is accompanie d b y phosphorylatio n o f p-catenin , a necessary cadheri n bindin g partner. Participatio n in this complex, i n turn, occludes interactio n o f N-cadherin wit h the acti n cytoskeleton . On e ca n imagin e that rapi d N-cadherin-mediate d outgrowt h concur rent with hig h Rig- 1 expression would suffic e t o permit progressio n throug h a n otherwis e neutra l midline t o th e poin t a t whic h Rob o concentration s increase, wher e axon s would disengag e fro m N-cad herin, turn , an d gro w longitudinall y by a differen t mechanism. Thus, severa l attractiv e and repulsive , short - and long-ranged cue s ac t hierarchicall y and occasionally collaborate t o generat e a fina l stereotype d targetin g outcome. Som e cue s ac t similarl y throughou t th e rostral-caudal exten t of the developing nervous system, but th e action s o f others appear t o diffe r acros s CN S regions. Additionally , certain molecule s ar e co-opte d during multiple epochs of development for slightly different purposes. The tempora l sensitivities of such cues are just beginning to be appreciated wit h the advent of conditional geneti c tool s an d improve d method s fo r transient transfections. These techniques ar e useful for determining th e virtuall y unknow n mechanisms tha t

56 NORMA L DEVELOPMEN T stop axon extension or stimulate the generatio n of terminal arbors for synapse formation.

and electrica l synapse s begin s wit h description s o f their matur e for m an d composition .

SYNAPTOGENESIS

Structure an d Composition of Mature Chemica l Synapse s

Sherrington coine d th e ter m synapse i n 189 7 t o de scribe the site s where axons contact th e dendrite s o r soma o f other neurons , i n keepin g wit h th e neuro n doctrine tha t nervou s tissue comprises separat e cells . Although thi s doctrine als o was espoused b y Ramon y Cajal ? Kôlliker , His, Forél, and severa l othe r contem poraries, Golgi an d other s ferventl y supported a n op posing reticular theory that the nervous system was an anastomosing network (Shepherd, 1991) . Sinc e arguments fro m bot h side s wer e base d mostl y o n Golgi impregnated sample s viewe d b y ligh t microscopy , unequivocal evidenc e supportin g the former doctrin e did not come unti l half a century later from a series of electrophysiological an d E M studies . Kuffle r (1942 ) and Fat t an d Kat z (1951 ) demonstrate d a t th e fro g neuromuscular junctio n tha t th e curren t underlyin g changes i n membran e potentia l a t th e postsynapti c muscle endplat e coul d no t deriv e directl y fro m th e presynaptic moto r axon , as the reticula r theory would have predicted . The n studie s b y Palad e an d Pala y (1954) an d D e Roberti s an d Bennet t (1955 ) i n th e mid-1950s provided clear, high-resolution EM image s of a cleft separatin g pre- and postsynapti c membrane s at chemica l synapses . Ironically , soli d evidenc e supporting th e existenc e o f a n electrotoni c synaps e came shortly thereafter, with the identificatio n o f gap junctions, wher e Bennet t (1963 ) an d other s demon strated tha t ioni c curren t ca n flow directly fro m on e neuron t o anothe r (Peter s e t al. , 1991 ; Shepherd , 1991). Subsequent electrophysiological , anatomical , an d biochemical findings continu e to challeng e our col lective notio n o f th e synaps e beyon d Sherrington' s general conceptualization . Thoug h characterizin g the matur e synaps e in differentiate d neura l networks has occupied neurobiologist s fo r more tha n a century, more recen t wor k indicate s tha t earlie r stage s o f assembly and differentiatio n ar e crucial for generating a normal network . I n thi s vein , the las t sections o f this chapter describ e ho w axona l and dendriti c morpho logies an d compositio n ar e modifie d t o assembl e synapses i n th e vertebrat e CNS . Sinc e maturatio n i s generally assesse d b y th e exten t t o whic h adul t fea tures are present, the following discussion of chemical

Ultrastructural Features The matur e chemica l synaps e is a polarized, adhesiv e junction formed mos t commonly between axon s and dendrites. In electro n micrograph s thi s synaps e (sometimes called a zonula adherens) i s characterized by rigidl y parallel pre - and postsynapti c membranes , separated b y a clef t o f 1 2 t o 2 0 nm, electron-dens e material withi n th e clef t and , i n most cases , thick , electron-dense materia l i n th e cytoso l subjacen t t o the postsynapti c membran e (Fig . 4-7) . Thi s synapse resembles othe r adhesive junctions , but can be distinguished b y th e presenc e o f synapti c vesicle s (SVs ) close t o the junctio n i n the presynaptic cytoso l (Gray , 1963). I n aldehyde-fixe d E M specimens , SV s ar e roughly 40-50 nm i n width , ca n appea r spherica l o r oblong (pleomorphic) , an d ca n hav e agranula r o r electron-dense core s (Grill o an d Palay , 1963 ; Val divia, 1971) . A t mos t synapse s the y ar e foun d i n a readily releasable poo l nea r the plasm a membrane or in a mor e remove d reserv e pool . Vesicle s within thi s pool ca n b e foun d docke d withi n a gridde d arra y of presynaptic cytomatri x component s or , i n photore ceptors, along a presynaptic ribbon that runs orthogo nal t o th e junctio n (Sjostrand , 1958 ; Gray , 1959a ; Bloom an d Aghajanian , 1966 , 1968) . Mitochondri a are ofte n foun d both pre - and postsynapticall y (Ibat a and Otsuka , 1968 ; Bodian , 1971) , an d a postsynapti c web of filaments is often subjacen t to the postsynapti c density (PSD ) (D e Roberti s e t al, 1961 ; Gray , 1963 ; Peters an d Kaiserman-Abramof , 1969 ; Stewar d an d Levy, 1982 ; Space k and Harris , 1997) . Although dendrodendritic , dendrosomatic, and all other conceiveable pairing s do occur in the vertebrat e CNS, th e larg e majorit y o f chemica l synapse s encountered ar e axosomati c o r axodendriti c (Peter s et al , 1991) . Axodendriti c synapses occur o n eithe r smooth dendriti c shaft s o r mushroom-shape d protru sions calle d spines tha t stu d th e dendriti c arbor s of most glutamatergi c neuron s (Nimchinsk y e t al , 2002). Spin e synapse s ar e typifie d b y a pronounce d PSD, a relativel y wide clef t (nea r 2 0 nm), polyribo somes i n clos e proximit y t o smoot h endoplasmi c reticulum, an d rounde d presynapti c vesicle s i n

NEURONAL DIFFERENTIATION: FROM AXONS TO SYNAPSE S 5 7

FIGURE 4- 7 Chemica l synapse . This electro n micro graph (left ) an d schemati c diagra m (right ) o f un stained tissu e section s take n fro m adul t ra t spina l cord depic t severa l ultrastructura l features typical of mature type I, asymmetric synapses. Among these features, abundant presynaptic vesicles and a pronounce d

postsynaptic densit y (PSD) ar e the mos t reliabl e an d conspicuous. Als o visible are th e electron-dens e col lection o f transmembran e an d matri x molecule s i n the synapti c clef t an d th e filamentou s we b subjacent to th e PSD . Mitochondri a ar e presen t i n bot h th e presynaptic terminal and the dendritic spine.

aldehyde-fixed tissues . By comparison, som e shaf t an d nearly all somal synapses have a much les s pronounced or absent PSD, a narrower cleft (near 12 nm), and con tain both rounde d and oblon g (pleomorphic ) vesicles. In Gray' s studie s o f cerebral cortex , h e terme d thes e synapses type s I an d II , respectively , althoug h Colon nier's distinctio n o f asymmetrica l an d symmetrica l synapses based o n the appearanc e o f the PS D i s more widely used (Gray , 1959b; Colonnier, 1968).

neurotransmitter an d neuropeptid e (Meiste r e t al. , 1986), an d som e 8 0 nm dense-cor e vesicle s can carr y catecholamines. Additionally , another typ e o f 8 0 n m dense-core vesicl e i s though t t o delive r protein s re quired a t nascen t synapse s (Ahrnar i e t al. , 2000 ; Bresler e t al. , 2001 ; Shapir a e t al, 2003) . Th e latte r also has been called a Piccolo-Bassoon transport vesicle (PTV) for the proteins use d t o identify it during purification (Zhai , 2001) . Converging technica l approache s hav e bee n use d to identif y an d characteriz e a bev y o f synapti c con stituents, suc h a s thos e involve d i n vesicl e traffickin g (Sudhof, 2004) . Synapsin s i n th e vesicl e membran e tether SV s to an acti n framewor k until the y ar e phos phorylated b y calcium-dependen t kinase s followin g Ca2+ influ x (Bahle r an d Greengard , 1987 ; Petrucc i and Morrow , 1987) . Membrane-boun d Rab 3 appear s to modulat e transpor t o f thes e liberate d vesicle s t o active zone s (Leender s e t al. , 2001) . SV s doc k a t these site s throug h th e bindin g o f solubl e N ethylmaleimide-sensitive fusio n protei n attachmen t protein receptor s (SNARES ) i n th e vesicl e mem brane t o target s a t th e plasm a membrane . Amon g these interactions , th e comple x forme d b y the vesicular SNARE S synaptobrevin/vesicle-associate d

Molecular Constituents Morphological distinction s betwee n synapse s reflec t functional differences : roun d SV s a t asymmetri c synapses usuall y release glutamate , an d pleomorphi c SVs a t symmetri c site s commonl y carr y inhibitor y neurotransmitters (Eccle s e t al , 1961 ; Uchizono , 1965). Smal l dense-cor e SVs , i n contrast , generall y carry catecholaminergi c neuromodulators , an d larg e dense-core vesicle s (-100-20 0 nm) carr y neuropep tides (D e Roberti s an d Pelligrin o d e Iraldi , 1961 ; Wolfe e t al. , 1962) . A s wit h mos t classificatio n schemes, thes e correlation s hav e notabl e exceptions . For instance , som e small , clea r SV s contai n neu ropeptides suc h a s oxytoci n o r a combinatio n o f

58 NORMA L DEVELOPMENT membrane protei n wit h it s target SNARES , synapto somal associated protein (SNAP) 25 and syntaxin , was the first identified and i s the most widely known (Sollner e t al. , 1993 ; Pevsne r e t al. , 1994) . Syntaxins , i n turn, ca n bin d t o eithe r Muncl S o r Muncl3 i n th e presynaptic gri d t o modulat e th e primin g event s for vesicle fusio n (Hat a et al., 1993 ; Asher y et al, 2000 ; Misura et al, 2000). SNAP-25 binding by synaptotagmin i n th e vesicl e membran e appear s t o regulat e subsequent fusio n por e openin g durin g calcium dependent exocytosi s (Jorgensen e t al. , 1995 ; Sugit a and Sudhof , 2000; Littleton e t al, 2001). ECM protein s such as laminins, collagens, and proteoglycans ar e found i n an d aroun d th e synapti c clef t (Dityatev an d Schachner , 2003) . As with othe r adhe sive junctions, the density within the synaptic cleft con tains CAMs such as Ng-CAM and N-cadherin (Chavi s and Westbrook , 2001; Phillips et al, 2001; Schuste r et al., 2001; Guan an d Rao , 2003). Members o f receptorligand families suc h a s Eph-ephrins and th e neurexin neuroligins ar e als o presen t (Scheiffel e e t al , 2000 ; Henderson e t al, 2001 ; Dean e t al, 2003; Grunwald et al. , 2004) . Transmembran e protease s suc h a s metalloprotease-disintegrins cleave the ectodomain s of these transynapti c molecule s t o attenuat e signalin g (Tanaka e t al , 2000 ; Matsumoto-Miya i e t al. , 2003) . Other extracellula r proteases such as plasminogen activators can degrade components o f the EC M i n a manner suspecte d t o permit morphological change s a t the synapse durin g development an d plasticit y (reviewed in Dityatev and Schachner, 2003 ; Berardi et al, 2004). In th e postsynapse , the PS D comprise s a numbe r of signaling and scaffoldin g molecule s for receptors in the plasm a membrane (Wals h and Kuruc , 1992 ; Walikonis et al, 2000; Kim and Sheng, 2004; Pen g et al, 2004; Phillip s et al, 2004 ; Collin s e t al, 2005). One quintessential componen t i s PSD-95 , a membe r o f the PSD/synapse-associate d protein/membrane associated guanylat e kinase family o f scaffolding mol ecules. I t is typically found at excitatory synapses an d directly bind s transmembran e component s suc h a s the N-methyl-D-aspartate receptor (NMDAR ) neuroligin, as well as molecules involve d in secondary signaling suc h a s neurona l nitri c oxid e synthetas e an d regulatory proteins suc h a s synaptic guanidine triphos photase activatin g protei n fo r Ra s (Iri e e t al. , 1997 ; Kim an d Sheng , 2004) . Similarly, glutamate receptor interacting protei n interact s with th e oc-amino 3-hydroxy-5-methyl-4-isoxazole propionat e recepto r (AMPAR) an d gephyri n wit h th e glycin e recepto r

(Meyer e t al , 1995 ; Don g e t al, 1997 ; Lev i e t al. , 2004). Th e cytoskeleta l protein s acti n an d spectri n concentrate a t bot h th e pré - and postsynapse . These proteins, alon g wit h tubulin , ar e though t t o under lie th e characteristi c filamentou s appearanc e o f th e postsynaptic we b an d ma y contribut e t o change s i n postsynaptic recepto r compositio n durin g develop ment and plasticit y (Matus et al., 1980 ; Fische r et al., 2000; Ki m an d Lisman , 2001 ; W u e t al , 2002 ; re viewed in Moss and Smart , 2001; Luscher and Keller, 2004; van Zundert e t al, 2004). Structural Correlates o f Synaps e Assembly and Maturatio n The pre - and postsynapti c elements o f developing cir cuits aris e principall y from interaction s betwee n ax onal an d somatodendriti c filopodia . Axona l growt h cones ma y for m termina l bouton s a s they d o a t th e neurornuscular junctio n (NMJ) , bu t predominantl y form e n passant ("i n passing") along the lengt h o f an axon (Bodian , 1968 ; Hind s an d Hinds , 1972 ; Skof f and Hamburger , 1974 ; Vaugh n et al, 1977 ; Vaugh n and Sims , 1978 ; Mason , 1982) . Coate d vesicle s an d smooth endoplasmi c reticulu m i n branchin g axon s and coate d pit s near the postsynapti c densitie s o f nascent contacts ma y participat e i n traffickin g o f synaptic component s o r membrane recyclin g durin g earl y periods o f synaptogenesi s (reviewe d i n Vaughn , 1989). Eighty-nanomete r granulate d PTV s als o hav e been foun d i n th e vicinit y of nascent synapse s (May and Biscoe , 1973 ; Ahmari et al, 2000). Another char acteristic featur e o f man y immatur e synapse s i s a reversed asymmetry, from th e earl y presence of presynaptic vesicle s i n th e absenc e o f a pronounce d PSD (Haye s and Roberts , 1973 ; Vaugh n e t al, 1977 ; Newman-Gage an d Westrum , 1984) . A s the synaps e differentiates, th e PSD thickens, a t least at type I sites, and presynapti c vesicle s steadil y increas e i n numbe r (Bloom an d Aghajanian , 1966 , 1968 ; Jones , 1983 ; Steward and Falk , 1986 ; Vaughn , 1989) . Mechanisms o f Synapse Initiatio n Spinal moto r neuro n dendrite s arboriz e extensively as they ente r th e margina l layer , a zon e occupie d pro fusely b y axonal terminal s (Vaugh n et al., 1988) . Ob servations suc h a s thi s on e an d th e branchin g o f dendritic filopodi a a t site s o f PSD-9 5 clusterin g suggest a "synaptotropic " branchin g o f dendriti c

NEURONAL DIFFERENTIATION : FRO M AXON S TO SYNAPSE S 5 9 arbors i n respons e t o presynapti c signal s (Vaugh n et al, 1988 ; Niel l e t al, 2004) . Axo n collateral s simi larly form a t sites of synaptobrevin II clustering during initial contact, consistent with a role for retrograde signaling i n presynapti c differentiation . Signal s promot ing earl y stages of synapse formation derive from bot h pre- an d postsynapti c sources , bu t th e sourc e an d identity o f the signa l initiatin g thi s cascad e o f event s are still the subjec t o f intense investigation. Som e initial clues have come fro m a series of biochemical an d genetic studie s a t th e NMJ , whic h demonstrat e tha t motor neuron-derive d agri n promote s clusterin g o f acetylcholine receptor s a t site s o f innervation , whereas a n unidentifie d retrograd e myotube-derive d signal i s required i n turn fo r normal differentiatio n of the presynapse (DeChiara et al., 1996 ; Gauta m et al., 1996). This bidirectional signaling during NMJ differen tiation i s recapitulated a t central synapses . Analogous to agri n a t th e NMJ , th e secrete d lecti n Nar p ca n cluster AMPA R an d promot e AMPAR-dependen t NMDAR clusterin g a t synapses i n culture s o f dissociated hippocampa l o r spinal neuron s (O'Brie n e t al, 1999; M i et al, 2004). Vesicles can be recruited to the presynapse either through th e bindin g of postsynaptic neuroligins t o presynapti c (3-neurexi n receptor s o r the presumabl y homophili c bindin g o f SynCA M (Scheiffele e t al, 2000 ; Biedere r e t al, 2002 ; Dea n et al. , 2003 ; Gra f e t al , 2004) . SynCA M signalin g also ca n promot e glutamatergi c transmissio n in non neuronal cell s partnere d wit h neuron s i n cultur e (Biederer et al, 2002). Whether the los s of SynCA M blocks o r attenuates synaps e inductio n remain s to b e demonstrated; down-regulatio n o r blockad e o f neu roligin isoform s attenuate s synaps e inductio n (Chi h et al., 2005). Additionally, blockade o f cadherin function o r ECM-integrin signalin g soon afte r synaps e assembly compromise s nascen t o r matur e synaps e structure, respectivel y (Togash i e t al. , 2002 ; Bozdagi et al., 2004). Blocking of ECM-integrin signaling similarly compromise s matur e synaps e structur e (Chavi s and Westbrook , 2001 ; Schuste r e t al , 2001 ; Cha n et al., 2003). Together these findings support th e ide a that severa l recognitio n an d adhesio n factor s initiat e and maintain synapses coordinately. Maturation of Synapse Composition Naive site s o f contact become competen t fo r neuro transmission throug h th e progressiv e transpor t an d

stable recruitmen t o f synapti c components . N-cad herin an d th e presynapti c gri d components Bassoon , Piccolo, an d Rab3-interactin g molecule appea r to be transported to presynaptic sites by PTVs (Wang et al., 1997; Richte r e t al , 1999 ; Ahma d e t al. , 2000 ; Bresler et al, 2001; Phillips et al., 2001; Altrock et al, 2003; Shapira e t al, 2003; Jontes et al, 2004). Postsy naptically, PSD-95 , NMDAR , an d AMPA R i n th è dendrite accumulat e at the synaps e ove r a more pro tracted tim e perio d covering a couple o f hours. Timelapse imagin g o f dissociate d hippocampa l neuron s grown fo r 8-1 2 day s i n cultur e shows tha t PSD-9 5 and NMDA R accumulat e a t synapse s gradually, implying tha t thes e component s ar e adde d piecewis e rather tha n i n large r increment s b y a fusio n particl e (Friedman e t al, 2000 ; Bresle r e t al , 2001 , 2004) . Similar work in dissociated cortical neurons grown for 2-4 day s suggest s tha t NMDA R an d AMPA R can b e delivered in packets akin to presynaptic transport vesicles, potentiall y implicatin g distinct mechanism s fo r receptor deliver y at different stage s of development o r in different neuro n types (Washbourne et al., 2002). Young synapse s exhibi t heterogeneit y i n recepto r composition. Fo r instance , man y immatur e gluta matergic site s contai n NMDA R cluster s bu t ar e thought t o lac k functiona l AMPAR . Ove r time , th e occurrence of these "silent " synapses diminishe s wit h the insertio n o f AMPAR a t thes e site s (Isaac , 2003). The NMDA R subuni t compositio n als o i s develop mentally regulated , a s th e NR2 A subuni t replace s NR2B a t synaptic sites (Shen g e t al, 1994) . Matura tion o f synapse receptor conten t an d recepto r subuni t composition i n thes e case s i s thought t o b e activity dependent (Ra o e t al. , 1998 ; Tova r an d Westbrook , 1999; Mos s an d Smart , 2001 ; Christi e e t al, 2002 ; Anderson e t al, 2004; Lusche r and Keller , 2004 ; van ZundertetaL, 2004) . Dale originally postulated tha t individua l neurons release onl y one typ e o f neurotransmitter t o accoun t for differentia l muscl e response s to preganglionic an d postganglionic sympatheti c fibe r stimulation . Thi s principle ha s been reinterprete d a s counterexamples , primarily in developing systems , have come to light. 1 Analogous t o th e aforementione d combinatio n o f neuropeptide- an d neurotransmitter-carryin g vesicl e populations in some adult terminals (De Robertis and Pellegrini d e Iraldi , 1961) , th e vesicula r glutamat e transporter-3 co-distributes wit h vesicular transporter s for glycine , y-aminobutyri c aci d (GABA) , acetyl choline, an d monoamin e neurotransmitter s durin g

60 NORMA L DEVELOPMEN T early brai n developmen t (Mclntir e e t al. , 1997 ; Chaudhry e t al, 1998 ; Takamor i e t al, 2000 ; Boul land e t al. , 2004) . I n a simila r vein , glycin e an d GABA are co-release d i n individua l vesicle s b y affér ents o f th e media l nucleu s o f th e trapezoi d bod y (MNTB) ont o th e latera l superio r olivar y nucleu s during a developmentally regulate d switc h i n neuro transmitter (Nabekur a e t al. , 2004) . Mos t o f thes e afférents ar e concurrentl y glutamatergi c durin g th e first postnatal week , bu t ove r tim e th e incidenc e o f this glutamatergic phenotype among MNTB neurons diminishes (Gillespi e e t al. , 2005) . Likewise , several lines o f evidence sugges t tha t glutamatergi c dentat e gyrus granule cell s ca n synthesiz e an d releas e GAB A and tha t th e appearanc e o f thi s GABAergi c pheno type is developmentally regulate d (Gutierrez , 2003) . Mechanisms of Synapse Maintenance and Disassembly AMuncl8-l nul l mutation abrogates vesicular neurotransmitter releas e (Verhage et al. , 2000 ; Voets et al. , 2001). Despit e th e apparen t rol e fo r presynapticall y derived signal s in earl y synaptogenesis , prenata l cytoarchitecture an d synaps e ultrastructure i n MunclS1 mic e appea r surprisingl y normal . Bu t b y birth , massive neurodegeneratio n ha s ensue d (Verhag e et al., 2000), which suggests that presynaptic release is not require d fo r synaps e formation bu t i s importan t for maintenance . Thyroi d hormon e deficiencie s similarly reduc e dendriti c spin e numbe r (Bicknell , 1998 ; Thompson an d Potter , 2000) . Th e augmentatio n o f spine numbe r b y estradic i an d termina l numbe r b y BDNF and glial-derive d factors is consistent with th e proposed role s fo r thes e factor s i n synaps e mainte nance a s well (McEwen , 1999 ; Mauc h e t al, 2001 ; Ullianetal.,2001,2004). An exuberanc e o f initial contact s i s thought t o b e pruned several-fol d ove r the cours e of normal develop ment (Knyiha r et al., 1978 ; Huttenlocher , 1979 ; Rakic et al. , 1986) , implicatin g mechanism s fo r selectiv e synapse maintenanc e an d elimination . Th e earl y F-actin dependenc e o f postsynaptic compositio n sug gests tha t synapse-specifi c factor s interactin g wit h th e actin cytoskeleto n ca n promot e th e maintenanc e o f a subset o f sites i n th e vertebrat e CN S (Alliso n e t al , 1998; Zhan g an d Benson , 2001) . Additiona l studie s suggest that stabilization of particular postsynaptic sites depends o n transmitter-evoke d recepto r activation , Ca2+ release , o r loca l protei n synthesi s (Fre y an d

Morris, 1997; Marti n et al, 1997 ; Engertand Bonhoeffer, 1999 ; Lohmann e t al., 2002; Hashimoto and Kano, 2003; Tashiro e t al, 2003 ; Nager l e t al, 2004) . Con versely, depressed o r blocked activit y leads to the elimi nation o f affecte d spine s i n developin g hippocampa l slices (Nagerl et al, 2004 ; Zhou et al, 2004) . Notably, th e effect s o f activity on synaps e numbe r in th e brai n appea r t o be developmentall y restricte d (Fiala et al, 2002 ; Gmtzendler et al, 2002 ; Nagerl et al, 2004) . Thi s windo w fo r activity-dependen t changes i n synaps e number, an d prunin g in general , has bee n propose d t o underli e critica l period s o f experience-dependent plasticit y in th e neocorte x an d cerebellum. Consisten t wit h thi s possibility , th e ef fects o f monocular deprivatio n o n ocula r dominanc e (OD) i n cortex ar e attenuated whe n protei n synthesi s is blocke d durin g th e critica l period . Furthermore , degradation o f chondroiti n sulfat e proteoglycan s i n the perisynapti c ECM, whic h ar e thought t o inhibi t axonal sprouting , reactivate s thi s O D plasticit y i n adult mic e (Berard i e t al, 2004) . Additionally, blocking of NMDAR activatio n during a critical period in creases climbing fiber innervation in the cerebellu m that correlate s wit h impaire d moto r coordinatio n (Kakizawa et al, 2000) . Cellular and Subcellula r Target Specificit y An axo n guide d t o it s target lamin a mus t b e abl e t o identify it s appropriate target cell typ e (e.g., GABAergic interneuro n vs . glutamatergic projectio n neuron ) and subcellula r domai n (e.g. , dista l vs. proximal den drite). This final stage o f axon guidance i s thought to be mediate d b y the cell - and domain-specifi c expres sion o f CAM s o r receptor-ligan d combination s wit h singular affinities . Fo r instance , GABAergi c basket cell axon s are guided alon g cerebella r Purkinje axons by a n ankyrinG-base d gradien t o f neurofasci n (als o called neurofilamen t 168) to form axo-axona l synapses at th e axo n initia l segmen t (Jenkin s an d Bennett , 2001; Ango e t al, 2004) . This hypothesis of subcellular targeting , thoug h widel y accepted , i s otherwis e untested excep t wit h N-cadherin , whic h appear s no t to function i n this capacity (Bozdagi et al, 2004) . A challenge fo r the developin g networ k i s how t o recruit o r selectivel y retai n synapti c component s a t specific sites of innervation. GluR6-containing kainate receptors ar e recruite d t o site s o f N-cadherin activa tion (Cousse n e t al , 2002) . Similarly , th e EphB 2

NEURONAL DIFFERENTIATION: FROM AXONS TO SYNAPSE S 6 1 receptor directl y clusters NMDAR when activate d by its cognat e ephrinB Z ligan d i n youn g hippocampa l neurons (Dalva et al., 2000). Neurexin can induce independent postsynapti c clustering of GABA receptor, NMDAR, gephyrin , an d PSD-95 , a s wel l a s neuroligins-1 and -2. In turn, neuroligin-2 can cluste r vesicles expressin g vesicula r transporter s fo r gluta mate, vesicula r GAB A transporter , o r th e GABA synthesizing enzym e glutamic aci d decarboxylas e 65 (Graf et al, 2004; Chih et al, 2005). The abundanc e of synapti c factor s wit h distinc t bindin g specificitie s and clusterin g activitie s suggest s that , i n combina tion, these factor s not only specify synaps e placemen t but als o encode synapse composition durin g matura tion and plasticity. Electrotonic Synapse s Whereas most of the literatur e on synapse s focuses o n the nature , modulation , an d assembl y o f chemica l synapses, ther e i s growing appreciation fo r th e func tional contributions o f electrical synapse s (also called gap junctions), Electrotoni c synapse s ar e forme d b y the abutmen t o f molecular hemichannel s called connexons (Fig. 4-8). Th e couple d connexons , which are integral membran e proteins , produc e a hermeti c

channel an d a synapti c ga p jus t 2 nm wid e (Goode nough, 1976) . Gap junction s often form betwee n th e same subcellula r domains , suc h as two dendrite s or two somata (Peinad o et al., 1993) . These synapses are commonly foun d i n lower vertebrates lik e the fro g o r chicken betwee n axon s an d dendrite s withi n th e spinal cor d an d som e brai n nuclei . I n mammals , these synapse s are widesprea d in th e retin a an d als o are foun d i n th e brai n an d spina l cor d (Paternostro et al, 1995 ; Ras h et al, 1998 ; Venanc e e t al., 2000) . Gap junction s ar e foun d wit h greate r frequenc y a t earlier developmental stages , and i t has been hypoth esized that strong electrical couplin g synchronizes activity an d stabilize s synapti c interaction s durin g development (Connor s e t al. , 1983 ; Peinad o e t al. , 1993). "Mixed" synapses, which contai n bot h electrotonic an d chemica l elements , ma y serve thi s stabilizing rol e i n lowe r vertebrates and th e ra t spina l cor d (Rash et al., 1998 , 2000) . Vertebrate connexon s ar e hexamer s o f connexi n isoforms (Goodenough , 1976 ; Segretai n an d Falk , 2004). Connexin compositio n determine s channe l gating properties and confer s gap junctions with selective permeabilities for distinct ion s and secon d messengers . Connexon compositio n i n th e nervou s system , a s i n other tissues, is cell-type specifi c an d developmentall y

FIGURE 4-8 Ga p junction . Gap junctio n connexons purified from mous e live r and visualize d by transmission electro n microscopy (left ) an d X-ra y diffractio n (middle) form orderl y lattices, indicative of their sixfold symmetry and composi tion. A schemati c representatio n o f ga p junction s i n th e plasm a membran e (right) depict s ho w th e abutmen t o f connexon hemichannel s i n th e lipi d bi layer o f each synapti c membran e produce s a hermeti c channe l an d a narrow synaptic gap. (Source; Image s modified from Caspa r and colleagues , 1977 , an d reprinted with permission from th e publisher.)

62 NORMA L DEVELOPMENT regulated (Bevan s et al, 1998 ; Suchyn a e t al, 1999 ; Bukauskas an d Verselis , 2004) . Thi s spatiotempora l control o f ga p junctio n gatin g an d permeabilit y i s likely to have consequences fo r nervous system assembly; however , the effec t o f connexin or ga p junction deficiencies on synapse formation, targeting, or stability have yet to be examined. The mechanism s guidin g spatial targeting of connexins or cell type-coupling specificity in the nervous system als o hav e ye t t o b e examined . Wor k i n non neural tissues , however , suggests that molecules typi cally associated with other types of junctions facilitate connexin targetin g to sites in th e plasm a membrane . For example , E-cadheri n overexpressio n ca n pro mote normal concentrations o f connexin targetin g to the plasm a membrane i n skin papilloma cells and alter couplin g preference s i n transfecte d melanoma s (Prowse e t al. , 1997 ; Hs u e t al , 2000 ; Hernandez Blazquez et al., 2001). Additionally, normal gap junction formatio n betwee n len s cell s i s disturbe d i n Nr-CAM-deficient mice an d b y antibodie s agains t N-cadherin (Frenzel and Johnson, 1996 ; Lustig et al., 2001). The tigh t junctio n component s zonula occludens 1 an d som e claudin s als o associat e wit h ga p junctions with as yet undetermined functional conse quences (Duffy e t al, 2002).

CONCLUSIONS

Neuroscientists hav e define d a serie s o f significant events o r stage s essentia l fo r norma l differentiatio n and integratio n of neurons into a functional network. Recent wor k links particula r stages of differentiation with the functio n of particular molecules o r molecular families, an d thi s work has provided the necessar y tools wit h whic h mechanism s ca n b e pursued . On e challenge fo r futur e experimenter s i s t o determin e how molecula r pathways of apparent overlappin g or converging functio n ar e parse d int o specifi c an d meaningful outcomes .

cAMP cycli c adenosine monophosphat e cGMP cycli c guanosine monophosphat e CNS centra l nervou s system CRMP collapsi n response mediator protei n DCC delete d i n colorectal cance r ECM extracellula r matrix EM electro n microscop y GABA y-aminobutyri c acid GDF growt h differentiation factor MAP microtubule-associate d protein MNTB media l nucleus of the trapezoid body Ng-CAM neuron—gli a cell adhesion molecule NMDAR N-methyl-D-aspartat e recepto r NMJ neuromuscula r junction Nr-CAM NgCAM-relate d cell adhesion molecule OD ocula r dominanc e PSD postsynapti c density PTV Piccolo-Bassoo n transport vesicle Shh soni c hedgehog S mo smoot h ened SNARES solubl e n-ethylmaleimide-sensitiv fusion protein-attachmen t protei receptors

e n

SNAP-25 synaptosom e associated protein-25 SV synapti c vesicle SynCAM synapti c cell adhesion molecul e

ACKNOWLEDGMENTS Th e author s than k loan a Carcea, Elizabet h Kichula , and Cynthi a Kwong for their careful readin g and critica l comments on this chapter and Alice Elste for her imag e of a chemical synapse. The au thors are supported by the Nationa l Institute s of Health (CDM, IHB , an d DLB) , Societ y fo r Neuroscienc e (TRA), and a n Irma T. Hirschl Award (DLB). Equal contribution s for th e writin g o f thi s chapte r from Mint z (polarity an d motility) , Bekiro v (guidance) , and Anderson (synaptogenesis) .

Abbreviations AMPAR ot-amino-3-hydroxy-5-methyl-4 isoxazole propionate recepto r BDNF brain-derive d neurotrophic facto r BMP bon e morphogeni c protein CAM cel l adhesion molecul e

Notes 1. "Dale' s principle/ ' base d o n argument s that transmitter functio n o f a neuron i s distinctive an d "un changeable" (Dale, 1935), actually was first defined by Eccle s (1957 ) t o state "that the sam e chemical transmitter is released from all the synaptic terminals

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5

Cell Death Stevens K. Rehen Jerold J.M. Chun

mechanism tha t plays a critical role i n specifi c stage s of development, sculptin g differen t organ s during the formation an d maturatio n o f tissue s (Elli s e t al. , 1991). Apoptosis is the bes t known form o f PCD, charac terized originall y by morphological change s observe d by microscopy (Kerr et al., 1994). Apoptosis is characterized by nuclear siz e reduction, blebbin g of the cel lular membrane, chromati n condensation , an d DN A fragmentation (Ker r et al, 1972 ; Wyllie et al, 1984) . In the absence of inflammation, the apoptotic corpses are usuall y eliminate d b y phagocytes (Arend s e t al, 1990), whic h us e th e phosphatidylserin e recepto r (PSR) to identify and remove the dying cells (Zhuan g et al, 1998 , Wan g e t al, 2003) . Apoptosis is considered a counterpoin t t o cel l deat h b y necrosi s (Ker r et al. , 1972) . Necrosi s i s distinguishe d b y earl y swelling and ruptur e o f both intracellula r organelle s and the plasma membrane. Although th e definitio n o f apoptosi s i s based o n morphological criteri a and PC D i s characterized as

Over the pas t several decades, the importanc e of cell death i n th e formatio n of the nervou s system has become wel l established . Indeed , understandin g cel l death i n th e developin g brai n ha s bee n on e o f th e most exciting areas of research in neuroscience . Th e present chapter focuse s on the mammalian fetal cerebral cortex as a paradigm of study.

HISTORICAL RECOGNITION O F PROGRAMMED CELL DEAT H AND APOPTOSI S

Embryologists hav e lon g recognize d tha t regressiv e events play a major rol e in shaping organ development (Clarke an d Clarke , 1996) . Abou t S O years ago , th e term programmed cell death (PCD ) wa s coined . I t evolved from detaile d studie s of the cellula r degenera tion durin g insec t metamorphosi s (Lockshi n an d Williams, 1965 ) and the morphogenesis o f avian limbs (Saunders, 1966) . PC D i s a geneticall y regulate d 73

74 NORMA L DEVELOPMEN T a physiologica l even t durin g development , bot h terms have been use d interchangeably sinc e thei r inception (Torne i étal., 1994) . This us e reflects an early consensus amon g scientist s abou t th e programme d nature o f apoptosis , eve n whe n triggere d b y a non physiological stimulus . I t i s important t o emphasiz e that the often-neglected distinctio n between apoptosi s and PC D ha s somewhat muddied th e field, delaying the appreciatio n o f other form s o f PCD tha t ar e also relevant to development of the nervou s system, such as autophagy (Dunn, 1994 ; Xu e et al., 1999 ; Guimarae s et al, 2003) . I n this chapter, the ter m PC D refer s t o all forms of cell death associate d with a developmen tal process .

contain fou r B H domain s (terme d BH1 , BH2 , BH3, and BH4) . There are two pro-death subgroup s o f the Bcl-2 family: the multidomai n subgrou p contains th e BH1 an d BH 2 domain s and include s Bax, Bak, Bok, and Bcl-xS ; th e BH3-onl y subgrou p include s Bini , Bid, Bcl-2-associate d deat h protei n (Bad) , death pro tein 5/hara-kiri , Puma, an d Noxa . Bcl-2 family mem bers ca n interac t wit h eac h othe r vi a thei r B H domains, and th e fina l fat e o f a cell ma y depend o n the intracellula r balance between anti- and pro-deat h proteins (Akhtar et al, 2004).

GENETIC CONTRO L O F PROGRAMMED CELL DEATH

During th e 1940s-1950s , Vikto r Hamburger , Rit a Levi-Montalcini, and Stanle y Cohen conducte d pio neering studies on mechanisms controlling cell death that culminate d wit h th e identificatio n o f nerv e growth facto r (NGF ; Cohe n an d Levi-Montalcini , 1957). I n 1986 , Levi-Montalcin i an d Cohe n wer e awarded th e Nobe l Priz e i n Physiolog y or Medicin e for thei r importan t discover y of NGF an d it s role i n both neurit e outgrowt h an d cel l survival . Researc h on the function of NGF lea d to the neurotrophi c theory, which postulate s tha t neurona l surviva l depends on competitio n fo r limite d amount s o f trophi c fac tor^) release d b y target s (Oppenheim , 1991) . Thi s early wor k ha s bee n th e impetu s fo r numerou s research group s investigatin g nove l neurotrophi c factors tha t regulate the surviva l of developing neural cells. Wit h th e clonin g o f the gen e fo r NGF (Scot t et al , 1983) , other relate d growt h factors wit h similar amino aci d sequence s hav e bee n discovered . This famil y o f proteins , collectivel y referre d t o a s neurotrophins, consist s o f NG F an d brain-derive d neurotrophic facto r (BDNF ; Scot t e t al , 1983) , neu rotrophin (NT)-3 (Ernfor s e t al, 1990) , NT-4/5 (Berkemeier e t al , 1991 ; Hallbroo k e t al , 1991) , an d NT- 6 and NT-7 (Gotz et al, 1994 ; Nilsson et al, 1998) . Two type s o f neurotrophi n receptor s hav e bee n identified o n th e basi s of their pharmacological properties: a low-affinity recepto r calle d p75 , and a famil y of high-affinit y receptor s (trks ) tha t ar e tyrosin e pro tein kinase s an d th e member s o f which bin d selec tively to differen t neurotrophin s (Barbacid , 1994) . I n general, NG F activate s trkA, BDN F and NT-4/ 5 ar e ligands fo r trkB , an d NT- 3 activate s trkC , althoug h there i s som e degre e o f cross-tal k amon g them .

The original concept of PCD suggests an event during normal developmen t tha t i s unde r geneti c control . Much o f what we kno w about thi s regulatio n arise s from th e pioneerin g wor k b y Sydne y Brenner, John Sulston, an d Rober t Horvitz , wh o wer e awarde d th e 2002 Nobel Priz e i n Physiolog y or Medicine fo r elucidating mechanism s o f cell deat h i n th e nematod e Caenorhabditis elegans. During nematode development, the generatio n of its 109 0 somati c cell s i s followe d b y th e deat h an d elimination o f 13 1 specified cell s (Ellis and Horvitz , 1986). Mos t o f these eliminate d cell s ar e neura l pre cursors. I n a serie s o f elegan t geneti c experiments , Horvitz an d colleague s define d the "centra l dogma " of PCD : tha t th e expressio n o f certai n gene s (ced-3 and ced-4 i n nematode ) i s require d fo r cel l death , whereas othe r gen e product s (ced-9) ca n bloc k th e phenomenon. In vertebrates, pro-death genes are the caspase family o f cystein e protease s an d th e apoptoti c protease activating factor (APAF) 1, and anti-apoptoti c protein s (including Bcl- 2 and Bcl-xL ) (se e Chapte r 6). The Bcl-2 family i s critically involved in th e regulatio n o f mammalian cel l deat h an d surviva l an d include s not only anti-apoptoti c proteins , bu t als o pro-apoptoti c proteins actin g throug h bot h caspase-dependen t and caspase-independent pathway s (Korsmeyer , 1999) . This famil y i s divide d int o thre e majo r subgroup s based o n th e patter n o f Bcl- 2 homolog y (BH ) do mains (Korsmeyer , 1999) . Anti-deat h Bcl- 2 famil y members, including Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 ,

NEUROTROPHIC THEORY AND CLASSICAL CELL DEATH

CELL DEATH 7 5

FIGURE 5- 1 Zone s in the developin g cerebral cortex. The anatom y of the ce rebral corte x change s a s developmen t proceed s (increasin g age fro m lef t t o right). A. The earl y proliferative ag e consists of a pseudostratified epithelium . B, C. With development , th e cerebra l wal l increases in width. In some cells , neurites processes stretch fro m th e ventricula r surface (blac k bar) to the pia i surface. C , D . With further development , mor e matur e neuron s (blac k triangular cells) begin to differentiate superficiall y i n the cortica l plate (CP) , alon g with the ingrowt h and outgrowt h o f axons in the intermediat e zon e (IZ ; lines parallel t o the blac k bar) . MZ, margina l zone; SZ , subventricular zone; VZ , ventricular zone. (Source: Adapte d from Cowan , 1979 )

Activation o f tr k receptor s initiate s intracellula r sig naling cascades tha t can produc e rapid, long-term ef fects o n neuro n growt h an d survival . Bindin g wit h p75 ca n promot e eithe r apoptosi s o r survival . I t ca n facilitate neurotrophi n signalin g throug h tr k recep tors promotin g cel l survival . Interestingly , p7 5 i s structurally related to the tumo r necrosis factor recep tors tha t regulat e th e onse t o f cel l deat h i n th e im mune syste m (Majda n e t al , 1997) . Constitutiv e expression o f the intracellula r p75 domai n i n trans genie mic e lead s t o cel l deat h i n severa l neurona l populations. Reader s wishin g t o lear n mor e abou t neurotrophic factor s and thei r receptor s ar e referre d to some o f the man y excellen t recen t reviews on thi s topic (e.g. , Huang and Reichardt, 2001 ; Dechant and Barde, 2002; Hempstead, 2002) .

DEVELOPING CEREBRA L CORTE X AS A MODEL FO R STUDYIN G MAMMALIAN NEURA L CEL L DEAT H

Neurons o f the mammalia n cerebra l corte x first arise during embryoni c developmen t fro m neuroprolifera tive zones that surround the cavities, or ventricles, that contain cerebra l spina l fluid . I n th e cerebra l corte x these proliferative regions are known as the ventricular zone (VZ) , which line s the ventricle , and th e subventricular zone (SZ) , whic h i s immediately suprajacen t to the VZ (Fig. 5-1). Developmental events in the VZ serve a s an archetypa l exampl e o f neurogenesi s else where in the neural tube, includin g the spinal cord . During neurogenesis , cell s underg o on e o f thre e general fates. They can continue to proliferate, become

76 NORMA L DEVELOPMEN T postmitotie, or die. Those neurons tha t becom e postmitotic, a step referred t o as the birth of a neuron, migrate ou t o f th e VZ , throug h a superficia l regio n termed th e intermediate zone (IZ) , until they reach a position i n the superficia l aspec t o f the anlag e o f the cortical gra y matter know n as the cortical plate (CP) . Once i n th e CP , neuron s differentiat e an d mak e synaptic connection s (Miller , 1988 ; Baye r an d Alt man, 1991 ) o r di e (e.g. , Finla y an d Slattery , 1983 ; Miller, 1995) . Death of Post-Mitotic Cells Historically, on e reaso n th e contributio n o f PCD t o development ha s no t bee n well-recognize d i s tha t apoptotic bodie s ar e susceptibl e t o rapi d clearanc e and phagocytosi s in intac t tissue. Thus, i t i s difficul t to study the presenc e of cell death i n static (fixed) tissue. A real-time approach coul d unequivocall y determine th e prevalence an d distribution of dying cells in situ, however , thi s metho d i s not currentl y practica l since e x viv o system s produc e artifactua l change s in cellular behavior and induce premature cell death. Approximately one-third or more of the post-mitotic neuronal populatio n undergoe s naturall y occurrin g PCD during development relate d to insufficient acces s to trophi c signal s (e.g. , NG F an d BDNF ) fro m tar gets an d afférent s (Oppenheim , 1991 ; Linden , 1994). Th e amoun t o f neurona l deat h varie s wit h time, location , an d cel l typ e (e.g. , Miller , 1995) . I n cortex, fo r example , th e incidenc e o f deat h amon g different population s o f post-migrator y neurons ca n vary fro m 10 % to 60% . Neurons i n th e deepes t an d most superficia l corte x ar e mor e likel y t o di e tha n those i n mid-cortex . Moreover , loca l circui t neuron s within a particular layer are twice as likely to die relative to laminar-cohort projection neurons. PCD play s a crucia l par t i n th e developmenta l strategy o f the cerebra l cortex . An overabundanc e of neurons i s generated t o create a transient scaffol d fo r the developin g corte x and i s subsequently eliminate d as th e corte x matures . Durin g developmen t o f th e mammalian telencephalon , th e genesi s o f neuron s destined for the si x layers of the cerebra l cortex is preceded b y generation o f a populatio n o f neurons tha t reside in the subplat e (an anlage of the white matter ) and the margina l zone (the anlage of cortical layer I). These neuropeptide-containin g neuron s (Chu n et al, 1987 ; Al-Ghoul and Miller, 1989 ; Ghos h e t al, 1990) ar e presen t i n larg e number s durin g

development an d functio n i n earl y synaptic circuitry (Chun an d Shatz , 1989a ; 1989b) . A s the brai n ma tures, their disappearance through neuronal death occurs in concer t wit h the invasio n o f the cortica l plat e by axona l projection s an d wit h th e eliminatio n o f synapses i n th e subplate . Thes e observation s sugges t that during development the subplate (a) is a transient synaptic neuropi l (Chu n e t al., 1987) ; and (b ) serves as a guidepos t fo r ingrowin g thalamocortica l axons subsequently eliminate d b y cel l death . Thi s ide a o f sculpting the cortex through a n initial overproductio n of neurons followed by paring back is in concert wit h the neurotrophic theory. Most studie s on PC D i n th e centra l nervou s system (CNS ) hav e focuse d o n th e period o f target in nervation (se e below) , whe n differentiatin g neuron s are sendin g ou t axon s and makin g synapti c connec tions with postmitotic neurons (Hamburger and Levi Montalcini, 1949 ; William s and Smith , 1993 ; Miller , 1995). An implicit assumption o f many of these studie s was that PC D playe d a minor rol e durin g embryoni c CNS development . Indeed , elaborat e mathematica l models have been constructed that assume there is no PCD durin g neuronogenetic phase s of developmen t (Cavinessetal., 1995) . Death of Proliferating Cells Death ca n affec t proliferatin g populations of cells, a phenomenon representin g a distinct for m an d phas e of cell death i n the developing CNS. Thi s populatio n is bes t represente d b y neura l progenito r an d ste m cells (NPCs). These cells, which have morphological and molecula r characteristic s o f radia l gli a (Nocto r et al., 2001), are located i n the VZ and SZ . They also share similarities with mitotic immune cell s such a s T cells of the thymi c cortex (Chun, 2001). Importantly, NPCs d o no t hav e synapti c contacts a t thi s stag e of development, an d ar e still proliferative . Thymic T cells are notable a s a comparison popu lation t o NPCs. Developin g T cell s underg o severa l distinct cell selection mechanism s (positive selection, negative selection , an d deat h b y neglect) tha t resul t in eliminatio n of 97% of the developin g T-cell popu lation (Egerto n e t al, 1990 ; Shortma n e t al. , 1990 ; Shortman an d Scollay , 1994) . Nevertheless , fo r de cades controversy existed following the proposa l of intrathymic cel l deat h (Metcalf , 1966) , becaus e o f th e absence o f histological evidenc e fo r intrathymi c cell death (Post e an d Olsen , 1973) . I t wa s not unti l th e

CELL DEATH 7 7 development o f nove l hybridizatio n techniques col lectively termed i n situ end labeling, or ISEL, that intrathymic cel l deat h wa s convincingly demonstrated (Surh an d Sprent , 1994 ; Chun , 2001) . ISEL identi fies breaks in double-stranded DN A produced during PCD b y attaching labele d nucleotide s t o fre e DN A ends. The labe l ca n the n b e visualized in tissu e sections (Gavrieli et al., 1992 ; Abrams et al., 1993 ; Wijsman e t al. , 1993 ; Woo d e t al. , 1993 ; Homm a e t al, 1994; Whit e e t al, 1994) . I n sit u end-labelin g plu s (ISEL+) improves the sensitivit y of the original ISEL techniques t o visualize PCD i n th e developing ner vous system (Blaschke et al, 1996 , 1998 ; Stale y et al , 1997; Pompeiano e t al, 1998 , 2000 ; Zhu an d Chun , 1998). Th e increase d sensitivity of ISEL+ ha s bee n validated i n severa l establishe d mammalia n model s of PCD . Fo r example , ISEL + reveal s norma l PC D and quantitative increases in PCD withi n the thymic cortex following dexamethason e treatment o f the thymus (Wyllie , 1980 ; Egerto n e t al , 1990 ; Shortma n et al 1990 ; Surh and Sprent , 1994) . Moreover, studies using ISEL+ i n the smal l intestinal villus, a model of PCD wher e th e entir e populatio n o f cells turns over every tw o t o fou r days , demonstrat e tha t apoptoti c DNA fragmentation can be identified in at least some dying cell s severa l day s befor e thei r actua l elimina tion (Pompeiano et al, 1998) . The searc h fo r PCD i n th e developin g cerebra l cortex wa s prompte d b y th e superficia l similarities between thymocyt e development an d cortical neurogenesis, particularly in ligh t o f the lac k o f histological evidenc e fo r cel l deat h i n th e thymus . Th e patterns o f labele d cell s reveale d b y ÏSEL + i n th e embryonic rodent brai n suggest that PCD occur s on a large scale throughout th e neuraxi s (Blaschke et al, 1998). As expecte d fro m previou s studie s o n target dependent PCD , dyin g cells are present in the anticipated postmitoti c neurona l compartment s o f th e cerebral corte x (I Z an d CP). Strikingly , a larg e amount o f PCD i s also present i n th e VZ . Th e tim e course o f cell deat h i n mous e cortex varies across development and i s related to the progressio n of the cel l cycle (Blaschk e et al , 1996 , 1998 ; Thomaido u e t al , 1997). Most PCD i s observed between gestational day (G) 1 2 and G18, when 25% to 70% of cells in the feta l cerebral corte x die (Fig . 5-2) . Pea k incidence o f cell death occur s abou t G14 . Prio r t o G1 0 an d i n th e adult, less than 1 % of cortical cells are dying. This pattern show s that ther e i s a substantia l amoun t o f cel l

FIGURE 5- 2 Distributio n of dying cells in the embryonic mouse cortex. In situ end-labeling plus (ISEL+) from gestationa l day (G)10-G18 and in the adult cortex (A, C, E, G, I, and K), along with nuclear staining by 4,6-diamino-2-phenylindole o f the sam e fiel d (B , D, F, H, J, and L). A, B. G10. Fro m this age through G14 (A-F) , th e neuroepitheliu m consist s o f a homogeneous-appearing proliferativ e layer (ppl). C, D. G12. E, F. G14. G , H. G16. Th e cerebra l wall at G16 ha s stratifie d int o additiona l layer s (th e mar ginal zon e [MZ] , cortica l plat e [CP] , intermediat e zone [IZ] , an d ventricula r zone [VZ] ) tha t ar e also apparent at G18 (I , J). K, L. Adult (Ad) cortex. Th e ventricle (v) is at the lowe r edge of each photograph . Note th e spars e ISEL + labelin g a t G1 0 tha t in creases through G14, and then declines with furthe r development. From G1 2 to G18, note the extensiv e labeling i n th e proliferativ e zone s (ppl, VZ) a s well as in th e postmitoti c zone s (MZ , CP , and IZ) . Onl y rare dyin g cells ar e detecte d b y ISEL+ i n th e adul t cortex. Thus cortical PCD i s developmentally regulated an d cease s by adulthood. Scal e bar s = 50 mm. (Source: Modifie d fro m Blaschk e an d colleagues , 1996)

death withi n proliferatin g NPC populations . Thes e cells hav e no t ye t sen t ou t axon s t o synapti c targets, hence, classica l neurotrophi c theor y canno t accoun t for thi s earl y phase o f cell death . Thi s phenomeno n represents a novel form of PCD an d is discussed below.

78 NORMA L DEVELOPMENT to th e eliminatio n o f dead cell s i n th e brain . Phagocytes, from mic e lackin g the gen e encodin g PS R are defective i n removin g apoptoti c cells . Thes e PCR deficient mic e hav e developmenta l abnormalities , such a s in the accumulatio n o f dead cell s in the lun g and brain . Som e o f these animal s also present a hyperplasic brai n phenotyp e resemblin g tha t o f mic e deficient i n th e cel l death-associated gene s APAF-1 , caspase-3, an d caspase- 9 (Fig . 5-3 ) suggestin g tha t phagocytes ma y also be involve d in promotin g apop tosis (L i e t al. , 2003) . Actually , i n th e developin g cerebellum, th e deat h o f Purkinj e neuron s ca n b e promoted b y microglia . This findin g suggest s tha t a form o f engulfment-related cel l death ma y link PC D to th e clearanc e o f dea d cell s (Marin-Tev a e t al. , 2004).

DIFFERENT PHASES OF

DEVELOPMENT, DIFFERENT MECHANISMS O F PROGRAMMED

CELL DEATH

FIGURE 5- 3 Examinatio n of caspase-9 and caspase-3 knockout mice. Capase-9 +/~ (A ) and caspas e 9~ /- (B) fetuses a t G16.5 , showin g th e large r an d mor phologically abnorma l brai n i n th e knockout . Scal e bar= 1.0mm. C-F . Caspase- 3 mutant s hav e a n expanded proliferativ e zone. C, D. BrdU immunolabeling i n a sectio n throug h th e developin g cerebra l cortex o f a hétérozygot e an d mutan t embryo , respec tively. E, E DAP I staining of the adjacent section i n a series fro m C an d D , respectively . I n th e caspase- 3 knockout fetus , th e corte x i s distorte d int o fold s and th e latera l ventricle i s identifiable only as a thi n slit in the center of the cortical mass. The thicknes s of the proliferativ e laye r relativ e to th e corte x looks very much alike in the tw o embryos. Statistics showed that the percentag e o f BrdU-labeled cells was only slightly increased i n th e -/ - fetuse s compare d t o th e +/ fetuses, v , latera l ventricle . Scal e ba r = 50 0 Jim. (Source: A, B: adapted fro m Kuid a e t al., 1998 . C-F : adapted fro m Pompeian o e t al., 2000)

Glial Death within the Developing Brain The rol e o f astrocytes or othe r gli a i n neurona l cel l death i s poorly understood. Interestingly , phagocyte s of macrophage lineages such as microglia contribut e

Using an earl y molecular marke r for post-mitotic neu rons, Weine r an d Chu n (1997) , foun d tha t PC D i n neuroproliferative zones does not become pronounce d until NPC s begi n t o differentiat e int o postmitoti c cells. This correlation suggest s that the initia l generation o f postmitotic neuron s ma y trigger th e onse t of PCD i n a given proliferative zone . The natur e of this link; however, is currently unclear. Similar to the developin g cerebral cortex, the reti na of newborn mice and rat s is composed o f two strata containing cell s i n variou s stage s o f development . One laye r of differentiate d cell s i s the ganglio n cel l layer an d th e othe r i s the neuroblasti c laye r (NBL). Like the cortica l VZ, the NB L is comprised o f NPCs (Alexiades and Cepko , 1997) . During differentiation, NPCs that are exiting the cell cycle, migrate from th e NBL to form other layer s of postmitotic neurons (Linden et al, 1999) . PCD ca n b e induce d i n th e NB L following inhibition o f protein synthesis . Interestingly , as the retin a matures, retinal cell s become less sensitive to protei n synthesis inhibitors, and i n adul t retinal tissue , thes e inhibitors hav e n o effec t o n cel l deat h (Rehe n e t al, 1996). Whe n BrdU , a marke r o f proliferatin g cells (Miller an d Nowakowski , 1988), i s used i n conjunc tion wit h protein synthesi s inhibitors, the vas t majority of cells undergoing PCD ar e not labeled with BrdU (Fig. 5.4) . This resul t suggests that the populatio n o f

CELL DEATH 7 9 newly postmitoti c neuron s i s particularly sensitive to PCD throug h inhibitio n o f protein synthesis . Contin uous synthesis of anti-apoptotic proteins appears to be important i n the regulatio n o f PCD durin g neuronal migration an d differentiatio n i n th e retin a (Rehen s et al , 1999) . Consisten t wit h thi s hypothesis , mous e embryos deficient for Bcl-x L, an anti-apoptoti c mem ber o f the Bcl- 2 family , exhibi t massiv e cell deat h o f immature neurons i n the cerebra l corte x (Motoyama et al, 1995) . Bcl-xL is up-regulated i n early postmitotic neurons a s they migrat e fro m th e V Z an d thi s hig h level o f expression is maintained throug h adulthood . On th e othe r hand , Bcl-x L expressio n is low in NPCs (Motoyama et al, 1995) . In summary , ISEL+ result s indicat e tha t PC D i s a commo n cel l fat e fo r NPC s throughou t th e neural tube . Studie s involvin g inhibition o f protein

FIGURE 5- 4 Identificatio n of newly postmitotic cell s sensitive to apoptosis induced b y inhibition of protein synthesis. Immunolabelin g for BrdU in th e prolifera tive zone o f a retinal expian t (arrows ) afte r inhibitio n of protein synthesis. Notice tha t the majorit y o f dying cells (arrowheads) , identifie d a s globula r apoptoti c bodies under differentia l interferenc e contrast , i s unlabeled wit h BrdU . Scal e ba r = 20 \im. (Source: Adapted from Rehe n e t al., 1999 )

synthesis i n th e developin g nervou s system an d tar geted deletio n o f the anti-apoptoti c gen e Bcl-x L suggest that durin g migration an d initia l differentiation, the PC D machiner y i s controlle d b y suppresso r proteins.

PROLIFERATIVE CELL DEATH, DNA BREAKS AND END JOININ G Thymic PCD occur s during the selection o f functionally an d molecularl y distinc t thymocyte s (Sur h an d Sprent, 1994), based on somatic DNA rearrangements encoding T cell receptor s an d immunoglobulins in T and B lymphocytes, respectively . This process, known as V(D)J recombination (Weaver and Alt , 1997) , i s responsible for the unparallele d degre e of immunologicai diversit y tha t i s fundamenta l t o th e immun e response. A s describe d above , a paralle l exist s be tween PCD i n the thymu s and the feta l brain , where cell death take s place i n conjunction with cell prolif eration (Shortman et al, 1990 ; Chun , 2001). ISEL+ result s suggest that a form o f cell selectio n occurs i n th e neuroproliferativ e regions o f the brai n and that the occurrence o f PCD i s associated with the maturation proces s (Blaschk e e t al. , 1996 , 1998) . I n the feta l brain , th e pea k o f cell deat h coincide s wit h the perio d whe n th e firs t neuron s destine d t o com prise th e matur e corte x ar e bein g generate d (Cavi ness, 1982) . Thi s spik e i n PC D migh t allo w th e selection o f the firs t cortica l neuron s havin g desired phenotypes whic h woul d serv e a s a templat e fo r th e selection o f later-generate d cells . Thi s hypothetica l scenario leave s open th e questio n o f how cells migh t be mechanistically selected . Nearly a decade befor e geneti c recombinatio n was formally demonstrated in lymphocytes, Dreyer and colleagues (1967 ) postulate d tha t recombine d gene s en coding cell-surface proteins might play a part in guiding the correct re-innervation of the goldfish tectum by retinal axons . These recombine d protein s would act a s a kind o f molecular cod e t o ensur e tha t th e topographi cally correc t retinotecta l mappin g i s achieved . Sinc e this idea was put forward, however, no gene undergoin g the requisit e recombination i n the nervou s system has been found . Thi s i s perhap s no t surprising , a s th e reagents that were instrumental in the discovery of lymphocyte V(D)J recombination—DNA sequences fro m a known candidate locus, and clonal cell lines in which the rearrangemen t even t takes place—do not currently exist for the nervous system.

80 NORMA L DEVELOPMEN T An indirect approach to this issue is to ask whether genes involve d i n immunologica l V(D) J recombina tion ar e expresse d i n th e nervou s system . I n fact , a common featur e share d by cortical thymocytes (Turka et al, 1991 ) and V Z NPC s (Chu n e t al., 1991 ) is expression o f a rang e o f gene s involve d i n th e initia l cleavage of recombined genes and th e rejoinin g of the free DN A end s t o complet e th e recombinatio n reac tion. Par t o f th e initia l cleavag e reactio n i n lympho cytes is driven by recombination-activating genes 1 and 2 (RAG 1 an d RAG2) , which ar e involve d in V(D)J recombination (Schat z e t al, 1989) . Ragl - knockou t mice, however , hav e n o obviou s brai n phenotyp e (Mombaerts et al, 1992) , and Rag 2 is not expressed in the CNS. It is notable that broken DNA ends that exist in cells that, like those produced b y recombinase cleavage, hav e blun t end s tha t ar e 5'-phosphorylated , al though mos t of these ends appear to be predominantly associated with cell death (Stale y et al, 1997). After cleavage , DN A en d joinin g tak e plac e b y means of several nonhomologous end-joining (NHEJ)

proteins including Ku70, Ku80, DNA-dependent protein kinas e catalyti c subuni t (DNA-PKcs) , XRCC4 , and DN A ligas e IV (Lig IV). Throug h gene-targetin g in th e mouse , i t has been show n that XRCC4 or Lig IV deficiency causes growth defects, premature senes cence, immun e respons e sensitivity , and inabilit y to support V(D)J recombination in primary murine cell s (Lieber, 1999). Strikingly, XRCC4 o r Li g I V deficiency i n mic e causes lat e embryoni c lethalit y accompanie d no t only by defective lymphogenesis but also by defective neurogenesis, manifested by extensive apoptotic deat h of newly generated postmitoti c neuronal cells (Gao et al, 1998) . I n the contex t o f Lig IV and XRCC4 defi ciency, embryoni c lethalit y an d neurona l apoptosi s likely resul t fro m a p53-dependen t respons e t o unre paired DN A damage (Fran k e t al, 2000 ; Ga o e t al. , 2000). Ku7 0 and Ku8 0 deficienc y results in dramati cally increase d deat h o f developing neuron s i n mic e though DNA-PKcs deficiency does not lead to any neuronal death phenotype. The Ku-deficien t phenotype is

FIGURE 5- 5 Massiv e cel l deat h o f newl y postmitoti c neuron s i n XRCC 4 knockout mice. A-F. Hematoxyli n an d eosi n stainin g of littermate XRCC4 +/~ and XRCC4~ /~ brai n sections . Horizonta l brai n section s fro m G14. 5 XRCC4+/- (A-C) an d XRCC4-7- (D-F) mutants at a comparable leve l reveal severe acellularit y (cavitation , lon g arrow ) an d thinne r cortica l plat e (CP , small arrows) in the cerebra l hemispher e o f XRCC4~/~ brain. Higher magnifications sho w relativel y normal ventricula r zon e (VZ ) of th e mutan t corte x (A, B vs. D, E) , wherea s in th e intermediat e zon e (IZ ) extendin g t o th e C P there are numerous pyknotic nuclei (dense hernatoxylin stain) (E, F). CP, cortical plate; S , striatal analge or the ganglioni c eminence; v, ventricle. Original magnifications: A and D xlOO ; B , C , E , an d F , x400 . (Source: Adapte d fro m Gao et al, 1998)

CELL DEATH 8 1 qualitatively similar to, but les s severe, than tha t associated wit h XRCC4 and Lig IV deficiency (Gu et al, 2000). Th e lac k o f a neurona l deat h phenotyp e i n DNA-PKcs-deficient fetuses , alon g wit h th e milde r phenotype o f K u deficienc y (vs . XRCC4 - o r Li g IV-deficient fetuses) , is likely related t o relativ e low fidelity of DNA end-joinin g in the backgroun d strain of thes e mutants , show n b y a V(D) J recombinatio n end-joining assay. A intriguin g aspec t o f these studie s i s the consis tency of abnormal cell deat h pattern s i n th e developing cerebra l corte x o f mice deficien t in DN A ligas e IV, XRCC4 (Barne s et al, 1998 ; Fran k e t al, 1998 ; Gao et al, 1998) , and Ku (Gu et al, 2000). Aberrant death i s not observe d a t th e earlies t embryonic ages , and i t is not unti l th e perio d whe n mos t postmitoti c neurons ar e generated tha t dea d cell s ar e clearl y observed (Fig . 5-5) . Thi s genera l timin g i s seen i n al l examined region s o f the neura l tube , includin g th e spinal cord (Gao et al, 1998) . These studie s provide the first evidence that evolutionarily conserve d gene s require d fo r completion o f V(D)J recombination in lymphocytes are required for normal neura l cel l surviva l i n th e developin g brai n (Gao e t al , 1998 ; Chun , 1999 ; Chu n an d Schatz , 1999). Severa l NHEJ proteins have roles in NPC sur vival durin g the onse t o f neura l differentiation . Th e synthesis o f thes e protein s coul d b e crucia l fo r th e normal behavior of newly postmitotic neurons during their migration from proliferativ e zones to final destinations in the CMS (Rehen e t al, 1996 , 1999) . The absenc e o f proteins important for the process of immunologica l V(D) J recombinatio n promote s cell death i n newly postmitotic neurons. This findin g suggests th e operatio n o f a nove l for m o f cell selec tion that distinguishes between neurons that make advantageous DN A rearrangement s o r othe r genomi c alterations and thos e tha t d o not. What kin d of DNA alterations coul d occu r i n NPCs within th e develop ing brain?

ANEUPLOIDY, CEL L SELECTION, AND PROLIFERATIV E CELL DEATH IN TH E BRAI N

As discussed above, there i s indirect evidence fo r so matic alteratio n o f th e genom e i n neuron s durin g neurogenesis base d o n a growin g lis t o f molecule s that functio n i n DN A recombination , repair , an d

surveillance (Ga o e t al , 1998 , 2000 ; Chun , 1999 ; Chun an d Schatz , 1999 ; G u e t al , 2000 ; Le e e t al , 2000; Rolig and McKinnon , 2000; Allen et al, 2001). Many o f these molecule s ar e als o implicate d i n th e genetic alteration s tha t ar e foun d i n cancers . On e prominent geneti c abnormalit y i n cance r cell s i s aneuloidy, a deviation from th e norma l diploi d num ber of chromosomes. Th e associatio n between cance r and neurodevelopmen t le d t o th e searc h fo r aneu ploidy a s on e expressio n o f genomi c chang e i n neurons. Direct assessmen t o f chromosome s i n dividin g cells ca n b e performe d wit h spectra l karyotypin g (SKY) which uses fluorescent labels to uniquely identify eac h chromosome . Thi s technique reveal s that a large fractio n o f NPC s harbo r chromosoma l abnor malities. Remarkably , one-third o f mouse NPC s have gained o r los t a t leas t on e o f their 4 0 chromosome s (Fig 5-6 ; Rehe n e t al, 2001). NPCs isolated fro m th e VZ o f the feta l mous e cerebra l corte x ar e ofte n ob served wit h on e o r mor e chromosome s displace d from the mitotic spindle. Suc h chromosom e displace ment suggest s tha t NPC s frequentl y mis-segregat e their chromosomes , whic h ma y represen t on e wa y that neuron s ca n becom e aneuploi d durin g development (Yan g et al, 2003). Aneuploidy ha s als o bee n detecte d i n th e adul t cerebral cortex , usin g interphas e fluorescen t i n sit u hybridization (FISH ) t o visualize individual chromosomes. I n adult male cerebra l cortex , FISH fo r X and Y chromosomes show s that approximatel y 1 % of neurons gained or lost a sex chromosome (Fi g 5-7) . Thi s implies tha t a t least som e fractio n o f aneuploid cell s that ar e produce d durin g neurogenesi s surviv e t o adulthood. Theoretically , th e overal l percentag e o f aneuploid neuron s i n th e adul t coul d b e muc h higher than 1% , assuming the rat e of gain/loss is similar fo r al l chromosome s (Kausha l e t al , 2003) , al though furthe r studie s ar e require d t o addres s thi s issue. Although some aneuploid cells survive, Aneuploid cells may account fo r a significant proportion of PC D during neurogenesis . Cell deat h i s likely a commo n fate fo r aneuploi d cells . I n cancers , man y cell s tha t are thought t o be dying have an altered genome. Fur ther, the hig h amount s of PCD in neuroproliferativ e zones supports the ide a o f a selection proces s against undesirable geneti c abnormalities . A s a first approximation, aneuploid y appear s t o b e generate d i n a stochastic fashion . On e ca n speculat e tha t rando m

82 NORMA L DEVELOPMEN T

FIGURE 5-6 Aneuploi d neural progenitor cell s (NPCs ) in the embryonic mouse brain. A. Spectral karyotyping (SKY) fro m a representativ e aneuploi d embryoni c NPC. Spectra l (left ) an d invers e DAPI (center) images of the chromosom e sprea d are shown , along with th e karyotype tabl e (right) . Not e tha t eac h differen t chro mosome ha s a uniqu e spectra l colo r (indicate d b y shades of gray). Euploid chromosom e numbe r i n mic e

is 40. The sprea d ha s an extr a copy of chromosome 2 , but ha s only one cop y of chromosomes 1 5 and 1 7 (39, XY, +2, -15, -17). B . Graphs sho w the percentag e of loss (left ) an d gai n (right ) of specific chromosome s i n NPCs. Bar s are code d o n th e basi s of spectral color of each chromosome (excep t for sex chromosomes). Spe cific chromosome s ar e los t a t rate s o f 1.6%—8.4 % o f cells analyzed and are gained at rates of less than 2%.

aneuploidy impart s diversit y and complexit y t o neu ronal populations , an d tha t aneuploi d NPC s ar e selected o n th e basi s of their genomi c contents , with one probable fate being cell death. The pervasiveness of adul t neura l aneuploid y may indicat e a selectiv e advantage acquire d b y som e aneuploi d neurons , a s has bee n note d i n certai n aneuploi d cance r cell s (Lengauer e t al , 1997) . Thus , variatio n i n chromo some numbe r ca n b e considere d a ne w mechanis m for generating cellular diversity in the CNS . The mai n conclusion from thes e data is that aneuploid cell s are found in the developing and adult ner vous system, demonstrating tha t brai n cell s can diffe r at th e leve l o f thei r genome . Whethe r DN A re arrangements ar e als o present i n thes e cell s remains an ope n question . Thes e somati c change s i n th e DNA of a cell could affec t a significant portion o f the genome, alterin g hundred s o f gen e copie s whic h would hav e important consequences fo r the physiol ogy of that cell. The globa l effect o f this process could

result i n a mature brai n that i s a unique geneti c mo saic, characterize d b y a euploi d populatio n inter mixed with a diverse aneuploid population of neurons (Rehen e t al , 2001 ; Kausha l e t al , 2003 ; Yang et al , 2003).

LYSOPHOSPHATIDIC ACID, CELL CYCLE EXIT, DECREASED PROGRAMMED CEL L DEATH, AN D INCREASES I N BRAI N DIMENSION S

A variety of extracellular factors hav e been show n t o influence NPC s an d youn g neuron s o f th e cortex . Neurotransmitters (LoTurco et al., 1995 ) and peptid e factors (Drag o e t al. , 1991 ; Ghos h an d Greenberg , 1995; Temple and Qian , 1995 ) affec t neurogeni c parameters o f developin g cortica l cells , althoug h i t i s generally unclea r ho w thes e signal s influenc e th e growth an d morpholog y of the intac t cerebral cortex.

CELL DEATH 8 3

FIGURE 5- 7 Aneuploi d cell s i n th e adul t corte x are neurons. A . Th e presenc e o f bot h microtuble associated protein (MAP ) 2-positive (large arrow) an d MAPZ-negative (smal l arrows ) cells i n a 1 0 Jim sec tion throug h adul t cerebra l corte x demonstrate s th e specificity o f MAP 2 labelin g a s a neurona l marker . Nuclei wer e counterstaine d wit h DAPI . B-G . Cell s in 1 0 ^Lm sections through the adul t male cortex were hybridized wit h X and Y chromosome paints . C , E , G. Cell s i n these sam e 1 0 \im sections were then im munostained for MAP2. B. The cel l o n th e lef t con tains bot h a n X an d a Y chromosome (note d b y a n asterisk), whereas the cel l on the righ t has an extr a Y chromosome. C. The cel l with two Y chromosomes is MAP2-positive. D, E. A cell i n motor corte x contains an extr a X chromosom e (D ) an d i s MAP2-positiv e (E). F, G. A cell i n moto r corte x contain s a n extr a Y chromosome (F ) an d i s MAP2-positiv e (G) . Scal e bars =10 Jim (D , F ) an d 5. 0 Jim (H , J) . (Source: Adapted from Rehe n e t al., 2001)

Some trophi c factor s suc h a s fibroblast growth facto r 2 (FGF-2 ) an d NT- 3 ar e als o know n t o regulat e th e transition fro m dividin g NPCs to terminally differen tiated neuron s i n th e CNS , (Ghos h an d Greenberg , 1995). Tha t said , factor s abl e t o modulat e PC D within the VZ are less well understood . A lipid molecule, lysophosphatidi c acid (LPA), has been implicate d i n th e regulatio n o f neurogeneti c processes, including proliferative cell death. Man y of the effect s o f LPA are mediated b y G protein-coupled receptors tha t ar e member s o f the lysophospholipi d receptor gen e famil y (Fukushim a e t al., 2001 ; Chun et al , 2002 ; Anlike r an d Chun , 2004 ; Ishi i e t al , 2004). A role for LPA signaling in nervou s system development wa s initially suggested b y the discover y of the firs t lysophospholipi d receptor , LPA 1? whic h shows enriche d expressio n i n th e V Z (Hech t e t al. , 1996). Subsequen t studie s hav e identifie d LPA 2 ex pression i n postmitoti c cell s o f the embryoni c cortex (Fukushima e t al, 2002 ; McGiffert e t al., 2002 ) and LPA3 expressio n withi n th e earl y postnata l brai n (Contos e t al. , 2000) . LPA , i s als o presen t i n th e brain (Da s and Hajra , 1989 ; Sugiur a et al, 1999 ) an d can b e produce d b y postmitoti c neuron s an d Schwann cell s i n cultur e (Weine r an d Chun , 1999 ; Fukushima e t al.,2000). In a n e x vivo brai n cultur e system , LPA exposure rapidly alters the organizatio n o f the developin g cere bral corte x (Kingsbur y et al. , 2003) . Afte r jus t 1 7 hours, LPA-treated hemispheres displa y striking cortical fold s an d a widenin g o f the cerebra l wall , com pared t o untreate d opposit e hemisphere s obtaine d from th e sam e animal s (Fig . 5-8 ) (Kingsbur y et al. , 2003, 2004) . Thi s expansio n o f cortica l thicknes s i s due t o a n increas e i n cell s withi n bot h proliferative and postmitoti c region s withou t a correspondin g change i n cel l density . Whereas LP A is a mitogen fo r many cell type s in culture (Moolenaar , 1995 ; Moolenaar e t al. , 1997) , LP A exposure appears to decreas e proliferation i n a n e x vivo brain cultur e syste m in favor of terminal mitosi s (Kingsbury et al., 2003). In addition, LPA exposure in thi s e x vivo system promotes the surviva l o f VZ cells . Therefore , th e increas e i n cortical thicknes s followin g LP A treatmen t i s du e to (1 ) th e increase d surviva l of man y NPCs , whic h enlarges the proliferatin g pool, an d (2 ) the inductio n of termina l mitosis , which increase s th e numbe r o f postmitotic neurons . Mos t importantly from a mechanistic standpoint , neithe r foldin g no r increase s i n

84 NORMA

L DEVELOPMEN T

FIGUR E 5- 8 Effec t o f lysophosphatidi c aci d (LPA ) o n cortica l development . Intac t cerebra l hemisphere s ex posed t o (LPA ) e x viv o exhibi t cortica l foldin g an d widenin g of the cerebra l wall compare d t o contro l hemi sphere s fro m th e sam e animal . Whole-moun t view s (A) and sagitta l section s (B ) fro m gestationa l da y (G ) 1 4 hemisphere s sho w dramati c cortica l foldin g followin g cultur e with LPA, compare d t o contro l medium . D, dorsal; R , rostral ; Ctx , cortex ; GE , ganglioni c eminence . Scale ba r = 0.50 mm . C . G1 4 cortice s labele d wit h th e nuclea r stai n DAP I sho w increase d thicknes s followin g

LPA-treatment . CP , cortica l plate ; IZ , intermediat e zone ; SZ , subventricula r zone ; VZ , ventricula r zone . Scale bar= 100μm . LP A promote s termina l mitosi s throug h a n increas e i n M-phas e cell s immunolabele d with anti-phosphohiston e H 3 antibod y (D) , a decreas e in S-phas e cell s (dat a no t shown) , an d a n increas e i n differentiatin g neuron s immunolabele d wit h anti-β tubuli n II I antibod y (E) . LP A decrease s th e numbe r o f dying cells immunolabele d for anti-activ e caspase- 3 an tibod y (F) . (Source: Modifie d fro m Kingsbur y e t al , 2004)

cortica l thickness , mitosis , an d cel l surviva l ar e ob served i n LPA 1/LPA 2 recepto r nul l mic e (Kingsbur y et al., 2003) , result s demonstratin g tha t th e effect s o f LP A in an ex vivo syste m ar e recepto r mediated . Th e rapi d increas e i n cortica l siz e followin g LP A exposure i s consisten t wit h studie s showin g tha t res cue o f V Z cel l death—likel y multipl e round s o f death , given th e ~2-hou r clearanc e tim e estimate d for dying cell s i n th e corte x (Thomaido u e t al. , 1997 ) — can hav e majo r consequence s fo r cortica l growt h (Kuid a e t al. , 1996 ; Pompeian o e t al. , 2000) . More over, th e increase d presenc e o f postmitoti c neuron s (β-tubulin-III-labele d cells ) i n th e VZ followin g LPA treatmen t ma y represen t prematur e differentiatio n of NPC s tha t contribut e to increase d VZ thickness . Th e switch t o a postmitoti c statu s coul d trigge r th e pro ductio n o f suppressor protein s (i.e. , Bcl-x L) able t o abrogate PC D i n cells migratin g fro m VZ t o CP . LP A signalin g ca n hav e majo r effect s o n cell s within th e embryoni c cerebra l corte x tha t are distinct

from cel l proliferation . Interestingly , th e foldin g pat tern observe d i n brain s treate d wit h LP A are superfi cially reminiscen t o f thos e observe d i n hyperplasti c brain s fro m knockou t mic e fo r caspase s (Kuid a et al. , 1996; Rot h e t al., 2000) or PS R (L i et al., 2003) . De spite thes e similarities , LPA-mediate d cytoarchitec toni c change s diffe r fro m thos e o f caspase deletio n i n several aspects . Disruptio n o f cel l deat h b y caspas e deletio n result s i n reduce d NP C deat h an d increase d VZ siz e withou t a marke d increas e i n C P thicknes s (Kuid a e t al., 1996 ; Hayda r e t al. , 1999b ; Pompeian o et al. , 2000). Augmente d cel l cycl e re-entr y b y over expression o f β-cateni n produce s fold s vi a th e tan gentia l expansio n o f cortica l surfac e are a withou t increase s in cortica l width (Chen n an d Walsh, 2002 ; Zechne r e t al. , 2003) . B y comparison , receptor mediate d LP A signalin g increase s cortica l thicknes s by alterin g bot h proliferativ e an d postmitoti c popula tions , an d furthe r produce s regularl y arrange d corti cal folds .

CELL DEAT H 8 5 Future studie s that involv e the inhibitio n o f specific G-protein s an d downstrea m effector s i n cortica l progenitors should provid e insight int o how receptor mediated LP A signalin g promote s termina l mitosi s and cel l surviva l i n th e developin g cerebra l cortex . Such studie s may also reveal links between cel l deat h and suppresso r proteins , includin g thos e relate d t o V(D)]/NHEJ an d Bcl- 2 family molecules , fo r the survival an d prope r functio n o f newl y postmitotic neu rons within the developing brain .

CONCLUSIONS Tissue homeostasi s i s currentl y viewe d a s a carefu l balance betwee n proliferatio n and cell death . Th e number, compositio n an d distributio n o f cells withi n both adul t an d developin g organs , includin g th e CNS, criticall y depend o n this balance. The dramatic increase i n cerebra l cortica l siz e acros s mammalia n evolution i s paralleled by increased cell number s an d an expansio n of cortical surfac e are a du e t o change s in th e regulatio n o f proliferation, differentiatio n and PCD withi n th e feta l brai n (Raki c an d Caviness , 1995; Caviness et al, 1995 ; Kuda et al 1996 ; Ceccon i et al , 1998 ; Yoshid a et al , 1998 ; Hayda r et al, 1999b ; Pompeiano e t al, 2000 ; Rot h e t al, 2000; Chenn an d Walsh, 2003 ; Li et al, 2003) . The presen t chapte r focused o n th e rol e o f PC D i n shapin g th e develop ment of the cerebral cortex . Collectively, th e result s discusse d i n th e presen t chapter indicat e that in the developin g CNS, PC D i s a significant cell fate fo r proliferating NPCs, and thi s PCD coul d b e responsibl e fo r a selectio n proces s based o n alteration s o f DNA , particularl y involving chromosomal aneuploidy . During migratio n and ini tial differentiation, cell death i s prevented o r delayed by th e expressio n o f suppresso r proteins , includin g members o f th e Bcl- 2 famil y an d th e V(D)J/NHE J factors, thu s allowing these newly postmitotic cell s to find their final positions and differentiate . Afte r reach ing thei r fina l destination , neural cell s star t to com pete fo r limite d trophi c facto r suppor t t o surviv e another roun d o f PCD. A newly identifie d group o f factors tha t affec t NPC s ar e th e lysophospholipid s — small lipi d signal s actin g throug h cognat e G pro tein-coupled receptors . LP A signalin g result s i n decreased cel l deat h an d induce d cel l cycl e exit , which ca n b e considere d new influences on cerebral cortical growth and anatomy. Future studie s will better

define th e mechanism s o f selectio n an d cel l deat h during nervous system development. Abbreviations APAF apoptoti c protease-activatin g factor BDNF brain-derive d neurotrophic facto r BH bcl- 2 homolog y BrdU bromodeoxyuridin e CNS centra l nervous system CP cortica l plat e DNA-PKcs DNA-dependen t protei n kinas e cat alytic subunit FGF fibroblas t growt h factor G gestationa l day ISEL i n situ end-labeling IZ intermediat e zon e Lig I V ligas e IV LPA lysophosphatidi c acid MAP microtuble-associate d protei n NBL neuroblasti c layer NGF nerv e growth factor NHEJ nonhomologou s end-joining NPC neura l progenitor cell NT neurotrophi n PCD programme d cel l deat h PSR phosphatidylserin e receptor SKY spectra l karyotyping SZ subventricula r zone trk tyrosin e protein kinase VZ ventricula r zone

ACKNOWEDGMENTS W e ar e gratefu l t o Am y Yang , Marcy Kingsbury, Brigitte Anliker, Christine Paczkowski, and Suzann e Peterson for the critical reading of this manuscript. This work was supported by the National Institute of Mental Health, National Institut e for , and Pe w Latin American Program i n Biomedical Sciences. References Abrams JM , Whit e K , Fessler LI , Stelle r H (1993 ) Programmed cell death during Drosophila embryogenesis. Development 117:29-43.

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6

Intracellular Pathway s of Neuronal Death Sandra M. Moone y George I. Henderso n

these populations di e after the y serve their role in cortical development . Th e subplat e neuron s provid e a temporary targe t populatio n fo r thalamocortica l ax ons that arriv e i n th e subplat e befor e th e neuron s of their targe t layer (cortical layer IV) have migrate d t o their final position (Ghosh e t al., 1990) . Cajal-Retzius neurons expres s reelin, whic h i s vital for normal mi gration of cortical neurons (Lamber t de Rouvroi t and Goffinet, 1998 ; Rice and Curran, 2001). This association is indicated b y the disrupte d cortical organization that occur s i n reeler mic e (Lamber t d e Rouvroi t and Goffinet, 1998 ) an d whe n Cajal-Retziu s neuron s are prematurely los t (Frotscher , 1998 ; Hartman n e t al. , 1999; Saftigetal, 1999) . Target-dependent neurona l deat h ma y b e th e most important proces s controllin g neurona l deat h in the CNS . Throug h thi s mechanis m th e nervou s system sculpts itself , matching th e numbe r o f neurons i n communicating part s o f a syste m fo r optima l outpu t and maximizin g efficien t function . Neurona l deat h occurs i f (a ) ther e i s a lac k o f input/innervation ,

Cell death i s critical to normal development i n multiple orga n systems . For example , it i s required durin g the formatio n o f digits and durin g th e generatio n o f the middl e ea r spac e an d th e vagina l openin g (e.g. , Saunders, 1966 ; Rodrigue z e t al. , 1997 ; Robert s an d Miller, 1998) . Likewise, in the centra l nervous system (CNS) o f th e norma l developin g mammal , a n esti mated 20%-80 % mor e neuron s tha n th e fina l num ber ar e generate d befor e th e surplu s i s eliminate d (Miller, 1995 ; Sastry and Rao , 2000). This loss of neurons varies by brain structur e an d b y specific neuro n populations. In som e brai n structure s there i s a wholesal e loss of transien t neurona l populations . Suc h cullin g o f populations i s required for proper developmen t t o ensue. Two exemplary populations ar e subplate neuron s (e.g., Kostovic and Rakic , 1980 ; Chu n et al., 1987 ; AlGhoul and Miller , 1989) , and Cajal-Retziu s neuron s (e.g., Dere r an d Derer , 1990 ; Meye r e t al. , 1998 ) i n neocortex (Marin-Padilla , 1998 ; Supe r e t al. , 1998 ; Sarnat an d Flores-Sarnat , 2002) . I t i s believe d tha t 91

92 NORMA L DEVELOPMENT (b) axon s reac h th e wron g plac e withi n th e targe t area, (c ) axons project t o the wron g target , (d ) ther e are simply too many axons terminating within the target, or (e) no target exists. One exampl e o f the latte r is the damage d trigeminal-somatosensor y system ; tran section o f the infraorbita l nerv e causes a los s of neu rons i n th e trigemina l brai n ste m nucle i an d th e ventrobasal nucleu s o f th e thalamu s (Klei n e t al. , 1988; Mille r e t al. , 1991 ; Wait e e t al. , 1992 ; Miller , 1999; Sugimot o et al., 1999 ; Bald i et al., 2000). It is important to note tha t programmed neurona l death i s a phenomenon mainl y confined to developing invertebrates . As the ter m implies , neuron s ca n die a s a consequence o f a genetic program . The bes t examples are provided by Caenorhabditis elegans (see Apoptosis, below). Thus, programmed cel l death mus t be distinguishe d fro m naturall y occurrin g neurona l death, whic h principall y represents target-dependen t neuronal death . Many developin g neuron s di e soon afte r the y ar e generated (se e Chapter 5) , perhaps a s a consequence of an error in DNA replication. Thi s adaptive mechanism allow s an organism t o protect itsel f from basin g its circuitr y o n a cadr e o f neuron s wit h mutate d o r damaged DNA .

EXPERIMENTAL MODEL SYSTEMS

This chapte r consider s in vitr o and i n viv o studie s of neuronal death . Eac h experimenta l approac h ha s its own advantages and limitations . A major advantage of in vitr o studie s is that a homogeneou s populatio n o f cells i s addressed . Thi s settin g i s crucia l t o studie s of the inductio n of neuronal death by xenobiotics, because cells within th e brai n diffe r widel y in their susceptibility (Watts et al., 2005). Additionally, the entir e population i s likely to respond t o a manipulation i n a similar manne r an d develo p synchronously . Thi s means that small changes i n transcript and protein expression o r post-translational modification s ar e mor e likely to be detectable. Disadvantage s t o this method are tha t cultur e condition s ca n influenc e th e path way^) activated , an d th e model ma y not b e physio logically relevant . I n contrast , i n vivo studie s us e a highly heterogenou s syste m i n whic h ther e ar e mul tiple cel l type s i n divers e developmenta l stage s a t a give n time . Thi s tempora l asynchron y make s i t harder to differentiat e transcrip t and protei n change s from a background of ontogenetic noise . In addition,

changes tha t ar e see n ma y be difficul t o r impossibl e to interpret . O f course , th e majo r advantag e t o thi s method i s that it is the mor e physiologically and clini cally relevant model . Direct evidenc e o f neurona l deat h i s difficul t t o amass because th e degenerativ e process ca n b e rapi d and th e debri s can b e remove d quickl y (Reddien and Horvitz, 2004) . Thus, cell s ca n expres s death-relate d proteins (e.g. , activ e caspas e 3 an d bax ) fo r a lon g time befor e the y di e (e.g. , Chen g an d Zochodne , 2003), an d no t al l cells rely on caspas e as an effecto r of cell deat h (se e Caspase-Independen t Cel l Death , below). Anothe r widel y use d metho d o f identifyin g dying cell s i s termina l deoxynucleotidy l transferas e mediated deoxyuridin e nic k end labelin g (TUNEL) , in which free , adenylate d end s o f degenerating DN A are labeled . Unfortunately , TUNE L ca n generat e false positiv e results; in a t least one mode l syste m th e amount o f TUNEL exceed s cel l los s (Gordo n e t al. , 2002). The onl y incontrovertible proo f of neuronal deat h is documentation o f a decrease i n neuronal number s in a longitudinal, population-base d study . Fina l neu ronal numbers ar e a function of three developmenta l events: generation, migration , an d death. A study performed a t a single time-point i n a mature animal cannot determin e th e relativ e contribution s o f thes e developmental events . A s a resul t o f th e potentia l of generatin g fals e positiv e results , a mathematica l method wa s devised to determine th e number s o f dying neuron s (Miller , 2003) . The grea t advantag e o f this approac h i s tha t th e estimat e o f cell deat h de pends on the documente d cel l numbers and the pro liferative activit y of the population .

MODES O F CEL L DEAT H

The mode s o f cell deat h includ e apoptosis , necrosis , and autophag y (e.g. , Gree n an d Reed, 1998 ; Lock shin an d Zakeri , 2004) . Apoptosi s i s a highl y con trolled mechanism , durin g whic h specifi c transcrip t and protein change s occur . Apoptosis ha s been calle d cellular suicide , a s it only affects th e cel l undergoin g it. I n contrast , durin g necrosis , cell s an d thei r or ganelles swel l and burst , releasin g enzymes int o th e local environment . A resul t o f thi s typ e o f deat h i s damage to neighboring cells, thus causing a cluster of dying cells. Autophagy involves lysosymal engulfmen t of damage d cell s o r cel l fragments . In man y model s

INTRACELLULAR PATHWAY S O F NEURONA L DEATH 9

of disease or injury , dyin g cell neighbor s ma y exhibit different mode s o f cell deat h an d a n individua l cel l may exhibit features o f different modes . One importan t poin t t o conside r i n cel l deat h i s that the lif e o f a critically damaged cel l ma y be tem porarily maintained, but i t will likely die. That is, the blocking of one mod e o f cell death ma y merely cause a cell to shift to another mod e o f cell death. Thus, the outcome i s that the cel l still dies, but i t may do so by a different sequenc e o f event s (e.g. , D'Mell o e t al, , 2000; Denecker et al, 2001; Zhou et al., 2005). Alternatively, blocking of cell death ma y allow cell survival, which i s not necessaril y a good thing. Fo r example, in mice deficien t i n apoptosi s protease-activatin g facto r (APAF) 1 , caspase 3 , o r caspas e 9 , ther e i s a lac k o f cell deat h i n th e neura l tube , an d thi s prevent s normal closur e o f th e tub e (Kuid a e t al. , 1996 , 1998 ; Cecconi e t al , 1998 ; Hake m e t al , 1998 ; Yoshid a et al, 1998 ; Honarpou r et al, 2000). It should als o be considered tha t enhanced cel l deat h i s not th e etiol ogy of a pathological state, rather, neuronal death is a process or end poin t associated with a given disease. Apoptosis The ter m apoptosis wa s coined t o describe a series of morphological step s throug h whic h dyin g cell s pass (Kerr e t al. , 1972) . With furthe r study , apoptosi s has become associate d with a metabolically active process (e.g., Wyllie , 1997 ; Putch a an d Johnson , 2004) . Accordingly, chromosoma l DN A i s cleaved , th e chro matin condenses , an d th e nucleu s i s broke n int o small pieces . Eventuall y th e cel l shrink s and break s into small , membrane-boun d apoptoti c bodie s tha t are engulfe d b y neighborin g cell s o r macrophages . This mechanism ensure s that proteolytic enzymes are contained withi n membrane-boun d entitie s an d tha t the apoptoti c cel l die s withou t causin g deat h o f neighboring cells. Much o f the initia l fundamental informatio n on the pathway s of apoptosi s cam e fro m studie s o f th e nematode C. elegans (e.g. , Horvitz et al., 1983 ; Hent gartner an d Horvitz , 1994 ; Reddie n an d Horvitz , 2004). O f the 109 0 cell s produce d durin g the devel opment of C. elegans, 13 1 die. Products o f two genes are essentia l fo r this death: Ced 3 and Ced 4 (Horvitz et al., 1983 ; Ellis and Horvitz , 1986). Los s or inactivation o f eithe r o f these gene s prevent s cel l death . I n the 95 9 cells that d o no t die , th e produc t o f another gene, Ced9 , bind s t o Ced4 , an d prevent s i t fro m

3

activating Ced3. Mammalian homolog s of these critical gene s hav e been identified . Ced3 i s homologou s with th e caspas e family , Ced 4 wit h APAF-1 , an d Ced9 wit h th e Be l family . Althoug h th e gene s an d gene product s involve d i n apoptosi s ar e highl y con served acros s species , th e mammalia n pathway s ar e considerably mor e comple x an d convolute d tha n those identifie d in C . elegans. For example, differen t pathways o f apoptosis ar e invoke d in cerebella r gran ule cell s dependin g o n whethe r th e cell s ar e pre - or postmigratory (Lossi et al., 2004). As state d above , us e o f th e ter m apoptosis ha s changed ove r the years . Th e definitio n ha s been re worked fro m a se t o f morphologica l feature s t o in clude molecula r an d biochemica l features . Putch a and Johnson (2004 ) suggest that apoptosis b e reserved for a caspase-dependen t cel l deat h tha t cause s th e morphological feature s described b y Kerr et al. (1972; see above) . They conten d tha t caspase-independen t cell deat h i s nonapoptotic , despit e acknowledgin g that i t would b e possibl e t o see apoptoti c morpholg y without caspas e activation . Man y researcher s us e apoptosis an d programmed cell death interchange ably, bu t thi s i s inappropriat e becaus e a program connotes geneti c determination , a s i n th e cas e o f C. elegans. In mammalia n systems , this type of genetic deat h i s rare , an d mos t apoptoti c deat h result s from environmenta l factors . I n thi s case , apoptoti c death i s bes t considere d a s naturall y occurrin g cel l death. I n thi s chapter, apoptosis include s morpholgi cal and biochemical events . Major Component s of Apoptotic Pathways Bel Family Bel protein s play pivotal roles i n cel l death . I n contrast to C. elegans, which only expresses a single gene, Ced9, many organisms have multiple member s of this family. Ther e ar e tw o functionall y distinc t group s within th e Be l family : thos e tha t induc e deat h an d those that protect agains t death. Bel proteins can als o be subdivide d structurally: there ar e three subgroup s defined b y th e numbe r o r typ e o f Bcl- 2 homolog y (BH) domains. Anti-apoptotic members o f the family , including Bcl-2 , Bcl-x L, Bcl-w , Mcl-1 , an d Al/Bfl-1 , contain fou r B H motifs : BH1 , BH2 , BH3 , an d BH4 . Pro-apoptotic member s ma y b e i n on e o f tw o sub groups: (1 ) thos e tha t contai n multipl e B H domain s

94 NORMA L DEVELOPMENT but lac k BH4-e.g. , Bax , Bcl-x s, Bak , and Bok/Mtd , or (2) the BH3-only subgroup tha t include s Bid , Bini/ Bod, Bad, Bmf, Bik/Nbk , Blk, Noxa, Puma/Bbc3, an d Hrk/DP5. Bel famil y members form dimers . Homodimers of two anti-apoptotic members , o r heterodimers formed by one pro-apoptotic an d one anti-apoptotic member , for example , Bcl-2-Bax , ar e pro-survival . I n contrast , homodimers o f pro-apoptoti c members , o r het erodimers o f tw o pro-apoptoti c members , resul t i n death. The BH 3 domain appears to be the prime suspect fo r inducin g apoptosis . Protein s wit h th e BH 3 domain have two functions: (1 ) to bind t o and inacti vate the survival-inducin g Bel proteins, and (2 ) to activate th e pro-apoptoti c Be l protein s wit h multipl e B H domains, suc h a s bax and ba k (Desaghe r e t al, 1999 ; Wei et al, 2000, 2001; Degterev et al, 2001). According to on e mode l o f apoptosis , Ba k i s a trans-membran e protein i n the outer mitochondrial membran e an d Bax is i n th e cytoplas m (Lucken-Ardjomand e an d Marti nou, 2005) . An apoptoti c signa l cause s a conforma tional change in both proteins that results in exposure of thei r N terminal s an d translocatio n o f Ba x to th e mitochondrial oute r membrane . Onc e i n th e mem brane, Bak and Bax form oligomer s that induce mem brane permeabilit y an d thu s allo w movemen t o f molecules suc h a s cytochrorn e C , HtrA2/Omi , an d Smac/DIABLO. Caspases Caspases ar e a group of structurally related enzyme s in mammals . Genes fo r caspases have homology with the C. elegans gene Ced3 (e.g., Jiang and Wang, 2004; Shiozaki an d Shi , 2004) . Lik e th e gen e produc t o f Ced3, caspase s are importan t fo r cel l death . I n con trast t o Ced3 , however , caspases are no t require d for death (se e below) . Caspase s ar e proteases ; a t leas t 1 4 caspases hav e been identified . They have cysteine s i n their activ e site s an d cleav e thei r targe t protei n adja cent to an asparti c aci d residue . Caspase s ar e synthe sized a s inactiv e isoform s an d ar e cleave d int o larg e and small subunits. Active caspases are tetramers com posed of two large and tw o small subunits. Caspases ar e groupe d int o thre e subfamilies : interleukin-lp-converting enzyme-lik e caspases , ini tiator caspases (e.g., caspase 8 or caspase 9), and effec tor caspase s (e.g. , caspas e 3 ) (Shi , 2002) . Initiato r caspases ca n cleav e themselves and effecto r caspase s are cleave d b y othe r enzymes , includin g initiato r

caspases. Activatin g initiato r caspase s ca n induc e a caspase cascade i n which effecto r caspase s are cleave d and the n othe r apoptosis-relate d downstrea m mole cules are activated. Caspase s ca n cleave mor e tha n 40 target proteins ; o f particula r importanc e ar e a n in hibitor o f DNases, nuclea r laminins , and cytoskeletal proteins. Inactivatio n o f DNas e inhibitor s allow s DNases to cut DN A into fragments that are multiples of 180-200 bp. The resul t of this serial snipping is the stereotypical DNA ladder on a Southern blot that is associated wit h apoptosis. Cleavage o f nuclear laminins results i n nuclea r fragmentation . Similarly, cleavage of cytoskeleta l element s result s i n membran e bleb bing and, ultimately, cell fragmentation. The importanc e o f caspase s i n norma l develop ment i s reflected in mic e tha t lack effective caspas e 3 or 9 . Bot h mutant s hav e craniofacia l malformation s and abnorma l brai n morpholog y (Kuid a et al., 1996 , 1998; Ceccon i e t al , 1998 ; Hake m e t al , 1998) . Specifically, thes e animals exhibi t enlarged prolifera tive zones , heterotopi c cluster s o f neurons , an d in creased number s o f neurons. Interestingly , caspas e 3 deficiency ca n b e offse t b y deficienc y i n th e anti apoptotic gen e Bcl-x L (Rot h e t al. , 2000) , implyin g that Bcl-x L ca n ac t throug h a caspase-3-independen t mechanism. Apoptosis Protease Activating Factor 1 APAF-1, homologous to Ced4, exists in the cytoplas m as a n inactiv e protein (Ferrar o et al, 2003 ; Shiozaki and Shi , 2004). Interaction with cytochrome C i n the presence o f adenosine triphosphate (ATP) results in a conformational chang e i n APAF- 1 tha t allow s seven molecules o f APAF-1 t o assembl e an d for m th e cen tral portio n o f the apoptosome . I n addition , th e con formational chang e expose s th e caspas e recruitmen t domain, whic h bind s procaspase 9 . In the absenc e of an inhibitory apoptotic protei n (IAP ) such as X-linked IAP (XIAP) , caspas e 9 i s activate d vi a a n unknow n mechanism. Activ e caspase 9 recruits and activates effector caspases 3 and 7 . APAF-1 knockou t mic e ar e embryoletha l (Cec coni e t al, 1998) . The y hav e craniofacia l an d CN S malformations tha t appea r t o resul t from insufficien t cell deat h durin g development . Interestingly , thei r limbs ar e differentiall y affected . O n gestationa l da y (G) 15.5 , the limb s are less mature those than i n wildtype animals, as evidenced b y the persistenc e of interdigital webbing . B y G16.5 , however , thei r limb s

INTRACELLULAR PATHWAY S O F NEURONA L DEATH 9

appear normal; the webbin g is lost after undergoin g a caspase-independent form o f cell death . Thi s findin g implies tha t som e tissu e ca n overcom e th e los s o f APAF-1, but th e CM S cannot, an d tha t deat h vi a the caspase-independent pathwa y takes longer . Cytochrome C Cytochrome C i s normally found betwee n th e inne r and oute r mitochondria l membrane s an d i s used fo r transporting electrons between th e Cytochrome B-C1 and cytochrom e oxidas e complexe s i n th e electro n transport chain. When th e mitochondria l permeabil ity transition pore i s compromised, substances i n th e intramembrane space , includin g cytochrom e C , ar e allowed t o move int o the cytoplasm . Once in th e cytoplasm, cytochrom e C interact s wit h APAF-1, ATP, and caspase 9 to form the apoptosome. Poly-Adenosine Diphosphate Ribose Polymemse Poly-adenosine diphosphate (ADP ) ribose polymerase (PARP) 1 is found i n the nucleu s an d mitochondria . It can b e activated i n response to DNA damage o r to oxidative stress (e.g., Hong et al., 2004; Nguewa et al., 2005). Followin g DNA damage , PARP- 1 becomes ac tive an d synthesize s poly(ADP-ribose ) an d catalyze s the addition of ADP-ribose to the DNA (e.g., Bouchard et al, 2003) . Followin g poly(ADP-ribosyl)ation, DNA repair enzyme s ca n b e recruite d an d activated . Gas pases 3 and 7 cleave PAR P in it s nuclear localization sequence, resultin g in an inactive form o f the enzyme that canno t ad d ADP-ribose , thus , th e DN A canno t undergo repair . During th e synthesi s o f poly(ADP-ribose) , nicotinamide-adenine dinucleotid e (NAD ) i s cleaved into ADP-ribos e an d nicotinamide . T o restor e NA D levels (an d thus the NAD/NAD H ratio) , the cel l con verts nicotinamid e t o NA D b y mean s o f tw o mole cules o f ATP , thu s depletin g th e energ y pool . Following severe DNA damage, the loss of cellular energy is likely responsible for a necrotic cel l death afte r excessive DNA damage (Szab o and Dawson, 1998 ; H a and Snyder , 1999 ; Fuerte s et al, 2003). Interestingly , the los s of energy may be necessary , but i s unlikely to be sufficient, for cell death (Wan g et al, 2004). Mitochondrial PARP- 1 ma y be involve d i n apop tosis via release of apoptosis inducing factor (AIF ) (Yu et al , 2002 ; D u e t al , 2003) . Genomi c damag e

5

causes poly(ADP-ribosylation) in the nucleus, resultin g in cellular NAD depletion, activatio n of mitochondrial PARP, AIF release, and subsequent cell death. Apoptosis Inducing Factor In normal , undamage d cells , AI F i s importan t fo r protection agains t oxidativ e stres s (Klei n et al. , 2002 ; Lindholm et al, 2004; Vahsen et al, 2004). Following PARP activation , AIF i s released fro m th e mitochon dria into the cytoso l (see Poly-Adenosine Diphosphate Polymerase, above). AIF translocates into the nucleu s where i t i s involve d i n condensatio n o f chromati n and DN A fragmentatio n (Hon g e t al , 2004) . Th e PARP-dependent releas e o f AIF i s a caspase-indepen dent mechanis m o f cell deat h (se e Caspase-indepen dent Cel l Death , below) . I n addition , caspase s ca n cause release of AIF, as can changes in mitochondria l membrane permeability , fo r example , b y Ba x an d Bak. Bcl- 2 ca n interfer e wit h AIF-mediated apoptosi s by blockin g th e cellula r redistributio n o f AIF tha t is necessary for death (Susi n et al., 1996 , 1999 ; Dauga s et al, 2000). Apoptosis-Related Receptors Ligands (e.g. , tumor necrosi s factor [TNF]-a , Apo2/ TRAIL, FasL , an d possibl y growth factors ) tha t inter act with receptor s o f the TN F famil y activat e the extrinsic apoptoti c pathway . TNF-oc binds t o th e TN F receptor, Apo2/TRAIL binds t o TNF-related apopto sis inducin g ligand-recepto r 1 (TRAIL-R1)/death re ceptor (DR) 4 and TRAIL-R2/DR5, and FasL binds to Fas(CD95,APO-l). Effectors o f Apoptosis

p53 p53 i s a multifunctiona l housekeepin g protei n in volved i n cel l cycl e regulation , recognitio n o f DN A damage and consequent repair , and cell death (Mille r et al, 2000 ; Morriso n e t al, 2002) . Although i t i s a transcription factor , i t als o respond s t o genotoxi c stress (e.g . Bertran d e t al. , 2004) . p5 3 activatio n ca n halt the cel l cycle and initiat e DNA repair or apoptosis (Harms et al, 2004). p53 usuall y has a short half-life (Ki m et al, 2004) . When stabilized, for example, through phosphorylation on serin e residues , p5 3 accumulate s i n th e nucleus ,

96 NORMA L DEVELOPMEN T where i t ha s transcriptiona l activities . p53-regulate d genes critica l for normal brai n development includ e cell cycl e proteins p21 and murin e doubl e minut e 2 , the DN A repai r protei n growt h arres t an d DN A damage-inducible gen e 45 , an d th e apoptosis-relate d proteins insulin-lik e growth facto r bindin g protei n 3 and Ba x (Levine , 1997) . I n addition , p5 3 regulate s the Fa s receptor (Somasundaram , 2000) and th e hor mone polyadenylat e cyclase-activatin g polypeptid e receptor 1 (e.g. , Cian i e t al , 1999 ; Johnson , 2001 ) (see Chapters 1 5 and 16) . p53 can play a direct role i n cell death by interacting wit h th e Be l famil y o f protein s (Chipu k e t al. , 2003, 2004 ; Mihar a e t al , 2003) , APAF-1 , an d apoptosis-related receptors . Thes e interaction s ar e pro-apoptotic. p5 3 down-regulate s pro-survival Bcl-2 and Bcl-x , and up-regulate s Bax, Bid, and APAF-1 . I t also up-regulate s TRAIL-R2/DR5 , Fas , an d FasL . I n addition, p53 represses c-Fos (Kle y et al, 1992) , thu s potentially interferin g with activato r protei n 1 and , hence, transcription. Oxidative Stress Oxygen, thoug h centra l t o life , ca n b e toxic . I n it s ground state , i t possesses two unpaired electron s with parallel spi n states. This setting makes a two-electron reduction kineticall y unlikely ; however , sequentia l one-electron reduction s ca n occur , hence generatin g oxygen fre e radicals , reactive oxyge n specie s (ROS) . In th e biologica l setting , th e initia l one-electro n ex change generate s th e superoxid e anio n radical . Th e protonated two-electro n reductio n produce s H 2O2 (the protonated for m o f the peroxid e ion), with the final protonate d four-electro n produc t bein g water . The oxyge n radicals resulting from thi s process generate a plethor a o f reactiv e specie s wit h othe r mole cules, suc h a s nitroge n an d iron , al l o f whic h ca n produce a pro-oxidan t environment i n cells , terme d oxidative stress. The centra l role s for ROS in apoptosis, bot h as initiators an d a s signalin g event s withi n th e apoptoti c process, ar e wel l documente d (Curti n e t al. , 2002 ; Fleury e t al , 2002 ; Polste r an d Fisku m 2004) . Although th e specifi c mechanism s by which RO S elici t and/or maintai n apoptosi s remai n undefined , thes e compounds hav e a n effec t o n a variet y o f cellula r components: proteins , DN A base s an d sugars , polysaccharides, an d lipids . On e ROS-relate d pathwa y that has been connecte d t o ethanol-mediated neuro n

apoptosis i s th e productio n o f pro-apoptoti c prod ucts o f lipi d peroxidatio n withi n mitochondri a (Ra machandran e t al , 2001 , 2003) . RO S reac t wit h unsaturated fatt y acids , initiatin g a self-perpetuatin g peroxidation o f membrane lipid s (Kappus , 1985) . I n addition t o direc t damag e t o biomembranes , thi s ubiquitous proces s generate s highl y reactive aldehydes, th e mos t studie d an d th e mos t toxi c bein g 4 hydroxynonenal (HNE ) (Esterbaue r e t al , 1990 ; Uchida e t al , 1993) . Importantly , HN E formatio n generates apoptoti c deat h o f neurons, and it s production, secondar y t o oxidativ e stress , ha s bee n com pellingly linke d t o neuro n deat h i n a variet y o f neurodegenerative diseases (Zarkovic, 2003) .

APOPTOTIC PATHWAY S

Caspase-Dependent: Intrinsic vs. Extrinsic Pathways of Apoptosi s The pathway s of apoptosis can b e divide d into intrinsic an d extrinsi c on th e basi s of activation sit e (Budi hardjo e t al , 1999 ; Shiozak i an d Shi , 2004) . Th e intrinsic, mitochondria-associate d pathwa y depend s on Bel proteins. Th e extrinsi c pathway is mediated by activation o f an apoptosis-relate d receptor . Th e path ways ar e no t trul y independent , a s ther e i s muc h cross-talk, and bot h can activat e caspas e 3 (Fig. 6-1) . In addition, activation of the extrinsi c pathway can activate a feedback loop to the intrinsi c pathway. Intrinsic Pathways The fundamenta l featur e o f the intrinsi c pathwa y i s the releas e o f pro-apoptoti c compound s fro m mito chondria. Thi s pathwa y i s activate d b y intracellula r stress signal s includin g bu t no t limite d t o oxidativ e stress or DN A damage . Thus, majo r effector s ma y b e ROSandp53. Mitochondrial permeabilit y i s affected b y th e Be l family o f proteins . Ba x homodimer s allo w io n flu x through th e mitochondria l membrane , an d this some how allow s movemen t o f cytochrom e C , possibl y through enlargemen t o f the voltage-dependen t anio n channels (Banerje e an d Ghosh , 2004 ) o r by openin g the mitochondria l permeabilit y transition por e (e.g. , Marzo et al., 1998) . Breach of the membran e cause s a loss of membrane potentia l and releas e of cytochrome c, AIF, HtrA2/Omi, and/or second mitochondria-derive d

INTRACELLULAR PATHWAY S OF NEURONA L DEAT H 9 7

activator of caspase/direct IAP binding protein, which has a low isoelectri c poin t (Smac/DIABLO) . I n nor mal cells , thes e substance s ar e sequestere d betwee n the inne r an d oute r mitochondria l membranes . Re lease int o th e cytoplas m initiate s apoptoti c pathway s (Fig. 6-1). It should b e note d tha t permeabilization of the mitochondria l membran e doe s no t necessaril y allow dumping of all intermembrane substances. Instead, there i s som e selectivity . For example , applicatio n o f

n-methyl-D-aspartate t o culture d primar y mouse corti cal neurons can caus e release of AIF and cytochrom e c (Wang et al. , 2004) . In contrast , oc-amino-3-hydroxy-5 methyl-4-isoxazoleproprionic aci d ca n induc e th e re lease of cytochrome c, but no t AIF. In response to DNA damage or ROS, p53 accumu lates in the mitochondria (e.g., Marchenko e t al., 2000; Erster e t al. , 2004) , wher e i t interact s with Bel-famil y proteins i n a transcriptional-independen t manner .

FIGURE 6- 1 Pathway s underlying neural cell death. Three cell deat h pathways tracing the flow of information from externa l effects t o nuclear response ar e de picted: intrinsi c and extrinsi c caspase-dependent pathway s and a caspase-inde pendent pathway . Variou s player s involve d i n thes e pathway s ar e shown . Positive an d negativ e effector s ar e depicte d b y line s endin g i n arrow s an d blunted tips , respectively . FADD, Fas-associate d deat h domain ; FLIP , FADD like interleukin-1-convertin g enzyme inhibitor y protein fo r other abbreviations see Abbreviations list. See text for details.

98 NORMA L DEVELOPMENT This interactio n explain s the rapi d response of a cel l to a deat h signal . P5 3 alter s expression of Bel famil y members i n a pro-apoptotic manner , i.e. , expression of Ba x is increased an d concurrentl y Bcl- 2 i s down regulated. I n addition , p5 3 up-regulate s APAF- 1 expression. Release d cytochrom e C form s a comple x with seven APAF-1 molecules, an d i n the presenc e o f ATP, caspase- 9 i s recruite d int o thi s complex , th e apoptosome, an d activated . Active caspase 9 cleaves and activate s effecto r caspase s 3 , 6 , and/o r 7 . Not e that at this point, the intrinsi c and extrinsi c pathways merge a s caspase s 8 an d 10 , activate d b y receptor s (see below), cleave and activat e effector caspases . Extrinsic Pathways The extrinsi c pathway is activate d b y factor s outsid e the cell membrane via ligand-receptor binding (Muzi o et al, 1996 ; Almasan and Ashkenazi, 2003; Choi and Benveniste, 2004). Binding of the ligand to the recep tor cause s recruitment o f cytoplasmic element s suc h as Fas-associate d deat h domai n an d TN F receptor associated deat h domai n tha t for m th e death inducing signalin g complex. Thi s proces s result s i n recruitment and activatio n of initiator caspases 8 and 10 through proteolysis . Caspases 8 and 1 0 then acti vate effecto r caspase s 3, 6, and 7 , and ca n als o cleave a membe r o f the Be l famil y o f proteins, Bid. At this point, th e intrinsi c pathwa y also come s int o pla y as Bid translocate s to the mitochondrio n an d cause s insertion o f Bax-Bax homodimers int o the mitochondr ial membrane, thu s allowin g release o f cytochrome c and th e resultin g downstrea m events , an d effecto r caspase activatio n (L i e t al. , 1998 ; Lu o e t al. , 1998 ; Wei et al., 2001; Scorrano an d Korsmeyer , 2003). Substrates of Caspases Substrates o f effecto r caspase s ma y numbe r i n th e hundreds (e.g. , Earnshaw et al., 1999; Nicholso n et al., 1999; Fischer et al., 2003; Fuentes-Prior and Salvesen, 2004). Thre e ar e o f particula r interest—Bid, PARP , and DFF40/CAD . Activated , i.e. , cleaved , Bi d translocates t o th e mitochondria , wher e i t interact s with othe r pro-apoptoti c member s o f the Be l family , specifically Ba x an d Bak , thu s causin g membran e permeability, and leakag e of cytochrome C (L i et al., 1998; Lu o et al, 1998 ; We i et al, 2001). Cleavage of PARP b y caspase s 3 an d 7 inactivate s the DN A re pair mechanism (Lazebni k et al., 1994) . Activation of

DFF40/CAD by caspase 3 results in enzymatic cleavage of double-stranded DNA, the hallmar k characteristic o f fragmente d DNA . Thi s structur e ca n b e visualized on a n agaros e ge l as a ladder o r can b e labeled i n vivo by means of TUNEL. Caspase-Independent Cell Death There i s some debat e a s to whethe r caspase-indepen dent cel l deat h i s a for m o f apoptosi s (Chipu k an d Green, 2004 ; Putch a an d Johnson , 2004) , however, pathways o f caspase-independen t cel l deat h ar e fre quently activated concomitant with caspase-dependent pathways (Ramachandra n e t al. , 2003) . Caspase independent cell death i s initiated b y the sam e effec tors a s thos e i n typica l apoptosis—fo r example , p5 3 and ROS (Hill et al, 2000; Godefroy et al, 2004), but the morphological outcom e differs. Caspase-indepen dent deat h doe s no t resul t i n DN A fragmentatio n or cell blebbing , no r d o th e mitochondri a los e mem brane potential. I n addition, the caspase-independen t pathway may be initiate d by overactivation of PARP-1 (Hong et al, 2004). As wit h caspase-dependen t pathways , the releas e of mitochondrial intermembran e substance s AIF, endonuclease G , HtrA2/Omi , and/o r Smac/DIABL O appears t o b e pivota l fo r caspase-independen t deat h (Susin e t al., 1999 ; Crega n e t al, 2002 ; Hong e t al , 2004; Yakovle v an d Faden , 2004) . Releas e o f AI F from th e mitochondri a results in activation of DNases that cause DN A fragmentation. Endonuclease G i s a mitochondrial nucleas e necessar y fo r replicatio n o f mitochondrial DN A (Cot e an d Ruiz-Carrillo , 1993) . If released int o the cytoplasm , it translocates into th e nucleus wher e i t causes internucleosoma l DN A frag mentation (L i et al , 2001) . HtrA2/Om i an d Smac / DIABLO als o appear to induc e caspase-independen t death, althoug h th e exac t mechanis m i s unknow n (Yakovlev and Faden , 2004). CONCLUSIONS AN D SUMMARY

Misleadingly referre d t o a s a regressiv e process, neu ronal deat h i s essentia l fo r norma l CN S develop ment. Withou t it , th e nervou s syste m ca n becom e overpopulated wit h poorl y integrate d neuron s an d circuitry that produces functiona l deficits. This observation i s supported b y studies of knockout mice defi cient i n caspas e 3 or 9 (Oppenhei m e t al, 2001) . I n

INTRACELLULAR PATHWAY S O F NEURONA L DEAT H 9 9 these animals , th e amoun t o f neurona l deat h i s re duced and learnin g an d memor y is compromised. Although neurona l deat h ma y b e describe d a s apoptotic o r necrotic , dyin g cell s commonl y exhibi t features o f bot h (Eva n an d Littlewood , 1998 ; Gree n and Reed , 1998 ; Lockshi n and Zakeri , 2004) . More attention ha s been directe d t o understandin g apoptosis . The pathway s activated durin g apoptotic degeneratio n are comple x an d ofte n appea r t o b e conflicting . The y include so-calle d intrinsi c an d extrinsi c systems , bot h of whic h culminat e i n th e activatio n o f caspas e 3 . Some cells , however , di e throug h a caspase-indepen dent mechanism. The curren t and futur e understand ing o f neuronal deat h ha s been an d wil l b e garnere d from studie s o f the action s o f toxins (e.g. , ethano l an d neurotransmitter blockers) , radiation , stress , an d othe r challenges. Suc h studie s provid e valuabl e insigh t int o the interpla y betwee n th e variou s modes of cell deat h and th e pathway s underlyin g thes e degenerativ e events. Abbreviations ADP adenosin e diphosphate AIF apoptosi s inducing factor APAF apoptosi s protease-activatin g factor ATP adenosin e triphosphat e BH Bcl- 2 homology CNS centra l nervou s syste m DIABLO direc t IA P binding protei n HNE 4-hydroxynonena l IAP inhibitor y apoptoti c protein NAD nicotinamide-adenin e dinucleotide PARP poly-adenosin e diphosphat e ribos e poly merase ROS reactiv e oxyge n specie s SMAC secon d mitochondrial-derive d activato r of caspase TNF tumo r necrosis facto r TUNEL termina l deoxynucleotidy l transferas e mediated deoxyuridine nick end labeling

ACKNOWLEDGMENTS Wor k o n thi s chapte r was supported b y th e Nationa l Institut e o f Alcohol Abus e an d Alcoholism.

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7

Developmental Disorders and Evolutionary Expectations: Mechanisms of Resilience Barbara L. Finlay Jeremy C. Yost Desmond T . Cheung

Every livin g organis m ca n trac e it s lineage bac k t o the unicellula r organisms that firs t populate d Earth . We are thu s th e descendant s o f creatures wh o hav e not onl y survive d but hav e successfull y reproduce d in the fac e o f gross atmospheric shifts, blast s of ionizing radiation, the impact s o f comets, ice ages, globa l warming, earthquake s an d hurricanes , plague s o f any numbe r o f viral an d bacteria l forms , prédation, starvation an d drought , th e toxin s of plants and ani mals, parasites, competing tribes, jealou s fathers, indifferent mothers , an d everyda y accident s o f al l kinds. Any survivor of this wildly improbable lineage is mad e of toug h stuff . The poin t of the presen t chapter i s t o tur n th e usua l discussio n o f develop mental vulnerability on its head and, rather than discuss the effect s o f an "environmenta l insult(s) " on a fragile infant , examin e th e desig n feature s o f th e tough stuf f o f whic h w e ar e made . W e argu e tha t only in this evolutionary context wil l the disorder s of development tha t sometime s d o emerg e mak e mechanistic sense.

EVOLUTIONARY DRIVES

Catastrophe Conservation o f th e buildin g block s o f th e embry o across the invertebrat e an d vertebrat e lineage s and th e resilience of that embryo have been part of a general rethinking of the characte r of the evolutionar y process to which ou r ancestors wer e subjected, along with greate r awareness of the effect s o f periodic globa l catastrophe s on th e natur e o f the genom e (Gerhar t an d Kirschner, 1997). Struggle for success among individuals in an evolutionary landscape o f potential niche s and thei r associated fitness peaks, success defined by better adaptatio n resulting i n greate r reproduction , i s the essentia l Dar winian view . The kind s of adaptations highlighte d b y this proces s ar e bot h increasingl y subtl e organis m an d environment fits and enhancement s importan t i n sexual selection, a central aspect of evolutionary change. Catastrophe, loca l o r global , provide s a forc e i n which all organisms, across radiations and species , ar e

104

DEVELOPMENTAL DISORDER S AND EVOLUTIONAR Y EXPECTATIONS 10 5 subjected t o a sever e filter ; succes s arise s fro m sur vival i n suddenl y change d environment s (Alvare z et al., 1980 ; Albritton , 1989) . Variation in gene trans mission durin g catastroph e ca n aris e from individua l competition a s well, but whole radiation s may be lost. The best-know n example , o f course, i s the extinctio n of the dinosaur s and surviva l o f mammals i n th e lat e Cretaceous period . Selectio n wil l favo r th e robus t and adaptabl e organism , able t o weathe r disaster , to relocate or create it s favored nich e o r get by in a ne w one, an d t o reproduc e a s muc h a s possible . Ou r genomes carr y th e histor y o f bot h type s o f change . Moreover, th e natur e o f geneti c chang e appear s t o make i t likel y tha t thi s histor y i s no t overwritte n i n such a way that it is inaccessible, but rathe r added to , as we will elaborate later. Evolvability "Evolvability" i s a related , bu t distinc t ide a tha t car ries impor t fo r ho w a genom e ca n respon d t o chal lenge. This idea has been used in two distinct ways in the allie d field s o f evolutionary biolog y an d artificia l intelligence an d robotic s b y mean s o f geneti c algo rithms. The first, and somewha t stricter , sens e o f this concept aros e fro m th e hypothesi s an d late r demon stration (in bacteria) that in times of extreme stress and crisis, "deliberate " additiona l variatio n and mutatio n would aris e as an adaptiv e mechanism, suppresse d i n favored environment s (Gerhart an d Kirschner , 1997 ; Radman e t al. , 1999) . Th e second , mor e general , sense is the observatio n that some types of genomic or informational structure s more readil y produce usable adaptive changes and the potentia l for evolution than others (Lipso n an d Pollack , 2000 ; Nolf i an d Flore ano, 2002 ; Baum , 2004) . Everythin g else equal , th e "evolvable" organism is more likel y to b e wit h u s today. Both the stric t and the genera l sense of evolvability will be importan t in understanding the respons e of organisms to developmental challenge .

the viru s a t th e cellula r leve l (antibodies) , make s its environment inhospitabl e (fever) , an d expel s i t (al l the variou s miseries o f flu) . Eac h o f these symptom s can be viewed as adaptive responses, no t as pathology. Of course , no t ever y response t o diseas e o r traum a is adaptation, an d th e urg e t o tell suc h "just-so " storie s must be strictly policed. Fo r example, exsanguination when a n arter y i s severed migh t cleans e th e woun d and quickl y lessen metaboli c load, but obviousl y it is not a n adaptiv e response. Conceptua l clarit y in thi s domain get s particularl y muddled whe n organism s are i n clos e competition . Fo r example , i t woul d ap pear that the enterprisin g rhinovirus has co-opted th e expulsion componen t o f the immun e respons e for its own adaptiv e purpose o f spreadin g itsel f around th e environment—whose adaptation is the sneeze? Evolutionary medicin e become s particularl y relevant t o the developmenta l respons e t o shortage s an d pathogens when the normal , tactical natur e of the response o f the adul t organis m t o challenges i s considered an d applie d t o developmen t (Bateso n e t al. , 2004; Gluckma n and Hanson , 2004) . The bes t and most elaborate d exampl e i n physiology is the balanc ing o f requirements fo r short- an d long-ter m surviva l in th e hypothalamic-pituitary-adrena l axi s i n re sponse to acut e and chroni c environmenta l stressor s (Sapolsky et al., 1986) . In order to survive an immedi ate threat , energy is mobilized b y metabolic change s that, if they persist long-term, will damage the animal. Alterations i n nervou s system s an d behavio r i n re sponse t o earl y environmenta l challenges , particu larly commonl y encountere d ones , migh t als o b e tactical adaptation s and shoul d no t automaticall y be construed a s pathology . Fo r example , perhap s i t i s a good be t to alter interest in foo d compare d t o general exploration i n perpetuit y i f earl y experienc e show s food t o be i n unusually short supply (Smart and Dob bing, 1977 ; Levitsky , 1979 ; Bateson e t al , 2004 ; Gluckman an d Hanson, 2004) . Environmental Expectatio n

Adaptive Response O r Pathology? The thir d conceptual thread t o be woven int o the argument for evolutionar y chang e is the recastin g of some state s usually viewed a s illnes s or patholog y a s adaptations b y th e emergin g fiel d o f evolutionary medicine (Ness e an d Williams , 1996) . A vim s co opting ou r genom e fo r it s ow n purpose s i s a ba d thing. Consequently , a n immun e respons e disables

The fourt h thread i s environmental expectation . Thi s is usually raised in the contex t o f predictable environ ments an d a genome predispose d t o lear n (Gree nough an d Black , 1992) : i f informatio n abou t th e future structur e of visual informatio n in th e worl d is well predicted b y present structure , and ther e i s time to learn it, why waste genome specifyin g it ? Considering on e o f th e classi c case s o f ethology , i f mother i s

106 NORMA L DEVELOPMENT the first large, moving thing any duck likely to survive sees, then learning the qualities of the first large, moving objec t i s an efficien t wa y to get th e information . On th e othe r hand , th e physiologica l armamentar ium availabl e indicate s tha t w e als o expec t trauma , infection, an d poo r nutritio n o f al l kinds . Ther e i s no reaso n to assum e tha t the brai n is not similarl y prepared. Recen t compariso n o f the genom e o f th e chimpanzee an d o f the human , showing rather mor e changes i n gene s involve d i n foo d metabolis m an d immune respons e tha n i n th e brain-relate d genes , has underline d tha t geneti c respons e t o often encountered challenge s may be fairly rapid, and "die t and pathogen s ar e dominan t selectiv e force s fo r al l species" (Olso n an d Varki , 2004) . Relevan t t o thi s chapter i s the cultura l history of alcohol. A s alcohol has served as a common sourc e of nutrition and foo d preservation i n Wester n Europe , thos e population s (compared t o Asian ones) have become equippe d t o metabolize alcohol , an d ther e i s some evidenc e tha t the incidenc e o f alcoholis m fall s th e longe r fer mented product s hav e been use d i n a particular cul ture (Goedd e e t al , 1992 ; Whitfield , 1997) . T o understand a developmental respons e t o an environ mental challenge , i t will make a great dea l o f differ ence whethe r th e challeng e i s "expected, " suc h a s alcohol, o r unprecedented—fo r example , a designe r drug such a s thalidomide that ma y mimic the for m of an early regulatory gene. A Warning and a Road Ma p In th e followin g section , we first examine a few cen tral cases of the developmenta l consequence s o f alcohol an d othe r substanc e abuse , an d tur n th e usua l style o f presentation upsid e down t o sho w the outra geous situations in which the typica l embryo can survive. A stron g cavea t i s necessar y here: a t n o poin t should a demonstration that th e "typical " infant ca n be born without defect—for example , following a persistent bath o f alcohol—be mistake n a s an argumen t endorsing alcoholis m i n pregnan t mothers . A sub stantial percentag e o f infants exposed t o ethano l d o have defects , a probabilit y n o prospectiv e paren t should ignore . No r ar e w e i n an y way attempting t o minimize the value of locating and ameliorating thos e defects cause d b y developmental insults described i n many of the other chapters of this volume. Rather, our point is that if we fail to appreciate the robus t nature of the developin g embry o an d consistentl y mislabe l

deviations as pathology instead o f adaptations (or jus t deviations), w e will b e i n a n explanator y real m tha t makes n o sens e i f we try to understan d th e patholo gies of development tha t can and do occur . Next, we will turn to some genera l desig n feature s of developmen t tha t ar e stabl e i n th e fac e o f chal lenge, and conside r som e particula r case s of nervou s system development tha t show those principles. Over all, the guidin g principal in this discussion is one tha t is true for all life sciences but i s often ignore d i n med icine: "nothin g i n biolog y makes sense except i n th e light of evolution." (Dobzhansky, 1973)

CONTEXTUALIZING TH E DEVELOPMENTAL EFFECT S OF ALCOHOL, NICOTINE , AND THALIDOMIDE

Alcohol The huma n bod y has a variety of mechanisms fo r the screening an d disposa l o f toxins. Alcohol i s no excep tion. The presenc e o f alcohol i n our evolutionary and developmental environmen t ha s le d t o specifi c en zymes responsibl e fo r it s metabolism : alcoho l dehy drogenase an d acetaldehyd e dehydrogenase . Ethano l is naturally encountered i n fermentation in fruit, an d also is a direct result of fermentation of starches in th e gut. I n man y huma n cultures , alcoho l i s sought no t only fo r th e obviou s psychoactiv e consequence s bu t also becaus e i t serve s a usefu l rol e a s a preservative and secondaril y as a source of calories (Goedde e t al., 1992; Whitfield, 1997 ; Dudley , 2000 , 2002). Alcohol can thus be considered not simply a completely alien disruptive element but also a normal part of the developmental environment . Thi s i s not t o downpla y th e dangers o f ethano l t o a developin g organis m bu t t o place it in the contex t of a regulating system. We not e in passin g tha t thi s evolutionar y histor y ma y hav e a significant impact o n the relevanc e o f animal mod els to human development , wher e frugivores an d omnivores migh t b e considerabl y bette r models tha n carnivores. With th e assumptio n o f the robustnes s o f the de velopmental syste m i n mind , w e tur n t o a recen t study fro m Martinez-Fria s an d colleague s (2004 ) o n the relationshi p betwee n alcoho l consumptio n dur ing pregnancy and congenita l anomalies , paying specific attentio n t o thos e anomalie s indicativ e o f feta l

DEVELOPMENTAL DISORDER S AND EVOLUTIONAR Y EXPECTATIONS 10 7 alcohol spectru m disorder s (FASD) . Fro m a corpu s of dat a collecte d ove r 2 4 years , Martinez-Fria s an d colleagues dre w a grou p o f 470 5 malforme d infant s and a comparable grou p of 4329 normal infant s whose mothers reporte d alcoho l consumptio n durin g preg nancy. These infant s wer e examine d i n th e firs t 3 days of lif e for a variet y of congenita l anomalies , including variou s nervou s syste m defect s an d a se t o f craniofacial anomalies. These facial anomalies, includ ing a hypoplastic nose , are indicativ e o f several cognitive impairment s (Swille n e t al. , 1999 ; Donna i an d Karmiloff-Smith, 2000) . In particular , they ar e typical of FAS D and , i n conjunctio n wit h materna l alcoho l consumption, ar e considered reliabl e predictor s o f the condition (Astle y and Clarren, 1997 ; Coles et al, 2000; Streissguth and O'Malley, 2000; Sood e t al, 2001). Subjects can be grouped b y maternal alcohol con sumption int o five categories, ranging from Grou p 1 , who experience d low , sporadi c exposur e throug h a range of daily doses, to Group 5 , who were exposed to over 92 g of absolute alcoho l per da y throughout ges tation, includin g severa l glasse s o f distille d spirits . Group 5 also included th e offsprin g o f those who self identified a s alcoholics. A n odd s rati o was calculate d for eac h birt h defec t t o indicat e th e effec t o f alcoho l consumption o n the ris k of developing that defect. To begin wit h the worst-cas e scenario, we shoul d expect infant s o f Grou p 5 mother s t o sho w sever e deficits. I n adults this level o f alcohol consumption i s associated wit h anoxia, the pathologica l deficienc y of oxygen, as well a s renal failure an d sever e nutritional deficiencies. As a result, it would be expected that this level of exposure would cause a variety of deficits i n a developing organism . Amon g 8 7 infant s expose d t o this amoun t o f ethanol, 6 7 had a t least on e physica l defect. Thus, only 23% were normal a t birth. Lookin g to th e nervou s system , however , 79 % o f th e infant s showed n o centra l nervou s system (CNS) defect s under standard infant neurologica l examinations . Given a level of alcohol exposur e known for its damaging effects on the physiolog y of adults, this is remarkable re silience. Becaus e th e neurologica l componen t of the suite of FASD-associated symptom s is not consistentl y detectable a t thi s age , facia l phenotype s alon e ar e used a s a proxy for FASD (Astle y and Clarren , 1997) ; fully 60 % of the infant s i n Group 5 had a normal facial phenotype. Although 40 % damage i s a tragic statistic, even i n thi s extrem e cas e o f shockingly irresponsibl e alcohol consumption , mor e tha n one-hal f o f the in fants show evidence o f being quit e normal .

Normality dominate s th e res t o f th e groups . I n Group 4 (th e second-highes t group), th e offsprin g of mothers wh o consumed betwee n 5 6 and 8 8 g ethanol per day , 95% of the infant s showed normal facia l phe notypes. According to the odds ratio, children of these mothers wer e twic e a s likel y t o develo p th e typica l FAS characteristics, but th e differenc e wa s not statistically significant . Onl y Grou p 3 , wit h lo w dail y con sumption, showe d a statistically significant relationshi p between alcoho l consumptio n an d thi s phenotype . At this consumption level , 97% of the infant s showe d the norma l phenotype . I n th e sporadi c alcoho l con sumption group s ( 1 an d 2) , no t onl y wer e FASD predictive anomalie s presen t i n onl y 3 % an d 2 % o f the infants , respectively , but the odd s ratio s suggeste d that alcohol consumptio n decrease d th e risk of anomalies, although thes e difference s wer e not statisticall y significant. An importan t consideration i n thi s study, and gen erally i n th e field , i s tha t o f socioeconomi c factors . Children of mothers o f lower socioeconomic statu s are at greate r risk , for a variety of reasons. Thes e mother s are mor e likel y to be expose d t o other ris k factors , in cluding lead (Malcoe et al., 2002; Morello-Frosch e t al., 2002), chemica l solvent s (Cordie r e t al. , 1992) , an d illegal drug s (Hans , 1999) . Thes e ris k factor s ar e als o associated with cognitive impairments. Hence, it is difficult to separate alcohol fro m othe r potential causativ e factors. Additionally , eve n give n equivalen t alcoho l and dru g exposure , childre n o f higher socioeconomi c status ar e mor e likel y to b e normal . A substantial re view o f neurobehaviora l studie s o f alcohol-expose d children (Mattson and Riley, 1998) confirms the prevalence o f low scores on cognitiv e tests in group s of low socioeconomic statu s (SES), bu t also includes startlin g examples of children who perform at normal levels and even abov e on cognitiv e tests (Streissgut h e t al, 1980 ; Fried an d Watkinson , 1988 , 1990 ; Frie d e t al., 1992) . Indeed, th e ter m low-risk group i s used synonymously with higher socioeconomic statu s in these studies. Notably, give n lo w to moderate consumptio n and a hig h level of maternal education , intelligenc e quotien t an d other cognitive scores fall well within the normal range , and occasionall y with mean scores a full standar d deviation above normal . Although thes e score s were lower than thos e achieved by children i n the sam e socioeco nomic leve l without fetal alcoho l exposure , i t i s clear that multipl e factor s i n th e developmenta l environ ment beyond alcoho l consumptio n ar e at work in chil dren who show substantial deficits .

108 NORMA L DEVELOPMENT Further, studie s o f th e effect s o f exposur e t o lo w (Forrest et al, 1991 ; Green e et al., 1991 ; Frie d e t al, 1992) or even moderat e (Man , 1980) amounts o f alcohol fail to find a cognitive deficit. Although thes e stud ies focu s o n exposur e t o lowe r amount s o f ethanol , they ar e significan t i n ligh t o f conventional wisdo m that an y an d al l alcoho l consumptio n durin g preg nancy is an unacceptable risk . Rather than being a silver bulle t capabl e o f derailin g norma l neurologica l development, alcoho l exposur e a t lo w level s ha s ef fects that are hard to demonstrate, which makes sense in term s o f the long-ter m presenc e o f alcohol i n ou r ancestry. Even at pathologically high levels it does no t have 100 % penetrance . Nicotine Nicotine i s certainl y les s commo n tha n alcoho l i n our evolutionar y history. Indeed, consumptio n o f any significant amoun t o f nicotin e i s a recen t develop ment i n huma n history . The suit e o f developmental effects cause d by smoking (the most typical way of using nicotine ) i s complex , involvin g respiratio n an d oxygen availabilit y directly, periphera l vasoactiv e ef fects, an d actio n o n centra l neurotransmitte r systems. Still, a numbe r o f it s effect s fal l withi n a well developed evolutionar y context i n development , tha t of reduced resourc e availability. Although w e canno t review this context and it s effects full y here , i t is well established tha t th e developmenta l progra m show s significant flexibility with regard to nutrient availability. Withi n limits , growth ca n b e scale d bac k o r de layed i n time s o f poo r nutrition , favorin g the brai n whose generatio n i s largely prenatal, an d caugh t u p when condition s ar e favorable (Lucas and Campbell , 2000). Tobacco exposur e restricts the availabilit y of nutrients t o th e developin g fetu s (Lamber s an d Clark , 1996). Carbo n monoxid e fro m tobacc o smok e binds with hemoglobin , th e oxygen-carryin g molecule i n blood, t o for m a stabl e carboxyhemoglobin, limiting oxygen transport in the mothe r an d thereby depriving the fetu s of oxygen a s well. Nicotin e increase s hear t rate an d cause s vasoconstriction , increasin g bloo d pressure i n the mother , reducin g uterin e bloo d flow, and therefor e decreasin g a fetus' s acces s t o al l nutri ents. Hypoxia , malnutrition , an d th e ensuin g meta bolic challenge s forc e th e fetu s t o mak e d o wit h inadequate resources. As such, it is not surprising that the mos t commo n effec t o f nicotine exposur e i s low

birth weigh t (Ellar d et al, 1996 ; Lamber s and Clark , 1996). Lo w birt h weigh t i s independently associate d with cognitiv e deficit s (Chaudhari e t al. , 2004 ; Cor bett and Drewett, 2004; Viggedal e t al, 2004), regard less o f th e causativ e agent . Indeed , a s a systemati c problem, i t is generally associated wit h growth deficits throughout th e body , includin g ostéopathi e effect s (Nelson et al., 1999) , yielding a generally underdeveloped organism . A s such , i t i s difficul t t o determin e whether an y cognitiv e deficit s ca n b e directl y attributed t o nicotine or tobacco o r if they secondarily stem from anoxi a and generall y decreased resources. The direc t effect s o f nicotine o n neurologica l de velopment ar e controversial, an d i n a way that is reminiscent o f th e comple x interaction s o f postnata l environment wit h prenata l malnutrition . A variety of studies indicat e n o cognitiv e deficit s i n infant s an d young children (Streissgut h et al., 1980 ; Forrest et al., 1991), an d ther e i s some evidenc e o f a transitiv e effect, with scores on the Bayle y Scales of Developmen t (BSID) tha t ar e lo w at 1 2 months comin g u p t o nor mal level s b y 2 4 month s (Frie d an d Watkinson , 1988). Ofte n test s tha t d o indicat e a n effec t i n chil dren of heavy smokers fai l to separate it from postna tal exposur e (Richardso n e t al. , 1995) , whic h ha s more reliabl y been associate d wit h decrease d score s on cognitiv e test s (DiFranz a e t al. , 2004) . Eve n so , this effect o n BSI D tes t scores is typically small when compared t o othe r factors , includin g number o f toys in th e household , SES , numbe r o f infan t illnesses , and eve n the identit y of the examiner . Essentially, the data indicat e tha t on e woul d b e bette r of f spending the cigarett e money o n toys , as a deficiency of toys in the child' s hom e i s a mor e reliabl e predicto r o f decreased cognitiv e score s tha n nicotin e an d tobacc o exposure. Embryological Silver Bullets In contras t to our relativel y robust developmental de fense agains t alcohol, fo r which we hav e evolved specific mechanism s dedicate d t o it s detoxificatio n and metabolism, an d agains t nicotine , whic h ma y be subsumed unde r mechanism s alread y available to buffe r resource limitations, there stands a new class of potential teratogens : designe r drugs . These drug s ar e ofte n evolutionarily novel , base d o n molecule s neve r en countered b y ou r ancestor s i n th e natura l environ ment. O r the y ca n b e simila r t o o r eve n base d o n molecules naturall y produced b y th e huma n body ,

DEVELOPMENTAL DISORDERS AND EVOLUTIONARY EXPECTATIONS 10 9 potentially bypassin g protective mechanisms, t o ma nipulate the developmenta l process directly. Though hardl y typical o f modern entrie s int o th e category, thalidomid e i s certainly the mos t infamou s example o f a designe r drug. Introduce d i n 195 7 as a sedative an d anti-nause a agent , i t wa s considered s o safe a s to b e regularl y prescribed t o comba t morning sickness an d insomni a i n pregnan t women . With drawn fro m th e marke t i n 1961 , i n les s tha n 4 years thalidomide ha d cause d a worldwid e epidemi c o f birth defects . Whil e n o accurat e censu s wa s eve r taken, it is estimated that between 10,00 0 and 20,00 0 babies wer e bor n disable d a s a resul t o f thalidomid e (Knightley e t al. , 1979 ; se e http://www.thalidomid e .ca, http://www.marchofdimes.com/professional/681 _ 1172.asp). Completely unknow n is the rat e of embryonic rejectio n i n th e fac e o f thi s drug . Thu s i t i s difficult t o estimate th e penetration o f the teratogeni c effects o f thalidomide. The method s o f action responsibl e for the terato genic effect s o f thalidomide ar e stil l unde r investigation nearl y 5 0 year s afte r it s introduction . Indeed , study has increase d drasticall y in recen t year s as ne w applications fo r th e dru g hav e bee n foun d i n treat ments fo r leprosy , lupus , rheumatoi d arthritis , an d other disorders. As a result, at least 3 0 different mech anisms have been propose d for the teratogeni c effect s (Stephens an d Fillmore , 2000) . Som e hav e been discredited, other s are contradictory, wherea s others ca n coexist handily. An affinity fo r DNA sequences rich in guanine has been established , suc h a s thos e i n th e GC-box , a hexanucleotide promote r i n th e huma n genom e (GGGCGG). Thalidomid e ca n inser t int o suc h se quences, interferin g wit h thei r functio n (Jonsson , 1972). Approximately 9% of the huma n genom e uses G-rich promoter s to the exclusio n of other commo n promoters such a s TATA and CCAA T boxe s (Bûcher , 1990). Additionally , thalidomid e ha s bee n linke d t o the fibroblas t growth factor (FGF ) famil y o f transcription factors . Wolper t (1976 , 1999 ) champion s th e idea tha t thalidomid e inhibits FGF-8 , a n initiato r of sonic hedgeho g (shh). Stephen s (1988 ) implicate d i t in th e insulin-lik e growt h facto r (IGF ) I/FGF-2 / angiogenesis pathway . A follow-u p t o thi s wor k (Stephens and Fillmore, 2000) details the interrupted pathway and it s promoter sequences, effectivel y tying together th e leadin g theorie s o n thalidomid e embry opathy to make a strong case for intersecting vulnera bilities in certain systems.

Within th e thalidomide-affecte d population , cer tain trend s i n th e abnormalitie s ar e relevan t t o ou r discussion. Som e defect s are much more commo n i n children tha t survive d to birth. Lim b outgrowt h defi ciencies ar e fa r and awa y th e mos t common , presen t in approximatel y 90% o f the group . Ea r an d ey e de fects ar e th e secon d mos t commo n pathologies , af fecting approximatel y 60 % of the grou p (Yan g e t al. , 1977; Quibell, 1981 ; Lenz , 1985) . In th e developin g limb , th e IGF-I/FGF-2/angi o genesis pathwa y i s necessar y fo r norma l outgrowth . Of 1 0 genes in the pathway , nine rel y on GC-box pro moters lacking TATA or CCAAT promote r sequences . This pathwa y i s particularl y vulnerabl e t o interca lation b y thalidomide . A simila r situatio n exist s fo r the vascula r endothelial growt h factor-integrin path way, whic h ha s bee n implicate d i n deformitie s o f the ear . Regardles s o f whic h o r ho w man y o f these mechanisms ar e eventuall y implicate d i n develop mental disorders , it i s notable tha t th e explanations , as a class , directl y implicate developmenta l contro l mechanisms.

FEATURES O F EVOLUTION AND DEVELOPMEN T THAT MAXIMIZE STABILITY

The prio r example s sho w tha t w e hav e a phenome non t o account for developmental stability of most infants i n th e fac e o f what woul d appea r t o b e sever e challenges, particularl y i n th e cas e o f "expected " challenges. Mos t o f the othe r chapter s i n this volum e give accounts o f abnormality; here we make some ar gument fo r forces fo r normality. A lis t o f the feature s o f the genom e an d develop ment tha t produc e "evolvability " and th e lis t of those that produc e stabl e solution s t o developmental chal lenges ar e quit e similar . Bot h problem s hav e th e defining featur e o f producin g a functiona l conse quence i n the even t of a deviation. In the cas e of evolution, the deviation to be assimilated comes from th e genome; i n th e developmenta l case s w e ar e dis cussing here, the deviatio n is introduced fro m the en vironment. The principa l difference i s that evolutio n lacks the tactica l nature of some developmenta l solu tions. Evolutio n ha s n o plan s fo r th e future , an d i n the genom e an d lif e historie s o f curren t organism s we se e the direc t evidenc e o f what i s evolvable. De velopment, however , mos t certainl y ha s intentio n

110 NORMA L DEVELOPMEN T and tactics , a s the y hav e bee n selecte d an d writte n into th e genome . N o developmenta l progra m ha s as its end poin t a perfect, nonreproductive infant. Trade offs an d fault s ca n b e gamble d o n t o produc e a n im perfect, reproductiv e adult. Duplicate and Vary An overal l quantitativ e feature of evolution i s that a s organisms becom e mor e complex , fro m prokaryote s to eukaryote s an d t o plant s an d animals , the y hav e more DN A an d mor e gene s (Lync h an d Conery , 2003). That an increas e in complexity should be partnered wit h an increas e i n genes a t first seems reason able o n it s face, unti l the amoun t o f conservatism in basic metaboli c pathway s is confronte d an d th e ac tual histor y of genetic chang e i s investigated (Szath mâry e t al. , 2001) . I t i s not clea r tha t increasin g th e number o f gen e product s pe r s e shoul d necessarily lead t o fancie r animal s —if Shakespear e ha d ha d a few mor e letter s o f th e alphabe t t o wor k with , fe w would argue that the plays would thereby b e deeper. The historica l record o f the genom e a s viewed in current animals does no t sho w linear increases of genetic materia l consisten t wit h th e notio n o f progressive additio n o f complexity . Rather , duplication s occur man y times over, gene duplicatio n followed by variation o f one o f the duplicate d genes , bot h a t th e level o f th e individua l gene an d a t th e leve l o f th e whole genome , a s wel l a s a t variou s intermediat e steps (Lync h an d Conery , 2000) . Below are a few examples. The trichomati c colo r vision of primates has arisen three separate times, by duplication of the "yel low" opsin followe d by one o r two minor amin o aci d changes i n on e o f th e gene s producin g a sligh t change i n wavelengt h selectivit y (Jacobs , 1998) . A whole-genome duplicatio n occur s a t th e chordate vertebrate boundary , implicate d particularl y i n th e subsequent elaboratio n of the cranium , jaw, and fore brain (Northcutt , 1996). The regulator y gene s controllin g bod y plan , which ar e conserve d acros s vertebrate s an d inverte brates, sho w evidenc e o f multipl e replication s i n evolutionary histor y (Gerhart an d Kirschner , 1997) . The us e o f this strateg y is clear—one gen e ca n con tinue it s essential roles , while a second ca n b e fre e t o vary, assum e ne w functions , or b e produce d i n different contexts . Th e abilit y t o duplicat e an d var y "semantic" aspects of the genome—tha t is, aspects of the genom e that relat e t o meaningful component s of

the organism s —is a featur e tha t allow s evolvability , and has been employed t o advantage in "genetic algorithms" i n compute r scienc e (Lipso n an d Pollack , 2000; Nolfi an d Floreano , 2002; Baum, 2004). Duplicate-and-vary b y it s natur e make s develop ment responsiv e t o local geneti c accident . On e frus trating aspect o f early genetic wor k (to geneticists ) i s that whe n i t becam e possibl e t o "knock out " singl e genes (i n th e cas e o f nonlethal omissions) , the usua l effect wa s often nil , largely because of the massiv e re dundancy o f gen e copies . I t shoul d b e emphasize d that extr a o r slightl y altere d gen e copie s di d no t evolve i n anticipatio n o f futur e accident . Onc e i n place, however , creature s wit h th e duplication s hav e had a developmenta l edge . Man y othe r version s of the duplicate-and-var y strateg y ca n b e see n i n addi tion t o gene duplicatio n an d variation . For example , the segmentai structure o f the vertebrat e an d inverte brate brai n an d bod y pla n i s anothe r versio n o f duplicate-and-vary, at a higher level of complexity. Large neura l structures , suc h a s th e corte x an d cerebellum, ma y als o b e case s o f duplicate-and-var y (or multiply-and-vary) , regardles s of whether the uni t duplicated i s a cel l assembly , a "cortica l column, " a region , o r a cortica l area . Cortica l area s hav e th e analogous strategi c feature s o f conserve d structur e throughout, suc h tha t cortical areas may be function ally substituted fo r each other , a s shown fro m myria d examples o f plasticity either earl y or lat e i n life , bu t particular cortica l areas , suc h a s visual or somatosensory cortex, appear to have functionally relevan t local features "wire d in " (reviewe d in Kingsbur y an d Fin lay, 2001 ; Pallas , 2001) . Finally , i t i s possibl e tha t some o f th e peculia r feature s o f ou r bilatera l bod y (and brain) plan migh t be placed i n this context. Th e exact calori c requirement s o f each bod y par t appea r to b e ver y tightly regulated . Fo r example , th e brai n size an d gu t lengt h (tw o metabolically expensiv e or gans) co-vary negatively with each othe r very precisely in primates with the resul t of keeping basal metaboli c rate unchange d (Aiell o and Wheeler , 1995) . Ye t the amount o f each organ we possess seems to be far from the bar e minimu m required , or eve n th e amoun t re quired fo r everyda y function. Jared Diamon d calcu lates from variou s successful surgeries in humans tha t we ca n ge t b y wit h on e kidney , hal f o f on e lung , a third of a liver, half the cerebra l cortex, and s o on (Di amond, 1994) . I t ha s no w bee n acknowledge d tha t creative use of brain lateralization, in the duplicate-and vary version, can b e found throughout th e invertebrat e

DEVELOPMENTAL DISORDERS AND EVOLUTIONAR Y EXPECTATIONS 11 1 and vertebrat e lineage , i n frui t fly , fish , birds , an d many arthropods , and , of course, ourselve s (Vallorti gara an d Rogers , 2005) . Humans d o quit e peculiarl y well with early, full-hemispheric deletions . Convergent Redundancy Frank duplication an d variatio n of identifiable devel opmental mechanism s see m t o hav e occurre d nu merous times , a s ca n b e see n i n th e man y isoform s and othe r variant s o f cel l recognitio n molecules , signaling molecules , an d trophi c molecules . A n ab solute hallmar k o f development , however , is redun dancy o f a differen t type , wher e multipl e distinc t mechanisms ar e al l directe d towar d th e sam e out come. Th e retinotecta l system , i n whic h th e twodimensional layou t o f cells i n th e retin a i s faithfull y transferred t o the two-dimensiona l surface o f the tecturn for the organizatio n of eye and bod y movements, has served as a "model system " for the investigatio n of axon guidance, synaptogenesis, and topographi c ma p formation sinc e Roge r Sperr y first investigated i t half a centur y ag o (Sperry , 1963) . Thi s mode l syste m provides th e cleares t instanc e o f mechanisti c re dundancy. Eac h propose d mechanis m ha s ha d it s researcher-champion, unti l th e realizatio n dawne d that evolution had take n no vow of mechanistic parsimony. There ar e a numbe r o f logica l possibilitie s o f mapping an arra y A-B-C-D ont o a secon d arra y A*-B*-C*-D*, eac h o f whic h alon e ca n solv e th e developmental puzzle . A could recogniz e A*; B recognize B*, and s o on, which is called "chemoaffinity, " Sperry's first hypothesis (Sperry, 1963) . Alternative to attraction, A could b e repelle d b y D*, and les s by C* (Bonhoeffer and Huf , 1992) . Or, A might stic k to B better than to C, preserving intrinsic order in the fiber array, and the n hav e some rul e about ho w to position itself relativ e to A*-D* (Fraser , 1980) . Or , tempora l order i n developmen t migh t b e exploited , havin g A and A* mature first, then B and B* , and s o on (Reese, 1996). Or , th e element s i n bot h array s could b e produced i n excess, connected at random, an d error s removed b y some secon d rule , a s cell deat h i s a majo r component of brain development (Oppenheim , 1991). Or, th e ma p coul d self-organiz e by a Hebbia n rule , using th e featur e that th e contras t leve l o f neighboring elements i n the visua l world is more highl y correlated tha n distan t element s (Schmidt , 1985 ; Wong, 1999). Th e answe r is, of course, tha t ever y single on e

of thes e logicall y separate mechanism s cooperate s t o produce orderl y maps. During development, i t is possible to disable one or more of these mechanism s alto gether, yet still preserve topographic map order, muc h as in gen e knockou t experiments , as residual mecha nisms rescue the map. These two separate forms of redundancy, duplicateand-vary and convergen t redundancy , contribut e t o a massively parallel , semimodula r architectur e o f de velopmental mechanism s livin g beneath th e surfac e of a n organis m maturin g ove r time . Startin g fro m scratch t o buil d a comple x organism , on e migh t b e predisposed t o a sequential , assembly-lin e mecha nism, producing one part at a time, and connectin g it up, bu t th e relativel y greater vulnerabilit y of such a system to loss of any component is obvious. I t is interesting t o observe the present-da y parallel evolutio n of manufacturing systems from th e strictl y serial Ford assembly lin e t o current , multiple-component "just-intime" manufacturing systems. The fac t tha t evolution must tinke r wit h existin g systems rather tha n desig n from scratc h i s ofte n give n a s a n argumen t fo r th e unique, redundan t natur e o f biological construction , but i t i s possible evolutio n ha s com e upo n solution s for efficien t biologica l constructio n tha t w e ma y no t yet be able to recognize. Catastrophe Curve s It will not d o to make a large fraction of an organism : it is a waste of energy for the parent s and certainl y n o good fo r th e offspring . Restated , partia l solutions ar e no goo d i n development—i f uncorrectabl e defect s are detected , n o mor e energ y shoul d b e spent o n th e embryo to compound th e metaboli c loss . For this reason, bailin g ou t i s perhap s th e mos t commo n re sponse t o developmenta l defect . Fo r man y species , particularly "reselected " animals who ar e specialize d for rapi d reproductio n i n challengin g environments , embryos ma y b e resorbe d unti l lat e developmenta l stages i n respons e t o ver y small change s i n environ mental stress , or the fetuse s may be aborte d an d cannibalized, a s an y researche r attemptin g t o stud y th e etiology of developmental disorder s in rodents knows. Even fo r primates , wh o spen d muc h energ y o n th e growth and car e of one o r two infants, a large proportion o f conception s ar e aborte d an d resorbe d i n th e first trimester; th e commo n figur e give n i n medica l advisories about pregnancy is "up to 50%," although i t is har d t o locat e th e empirica l basi s o f thi s claim .

112 NORMA L DEVELOPMEN T Even i f the numbe r i s muc h lower , th e recognitio n of developmenta l disorde r and terminatio n o f devel opment, particularl y i n earl y stages , ar e a s muc h an importan t face t o f developmen t a s constructiv e mechanisms. If a commitment t o the offsprin g i s unavoidable — for example , i f it is the onl y chance a t reproduction , or a life-threatenin g metaboli c commitmen t ha s al ready bee n made—the n ever y subsequent choic e t o preserve the offsprin g shoul d be mad e a s is physically possible. Particularl y in shortag e situations , clearl y strategic choice s ar e made—fo r example , t o preserve the brai n o f the infant , which i s generated onl y early in development, ove r and against the musculoskeletal system, whos e growt h ca n b e delayed , o r to favo r th e infant's nutritiona l need s ove r th e calori c require ments o f the mothe r (Marty n e t al., 1996 ; Luca s an d Campbell, 2000) . One "difficulty " i n researc h o n de velopmental disorders , which ma y not be entirely obvious to those workin g in well-establishe d paradigms, is to be abl e t o fin d jus t those regime s that li e on th e cusp betwee n th e plateau s o f embryoni c rejectio n and death , an d ful l normality . Analogous strategi c similaritie s can be seen in various developmenta l mechanism s i n th e nervou s sys tem, fo r example , a certai n numbe r o f neurons , a certain convergenc e rati o between neurons , or a feature o f physiology. Central mechanism s ca n gir d th e system agains t environmental stressors , but a t a critical value, this defense will suddenl y and catastrophi cally collapse. For example, if the numbe r o f neurons in th e corte x i s progressively depleted b y the mitoti c inhibitor methylazoxymethanol during earl y develop ment, the thalamus projectin g to that cortex will show no los s o f neuron s unti l mor e tha n 70%-80 % o f its target i s gone, an d the n collaps e itsel f (Wo o e t al. , 1996). Simila r event s ma y b e a t pla y wit h th e anti mitogen ethanol. Prenatal exposur e to ethanol cause s a <3S% reductio n i n th e numbe r o f cortical neuron s (Miller an d Potempa , 1990 ) an d n o los s o f thalamic neurons (Moone y an d Miller , 2000) . Unfortunately, no studie s hav e generate d feta l damag e sufficien t to caus e catastrophi c los s o f cortical neurons . I f th e convergence rati o i n th e retinotecta l syste m i s challenged, synapti c organizatio n reconform s t o simulta neously produc e norma l single-neuro n respons e properties and norma l gross topography, and a t a critical point , bot h aspect s o f organizatio n fai l togethe r (Xiong and Finlay , 1996) . The belie f that developin g organisms should pro -

duce a linea r respons e i n degre e o f malfunctio n t o linear increase s i n environmenta l stressor s is written very deepl y int o ou r experimenta l paradigms , an d a t best is a hypothesis to be demonstrated. Th e natur e of malfunction-functions wil l depend o n th e evolution ary expectation s an d consequen t strategi c develop mental choice s o f each separat e species. Self-Initiate, Self-Terminat e The natur e o f contro l an d monitorin g regime s t o move animal s through developmenta l stage s shoul d be evaluate d routinel y for developmental robustness . In the following sections, w e examine som e aspect s of early neura l development , particularl y neurogenesis , neuron typ e specification , critica l periods , an d th e features thes e period s appea r t o hav e t o stabiliz e de velopmental outcomes . Th e botto m lin e i s that mos t developmental mechanism s contai n numerou s (an d redundant) logica l checkpoint s t o determin e i f th e correct complemen t o f cell s i s bein g produced . Feedback-free, " I Love Lucy" assembly lines are virtually nonexistent . Developmental mechanism s ar e initiate d whe n multiple condition s fo r initiatio n ar e satisfied , an d are terminate d whe n th e proces s initiate d i s com pleted, not wit h respec t t o arbitrar y clocks. In evolu tion of mammals, in which the time take n to produc e a brai n scale s b y a facto r wel l ove r 10 , mechanism s like these simply allow graceful scalin g (Clancy et al., 2001). As brains enlarge durin g evolution, investigators begi n t o observ e "waitin g periods" i n th e devel opment o f certai n properties , a s a se t o f cell s o r axon terminal s g o quiescen t unti l appropriat e envi ronmental conjunction s appea r to be reached. In the case of developmental challenges , th e same abilit y to pause o r dela y ma y b e employe d t o amas s enoug h material o r wai t for th e righ t condition s t o occu r t o proceed.

ROBUSTNESS AND VULNERABILITIE S IN FUNDAMENTA L DEVELOPMENTAL PROCESSES

Having introduce d severa l feature s of developmen t through whic h evolutio n an d developmen t produc e stability, w e no w examin e i n detai l instantiation s of these principle s a t variou s developmenta l stage s i n the vertebrate nervous system.

DEVELOPMENTAL DISORDERS AND EVOLUTIONAR Y EXPECTATIONS 11 3 Neurogenesis and the Process of Cell Type Specificatio n Neurogenesis comprise s th e productio n o f neuron s and gli a tha t wil l resid e i n a structure ; thi s i s analogous to organogenesis, which is the productio n of cells that wil l constitut e a n organ . Thus, neurogenesi s in cludes th e proliferatio n o f neural precursors, the fat e decision, th e migration , differentiation , an d some times deat h o f th e cells . Mos t neurona l precurso r cells are generated i n the proliferativ e zone s that line the inne r surface o f the neura l tube (se e Chapters 2 , 11, an d 12) . In thes e zones , precurso r cells underg o mitosis, wit h "symmetric " divisions , producing more precursors. Eventually, some precursor s begin "asym metric" divisions , with on e daughte r cel l exitin g th e cell cycl e and the proliferativ e zone , and the n begin ning th e transformatio n into particula r types of neu rons o r gli a (Cavines s e t al. , 1995 ; Ohnum a an d Harris, 2003). The origina l size of the progenito r poo l combined wit h th e duratio n o f neurogenesi s o f an y given par t o f th e brai n predict s it s siz e (Finla y an d Darlington, 1995 ; Finlay et al, 2001). Not every precursor cel l i n the nervou s system differentiates. In some regions of the brain , stem cell s remain undifferentiate d throughou t th e lif e o f th e animal, an d recen t wor k suggests that these cell s can be presse d int o servic e to effec t repair s for damage d neural circuit s even i n th e adul t brain (e.g. , AlvarezBuylla and Garcia-Verdugo, 2002; Gage, 2002 ; PicardRiera e t al. , 2004) . That is , neuronal ste m cell s may provide a substrat e fo r compensator y plasticit y i n some systems of the brain s of adults. We concentrat e her e o n mechanism s tha t con trol th e assignmen t o f cel l identity , wit h particular attention pai d t o ho w th e proces s may regulat e itself if disturbance s occur . Researcher s firs t frame d th e question o f cel l specificatio n in term s o f wher e cel l specification occurs . Conceivably , thi s decision i s (a) intrinsic, suc h tha t specifi c precurso r population s give ris e t o particula r subpopulation s o f cell s only ; (b) determine d b y a cloc k producin g a certai n cel l type after a required number o f precursor cell subdivisions or with regard to some externall y specified stage; or (c ) extrinsic , defined b y the externa l milieu of th e precursor cells , i n eithe r th e proliferativ e zone s o r their eventua l destination. The answe r that ma y vary from on e brai n regio n t o anothe r i s likely th e usua l developmental answer: all of the above . We give a few specific examples , an d conside r th e implication s of

hybrid mechanism s fo r producing norma l structure s in response t o challenge. In vivo and i n vitro experiments demonstrat e tha t the orde r of neurogenesis and cel l type are correlated . For example , i n th e vertebrat e nervou s system, mos t glia ar e produce d afte r th e completio n o f neurona l generation (Ohnuma an d Harris , 2003). In the retina, the si x types of cells are produced i n order , although in al l cases , th e productio n o f cel l type s overlap s (Policy e t al , 1989) . Evidenc e suggest s tha t a com bination of clock-like initiation of specification o f particular cel l type s an d feedbac k processe s tha t asses s how man y cells o f a clas s have been produced . On e of these, p27 KiP1, a cel l cycl e inhibitor , gradually increases in progenitor s until a critical level is reached , whereupon cell s designate d a s oligodendrocyte s exi t the cel l cycl e and differentiat e (Freeman , 2000 ; Dyer and Cepko , 2001 ; Ohnuma and Harris , 2003). Thus, the gradua l accrua l o f p27KlP1 i s a negativ e feedbac k mechanism tha t tells precursor cell s when t o start differentiating into glia as opposed to neurons . Injury i s capabl e o f resettin g th e neurogeneti c mechanism. I n frog s an d fishes , fo r example, Mulle r glia ca n re-ente r th e cel l cycl e i n respons e t o retina l injury, an d ca n produc e progen y tha t differentiat e into neurons (Reh and Levine , 1998) . I n mature glia, p2yKipi j s expresse d a t hig h levels . Following retinal injury, however , p27 Klpl i s down-regulate d i n cells that re-ente r th e cel l cycle , allowin g for the genera tion o f ne w neurons . Regulatio n o f specificatio n of cell classe s ha s als o been show n t o occu r a t a mor e detailed level—fo r example , i f the retin a i s deplete d of a particular class of amacrine cells while retinogenesis is ongoing, the subsequen t production o f that cell type is up-regulated (Reh , 1987) . Negative feedback control of the rat e of histogenesis provide s flexibility and robustnes s in th e develop ing organis m relativ e t o mor e rigi d developmenta l schemes tha t migh t plausibl y hav e evolved . Fo r in stance, suppos e tha t onl y gli a ar e produce d o n a set day following the onset of neuronogenesis, or that glia are produce d onl y afte r a certai n numbe r o f cell cy cles wit h n o feedbac k regulation . I n eithe r o f these cases, if anything interfere s with cel l productio n at a certain tim e point , a n essentia l cel l grou p migh t never be generated. O n th e othe r hand, if progression through cel l classe s is regulated onl y by feedback, in the cas e o f lo w resourc e availability , neurogenesis could stal l a t a n earl y point an d neve r produc e late generated cel l groups . Intrinsi c clocks , overlappin g

114 NORMA L DEVELOPMENT distributions of cell production , an d feedbac k regula tion of the amoun t of eac h cel l typ e in concer t together virtually guarantee that some members of each cell clas s are generated , an d thei r norma l ratio s defended. Cell Migratio n The migratio n of developing neuron s to their adult positions in the brain is a critical step in the developmen t of the nervou s system. Severa l distinc t developmenta l mechanisms, suc h a s radia l an d tangentia l migratio n and cellular adhesion molecules, are thought to govern this proces s (Rosenwei g et al , 1999 ; Corbi n e t al , 2001). Thi s i s a proces s tha t ca n g o wrong , and "ec topia," cel l group s lyin g elsewher e tha n thei r norma l terminal site, is one o f the principa l morphological deviations associate d wit h retardatio n (Evrar d e t al. , 1989a, 1989b) . Eve n i f migration abnormalitie s resul t in misplaced cells, however, all is not lost. One o f the mos t striking demonstrations o f developmental robustnes s comes fro m on e mode l syste m of perturbed cell migration, in reeler mice. Reelin is a protein secreted b y several neuronal populations during development, an d i s vital fo r allowing neurons t o complete migratio n an d adop t thei r ultimat e posi tions i n a numbe r o f laminar structures in th e CN S (Rakic an d Caviness , 1995 ; Ric e an d Curran , 2001) . Mice lackin g reeli n (reeler mice , s o named fo r thei r staggering gait , whic h result s primaril y from a cere bellar anomaly ) sho w abnorma l pattern s o f cerebra l cortical lamination. The arrangemen t of the si x layers of the cerebra l corte x is inverted, so that th e earliest generated cell s form th e most superficial layer s of the cortex an d late-generate d neuron s ten d t o be distributed i n dee p cortex . Despit e thi s perturbation, affer ent projections to the visual, somatosensory, olfactory , and moto r cortice s fin d thei r correc t targe t cells . I n addition, th e overal l organizatio n o f majo r system s and the physiologica l response s o f individual neuron s in reeler mic e ar e comparable t o those in the norma l brain. Reeler mice have other anatomical abnormalities relative to wild-type mice. These include change s in synaptic density, distribution, and topology that are present in a number o f brain structures including the hippocampus, piriform cortex , and cerebellum . Nev ertheless, althoug h morpholog y i s grossl y abnormal, function i s spared. This result suggests cautio n i n any assumption tha t disturbe d morpholog y ha s negativ e functional consequences .

Reeler mic e provid e a n additiona l demonstratio n of the robustnes s of the "duplicate-and-vary " strategy. Only mic e homozygou s fo r the mutan t reelin allele show the neuronal abnormalities listed above. Studies of mice heterozygous for reelin have neuronal and be havioral phenotype s virtuall y indistinguishabl e fro m those o f wild-type mice (Salinger et al., 2003). Maturation o f Cell Types durin g Later Development The maturatio n o f neurona l morpholog y provides an example o f convergen t redundancy . I n vitr o experi ments sho w tha t granul e an d Purkinj e cell s isolate d from thei r norma l connections gro w in a typical manner (Sei l e t al, 1974) . This finding implies that signals intrinsic to cells can regulat e aspects of neuronal differ entiation. The neura l environment of developing cells, however, also influences cell morpholog y an d connec tivity. For instance, som e spinal cells are directed t o become moto r neuron s unde r th e influenc e o f cell s ventral to the notochor d (Roelin k et al., 1994) . I f a second lengt h o f notochord i s inserte d abov e th e spina l cord, cells begin to differentiate a s motor neurons on either side of the notochord. Cell-cel l interactions of this sort, in whic h cells influenc e the fate s o f adjacent precursor cells, are referred t o as induction, and have been documented extensivel y in the vertebrate brain (Rosenweig et al., 1999). The abilit y of extrinsic cell-cell interactions to regulate cel l fat e is not restricted t o precurso r cells. Cortical transplan t studie s consistentl y sho w tha t sensory and motor cortex cells grafted ont o distant cortical region s ca n assum e morphological , connectional , and functional properties appropriate to that region. For instance, fetal visua l corte x of rats transplanted into ra t neonatal somatosensor y cortex develops the barrel-like whisker representation s characteristi c o f primar y so matosensory cortex (Schlaggar and O'Leary, 1991), an d the expressio n o f the limbi c system-associate d mem brane protei n tha t mark s specific functional region s of the cerebra l corte x can b e regulate d b y environmental stimuli (Levitt et al, 1997) . Patterned sensor y activity can alte r cell fate. Cross modal rewirin g o f retina l axon s int o ferre t auditor y thalamus provides visually patterned activit y to the au ditory cortex (Pallas et al., 1999). Primary auditory cortex provide d wit h earl y visual input resemble s visual cortex topographicall y (Ro e e t al. , 1990) , physiologi cally (Roe et al., 1992) , and perceptuall y (vo n Melchner e t al. , 2000) . Neuron s i n thi s altere d auditor y

DEVELOPMENTAL DISORDER S AND EVOLUTIONARY EXPECTATIONS 11 5 cortex no t onl y assum e th e functiona l propertie s an d arrangement o f neurons i n th e visua l corte x but als o are capabl e o f mediatin g visuall y guide d behavio r (von Melchner et al, 2000). Hence, intrinsi c factors suc h a s gen e expressio n and extrinsi c factor s suc h a s induction , neuron neuron interactions , and patterne d activit y are all capable of regulating cell fate at various (and sometimes overlapping) point s i n development . No t al l o f thes e mechanisms ar e necessaril y invoke d o r eve n neces sary during the production o f any given neuron . Postnatal Development and Critical Period s Critical periods ar e tim e window s i n postnata l de velopment durin g whic h neuron s an d circuitr y ar e particularly receptive to acquiring certain kinds of information critica l fo r norma l developmen t (Hensch , 2004). Critica l period s hav e bee n extensivel y doc umented fo r sensor y systems , moto r systems , an d multimodal function s suc h a s imprinting , birdson g learning, soun d localization , an d huma n languag e learning. Ho w are critica l periods useful fo r ensuring normal development , when i t seems that the unlucky absence o f a particular experience at a particular time might permanently derai l normal development ? Th e answer i s that th e critical-perio d onset , duration , and termination ar e not regulated simpl y by age, but rathe r by experience . I f appropriate neura l activatio n i s no t provided a t all, then developing circuit s often remai n in a waiting state until such inpu t i s available. For instance, the segregatio n o f ocular dominance column s depends o n visua l experience . I f all visual experienc e is denied, the representatio n o f the tw o eyes does no t begin it s segregation an d th e specia l neurotransmit ters an d receptor s tha t ar e responsibl e fo r this struc tural chang e ar e hel d i n thei r initia l state (Kirkwoo d et al., 1995) . Within certai n constraints , whe n experi ence i s reinstated, anatomical , pharmacological , an d physiological event s then progress as they would hav e independent o f the ag e of the animals . A similar phenomenon ha s als o been observe d i n bird s tha t lear n their songs from tutors—fo r th e unfortunat e nestlings born to o lat e i n th e seaso n t o hear an y o f the sprin g songs that establish territory, the critica l period is held over unti l nex t spring , whe n singin g begin s agai n (Doupe an d Kuhl , 1999) . Critica l period s hav e vul nerabilities. I f activation i s only partial, i t may initiat e the progressio n o f the critica l period , stabilizin g a n

abnormal state , a s has been seen i n the productio n o f ocular dominanc e columns : i f both eye s ar e closed , the critica l period i s delayed. O n th e othe r hand , i f just on e ey e i s closed , th e critica l perio d proceeds , dedicating al l resource s t o th e ope n ey e (Kat z an d Shatz, 1996) .

ETHANOL AND THE DEVELOPING

NERVOUS SYSTEM: EVOLUTIONARY CONSIDERATIONS

Here we look at a few of the cases in which the effect s of ethanol o n developin g system s have been investigate d and consider them in the context of self-regulation. The effects o f ethanol o n th e nervou s system—at any stage of development—are know n t o be numerou s an d varied (se e Chapter s 11-18) . Fo r instance , ethano l ca n both inhibi t an d stimulat e neuro n proliferation , de pending o n th e physica l locatio n o f the cells , an d th e concentration an d eve n timin g (da y or night) o f expo sure (Luo and Miller , 1998; see Chapter 11) . Given tha t th e effect s o f ethanol o n th e develop ing nervous system are numerous, complex , an d con tingent upo n man y factors , ca n w e b e sur e tha t al l these effect s ar e malignant? To be sure , some ar e un ambiguously deleterious . Ethano l ca n induc e neu ronal deat h i n vitro and i n vivo, oxidative stress, an d excitotoxicity (Chapter s 1 5 an d 16) ; interfer e wit h glucose transpor t an d uptake ; an d reduc e th e expres sion an d adhesiv e propertie s o f cel l adhesio n mole cules (Chapter 13) . Other effects o f ethanol, however, may no t necessaril y be maladaptive . Fo r instance , i n addition t o causin g neurona l deat h i n developin g cerebellum (L i e t al. , 2002 ) an d corte x (Jacob s an d Miller, 2001) , ethano l ca n slo w th e productio n o f developing cerebella r (L i e t al. , 2002 ) an d cortica l (Miller an d Nowakowski , 1991 ; Miller , 2003 ) neu rons, an d i t can increas e th e tim e durin g which post mitotic cortica l neuron s remai n i n th e proliferativ e zone befor e commencin g thei r migratio n whil e als o decreasing th e rat e o f neurona l migratio n (Miller , 1993). Give n tha t developmen t i s dynamically regulated an d sensitiv e t o environmenta l cues , i t i s no t implausible that these delay s might be an adaptiv e response t o a hostile developmenta l environmen t (hig h concentrations o f ethanol ) i n whic h cel l productio n and migratio n ar e retarded unti l a more favorabl e environment becomes available. This interpretation gen erates testabl e predictions . I f cell-cycle an d migratio n

116 NORMA L DEVELOPMENT delays ar e a respons e t o a hostil e environment , then we should expec t t o se e a restoratio n o f these activi ties t o norma l (o r even above-normal ) rate s once th e normal developmenta l environmen t i s restored . I n practice, we migh t expec t t o se e resumptio n o f neu ron proliferation and migration after transient exposur e to ethanol (afte r the restoratio n o f normal cellular environment). Certainly , researc h ha s demonstrate d tha t the nervous system can and does recover from ethanol related developmenta l insult s (Ander s and Persaud , 1980; Riley, 1990 ; Popova, 1997 ) but the precise mech anisms mediatin g recover y are poorl y characterized . We suggest that some of the observe d ethanol-induced changes i n the developing nervous system may not be disorders, bu t remnant s o f processes tha t hav e medi ated recovery.

Finally, w e need to establish bette r causa l links in developmental model s tha t g o fro m fundamenta l physiology to morphology t o behavior, i n order to discriminate pathologica l chang e fro m compensator y change and from simpl e deviation. Evolutionar y mod els ca n provid e scaffoldin g fo r suc h understandin g that pathology-based researc h ca n never provide . Abbreviations BSID Bayle y Scales of Developmen t CNS centra l nervous system FASD feta l alcohol spectru m disorde r FGF fibroblas t growt h factor IGF insulin-lik e growth facto r References

SUMMARY AND CONCLUSION S Development ca n go wrong. One nee d onl y consider the appearanc e o f a poorl y placed o r underfertilize d garden plan t compare d t o it s well-nourished siblings to counteract th e ros y glow that thi s chapter , wit h its relentless underscorin g o f mechanism s designe d fo r developmental stability , may hav e produced . Never theless, ther e ar e man y reason s fo r takin g a vie w of pathology that begins with evolution, rather than with human developmen t i n isolation. First, althoug h du e cautio n shoul d b e employe d in advisin g pregnan t mothers , communicatin g th e wild improbabilit y o f gros s developmenta l disorder s from tin y infraction s in alcoho l consumptio n durin g pregnancy would reliev e unnecessar y anxiety . Second, animal "models " should b e examine d fo r their appropriateness t o understanding huma n pathol ogy. Vertebrate nervous systems are quite conservativ e in structure; curren t wor k comparing th e genome s of different specie s suggest s that thei r defensiv e packaging is quite different, dependin g o n such factors as "r" and "k " selection an d th e dietar y and pathoge n expo sure i n th e evolutionar y history o f th e species . Th e kind of modeling of development w e do must chang e and is , in fact, i n th e proces s of change—from th e assumption o f linear dose-response curve s to pathogens and teratogens , t o multicomponen t chaoti c system s in whic h "normality " i s the stronges t attractor , o r i n which severa l attractors, each representin g a developmentally stable strategy, may exist.

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8

Prenatal Alcohol Exposure and Human Development Claire D. Coles

Although a relationshi p betwee n materna l drinkin g and reproductiv e problem s ha d bee n suspecte d lon g ago (Warner and Rosett , 1975 ; Abel, 1984) , it was the description o f fetal alcoho l syndrom e (FAS ) in 197 3 (Jones and Smith , 1973 ; Jones et al, 1973 ) that initi ated comprehensiv e studie s o f the effect s o f prenatal alcohol exposur e o n huma n development . Researc h over th e las t 3 0 years ha s confirme d the origina l observations that affected childre n sho w growth retardation; birt h defects , particularly a characteristi c facia l dysmorphia; and behaviora l deficits indicative of central nervous system (CNS) damag e (Fig . 8.1). Animal models as well as epidemiological an d clinica l studies in human s hav e provide d evidence o f the effect s o n both physica l an d behaviora l characteristic s o f ex posed offspring , bu t th e CN S effect s an d thei r per sonal and social consequences hav e received the mos t study in human samples . There have been a number of comprehensive reviews of the result s of prenatal alcohol exposur e on development , particularl y in earl y childhood (e.g. , Coles , 1992 , Stratto n e t al. , 1996 ;

Streissguth, 1997 ; Mattso n an d Riley , 1998 ; U S De partment o f Health an d Huma n Services , 2000). Recently, publication s i n thi s are a hav e increase d s o much tha t it is now impossible to review the entir e literature in a single chapter. For that reason, this chap ter refer s briefl y t o earlie r findings and deal s i n mor e detail with recent studies. A decad e ago , th e followin g area s wer e amon g those tha t seeme d t o hav e th e mos t researc h poten tial: threshold effects , neuroimaging , long-term development i n affecte d individuals , specific versus global effects, an d wha t i s now calle d "translation " amon g the differen t discipline s studying this problem (Coles , 1992). These areas have remained active. In addition, several other issues have come to the fore. With better diagnosis and identificatio n of affected individual s by professionals an d increasin g demand fo r services and resources by families, th e nee d fo r improving education an d remediatio n o f affecte d individual s has be come evident . Also , a s clinica l an d longitudina l research cohort s hav e matured , differen t area s o f

123

124 ETHANOL-AFFECTE D DEVELOPMENT

FIGURE 8- 1 Rang e o f effects o f prenatal alcoho l exposur e o n th e centra l ner vous system.

behavior hav e becom e salient . Fo r instance , socia l and adaptiv e function s hav e bee n identifie d a s im paired i n individual s with FA S (Kell y e t al. , 2001) , and patient s fro m clinica l sample s ar e reporte d t o have a hig h frequenc y o f lega l an d menta l healt h problems (Streissgut h et al, 1996 ; Fam y et al, 1998 , Fast an d Conroy , 2004) . Thes e issue s ar e discusse d below, i n additio n t o recen t researc h o n neurodevel opmental outcomes in affected individuals .

METHODOLOGICAL CONSIDERATIONS

Despite 3 0 year s o f study , ther e remai n significan t disagreements i n the literatur e an d amon g th e pub lic and man y professionals about the effect s o f prenatal alcoho l exposure . Some of these discrepancie s can be accounte d fo r b y difference s i n methodologie s used to study this phenomenon. Before undertaking a review o f the researc h literatur e i n thi s area, i t is important t o understan d ho w methodolog y affect s out comes an d t o acknowledg e tha t thes e issue s are no t specific t o this field. It i s obviousl y impossible , ethicall y an d practi cally, to carry out tru e experimenta l studie s of the effects o f prenatal alcohol exposur e o n huma n growth , behavior, and cognition . For those aspects o f development tha t canno t b e modele d i n anima l studies , research mus t b e don e i n eithe r clinica l sample s o f already affected individual s o r i n cohort s of offsprin g

of wome n wh o drin k durin g pregnancy . Eithe r ap proach ha s rea l value an d rea l difficulties . A s experimental contro l o f the "independent " variabl e i s no t possible, suc h studie s mus t b e correlationa l an d de scriptive. In addition, it is usually not possibl e to con trol completely the confounder s o r effect modifier s in such situations . When conductin g studie s with sam ples o f individual s draw n fro m clinica l settings , re searchers mus t tak e int o accoun t systemati c biase s and confounders . Childre n diagnose d wit h FA S or other effect s o f prenatal alcoho l exposur e come to th e attention o f professionals because o f problems i n be havior or development. Thus, it is inappropriate to use such sample s t o evaluate the relationshi p betwee n al cohol exposure and these particular outcomes. Nevertheless, stud y o f suc h childre n ma y b e valuabl e fo r other researc h purposes . Also, many clinicall y identified alcohol-affecte d childre n hav e negativ e earl y caregiving histories resulting from materna l substanc e abuse, out-of-hom e placement , an d othe r caregiving failures, an d researc h wit h suc h group s mus t includ e similar contras t group s to avoi d confusing the effect s of pre- and postnata l environment s on th e outcome s of interest. In addition to being very expensive and tim e con suming, exposur e samples also have methodological limitations. Wome n wh o drin k durin g pregnanc y usually also smoke tobacco and ma y us e other drugs (Day e t al , 1993 ) (se e Chapte r 19) , introducin g other confounder s into the research . They may have

PRENATAL ALCOHO L EXPOSUR E AND HUMA N DEVELOPMEN T 12 5

a geneti c predispositio n to alcoholism an d associate d mental healt h problem s (Zucke r et al. , 1995) , have a family histor y characterized b y substance us e an d it s social correlates, and b e selected fro m a low socioeconomic status (SES) environment , a s such wome n ar e more likely to be availabl e for recruitment int o exposure studies. Another limitatio n o f the use of exposure samples i s tha t mos t sampl e population s ar e com posed o f wome n drinkin g i n th e lo w t o moderat e range (e.g. , Da y et al, 1993 ; Jacobso n e t al, 1998) . Although i t is valuable to have informatio n abou t th e effects o f lower amounts of ethanol exposure. The rel ative absence o f exposure studies of infants of heavier drinkers i s du e i n som e par t t o th e difficult y i n re cruiting such women . For these reasons , when tryin g to understan d th e effect o f alcohol exposur e on offsprin g development , it i s important to tak e int o consideratio n th e charac teristics o f the researc h sampl e an d th e resultan t im plications. An y singl e stud y ma y no t b e sufficien t to understan d thes e teratogeni c effects , wherea s th e body of literature as a whole ca n provid e considerable insight.

THRESHOLDS AN D DOSE-RESPONSE EFFECTS OF PRENATAL ALCOHOL EXPOSUR E

There i s a great deal o f interest i n th e questio n o f th e "dose" o f alcoho l necessar y t o caus e negativ e out comes. Fo r public health reasons, i t would be valuable to kno w i f there i s a "safe " level of prenatal exposure. This is a complicated question . Some researcher s posit that the construc t of threshold i s not usefu l becaus e i t oversimplifies th e rea l relationship between th e terato gen an d th e multipl e outcome s tha t ar e measure d (Sampson et al., 2000). Sampson an d colleague s argue in their review that there are no meaningful difference s in behavior , standard scores, academic outcomes , an d behavior betwee n thos e individual s labele d a s having FAS and thos e characterize d as having alcohol-related neurodevelopment disorder s (ARND) . The y believ e that behavioral outcome doe s not vary with variation in physical teratogeni c effects . I n addition , thes e re searchers present evidenc e from thei r large prospectiv e exposure stud y suggestin g tha t th e relationshi p be tween prenatal exposur e and neurodevelopmental outcomes i s monotonie, o r without meaningfu l threshol d

effects "whe n dos e an d behaviora l effect s ar e quanti fied appropriately" (p. 421). Other investigator s have identifie d threshol d ef fects in their samples. Jacobson and colleague s (1998) found tha t aspects of infant cognition sho w threshol d effects. A t 2 6 month s i n thei r cohort , psychomoto r functioning an d aspect s o f fin e moto r functionin g are affecte d onl y i n th e offsprin g o f heavie r drinker s (Kaplin-Estrin e t al, 1999) , althoug h non e o f these children exhibi t physica l dysmorphia . I n anothe r sample, Da y and colleague s (1991 , 1999 , 2002 ) show that growt h i s affecte d i n a dose—respons e fashion . Growth i s more affecte d i n individual s whose moth ers drank most heavily (>1 drink/day); they average 14 pounds les s tha n controls . Simila r relationship s ar e evident fo r cognitio n an d achievement . Whe n thi s cohort was 6 years old, a threshold o f exposure to on e drink dail y i s foun d fo r academi c achievemen t i n reading and spelling , whereas effects o n mathematic s skills are linear (Goldschmidt et al., 1996) . In addition, as discussed below, the morphologica l outcomes characteristi c of prenatal alcoho l exposur e appear t o b e affecte d i n a dose-respons e manne r (Lynch e t al. , 2004) . All of these report s sugges t that heavier drinkin g i s associate d wit h greate r physica l and neurodevelopmenta l effects . Th e exten t to which results can b e describe d a s monotonie or reflective of "thresholds" i n alcoho l us e ma y depend o n th e typ e of sample s recruite d an d th e amoun t o f exposure i n the sampl e a s wel l a s b y th e method s use d i n th e study and i n the data analysis.

PHYSICAL EFFECTS O F PRENATA L ALCOHOL EXPOSUR E

Facial Dysmorphi a The facia l dysmorphi a associate d wit h heav y expo sure is one o f the thre e diagnostic features use d to define FAS . These features include midface hypoplasia (anteverted nares) , shor t palpebra i fissures, and flattened o r indistinc t philtrtim , usuall y associated wit h a thi n o r flattene d uppe r vermillio n border . Othe r minor anomalie s als o ar e commonl y foun d i n af fected children , includin g epicanthal folds, lo w nasal bridge, ea r anomalie s (lo w set and rotated) , an d mi crognathia. The craniofacia l anomalie s ar e believe d to be the resul t of disturbances o f cell migration dur -

126 ETHANOL-AFFECTE D DEVELOPMENT ing organogenesi s (U S Departmen t o f Healt h an d Human Services , 2000; see Chapter 17) . In practice, assessment of these anomalies is complicated b y variance associate d wit h ag e an d ethnicity . Whe n af fected individual s are followe d ove r childhoo d int o early adulthood, som e o f the individua l features tha t are salien t durin g infanc y an d earl y childhoo d be come harde r t o identify , particularl y afte r pubert y (Streissguth, 1997) . Several differen t "systems " of diagnosis are use d i n clinical diagnosi s an d researc h o n FAS . Astle y an d Clarren (2001 ) hav e propose d a syste m tha t focuse s on "sentine l features, " whic h the y hav e identifie d through analysi s of a large sample o f children wh o applied for diagnostic services. In this large clinical sample, severa l facia l feature s hav e bee n foun d t o b e associated with the diagnosi s at each stag e of development. Thes e feature s ar e (1 ) a n absen t o r indistinc t philthrum, (2 ) a thinne d uppe r vermillion , an d (3) shortene d palpebra i fissures . I n thei r four-digi t code metho d for diagnosis, these facia l feature s consti tute on e dimensio n necessar y fo r diagnosi s (Astley , 2004). Othe r dimension s ar e growt h (les s than thir d percentile), materna l alcoho l use , an d evidenc e o f CNS involvement . The Collaborativ e Initiativ e o n Feta l Alcoho l Spectrum Disorders , sponsore d b y the Nationa l Insti tute o n Alcoholis m an d Alcoho l Abuse , ha s devel oped a Dysmorphia Cor e Physica l Exam that i s used in internationa l studie s to diagnosis participants of all ages and ethnicitie s (Ma y et al, 2000 ; Hoym e e t al, 2005). Thi s method , develope d b y Jone s an d col leagues (1973) , i s based o n th e origina l description s of FAS and i s a modification o f the criteri a offere d b y the Institut e of Medicine (Stratto n et al, 1996) . A s in other systems , th e chil d i s measure d an d examine d for physica l an d facia l anomalies , a s wel l a s height , weight, head circumference , and heart and neurolog ical problems . The n a determinatio n i s mad e a s t o whether th e chil d ca n b e rate d a s (1 ) havin g FAS , (2) no t having FAS, or (3) deferred. The classification "deferred" indicate s tha t som e characteristic s ar e present bu t tha t th e diagnosi s cannot b e mad e with out mor e informatio n (e.g., neurodevelopmenta l tes t results). Thi s methodolog y ha s bee n use d i n Russi a and Sout h Afric a a s well as the Unite d State s (Hoym e et al, 2005). Coles an d colleague s (Fernhoff e t al., 1980 ; Black ston e t al , 2004 ; Lync h e t al. , 2004 ) hav e use d a

slightly differen t methodology . I n a longitudinall y followed exposur e sample report , a dose-response pattern between alcoho l exposur e and physical characteristics can be identified both in the neonatal perio d an d later i n development . Th e sam e dysmorphi c features, however, ar e no t alway s salien t ove r development . Rather, it is the total "dysmorphia score" that is reliable from infanc y through middle adolescence. Mor e longi tudinal researc h is needed t o determine th e continuit y of physica l characteristics , particularl y facia l anom alies, to provide guidelines for making the diagnosi s in older childre n an d adults . This kind of research i s important als o because ther e may be ethnic differences in facial characteristics tha t can affect diagnosis. Finally, it is necessar y t o us e a longitudina l approac h becaus e use o f clinical sample s i n a cohor t desig n exaggerates the salienc e o f feature s tha t ar e expecte d t o b e par t of th e criteri a an d ignore s individual s whose features are not consistent with these expectations . Effect of Alcohol Exposur e on Growt h Growth retardatio n i s useful fo r the identificatio n of FAS during infanc y an d th e preschoo l period , bu t i t is les s consistentl y eviden t i n olde r childre n an d youth (Streissguth , 1997 ; Cole s et al, 2002). It is difficult t o evaluat e thi s facto r i n clinica l sample s because th e argumen t i s necessaril y circular . Thi s criterion i s use d i n makin g a clinica l diagnosis , hence suc h individual s ar e necessaril y growt h re tarded. Expose d individual s who presen t fo r diagno sis do no t receiv e i t i f they ar e no t growt h retarded . Long-term follow-u p o f childre n identifie d earl y i n life i s necessary to confir m persistent growt h deficits . For thi s reason , exposur e studie s provid e a bette r method fo r evaluatio n o f thi s outcome . Generally , children expose d t o alcohol appea r to be statistically smaller tha n control s o r tha n nationa l norm s (Da y et al, 1999 , 2002) . Ofte n mean s ar e withi n norma l ranges, however , s o althoug h thi s characteristi c i s statistically "real, " i t ma y o r ma y no t b e o f clinical significance, an d i t is not necessaril y helpful in iden tification o f specifi c individual s if prenata l alcoho l exposure i s no t known . I t i s no t ye t establishe d whether lowe r birt h weight , height , an d hea d cir cumference associate d wit h prenata l alcoho l expo sure (and , perhaps, associate d polydru g use) ar e ris k factors fo r other condition s occurring later in life .

PRENATAL ALCOHOL EXPOSUR E AND HUMA N DEVELOPMEN T 12 7 NEURODEVELOPMENTAL EFFECT S

OF PRENATAL ALCOHOL EXPOSURE

Effects on Brain Structure : Information fro m Neuroimaging studies The followin g sectio n briefl y review s neuroimagin g studies o f the effect s o f prenatal alcoho l exposur e o n brain structure. Brain morphology could b e described as a "physica l effect " o f prenata l exposure , a s well ; a fulle r discussio n o f thi s subjec t i s provide d i n Chapter 9 . Both huma n studie s of behavior and anima l mod els leave no doub t tha t alcoho l i s a teratogen that has toxic effects o n the brain . Until recently, it was impossible t o evaluat e directl y it s effec t o n huma n brai n structure an d functioning , but i n the las t decade, im aging studie s o f alcohol-affecte d patient s hav e bee n conducted. Severa l researc h group s usin g clinica l samples o f patients with feta l alcoho l spectrum disor ders (FASD ) hav e reporte d effect s o n th e brai n i n older childre n an d adults . Mos t o f thes e publishe d studies hav e use d structura l magneti c resonanc e im aging (sMRI ) an d hav e focuse d o n morphology . Sev eral studie s (Bookstei n e t al , 2001 , 2002 ; Sowel l et al , 200lb ) hav e correlate d structura l outcome s with neurobehaviora l data. Although functiona l magnetic resonanc e imagin g (fMRI ) i s being develope d as a researc h too l i n thi s area , n o studie s hav e been published a s of this writing. In sMR I studies , microcephal y ha s been th e mos t consistent finding , wit h FAS patients having a general reduction i n brai n volum e compare d t o tha t o f con trast groups (Riley et al, 1995 ; Archibald et al, 2001; Sowell e t al , 2001a , 2001b ; Bhatar a e t al , 2002) . More specifically , relativ e reductions i n whit e matte r have been noted th e corpus callosum and the parietal lobe (Riikone n e t al, 1999 ; Archibal d e t al , 2001 ; Sowell et al, 200lb ) in patients with FAS. The corpu s callosum ha s bee n a focu s o f interes t becaus e i t i s a midline structure an d i s readily identifiable. Agenesis and hypoplasi a o f thi s structur e hav e bee n reporte d repeatedly, particularly in extremel y dysmorphic individuals (Rile y e t al , 1995 ; Johnso n e t al , 1996 ; Swayze e t al , 1997 ; Riikone n e t al , 1999 ; Sowel l et al , 2001b ; Bhatar a e t al , 2002) . Sowel l an d col leagues (20 0 lb) reporte d reduction s an d displace -

ments i n th e anterio r regio n o f the corpu s callosu m as wel l a s i n th e splenium , whic h correlate d wit h deficits i n verba l learnin g an d memor y i n th e sam e patients. Bookstei n an d colleague s (2001 , 2002 ) re ported tha t ther e i s greater variabilit y in th e shap e o f the corpu s callosu m amon g alcohol-affecte d patient s and tha t thicke r o r thinne r structure s ar e relate d t o specific pattern s o f neurodevelopmenta l deficits : a thicker corpu s callosu m i s associated wit h deficit s i n executive functioning , whereas a thinner on e accom panies motor deficits . The cerebellu m ca n be affecte d b y prenatal expo sure t o ethanol . I t i s smalle r i n childre n wit h FA S (Riikonen e t al, 1999 ; Autti-Ram o e t al, 2002) , particularly i n th e anterio r vermis (lobule s I-V ) (Sowel l et al, 1996) . These findings are supported b y animal studies (e.g. , Goodlet t an d Lundahl , 1996) . Man y studies report other specifi c deficits i n CNS morphol ogy, although non e a s consistently as those o f the cor pus callosum an d the cerebellum . Studies o n th e structura l effec t o f prenata l expo sure on the brai n indicate that significant morphological change s ca n b e found . I t i s no t clear , however , that a specific area of deficit i s present i n all cases. This finding suggest s tha t th e teratogeni c effec t i s mor e general. Severa l candidate s fo r further researc h hav e emerged fro m thes e data , bu t eve n th e corpu s callo sum, whic h ha s been implicate d frequently , does no t show specific macrostructural patterns relate d to alcohol exposur e across all studies. For instance, althoug h callosal agenesis has been reporte d (Rile y et al, 1995 ; Johnson et al, 1996 ; Bhatar a et al, 2002) , other stud ies fin d thinnin g o f these area s o r posterio r displace ment (Sowell et al, 200lb) , and yet other studie s find macrostructural anomalie s onl y i n a minority of individuals with FAS (Autti-Ramo et al, 2002) . This situation i s further complicate d b y a stud y i n nonhuma n primates showin g that repeated , episodi c exposur e t o ethanol durin g pregnancy can result in an increas e i n the siz e o f the corpu s callosum , especiall y th e ante rior segmen t (Mille r e t al , 1999) . Thes e outcome s suggest that, particularl y in individual s whose alcoho l effects ar e subtle, gross anatomical studie s may not b e observable. A complementar y imagin g techniqu e t o sMR I i s diffusion tenso r imagin g (DTI) , whic h allow s th e measurement o f whit e matte r integrity . I n FA S pa tients, sMR I studie s frequentl y repor t effect s o n in tegrity o f whit e matte r tract s (Johnso n e t al , 1996 ;

128 ETHANOL-AFFECTE D DEVELOPMEN T Swayze e t al , 1997 ; Clar k e t al , 2000 ; Archibal d et al, 2001; Sowell et al, 2001b; Bhatara et al, 2002). Through DTI , M a an d colleague s (2005 ) foun d tha t fractional anistrop y in th e gen u an d spleniu m o f th e corpus callosum o f alcohol-affected youn g adults was significantly lowe r than tha t of age-matched controls . This suggest s a n associatio n o f teratogenic exposur e and white matter integrity. Although i n a n earl y stag e o f exploration , neu roimaging procedure s hol d promis e fo r understand ing th e teratogeni c effect s o f prenata l exposure . When fMR I studie s ar e publishe d an d functiona l outcomes ar e correlate d wit h behaviora l outcomes , knowledge o f structure-function-behavio r relation ships in these patient s will be greatly advanced. Motor and Neuromotor Effects Alcohol effect s o n psychomoto r functio n hav e bee n observed regularl y i n infant s an d toddler s (Cole s et al, 1985 ; Coles, 1993 ; Jacobson et al, 1993 ; see discussion o f Jacobson e t al., 1988 , above) . Problems i n motor functionin g in older children ar e less well doc umented bu t ar e reporte d (Aronso n e t al. , 1985 ; Kyllerman e t al, 1985 ; Janzen e t al., 1995) . Descrip tive studie s o f childre n wit h FA S consistentl y not e deficits i n motor skills and coordinatio n (Mattso n an d Riley, 1998) . Among quasi-experimenta l researc h de signs with children diagnose d with FAS are deficit s i n visual-motor integratio n (Conry , 1990 ; Cole s e t al. , 1991; Mattso n e t al, 1993 ) an d fine-moto r strengt h and coordinatio n (Bar r e t al. , 1990 ; Conry , 1990) . Kyllerman and colleague s (1985 ) found tha t childre n (mean ag e 70 months) bor n t o women wh o abuse alcohol ha d lowe r moto r developmen t score s an d di d more poorl y on test s o f motor coordinatio n tha n di d those i n a comparison grou p who were not prenatally exposed. Children wit h FAS also show deficits i n balance (Marcus , 1987 ; Roebuc k e t al, 1998a , 1998b) , increased clumsines s (Steinhause n e t al. , 1982) , ab normal gai t (Marcus, 1987 ; Conn-Blower , 1991 ) an d tremors (Aronso n e t al, 1985 ; Marcus , 1987) . Roe buck an d colleague s (Roebuc k e t al, 1998a , 1998b ) suggest tha t damag e t o th e cerebellu m fro m heav y prenatal alcohol exposur e may interfere with efficien t use o f visua l an d somatosensor y syste m cues . Usin g electromyography, thes e researcher s conclude d tha t this defici t wa s th e resul t o f damag e t o th e CN S rather tha n t o the periphera l nervou s system control ling muscles or vestibular system.

Peripheral systems are affected b y prenatal exposure to alcohol . Avari a an d colleague s (2003 ) examine d nerve conductio n i n median , ulnar , peroneal , an d tibial nerve s i n newborn s wh o wer e re-examine d a s toddlers at 1 2 and 1 4 months. Ulna r and tibia l moto r nerve velocitie s ar e slowe r an d dista l amplitude s are lowe r i n ethanol-expose d children . The observe d changes differe d fro m thos e i n alcoholi c periphera l neuropathy o f adult s an d reflecte d bot h myeli n in volvement (reduced velocity) and axona l damage (de creased amplitude) . In contrast, assessment of sensory nerve conductio n doe s no t identif y significan t differ ences betwee n alcohol-expose d infant s an d thos e i n the contro l group . N o clinica l correlates are noted i n either group of infants. Although researc h i n thi s are a i s limited , thes e studies and persisten t clinical report s suggest that further exploratio n o f effect s o n moto r an d sensory / motor developmen t i s warranted. Deficits in these areas hav e a significan t effec t o n earl y life, potentiall y affecting physical , cognitive, an d socia l development . In addition , earl y motor deficit s ar e ofte n correlate d with specifi c learnin g an d academi c problem s a t school ag e and later . Global and Specific Effects o n Neurocognitive Functioning Global Cognitive Effects Global intellectua l deficit s ar e th e primar y neurodevelopmental outcom e associate d wit h prenata l alco hol exposure . Thi s i s consisten t wit h th e result s o f other earl y neurologica l insult s that , contrar y t o th e adult pattern , ten d t o b e mor e globa l tha n specific . Deficits i n genera l intellectua l functio n (tha t is , scores on intelligence o r other abilit y tests) have been observed i n sampl e population s compose d o f chil dren diagnose d wit h FA S and i n thos e draw n fro m longitudinal studies of heavy gestational alcohol expo sure. Children meetin g the diagnosti c criteria for FAS show a relatively broad rang e of intellectual outcome s with sampl e estimate s rangin g fro m sever e intellec tual deficienc y to averag e levels o f functioning (e.g. , IQs rang e fro m 2 0 t o 120 ; Mattso n an d Riley , 1998 ; and fro m 1 6 to 105 ; Streissgut h e t al, 1978) . Mea n performance o n standardized measures of intellectual function typicall y hav e bee n i n th e borderlin e t o mildly deficien t rang e (IQ s rang e fro m 6 5 t o 75 ) (Stratton e t al. , 1996) . I n larg e retrospectiv e an d

PRENATAL ALCOHOL EXPOSURE AND HUMA N DEVELOPMEN T 12 9 prospective studies , hal f o f th e childre n wit h FA S meet criteri a for mental retardation , an I Q < 70 on a standardized measur e o f intelligenc e (Abel , 1998) . There has also been discussio n of the effec t o f prenatal exposur e to low or moderate level s of alcohol an d most researc h studie s hav e reporte d tha t i n suc h co horts there is a decrement o f several IQ points (4 to 6) attributable t o th e exposur e (e.g. , Streissgut h e t al. , 1993). Specific Cognitive Effects In additio n t o moto r problems , function s tha t hav e been examined an d appea r t o be affecte d b y prenatal alcohol exposur e includ e specifi c deficit s i n visual spatial processing, arousal and regulatio n of attention, w;orking memory , plannin g an d organizationa l skills, mathematical achievement , an d behaviora l regula tion. In reading the literature , it is important to examine th e exten t t o whic h globa l deficit s hav e bee n taken into consideration i n the interpretatio n o f "specific" deficit s i n outcome . Individual s showing mental retardatio n o r borderlin e intellectua l functionin g (IQ<85) canno t b e compare d t o norma l control s (IQ=100) o n specifi c neurocognitiv e task s withou t accounting fo r global deficits . Those studies that hav e controlled fo r overall IQ o r that have ascertained appropriate contras t group s ar e mor e convincin g tha n those that have not. Visual-Spatial Processing . Evidenc e fo r a specifi c deficit i n visual-spatial processin g (Spoh r et al., 1993 ; Ueckerer an d Nadel , 1996 ; Cole s e t al , 2002 ) ha s been found . Mattso n an d Rile y (1998 ) foun d tha t mean verba l I Q wa s 61.0 0 ±12.82, wherea s mea n performance I Q wa s 55.3 3 ±13.45. Thi s patter n i s similar t o tha t see n i n individual s wit h nonverba l learning disabilities (Rourke, 1995) . Deficit s in visual memory (Carmichael-Olso n e t al. , 1998 ; Kaeming k and Paquette, 1999 ; Platzma n et al., 2001), visual perceptual skill s (Steinhause n e t al. , 1982 ; Aronso n et al, 1985 ; Morse et al, 1992; Aronson and Hagberg, 1998), visual-moto r integratio n (Janze n e t al. , 1995 ; Mattson e t al, 1998 ; Kaeming k and Paquette , 1999) , and spatia l memory (Ueckerer and Nadel , 1998 ) have been reported . A specific deficit i n visua l processing, which is independent of the global intellectual deficit, is suggeste d by findings fro m a cohor t of alcohol exposed individual s seen durin g adolescenc e (Cole s et al, 2002). In this study, visual and auditory sustained

attention wer e compare d and adolescent s wit h dys morphic (i.e. , FAS ) and nondysmorphi c FAS D wer e less efficien t a t processin g visua l tha n auditor y in formation. Sensitivit y t o th e visua l stimul i wa s sig nificantly lowe r i n alcohol-affecte d youth, a findin g suggesting tha t th e neurocognitiv e impac t o f alcohol exposure i s more significan t fo r task s that involv e visual stimuli than fo r auditory stimuli. There ar e inconsistencie s i n reporte d outcome s on th e effect s o f ethanol o n sensor y processing . No t all studies have found a specific or consisten t impairment i n visua l o r spatia l processin g o f information , and som e repor t that auditory information processing is mor e impaire d (e.g. , Janze n e t al. , 1995 ; Kern s et al, 1997; Conno r et al, 1999) . Certainly, there may be a real difference a s a function o f exposure in differ ent group s o f individuals ; however, methodologica l differences amon g studie s an d i n analysi s strategies may also contribute t o the variability . On th e basi s of a revie w of retrospective and prospectiv e longitudinal studies and individua l case studies, Mattson an d Riley (1998) sugges t that relativ e deficits in visua l an d ver bal learning are equivalent. They compared studie s of children wit h FASD and studie s of children with low to moderate alcohol exposur e for whom n o diagnosis could b e made . Thes e group s o f children ma y have different pattern s o f deficits . Studie s of diagnosed in dividuals also vary in the exten t to which they attemp t to equat e th e discriminativ e powe r o f th e task s b y modality (auditor y or visual ) an d whethe r the y con trol fo r overal l ability in th e analyses . Differences in SES may also contribute t o discrepancies i n outcome across studies. Children wit h FAS from lo w SES environments hav e verba l IQ s tha t ar e lowe r tha n thei r performance IQ s (Conry , 1990 ; Kern s e t al, 1997) . Conry (1990) , wh o include d a lo w SE S contras t group, conclude d afte r examinin g discrepancy score s that "alcohol-involvemen t appeare d t o hav e greate r effects o n visual/spatia l problem solvin g than o n ver bal effects " (p . 654). Arousal an d Attention. Th e regulatio n of attention in prenatall y expose d childre n ha s bee n a matte r o f persistent interes t (Kopera-Fry e et al., 1997) . S o com monly i s attentio n defici t hyperactivit y disorde r (ADHD) diagnose d i n childre n wit h FA S and associ ated disorder s that som e hav e suggeste d tha t ADHD is a hallmar k o f a behaviora l phenotyp e associate d with exposure (O'Malley and Nanson, 2002). Leaving aside th e issu e of the existenc e o f such a phenotype ,

130 ETHANOL-AFFECTE D DEVELOPMENT the regulatio n o f attentio n i s o f grea t importance , as master y of arousa l and attentio n i s a prerequisit e for learnin g an d socia l developmen t (MacKintosh , 1975) and thus has the potential t o mediate a number of long-ter m neurodevelopmenta l outcomes . Man y children wit h FAS have difficulties wit h regulation of arousal; thi s proble m ma y b e associate d wit h thei r ADHD diagnosis (Streissguth e t al., 1986 , 1995 ; Nanson an d Hiscock , 1990 ; Kopera-Fry e e t al , 1997 ; Oesterheld an d Wilson , 1997 ) o r other alteration s i n attention (Streissgut h et al, 1984; Cole s et al, 1997) . Investigations int o th e effec t o f prenata l alcoho l exposure o n attentiona l regulatio n tha t hav e use d prospective longitudina l design s hav e produce d in consistent results . Streissgut h an d colleague s (1984 ) found tha t prenata l alcoho l exposur e resulte d i n more errors and slowe r reaction times on a task of vigilance an d sustaine d attention i n preschoo l childre n with moderat e alcoho l exposure . I n the sam e cohort , observations o f children' s behavio r b y traine d ob servers an d rating s b y parent s sugges t tha t expose d children ar e les s attentive, less compliant, an d mor e fidgety (Landesman-Dwyer and Ragozin, 1981) . These results hav e no t alway s bee n confirme d b y othe r prospective studie s using exposure samples (i.e. , Boyd et al, 1991; Cole s étal, 1997) . Brown and colleagues (1991) als o sho w an associatio n betwee n gestationa l alcohol exposur e and regulation of attention, but they attribute their results to the effect s o f the postnatal en vironment. A four-factor, multidimensiona l mode l o f attentional regulatio n (Mirsk y et al, 1991 ) wa s used to asses s 7-year-old s prenatall y expose d t o alcoho l (Coles et al., 1997). These children diffe r significantl y from a contras t grou p o f childre n diagnose d wit h ADHD i n thei r abilit y t o attend . Alcohol-affecte d children hav e mor e difficult y encodin g informatio n and in flexibility in problem solving, but they are similar t o control s i n thei r abilitie s to focu s an d sustai n attention. Th e childre n diagnose d wit h ADHD have a contrasting pattern. Hypothesizing that earl y difference i n arousa l levels note d i n alcohol-expose d infant s (Cole s e t al. , 1985) might be associated wit h later problems i n self regulation and attention , Kable and Cole s (2004 ) further examine d th e relationshi p o f alcoho l exposur e and informatio n processing i n a differen t sampl e o f high-risk, low-birth-weigh t infant s wh o ha d bee n ex posed t o moderate amount s o f alcohol. Behaviorally , at 6 and 1 2 months, these infant s sho w small but significant developmenta l difference s i n menta l an d

psychomotor development a s well as in behavior reg ulation (Coles et al., 2000). When they were 6 months old, th e infants ' attentio n wa s evaluated b y us e o f a habituation paradigm with cardiac orienting response as outcome . Infant s with mor e alcoho l exposur e re spond mor e slowl y than "low-risk " controls to presen tation o f bot h visua l an d auditor y stimul i an d ar e rated a s significantly higher i n arousal level. This difference i n information-processin g speed i s consistent with earlie r work (Jacobson e t al., 1994 ) showin g tha t fixation duration (lookin g time) i s longer i n alcohol exposed infants . The implicatio n i s that alcohol i s related to the spee d o f information processing. The relationshi p betwee n attentio n an d arousa l regulation i s suggestive. The abilit y to focu s an d sus tain attentio n depend s o n th e abilit y t o moderat e arousal to allow information to be processed efficientl y (Ruff an d Rothbart , 1996) . I t ma y b e tha t alcohol exposed infant s canno t modulat e arousa l effectivel y and ar e therefor e less efficient i n processin g environmental information . Findings amon g adolescents an d adults o f inefficiencie s i n informatio n processin g i n the visual modality (e.g., Coles et al., 2002) or in both auditory an d visua l modalities (Conno r e t al. , 1999 ) may be influence d by arousal leve l as well. This suggestion is supported by findings from a study of the ef fects o f maternal substanc e abus e on attention . Sues s and colleague s (1994 ) foun d tha t prenata l exposur e to alcohol , but no t opiates , influence d attention an d psychophysiology (cardiac functioning and vagai tone) at schoo l age . Ho w thi s compromise i s related t o de velopmentally later and more comple x cognitiv e functions and attentio n an d behaviora l regulation has no t yet been investigated . Executive Function Skills . Executiv e functio n i s a construct referre d t o as, "perhaps the mos t appealing , yet least understood, aspect of cognition and metacog nition" (Borkowsk i and Burke , 1996 , p . 235) . Executive function involve s higher-order cognitive processes that range from attentiona l regulation , workin g mem ory skills , planning an d organizationa l thinkin g an d problem solvin g (Lyon, 1996; Morris, 1996) . From an information-processing prospective , attention , mem ory, an d executiv e functio n ma y be interrelate d pro cesses that affec t cognitiv e functioning and th e ability to carr y out goal-oriente d behavior s (Borkowsk i an d Burke, 1996). Both clinica l description s o f childre n wit h FA S and longitudinal exposure studies implicate ethano l i n

PRENATAL ALCOHOL EXPOSUR E AND HUMA N DEVELOPMEN T 13 1 compromising executiv e function . Investigator s have approached thi s construc t fro m severa l theoretica l positions. Som e investigator s aggregate measure s t o obtain a n estimat e o f executive functio n (e.g. , Samp son et al, 1997 ; Conno r et al, 2000), whereas other s evaluate specifi c area s thought t o represent aspects of executive functio n (e.g. , Kern s et al, 1997 ; Mattso n et al, 1999 ; Schonfel d e t al, 2001) . It i s difficult t o compare studie s becaus e th e construc t pe r s e i s no t well defined. Therefore, th e followin g review will examine the effec t o f prenatal alcoho l exposur e o n various component s tha t ar e usuall y understoo d t o b e part of executive functioning . Memory an d Metamemory . Memor y i s a broa d field o f study, an d onl y som e aspect s o f memory ar e considered to be part of executive function. Childre n with FAS exhibit deficits in memory skills, but the im pairments are not always stable and their extent seems to be influence d b y modality of the tas k used an d b y the specifi c cognitive demands o f tasks (Mattson an d Roebuck, 2002) . Fo r instance , a n interactio n o f age and tas k characteristic s ma y determin e whethe r effects ar e shown . Usin g th e McCarth y Scale s o f Children's Abilitie s (McCarthy , 1972) , Janze n an d colleagues (1995 ) d o not fin d difference s i n memor y function betwee n youn g childre n diagnose d wit h FAS and a contrast group selecte d fro m day-car e settings. This finding contrasts with a study by Coles and colleagues (2004) , wh o use d a differen t test . The y find that childre n i n the sam e ag e range d o have spe cific memor y deficits. The effec t o f presentatio n modalit y (i.e. , visual auditory) on memory has been investigated. Learnin g and recal l o n task s presented visuall y o r i n narrative form w ;ere compared (Platzma n et al., 2001). Adolescents wit h FA S have poorer recal l o f visual element s than control s an d age mates i n special educatio n pro grams, wherea s long-ter m recal l o f auditoril y pre sented item s i s not impaired . Short-ter m memor y for auditorily presented item s (i.e., random string s of digits; Wechsler Intelligenc e Scal e for Children-Revised, Digit Span) is also similar to that of contrast groups. In a differen t sampl e o f clinically referre d children, age s 3 to 9 years old, wit h a diagnosi s o f FASD, a similar auditory tas k an d a visual/spatia l analo g wer e contrasted. Relativ e deficit s for spatia l spa n ar e evident , whereas performance on th e digi t span tas k is consistent wit h overal l abilit y leve l (Cole s e t al , 2004) . These findings suggest specific deficits in memory with

language-related o r auditorily presented material s relatively spared . Thi s patter n o f performanc e i s als o noted b y Mattson an d Roebuck (2002) among schoolaged an d adolescen t childre n diagnose d wit h FA S or having ha d heav y prenata l exposure . I n thi s group , memory performanc e is compared t o tha t o f normal controls o n fiv e standardize d measure s o f learnin g and memory : a verbal learning task (California Verbal Learning Test-Children' s Versio n [CVLT-C] ; Deli s et al., 1987) , the Biber Figure Learning Test, and three subtests fro m th e Wid e Rang e Assessmen t o f Memory and Learnin g (WRAML ; Sheslo w an d Adams , 1990) . These subtest s ar e Verba l Learning , Visua l Learning , and Soun d Symbol . The constructio n o f the CVLT- C is important for understanding the result s of these studies. This tes t i s a verbal learnin g tas k wit h orall y pre sented lists of words in several categories (i.e., clothing, fruits, an d toys) . Due t o the lis t structure, a n effectiv e memory strategy (grouping by semantic category ) can be used to aid learning and recall . Through th e us e of tests of verbal and visual memory, i t has been show n tha t alcohol-affected childre n learn fewe r item s (bot h word s an d figures ) overal l than control s (Mattso n an d Roebuck , 2002) . After a standard dela y period , however , th e percentag e re called (o f the materia l tha t ha d bee n learned ) i s no t different betwee n th e group s fo r verbal material . O n the nonverba l material , th e alcohol-affecte d grou p show7 relativ e deficit s in delaye d recall . Thus , verba l memory i s les s affecte d tha n nonverba l memory . Data fro m thes e studie s hav e bee n reanalyze d t o examine th e effec t o f stimulus differences i n tw o of th e verbal memory tasks (Roebuck-Spencer an d Mattson , 2004). Accordingly, children wit h FASD demonstrat e semantic clusterin g on th e CVLT-C , indicatin g tha t they are able to take advantag e o f the lis t characteris tics t o ai d recall . The y perfor m bette r o n thi s tas k than o n th e WRAM L verba l memor y task , whic h does no t offe r th e sam e opportunit y t o us e th e "im plicit" memor y strateg y inheren t i n the stimulu s ma terial. O n th e basi s o f thes e findings , th e author s argue tha t previousl y observe d difference s i n verba l and nonverba l memory ma y resul t from us e o f more effective memor y strategie s o n verba l learnin g tasks . Other wor k (i.e. , Platzma n e t al, 2001 ; Cole s e t al. , 2004, se e discussion above) does no t suppor t this explanation becaus e visuall y and auditoriall y presente d materials are equivalent in these studies. Various studie s repor t tha t childre n with FAS D have memory deficits on verbal learning tasks (Mattson

132 ETHANOL-AFFECTE D DEVELOPMENT et al, 1996; Kern s étal, 1997 ; Schonfel d étal , 2001; Willford e t al, 2004). A longitudinal follow-u p study of 14-year-old s shows that verbal-auditory rathe r tha n visuospatial memor y i s affected wit h learning ; short and long-ter m memor y deficit s on a paire d associat e task ar e detected (Willfor d e t al, 2004) . I n a sampl e of adolescent s an d youn g adult s classifie d a s having an "averag e IQ " o r "belo w averag e IQ, " Kern s an d colleagues (1997 ) show that individual s with FAS perform belo w expectation o n the CVLT-C. The greates t difference i s not o n initia l learnin g trials, bu t o n th e fifth trial. These results suggest tha t affected individu als are not able to use semantic memor y strategies appropriately t o maintai n a comparabl e learnin g slop e over repeate d trials . That is , the individual s with FAS do not use the categorie s inheren t i n the lists to organize an d facilitat e their recal l an d thei r response s do not sho w the clusterin g o f semantically relate d item s that i s usually found i f effective memor y strategies are used. Thi s findin g contrast s t o a lack of difference i n primacy effec t an d seria l clusterin g scores , whic h re lies less on strategy use . Mattson an d Rile y (1999) contras t intentiona l an d incidental memor y b y measurin g recal l o f informa tion (a ) whe n childre n wit h FA S ar e awar e o f th e memory tas k involved an d (b ) when the y lear n infor mation usin g effective memor y strategies provide d by the investigator s withou t bein g awar e o f the nee d t o learn the informatio n in expectation o f recall. The in vestigators conclude tha t incidenta l memor y i s not affected b y prenatal alcohol exposure . Coles an d colleague s (1997 ) gav e a n alcohol exposed an d a contras t grou p o f 7-year-olds a paire d associate tas k requirin g tha t th e childre n continu e with th e list-learnin g process unti l the y achiev e mas tery. At both immediat e an d delaye d recall , alcohol affected childre n ar e no t differen t fro m control s with this methodology . Th e numbe r o f repetition s re quired t o achiev e lis t mastery , however , i s signifi cantly higher for alcohol-affected children . Cumulatively, the abov e findings suggest that on e aspect o f th e memor y defici t observe d i n alcohol affected individual s involves employing strategies. That is, they may not be exhibiting effective "metarnemory " (an importan t aspec t o f executive functioning) in tha t they ma y hav e mor e difficult y i n selectin g an d em ploying effective learnin g strategies and the y ma y no t be aware of the leve l of effort require d t o achieve mas tery o n a particula r task. It ma y als o be tha t becaus e alcohol-affected individual s process informatio n mor e

slowly, learning time i s spent less efficiently an d mor e trials ar e required. Whethe r thes e deficit s are uniqu e to individual s with FAS D o r are characteristi c of slow learners i n general i s not clear and shoul d b e the sub ject of further study . An aspect o f memory tha t i s considered a factor i n executive processin g i s active workin g memory. Thi s process involve s menta l manipulatio n o f element s stored briefl y i n short-ter m memor y and ha s been re ferred t o as a "mental scratc h pad " (Ashcraft , 1995) . It requires tha t informatio n be store d an d processe d i n meaningful "units " at a given momen t in time. Man y of the cognitiv e task s comprising neurodevelopnien tal tes t batterie s includ e us e o f active workin g mem ory because higher-order processing or problem solving requires tha t severa l unit s o f informatio n b e main tained simultaneously . In alcohol-affecte d individuals , problems i n activ e working memor y hav e bee n identifie d earl y i n in fancy. Usin g a visua l expectanc y paradig m (Hait h et al. , 1988 ; Jacobson e t al. , 1992 ) as a measur e o f working memory, Was s and Hait h (1999 ) show that 3month-old infant s exposed t o alcohol prenatall y have more difficult y tha n nonexpose d control s i n main taining an d manipulatin g three item s o f information simultaneously, a result implying a reduction i n working memory capacity . On th e sam e task, these infant s have difficultie s shiftin g fro m on e rul e t o anothe r (that is , learning t o anticipat e wher e item s woul d b e presented next ) an d wit h masterin g comple x spatia l sequences. Kodituwakk u an d colleague s (1995 ) use d several neurodevelopmental tasks tapping activ e working memory in older individuals. They conclude tha t a problem i n managin g goal s i n workin g memory i s the mechanism underlyin g much o f the cognitiv e impairment see n i n childre n wit h FAS . I n practica l terms , such a defici t wil l affec t dail y functionin g an d academic performanc e a s well a s results o f neuropsychological tests. For instance, children diagnosed wit h FAS consistently sho w impairment s i n doin g mathematics , as demonstrate d b y performanc e o n th e arithmeti c subtests from the Wechsler series (e.g., Wechsler, 1991) of intelligenc e tests . Thes e test s rel y heavil y o n activ e working memor y skill s b y requiring th e manipulatio n of number s an d arithmeti c operation s i n short-ter m memory to generate correct answers . Planning an d Organization . Peopl e wit h FAS D are describe d a s having impairments i n planning , or ganization, an d problem-solvin g aspect s o f executiv e

PRENATAL ALCOHOL EXPOSURE AND HUMA N DEVELOPMEN T 13 3 functioning (Streissguth , 1997) . Tw o task s tha t hav e been use d to evaluate functioning in this area are progressive plannin g task s (e.g., Tower o f London; Shal lice, 1982 ) and task s assessing learning an d reversa l or shifts i n discriminativ e stimuli (i.e., Wisconsin Car d Sort Task [WCST] ; Heato n e t al, 1993) . Bot h kind s of tasks were originally used t o measur e th e effect s o f frontal lobe damag e i n adult s (Shallice , 1982) . Pro gressive plannin g task s requir e tha t th e perso n bein g tested replicat e a visuall y presente d desig n b y rear ranging circular disks or balls that are placed o n vertical pegs. There ar e rule s restraining the numbe r an d type o f move s permitted . Th e tas k require s tha t th e subject retai n thes e rule s a s well a s the sequenc e o f moves in working memory to correctly solve the prob lem. Initially , th e tas k i s simpl e an d require s littl e planning, bu t a s task complexit y increases , th e indi vidual mus t be abl e t o visualize a multistep sequence of moves to complete th e tas k successfully . Hie WCST and similar tasks require the individual to identif y o r sor t according to specifi c characteristics of the stimul i (i.e. , color, number , o r shape) presente d to them, wit h feedbac k provided to facilitat e learning. For th e initia l se t o f trials , th e correc t dimensio n may be color , so that all responses that matc h i n color are rewarded . Onc e th e correc t dimensio n i s reliably learned, a differen t dimension become s "correct " s o that, fo r instance , numbe r become s th e correc t solu tion an d i s rewarded. This task requires cognitive flexibility, becaus e th e individua l mus t inhibi t previously learned response s an d shif t set s o r rule s learne d fro m previous trials. Using both kinds of tasks, children wit h FAS have been foun d to perform more poorly than control s and to mak e perseveratio n error s (repetitio n o f response s that ar e incorrec t o r no t reinforced ) (Kodituwakk u et al , 1995 ; Cole s e t al. , 1997 ; Kern s et al, 1997) . These findings suggest tha t the y have difficulty incor porating environmental feedback to correct a response. Children wit h FA S typicall y hav e mor e difficult y learning th e shifts , particularl y reversa l shifts , tha n would b e expecte d (Kodituwakk u et al., 1995 ; Cole s et al, 1997 ; Kern s et al., 1997) , a pattern implying difficulties wit h inhibitio n o f learne d response s o r a n inattentiveness to new information. Kodituwakku an d colleague s (2001 ) used anothe r paradigm t o discriminat e emotion-lade n learnin g from conceptua l se t shiftin g (th e reversa l shif t task s discussed above ) t o furthe r document neurodevelopmental impairment s associate d wit h FAS . These au -

thors expecte d performanc e o f alcohol-exposed indi viduals to be impaire d i n tasks that involve d an emo tional component . I n thi s study, performance on th e WCST wa s contraste d wit h tha t o n a differen t tas k that assessed visual-discrimination reversal learning as well a s extinctio n o f reward-respons e association s (Rolls e t al., 1994) . This second tas k was assumed t o measure emotion-relate d learnin g becaus e a reward was provided rather than th e feedback provided in the WCST. Afte r controllin g fo r conceptua l se t shiftin g (performance on the WCST) and for general intellec tual abilities , school-age , alcohol-expose d childre n perform mor e poorl y tha n th e referenc e sampl e o n emotion-related learning; they achieve fewer reversal s and sho w mor e variabilit y in extinction . I n addition , there i s a significant relationship between thes e mea sures of emotion-related learnin g and conceptua l shift ing and parent-reported behavio r problems. Th e ability to regulat e arousal may hav e affecte d performanc e on this learnin g task , although thi s issu e i s not addresse d in this studv.

ACADEMIC ACHIEVEMENT

Given th e neurocognitiv e problem s identifie d i n exposed individuals , i t i s no t surprisin g tha t academi c achievement i s frequentl y affected . Learnin g prob lems, schoo l failure , repeatin g grades , an d droppin g out of school have all been cite d as negative outcome s associated wit h FA S in clinica l sample s (Streissgut h et al, 1996 ; Autti-Ramo , 2000). It is important to bear in min d tha t fo r alcohol-expose d a s well a s for othe r children, academi c achievemen t i s determine d b y complex interaction s betwee n th e child' s neurodevelopmental status, environmental supports for academic success, an d emotional stability. Overall academi c achievemen t i s predicted b y th e individual's genera l intellectua l abilit y an d b y SES . Academic problem s consisten t with globa l abilit y deficits ar e typically seen. There ma y also be specifi c areas of academic weaknes s for the chil d wit h FAS beyond those associate d with cognitive deficit an d social disadvantage. Researc h suggest s that , relativ e t o gen eral intellectua l ability , alcohol-exposed childre n an d adolescents hav e specific learning problems with mathematics (Spoh r an d Steinhausen , 1984 ; Streissgut h et al., 1994) . Deficit s in mat h achievemen t associate d with prenata l alcoho l exposur e hav e bee n identifie d regularly i n bot h longitudina l an d clinica l studie s

134 ETHANOL-AFFECTE D DEVELOPMENT (Streissguth e t al , 1994 ; Kodituwakk u e t al , 1995 ; Goldschmidt e t al , 1996 ; Mattso n e t al , 1996) . Streissguth an d colleague s (1993 ) repor t tha t arith metic disabilitie s are related t o prenatal alcoho l expo sure i n thei r longitudina l cohort , particularl y whe n there wa s "massed" o r heav y drinking. Thes e investigators also not e that thei r findings in the longitudina l cohort ar e consisten t wit h observation s fro m thei r older, clinical sample o f patients diagnosed wit h FAS. Other longitudinal sample s hav e also found evidenc e of specifi c deficit s in thi s area . Cole s and colleague s (Coles e t al, 1991 , 1997 ; Howel l e t al, 2006 ) not e specific deficit s i n mat h functionin g i n a cohor t o f African-American schoo l childre n eve n whe n globa l delays ar e controlled . I n thi s population , thes e prob lems appear to be related to deficits in visuospatial and visual-motor functioning . Jacobson (1999 ) note prob lems i n mat h i n a Detroit longitudina l cohor t a s well and attribut e these difficultie s t o problems with working memory and executive functioning skills. In a sampl e populatio n includin g bot h inner-cit y and suburba n 4V2-year-ol d childre n wit h lo w birt h weight, specific deficit s in preacademic mathematica l skills ar e identifie d among alcoho l expose d childre n (Kable e t al, 1999) . Highe r score s o n a cumulative risk inde x relate d t o alcoho l an d othe r dru g us e ar e significantly relate d t o poore r performanc e o n th e Test of Early Mathematics Ability , 2nd Editio n (Gins burg an d Baroody , 1990) , o n a numbe r o f specifi c preacademic skills , includin g cardinality , constancy , counting, an d visua l recognitio n o f numbers. Apparently, problem s wit h workin g memory , visua l per ception, an d executiv e functionin g underli e thes e functional problems . Adult s wit h FA S als o showe d mathematics-related difficulties , includin g computa tion, an d i n solving problems requirin g estimation o f magnitude (Kopera-Fry e et al. , 1996) . Thus , there i s considerable evidenc e tha t thi s academi c are a i s a problem i n affecte d individuals , due, n o doubt , t o deficits i n cognitiv e processe s tha t suppor t mat h achievement. Som e candidat e processe s are those areas discusse d abov e i n thi s chapter—i.e. , attention , executive functioning , and workin g memory a s well as visuospatial skills (Geary, 1993 ; Ashcraft, 1995) .

SOCIAL AND EMOTIONA L FUNCTIONING

Problems wit h behavior and emotiona l regulatio n ar e the most frequent reason for clinical referral of children

suspected o f fetal alcoho l exposur e (e.g. , Steinhause n et al, 1993;Janzenetal, 1995; Kopera-Frye et al, 1997 ; Oesterheld an d Wilson , 1997 ; Roebuc k e t al, 1999 ; Mattson an d Riley , 2000 ; Kodituwakk u et al, 2001) . Social an d emotiona l problem s ar e s o commonly re ported b y parents an d i n clinica l studie s i t has been suggested that "people wit h FAS appear to differ fro m the mentall y retarde d populatio n becaus e o f addi tional problem s i n th e socia l domain " (Kell y e t al. , 2001, p . 143) . In a survey of a clinica l populatio n of individuals with FAS and ARND, Streissgut h an d col leagues (1996 ) repor t a hig h frequenc y o f menta l health, legal , and social problems among the second ary disabilities in thi s group. Th e vas t majority (94% ) of individual s ar e reporte d t o hav e menta l healt h problems o f some kind , wit h th e mos t commo n psychiatric problem s bein g ADH D an d depression . These report s ar e certainl y a concern, but i t may be too earl y to interpret thes e outcome s a s the direc t results o f th e teratogeni c exposure , particularl y a s thi s survey di d no t includ e a compariso n grou p an d wa s drawn fro m a clinicall y referre d populatio n tha t should be expected t o have such problems . In interpretin g report s fro m clinica l sample s o r from sample s tha t ar e self-referre d (e.g. , O'Conno r et al., 2002), some othe r factors that migh t influenc e behavior shoul d b e take n int o account . Man y chil dren wit h FAS D experienc e environmenta l factor s known t o contribut e t o behavioral , social , an d emo tional problems, an d i t is disingenuous to ignore thes e issues t o attribut e problem s t o prenata l exposure . Children diagnose d wit h FA S often com e fro m low income or socially disadvantaged families an d usually have limited acces s t o educational o r other resources . Caregiving disruption s are the nor m rathe r tha n th e exception i n children referre d to clinical settings, an d many children ar e in foster car e following a history of neglect and abuse . To examine the frequenc y o f behavior concerns i n a clinica l sampl e o f alcohol-affected children , Cole s and Kabl e (unpublishe d results ) evaluate d 28 7 pa tients (mea n age , 4.5 years) referred for evaluation t o a FA S specialty clinic. Of the 10 9 children diagnose d with FAS , 54.7 % wer e describe d a s "to o active, " 30.5% had difficultie s wit h tantrums, and 41.1% were described a s having problems wit h aggression. Whe n these childre n ar e compare d t o those referre d t o th e same clini c wh o hav e n o evidenc e o f dysmorphia o r growth problem s an d t o those wit h no histor y of prenatal alcoho l exposure , th e frequenc y o f th e sam e

PRENATAL ALCOHOL EXPOSURE AND HUMAN DEVELOPMEN T 13 5 problems i s 53.1% , 25.0% , an d 43.8% , respectively . Upon furthe r examinatio n o f the clini c sampl e an d dividing it into four groups (FAS, ARND, nondysmorphic FASD , an d unexposed) , n o significan t relationship betwee n diagnosti c categor y an d behaviora l o r emotional outcome s i s evident . Thes e finding s sug gest that problems wit h emotional an d behavioral reg ulation, commo n i n childre n wit h FAS , is associated as muc h wit h clinica l referra l statu s o r advers e early experiences as with prenatal exposure. The relationshi p betwee n prenata l alcoho l expo sure and conduct disorde r and delinquency in adolescence i s an area of interest. Some researchers and FAS advocates hav e suggeste d tha t prenata l alcoho l expo sure i s associated wit h delinquen t o r criminal behav ior during adolescenc e (e.g. , Streissgut h e t al., 1996 ; Fast e t al. , 1999 ; Fas t an d Conry , 2004) . Thes e con clusions ar e ofte n base d o n studie s o f adolescents al ready referre d t o clinic s fo r treatmen t o r i n th e juvenile justic e system. I n a surve y o f secondary disabilities, Streissgut h an d colleague s (1996 ) describ e that caregiver s repor t tha t a majorit y o f yout h an d young adults with FASD are involved in "delinquent" or problematic behaviors. Of 25 3 patients 1 2 years of age o r older , 60 % have disruption s i n schoo l experi ence (e.g. , suspensions), 50 % have demonstrate d "in appropriate" sexua l behavior , an d 30% , alcohol an d drug problems. Fas t and colleague s (1999 ) als o repor t an associatio n betwee n prenata l exposur e and crimi nal behavior . In a sample of 287 adjudicate d adolescents remande d fo r inpatien t forensi c psychiatri c assessment, 23.3 % were found to have some prenata l alcohol-related diagnosis. In studie s of the relationshi p o f prenatal exposure and behavio r i n les s high-risk groups , th e result s ar e more mixed . The Achenbac h (1991 ) Child Behavio r Checklist an d th e teache r version , th e Teache r Re port Form, hav e often been used to measure behavio r problems. Elevate d score s o n proble m indice s hav e been reporte d in some studies (i.e., Olson e t al., 1997 ; Mattson and Riley , 2000) but no t others (Steinhausen et al. , 1993) . Eve n whe n score s fo r childre n wit h FASD ar e statisticall y highe r tha n thos e o f age matched contro l groups , th e score s d o no t alway s reach clinicall y significan t levels (e.g. , Brow n e t al, 1991). Lync h an d colleague s (2003 ) specificall y examine th e question o f delinquency an d conduct problems i n alcohol-expose d adolescent s compare d t o contrast group s fro m th e sam e populatio n o f low income, urba n youth . The y sho w wit h a sampl e o f

248 children , mea n ag e o f 1 5 years , male s ar e mor e likely than females to report delinquent behavior, but there i s no relationshi p between prenata l alcoho l exposure and outcomes . Delinquen t behavio r is related to parenting factor s an d environmenta l stress . In th e same grou p o f youn g people , examinatio n o f aca demic an d schoo l record s indicate s n o highe r inci dence of conduct problems, attendanc e problems , o r suspensions i n alcohol-expose d yout h (Howel l e t al. , 2006). Methodological factor s mus t be considere d whe n assessing th e result s o f research . Interpretatio n o f reports fro m clinica l studie s i s particularl y speculative i n th e are a o f behavio r an d social/emotiona l functioning. Investigator s mus t b e ver y carefu l t o identify appropriat e contras t group s a s well a s the ef fects o f confoundin g an d mediatin g factor s whe n attempting t o understan d observe d outcomes . Fo r the mos t part , thes e methodologica l restraint s have not bee n observe d i n thi s area of study, and fo r these reasons, i t i s to o earl y t o dra w conclusion s abou t the effec t o f prenata l exposur e o n thi s are a o f func tioning.

CURRENT ISSUES AND SUGGESTION S FOR FUTUR E RESEARC H

Despite greatl y increased understandin g o f the con sequences o f prenatal exposure , som e issue s are stil l being debated . These area s of controversy and explo ration sugges t th e directio n fo r researc h durin g th e next decade. Description of Characteristic s Over Time Although som e sampl e population s an d a number o f exposure studie s hav e bee n followe d longitudinally , there remain s limited informatio n about th e trajectories o f developmen t resultin g from prenata l alcoho l exposure amon g thos e wit h eithe r FA S o r ARND . Even objectiv e issues such a s facial dysmorphi a have not been studie d i n the sam e individuals over time to evaluate th e salienc y of features at different point s i n development. Outcome s from longitudina l cohort s i n which repeate d wave s of data hav e been collected o n the sam e individual s (Streissgut h e t al. , 1993 ; Da y et al, 2002) have often bee n reporte d by time period , as i f thes e wer e cross-sectiona l cohorts , instea d o f

136 ETHANOL-AFFECTE D DEVELOPMENT examining them ove r time to evaluate developmenta l trajectories. Sinc e thes e database s do exist , investigators hav e th e opportunit y t o investigat e th e longi tudinal aspect s o f developmen t i n alcohol-affecte d children and , whe n the y hav e don e so , to greatly increase understandin g in this area. Behavior and Conduct Disorder s A largel y unresolve d questio n i s that o f the relation ship betwee n prenata l exposur e an d late r behaviora l and emotiona l problems . Man y o f these problem s a s well a s psychiatri c disorder s (Fam y e t al. , 1998 ; O'Connor et al., 2002 ) have been identifie d i n clini cally referred samples. I t is likely that the nex t decad e will produc e a numbe r o f studie s o n thes e relation ships an d furthe r examinations o f the effec t o f expo sure o n th e ris k fo r lega l problem s an d substanc e abuse. I f studies are carrie d ou t wit h th e appropriat e methodologies and contro l fo r the multipl e confounding factors associate d wit h materna l substanc e abuse , the contributio n o f prenata l exposur e t o suc h out comes ma y be better understood . Behavioral Phenotypes In th e rea l world , i t i s ofte n difficul t t o ascrib e ob served outcome s t o materna l alcoho l use . Ther e ha s been a goo d dea l o f interest i n identifyin g a "behav ioral phenotype" that characterizes peopl e wh o are affected b y prenata l exposure . Suc h a constellatio n o f behavioral outcome s i s believed t o characteriz e bot h those wit h th e ful l FA S and thos e wh o d o no t hav e identifying facia l features . Clinician s woul d lik e t o have a checklist of such behaviors that can b e used by teachers, parents , an d othe r observer s to discriminate affected fro m unaffecte d children an d t o facilitate referral. Th e searc h fo r such a phenotype does no t take into accoun t th e multipl e factor s tha t affec t develop ment an d th e man y etiologie s tha t ca n hav e a final common pathwa y in behavioral terms. Intervention an d Standards o f Care The mos t neglecte d are a o f researc h o n th e behav ioral effect s o f alcohol i s on method s fo r interventio n to improve outcomes fo r affected individuals . Parent s and caregiver s o f affecte d childre n stat e tha t thei r most urgen t nee d i s appropriat e interventions . I n

1996, a repor t b y the Institut e o f Medicine (Stratto n et al , 1996 ) recommende d tha t studie s b e don e i n this are a an d tha t standard s of car e fo r affecte d indi viduals be written. To date, n o report s of intervention studies i n huma n sample s hav e bee n published , al though ther e are currently five such studies being car ried ou t unde r th e aegi s o f th e Center s fo r Diseas e Control an d Prevention . N o standard s o f car e hav e been propose d b y any agenc y o r professiona l organi zation. Althoug h i t is apparently difficul t t o carry ou t work in this area, i t seems likel y that as more information i s accumulate d abou t cognitiv e an d behaviora l consequences of prenatal exposure , interventio n an d treatment wil l be a focus of research i n the future.

Abbreviations ADHD attentio n deficit , hyperactivity disorder ARND alcohol-relate d neurodevelopmenta l dis order CNS centra l nervou s system CVLT-C Californi a Verba l Learnin g Test children's version DTI diffusio n tenso r imagin g FAS feta l alcohol syndrom e FASD feta l alcoho l spectru m disorder s fMRI functiona l magnetic resonance imagin g SES socioeconomi c status sMRI structura l magnetic resonanc e imagin g WCST Wisconsi n Car d Sort Task WRAML Wid e Rang e Assessmen t o f Memor y and Learnin g

ACKNOWLEDGMENTS Th e autho r woul d lik e t o ac knowledge th e continuin g contributions of th e facult y and staf f o f th e Materna l Substanc e Abuse and Chil d Development Laboratory, Department of Psychiatry and Behavioral Sciences , Emory University School of Medicine, an d thos e o f th e researc h participant s an d thei r families wh o make research o n thes e problems possible. Some o f th e wor k referre d t o i n thi s chapte r was supported b y awards to the autho r fro m th e followin g agencies: th e Georgi a Departmen t o f Huma n Resource s (DHR), Maternal Substance Abuse Prevention Program, the National Institute of Alcohol Abuse and Alcoholism, and the Centers for Disease Control and Prevention.

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142 ETHANOL-AFFECTE D DEVELOPMEN T function i n childre n wit h moderat e prenata l holisms alcohol exposure . Alcohol Cli n Ex p Re s 28:497 - theor 507. Cohe Zucker RA , Fitzgeral d HE , Mose s H M (1995 ) Emer - Risk, gence o f alcoho l problem s an d th e severa l alco - York

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9

Influence of Alcohol on Structur e of the Developing Human Brain Susanna L . Fryer Christie L. McGee Andrea D. Spadon i Edward P. Riley

A diagnosis of fetal alcohol syndrome (FAS) requires a combination o f three cluster s o f symptoms: (1 ) a distinct craniofacia l appearance, (2 ) growt h deficiency , and (3 ) centra l nervou s syste m (CNS ) dysfunction . Characteristic FAS-relate d craniofacia l dysmorphol ogy includes short palpebrai fissures (eye openings), a smooth philtru m (th e area above the upper lip) , a flat nasal bridge an d mid-face , and thinnes s o f the uppe r lip. Th e growt h deficienc y related t o FA S manifests either prenatall y and/o r postnatally , and ca n necessi tate neonatal car e for failure to thrive. The thir d clas s of symptoms , CN S dysfunctio n an d accompanyin g structural brai n changes , ar e th e subjec t of the pres ent chapter . The effect s o f prenatal alcoho l exposur e on cognitio n an d behavio r ar e variabl e an d wide reaching. Commo n difficultie s includ e attentio n deficits; decrement s i n genera l intelligenc e quotien t (IQ); behaviora l problems ; visual , auditory, and per ceptual disturbances ; fin e an d gros s motor problems ; and learnin g disabilitie s (Streissgut h an d Connor , 2001). Indeed , th e syndrom e ha s bee n cite d a s th e

leading preventable caus e of mental retardatio n (Pulsifer, 1996) . The stud y o f brai n structur e i n alcohol-expose d individuals offer s a means t o understand th e etiologi cal relationship s tha t underli e th e neurobehaviora l abnormalities observed i n thes e individuals . By examining relationships between brai n structure and behavior, we gain insight s int o th e mechanism s underlyin g alcohol teratogenicity . Before delving into a discussion of brain structura l changes arisin g from feta l alcoho l exposure, i t i s important t o ad d a not e o n terminol ogy. In recognitio n of the wide-rangin g effect s of prenatal alcohol exposure , the term fetal alcohol spectrum disorders (FASD ) ha s bee n adopte d t o describ e the entir e rang e o f outcome s fro m prenata l alcoho l exposure (Bertran d e t al. , 2004) . Thes e effect s var y from th e pronounced facial , growth, and CN S abnor malities traditionall y ascribed t o FAS , to subtl e neu robehavioral, growth , o r physica l deficit s tha t ma y also occu r a s a resul t o f suc h exposure . Usin g thi s classification, FA S corresponds t o dysmorphi c FAS D

143

144 ETHANOL-AFFECTE D DEVELOPMEN T (i.e., al l thre e o f the diagnosti c criteri a ar e met , in cluding th e combinatio n o f spécifi e dysmophi a re quired t o mee t FA S criteria) . Therefore, individual s with a history of prenatal alcohol exposur e and deficits thought to be related t o the alcohol exposure , bu t without th e necessar y criteria for an FA S diagnosis, would be considere d a s nondysmorphi c FASD . Th e termi nology use d throughou t th e presen t chapter refer s to alcohol-exposed individual s as having FAS when the y meet th e thre e majo r criteri a fo r th e diagnosis , an d nondysmorphic FAS D whe n the y do not . I t is important to note that although there is a general consensu s on th e wide-rangin g an d variabl e effect s o f feta l alcohol exposure , diagnosti c an d classificatio n terminology remain s controversial . Thus , honing the diag nostic criteri a tha t describ e feta l alcoho l effect s i s currently a priority for the fiel d (Rile y et al., 2003).

AUTOPSY STUDIES

Dysmorphic Fetal Alcohol Spectrum Disorde r Autopsy, which literall y means, "t o see for oneself," in volves examinatio n t o determin e th e exac t caus e o f death. Autopsies are usefu l i n the stud y of brain disease processes. Postmorte m examinatio n permit s a mor e direct stud y of neuroanatomy tha n i n vivo techniques . Examination of expired tissue, however, presents a confound whe n attemptin g t o generaliz e th e finding s t o live cases. In other words, autopsies allow for a detailed description no t possibl e wit h les s invasiv e techniques, but the data they provide are not necessarily representative o f living cohorts. Historically , autops y cas e report ing wa s on e o f th e initia l method s use d t o examin e teratogenic effect s o f alcohol o n brai n structure. In th e autopsy case s o f children wit h FAS , death usuall y occurred because of major CNS o r cardiac dysfunction. The handfu l o f subsequent autops y report s sho w that developing brain s sufficiently expose d t o alcoho l have a host of structural abnormalities. These include gross microcephaly , cellula r disorganization , an d anomalies of specific brain structures such a s the cor pus callosu m an d cerebellum . Th e firs t autops y o f a FAS case wa s o f a n infan t wh o die d a t 5 days of ag e (Jones, 1975) . Th e infant' s brain exhibite d enlarge d lateral ventricles an d agenesis (absence ) o f the corpu s callosum. I n addition , neuroglial heterotopias , a type of microdysplasia i n which abnormal neural and glia l

tissue cover parts of the brai n surface, were described . Notably, suc h microdysplasia s ar e though t t o resul t from aberran t cortical neura l migratio n and ar e com mon i n autops y case s o f prenata l alcoho l exposure . For instance , i n a n autops y repor t o f five cases, het erotopias wer e note d i n eac h case , althoug h th e amount o f abnormal tissu e wa s variable among indi viduals (Wisniewsk i e t al. , 1983) . Anothe r autops y study tha t examine d brain s fro m fetuses , infants, an d one chil d corroborate d th e presenc e o f microdys plasias and commente d o n th e diversit y of malformations observed (Peiffe r e t al., 1979) . Furthermore , th e authors suspecte d tha t dosage , i n additio n t o tim e course o f exposure, likel y influences the degre e an d nature o f structural damag e t o the developing brain. Recent neuropathologica l studie s associat e FA S with specific types of complex cerebral malformations (Coulter et al., 1993) . Evaluatio n o f a 2-month-old infant expose d t o a bing e patter n o f alcoho l exposur e during th e firs t trimeste r o f pregnanc y wa s th e firs t observation associatin g feta l alcoho l exposur e wit h midline cerebral dysgenesis. Due t o elements o f septooptodysplasia, midlin e cerebra l dysgenesi s i s a typ e of midlin e malformatio n characterize d b y a lac k o f the septum pellucidum, optic nerv e damage , an d endocrine abnormalities . I n additio n t o thi s midlin e damage, th e cas e showe d genera l microcephal y an d cerebellar Purkinj e neuro n disruption . Specifically , the cerebellar neurons were unusually positioned an d had abnormal dendritic structure. Non-Dysmorphic Feta l Alcohol Spectru m Disorde r An earl y cas e stud y tha t examine d th e brain s o f four neonates include d case s o f bot h dysmorphi c an d nondysmorphic FAS D (Clarre n e t al., 1978) . Disrup tions t o brain structur e wer e note d i n addition t o microcephaly, includin g heterotopia s an d histologica l structural aberration s associate d wit h error s i n neu ronal an d glia l migration . Th e degre e o f structura l damage t o th e alcohol-expose d brain s prompted th e authors to conclude "tha t problem s o f brain morpho genesis can occur a s the predominan t effec t o f ethanol exposure i n utero" (p . 67). Brain alterations may more directly indicat e damag e fro m alcohol-relate d effect s than facia l dysmorphia . As th e consensu s o f initia l autops y report s indi cating extensiv e an d diffus e damag e throughou t th e alcohol-exposed brai n mounted , investigator s bega n

ALCOHOL AN D THE DEVELOPIN G HUMA N BRAI N 14 5

Medical imagin g technologie s detec t inheren t differ ences i n biological tissu e density (e.g., gray brain matter vs. white brain matter vs. bone), displayin g them a s images with contrast differences. Imagin g technologie s offer th e abilit y to examine structural brain damage i n vivo an d includ e larger , more representativ e sample s than th e descriptions of brain structure provided by autopsy. Give n tha t CN S deficit s ar e a hallmark feature of FASD, brain matter quantification through imagin g studies provide s a crucia l insigh t int o specifyin g ho w normative brain-behavio r relationship s migh t b e af fected b y alcohol exposure. In contrast to autopsy, magnetic resonanc e imagin g (MRI ) an d othe r i n viv o techniques provid e information, albeit indirectly, of living tissue. Moreover , a s there i s no inheren t selectio n bias, the findings of these studies ar e likely more repre sentative of the populatio n o f interest . Structural brai n imag e analyse s revea l tha t chil dren an d adolescent s prenatall y expose d t o alcohol , with o r withou t dysmorphi c facia l features, hav e pat terns of brain structure malformation s consistent with the neuropsychologica l an d behaviora l effect s foun d in thi s population . Mos t o f the existin g researc h o n neurostructural change s associate d wit h prenata l al cohol exposur e relies on structural MRI techniques .

Reliably, quantitativ e volumetri c analyse s hav e con firmed th e overal l reductions i n siz e of the brai n an d the cerebra l vaul t (Swayz e e t al. , 1997 ; Archibal d et al, 2001 ; Autti-Ramo e t al, 2002) . Analytic tech niques examining regional variations in brain size and shape hav e suggested tha t feta l alcoho l exposur e pro duces a pattern o f differential brai n damage . Specifi cally, th e corpu s callosum , cerebella r vermis , basa l ganglia, and parietal regions may show particular sensitivity to th e effect s o f prenatal alcohol . A discussion of findings from specifi c studies follows. A series o f imaging studies from a group o f collab orators in San Diego has focused on image s collected from a sample o f children an d adolescent s prenatall y exposed t o alcohol , bot h wit h an d withou t dysmor phic feature s (ALC ) (Archibal d e t al., 2001). Analysis of total brain volume show s that there are lobar differ ences betwee n AL C and contro l groups . After statisti cally controllin g fo r overal l brai n reduction s i n th e ALC group , the parieta l lobe i s disproportionately reduced, suggestin g tha t thi s regio n i s particularly vulnerable t o alcoho l exposur e durin g development . Such dat a parallel findings in the mature rat, in which parietal corte x i s reduced b y one-thir d followin g prenatal exposure to ethanol (Mille r and Potempa, 1990 ; Mooney an d Napper , 2005) , but occipita l corte x i s unaffected. The regiona l tissue composition i s affected b y prenatal exposure to ethanol. Ra w volume reduction s are evident for both gra y and whit e matte r whe n FA S individuals are compared with controls . Yet , when over all reduction s i n brai n volum e wer e statisticall y accounted for , only white matte r reduction s reache d statistical significance . Thus, global whit e matte r hy poplasia appear s t o b e mor e sever e tha n globa l gra y matter hypoplasi a i n brain s o f individual s wit h FAS . In addition, exploratio n o f proportional tissu e composition i n eac h lob e reveale d tha t parieta l gra y an d white matte r volume s ar e disproportionatel y reduce d in individual s with FA S relative t o controls , wherea s occipital lob e whit e matte r i s proportionally larger in FAS subjects. These finding s sugges t relativ e sparing of white matter i n this region.

Total Brain Volume and Shape

Voxel-Based Morphometry

to speculat e tha t n o particula r patter n o f behavioral or intellectual functioning is characteristic of individuals with FAS (Clarren, 1986) . Yet, neuropsychological studies suggest a syndrome-specific pattern o f cognitive and behaviora l deficits associated with fetal alcoho l exposure (Mattso n an d Riley , 1998) . Th e subject s tha t comprise autops y studie s likely represen t th e mos t severe cases of prenatal alcohol exposure, i.e. , those with damage incompatibl e with life . Thus , finding s fro m autopsy studies may not be representative of the major ity of individuals with fetal alcohol effects. This is especially true in the case of FAS, because mos t of the brai n damage cause d b y fetal alcoho l exposur e doe s not pre clude viability (Clarren, 1986) .

IMAGING STUDIE S

Early MR I Studies Most MRI studies of individuals prenatally exposed t o alcohol hav e focuse d o n measure s o f brain volume .

Whole-brain voxel-base d morphometr y (VBM ) aim s to localiz e cortica l abnormalitie s by examining eac h voxel, o r point, o n th e brai n image . Thi s methodol ogy avoid s th e nee d t o defin e boundarie s o n eac h

146 ETHANOL-AFFECTE D DEVELOPMENT image, as is often necessar y with standard volumetri c brain imagin g methods . Thus , VB M allow s for th e study of brain regions without clear gyral or structural boundaries. As voxel intensity values are considered t o be a proxy for tissue density, in VBM, statistica l parametric map s depictin g average d tissu e densitie s are created fo r both gray and white matter. Results from a VBM analysi s of children an d ado lescents with prenatal alcoho l exposure (Sowell et al., 200 lb) complemen t earlie r volumetri c finding s o n the sam e sampl e (Archibal d et al, 2001) . The VBM study reveal s prominen t abnormalitie s i n th e peri Sylvian cortice s o f th e lef t tempora l an d parieta l lobes. Specifically , alcohol-exposed subject s have excess gra y matte r densit y and decrease d whit e matte r density i n thes e regions , compare d t o age-matche d controls. Shape Analysis Shape analysi s determines th e siz e o f th e brai n b y measuring th e distanc e fro m th e cente r t o variou s landmark point s o n th e brai n surface . Accordingly, the averag e brai n surfac e exten t i s smalle r fo r th e alcohol-exposed subject s tha n fo r control s (Sowel l et al. , 2002a) . This resul t i s consisten t wit h myria d studies showin g tha t prenata l exposur e t o alcoho l causes microcephaly . Furthe r examinatio n reveal s prominent regiona l pattern s i n alcohol-induce d siz e and shap e differences. Specifically , large group differ ences ar e eviden t i n th e peri-Sylvia n an d parieta l regions, wit h characteristi c narrowin g observe d i n th e alcohol-exposed subject s and increase s in gray matter density. I n addition , portion s o f the anterio r an d or bital fronta l cortex , particularl y i n th e lef t hemi sphere, ar e affecte d i n ALC subjects . These pattern s confirm tha t alcohol-induce d abnormalitie s ar e spe cific rathe r tha n reflectin g diffus e damag e o r globa l microencephaly. The surfaced-base d analyti c method s use d b y Sowell and colleagues (2002a) allow researchers to locate relatively small regions of gray matter volume in creases masked by reductions i n gray matter. I n othe r words, gros s volumetri c studie s hav e misse d subtl e anomalies b y taking the tota l brai n volum e int o ac count. Regiona l shap e abnormalitie s ma y occu r i n alcohol-exposed subject s because of abnormal myeli n deposition tha t (a ) prevent s norma l growt h an d (b) leads to cortical thinnin g in select areas. Evidence of fronta l lob e abnormalitie s ar e consisten t wit h

findings from th e cognitiv e an d behaviora l literatur e citing deficit s i n executiv e function s suc h a s plan ning, cognitive flexibility, and respons e inhibitio n following prenata l exposur e t o alcohol (Mattso n e t al, 1999; Conno r et al, 2000; Kodituwakku et al, 2001). Brain shape has been explore d with surface-based image analysis techniques designe d t o match cortica l anatomy betwee n hemisphere s base d o n delineate d landmarks (Sowel l e t al, 2002b) . From thi s stud y of differences i n brain shape and gray matter density, the authors not e tha t th e lef t hemispher e appear s t o b e more affecte d tha n th e righ t hemispher e an d specu late tha t AL C subject s migh t hav e altere d hemi spheric symmetr y compare d t o th e patter n see n i n typically developing subjects. Specifically, gray matter asymmetry i s mor e prominen t i n th e tempora l lob e for nonexpose d controls , wit h th e righ t hemispher e having more gra y matter tha n th e left . Thi s asymmetry i s significantl y reduce d i n alcohol-expose d sub jects. The suggestio n o f altered brai n asymmetry as a consequence of prenatal alcoho l exposur e is provocative, althoug h furthe r studie s ar e neede d befor e thi s claim ca n b e substantiate d an d it s implication s fo r possible behavioral effects understood . Taken together , dat a fro m th e Sa n Dieg o grou p show tha t asid e fro m globa l reduction s i n brai n volume, parieta l region s ar e disproportionatel y af fected an d whit e matte r i s mor e affecte d tha n gra y matter. Cerebellum Several MRI studies describe cerebellar hypoplasia in individuals prenatall y expose d t o alcoho l wit h an d without FA S (Mattson e t al, 1996 ; Riikone n e t al, 1999; Archibald et al, 2001; Autti-Ramo et al, 2002). Both cerebella r gra y and whit e matter are reduced i n alcohol-exposed individual s (Archibald et al, 2001) . Reductions ar e particularl y evident i n verma l corte x of earlier maturing lobules (Sowel l et al., 1996) . Th e methodology use d t o generat e thes e result s i s as follows. For each subject, the midsagitta l section i s identified an d the vermis is divided into three regions : the anterior vermis (lobules I-V), lobules VI and VII, and lobules VIII-X. Alcohol-induced difference s ar e only evident i n th e anterio r vermis , wher e th e alcohol exposed childre n showe d significan t reductions. Thi s pattern o f cerebellar vermal dysmorphology is distinct from th e pattern s foun d in childre n wit h other disorders, suc h a s autism, Willia m syndrome , an d Dow n

ALCOHOL AND THE DEVELOPIN G HUMAN BRAI N 14 7

syndrome (Ra z et al, 1995 ; Courchesn e e t al, 2001; Schmittetal.,2001). Animal model s o f feta l alcoho l exposur e consis tently sho w abnormalitie s o f th e cerebella r vermi s and hemispheres . Variou s investigator s hav e exam ined th e effect s o f episodic ethano l exposur e during the firs t postnata l wee k (se e Goodlet t e t al. , 1990 ; Light e t al. , 2002) . Followin g suc h exposure , th e number of Purkinje neurons is reduced. As in huma n studies, the greates t differences ar e apparent in earlymaturing region s o f th e cerebella r vermis . Thus , ethanol-induced change s i n th e anima l studie s no t only concu r wit h th e finding s i n human s bu t als o show that th e Purkinj e cel l i s a key target of ethano l toxicity. Corpus Callosum The corpu s callosu m i s a bundle o f fibers that con nects th e tw o cerebral hemispheres . I t develops dur ing the secon d trimester of gestation. Autopsy reports document agenesi s o f thi s structur e i n infant s ex posed t o alcohol i n utero. Partial or complete agene sis i s describe d i n severa l imagin g studie s (Rile y et al, 1995 ; Johnson et al, 1996 ; Swayz e et al, 1997 ; Riikonen e t al, 1999 ; Clar k e t al, 2000) , and i t is suggested tha t prenatal alcoho l exposure is a leadin g cause o f this condition (Jere t et al., 1986) . Mos t indi viduals expose d t o alcoho l prenatall y d o no t hav e such sever e alterations. Careful evaluation indicates that alcohol induce s subtle yet significant changes i n the siz e an d shap e o f this structure . Studie s o n th e morphology of the corpus callosum reveal that anomalies within specific cortica l regions often correspon d to particular callosal subregion s through whic h thos e regions send white matter fibers (Barkovich and Nor man, 1988) . Authors of the first study to systematically evaluate the corpu s callosum divid e a midsagittal are a sectio n of th e callosu m int o fiv e subregion s (Rile y e t al. , 1995). Afte r excludin g tw o subject s with complet e agenesis and controllin g for overall brain size, group differences emerge i n three callosa l subsections . Th e alcohol-exposed grou p display s disproportionate are a reductions i n th e gen u (mos t anterio r section ) an d two areas that correspond to the spleniu m (mos t posterior region) . These data ar e consistent wit h a study by Autti-Ramo and colleague s (2002) , who show that the spleniu m o f corpus callosum i s reduced i n area , length, an d diamete r i n childre n prenatall y exposed

to alcohol . Strikin g differences ar e eviden t o n map s charting th e averag e corpus callosu m displacemen t in alcohol-expose d subject s (Sowel l e t al. , ZOOla) . Specifically, the posterior portion o f the corpu s callo sum i s the sectio n mos t significantl y displaced , lying more inferio r an d anterio r i n th e alcohol-expose d subjects. Thi s alcohol-induce d chang e i n callosa l placement correlate s wit h performanc e o n a verba l learning task . Indeed , displacemen t i s a bette r pre dictor o f performanc e than testin g o f verbal IQ . I n other words , individual s wit h greate r callosa l dis placement exhibi t increase d verba l learnin g impair ments. A series of studies on a large sample of adolescents and adults exposed to alcohol prenatally evaluates the corpus callosu m b y mean s o f landmar k strategie s (Bookstein et al, 2001, 2002a , 2002b). This work develops usefu l discriminatio n protocols t o distinguis h ALC fro m contro l subjects , a goa l distinc t fro m tha t of othe r imag e analysi s research , whic h analyze s mean grou p difference s fo r th e purpos e o f descrip tion. Variabilit y in th e corpor a callos a i s greate r i n adolescents (Bookstei n e t al. , 2002a ) an d adult s (Bookstein e t al, 2001 ) tha t wer e prenatally expose d to alcohol tha n i n age-matched controls . The author s suggest tha t hypervariatio n i n callosa l shap e withi n the AL C grou p i s a functio n o f th e timin g an d amounts o f exposure durin g th e developmen t o f this structure. Thus, differences i n the manne r of the feta l alcohol exposur e may explain the numerou s patterns deviating from norma l callosa l variation . In addition , the dysmorphi c an d nondysmorphi c FAS D group s are indistinguishabl e wit h regar d t o callosa l hyper variance. Thi s findin g i s consisten t with researc h i n the behaviora l domai n (Mattso n an d Riley , 1998 ; Connor e t al., 2000 ) and speak s to the utilit y of augmenting the us e of facial features associated with FAS with MRI findings as markers of alcohol-related brain damage. Two callosa l summar y score s an d fou r land mark point s ca n b e use d t o creat e a classificatio n algorithm tha t discriminate s th e sampl e (alcohol exposed vs . nonexposed ) wit h hig h sensitivit y (10 0 out o f 11 7 subjects ) an d specificit y (4 9 ou t o f 6 0 subjects) (Bookstei n et al. , 2002a) . This typ e o f discrimination protoco l ma y b e especiall y usefu l a s a diagnostic too l fo r th e ag e rang e studie d a s man y conventional sign s used i n th e diagnosi s o f youn g children becom e les s apparen t by adolescence an d adulthood.

148 ETHANOL-AFFECTE D DEVELOPMEN T A subsampl e fro m th e Seattl e studie s wa s use d to examin e th e relationshi p betwee n th e alcohol induced callosa l hypervarianc e an d decrease d neu ropsychological performance (Bookstein et al., 2002b). The relatio n o f callosal shap e an d neuropsychologi cal performanc e wa s analyze d usin g partia l least squares (PLS) analysis . Briefly , a PLS analysi s applies traditional multipl e regression methods t o latent variables (LVs), which are variable s created fro m a facto r analytic procedure to combine an d summarize multiple measure s o f a construct . Th e dat a reductio n ca pabilities o f PL S ca n b e use d t o comba t bot h th e complexity brough t abou t b y larg e number s o f out come variables and the statistical problems (e.g. , multicollinearity) associate d wit h multiple , indirec t measurement o f complex constructs . On th e basi s of this analysis , exces s shap e variatio n correlate s wit h two different profile s of cognitive deficit that are unre lated t o I Q o r th e dysmophic/nondysmoprhi c grou p distinction. A relativel y thick callosa l trac t i s associ ated with deficits in executive function, whereas a relatively thin callosum i s related to motor deficits . The effec t o f ethanol o n th e corpu s callosu m ha s been examine d in nonhuman primate s (Miller et al., 1999). MRI studies and analyse s of postmortem tissue show tha t th e callosu m i s large r i n som e ethanol exposed animals, the mos t affected segmen t being the anterior par t (includin g th e rostrum) . This segmen t interconnects th e fronta l cortice s an d i s involved in executive function . Moreover , th e numbe r o f axons in th e anterio r callosu m i s increase d i n ethanol exposed monkeys . I t i s importan t t o not e (a ) tha t these ethanol-inducec l change s ar e dose-dependen t and (b ) that they are evident in monkeys with dysmorphic and nondysmorphi c FASD .

Hippocampus The hippocampu s i s associated wit h short-term mem ory an d learning . Accordin g t o neuropsychologica l studies of children with FASD, i t appears that the hip pocampus i s damaged by prenatal alcohol exposure. In a recent study, evaluating spatial learning and memor y in childre n wit h feta l alcoho l exposur e (Hamilto n et al. , 2003) , a virtual Morri s maz e tas k based o n ap proaches routinel y used in animal studies was used as a measure o f hippocampal functio n (Sutherlan d e t al. , 2001; Johnso n an d Goodlett , 2002) . Alcohol-exposed children hav e impaired place learnin g relative to con trols bu t ar e equall y proficien t durin g th e cue navigation phase . Thu s th e result s o f th e huma n studies, like those with animals, implicate tha t ethano l interferes with hippocampal-mediated place learning . Imaging studie s sho w hippocampa l damag e i n alcohol-exposed subjects , althoug h no t al l studie s confirm thi s finding . I n a smal l sampl e o f Finnis h adolescents prenatall y exposed to alcohol, som e chil dren hav e hippocampa l abnormalitie s includin g hy poplasia an d regiona l thinnin g (Autti-Ram o e t al , 2002). Moreover , i t appear s tha t th e hippocamp i i n these individuals is asymmetrical; specifically, the right hippocampus are significantly larger than the lef t hip pocampus (Riikone n e t al., 1999) . N o lateralizatio n i s evident in controls. In contrast, another study describes relative sparing of the alcohol-exposed hippocampu s i n an otherwise hypoplastic brain (Archibald et al., 2001). Attempts to replicate existin g MRI studies are neede d to clarify suc h conflicting findings. Particularly, longitudinal studie s will assis t i n assessin g developmental trends; i t i s difficul t t o meaningfull y compare cross sectional sample s that differ o n crucia l variables such as age.

Basal Gangli a MRI studies show that the volume of the basal ganglia is reduce d i n individual s prenatally expose d t o alco hol (Mattson et al, 1996) . Although both th e caudat e and the lenticular nuclei are reduced i n volume, only the caudat e i s reduced afte r brai n siz e i s taken int o account. A larger study that further delineate s subcortical structure s (Archibal d et al. , 2001 ) describe s sig nificant difference s i n childre n wit h FAS . N o differences ar e eviden t i n childre n wit h nondysmor phic FASD . Th e latte r dat a concu r wit h findings of a MR I analysi s o f th e basa l gangli a showin g n o abnormalities i n childre n wit h dysmorphi c an d nondysmorphic FASD (Autti-Ramo et al., 2002).

Optic Nerv e Eye abnormalities are frequently documented i n individuals wit h FAS . Optic nerv e hypoplasia i s the mos t frequent for m o f ocula r dysmorpholog y associate d with prenata l alcoho l exposur e (Stròmlan d an d Pinazo-Durân, 2002) . Ten o f 1 1 childre n diagnose d with FA S showed evidenc e o f optic nerv e hypoplasia when evaluated wit h MRI, ophthamological examinations, an d electroretinogra m (ERG ) (Hu g e t al. , 2000). In additio n to the structura l damage to the optic nerve , visua l acuit y was decreased i n al l bu t on e subject. Report s o f th e frequenc y o f visio n problem s in FA S vary ; ove r hal f o f th e childre n studie d i n

ALCOHOL AND THE DEVELOPIN G HUMA N BRAI N 14 9 a Swedis h sampl e ha d visua l impairment, an d >10 % showed sever e acuit y problems (Strômland , 1985 , i n Strômland an d Pinazo-Durân , 2002) . Thi s findin g indicates tha t prenata l alcoho l exposur e ha s detri mental consequence s o n th e developin g visua l system, a conclusion corroborate d b y ERG results . Ten of 1 1 subject s i n th e stud y b y Hu g an d colleague s (2000) ha d abnorma l ER G results , attributabl e t o a lack o f sufficien t retina l sensitivity . Data fro m oph thalmological studie s o f FA S subject s sugges t tha t ocular deficit s shoul d b e considere d amon g th e con stellation o f presentin g symptom s o f a n alcohol exposed individual . Thus, a n ey e examinatio n ma y be o f diagnosti c us e i n identifyin g individuals wit h prenatal alcoho l exposur e (Strômlan d an d Pinazo Durân, 2002).

OTHER IMAGING APPROACHES Ultrasonography In ultrasonography , sound wave s are recorde d t o pro duce image s of internal organ s and bod y tissues. This imaging modality 7 has been use d t o examine the con sequences o f prenata l alcoho l exposur e o n th e feta l development o f the fronta l cortex (Was s et al., 2001). The sampl e consist s o f 16 7 women , almos t hal f o f whom consume d varyin g amounts o f alcoho l whil e pregnant. Result s fro m thi s stud y represen t th e spec trum of fetal alcohol exposure effects; th e prospectively identified sampl e relie s o n neithe r spontaneousl y aborted fetuse s nor severel y affected childre n identi fied throug h retrospectiv e methods , scenario s tha t overrepresent case s o f heav y exposure . Th e fronta l lobe measuremen t wa s operationalized a s th e linea r distance fro m th e posterio r cavu m septu m pellu cidum to the inne r surface o f the calvarium . It should be note d tha t this measuremen t i s logically less com prehensive and precis e tha n are a or volume measure ments o f brai n structure . Regressio n analyse s wer e used t o determin e whic h o f several variable s studie d were predictiv e o f i n viv o feta l fronta l lob e size . I n general, alcoho l exposur e i s a significant predictor o f reduced fronta l lob e size . Interestingly , othe r brai n measurements take n fro m th e ultrasonographs , suc h as th e distanc e betwee n th e posterio r thalamu s an d the inne r calvarium, are less sensitive to alcohol exposure than th e fronta l cortex . There is an interaction between th e effec t of alcohol exposure on the developing frontal corte x and materna l

age (Was s e t al. , 2001) . Withi n th e alcohol-expose d women, a maternal age of greater tha n 30 years old increases the ris k of a fetus having a significantly smaller frontal lobe . Thi s findin g i s consistent wit h previou s research identifyin g advance d materna l ag e a s a ris k factor for having a child born with FAS (May and Gos sage, 2001) . Thus , i t appear s tha t th e deleteriou s ef fects o f alcoho l exposur e o n th e developin g fronta l cortex are exacerbated with increased materna l age. Emission Computed Tomography Emission compute d tomograph y (CT ) technologies , such a s positron emissio n tomograph y (PET ) an d sin gle photon emissio n compute d tomograph y (SPECT) , can b e use d t o measure cerebra l metabolism . Accord ingly, a radiotracer i s injected int o a subject's body an d images o f a nonsedate d individual , wh o migh t b e asked to perform a behavioral task, are taken. The im ages are reconstructed t o appreciate th e activity within nuclei o f the brain . Fo r the purpose s o f the researc h discussed i n th e presen t chapter , measurement s ob tained in this way are a proxy for brain function in that increased metaboli c rate s withi n a brai n regio n indi cate more neura l activity in that region . The effec t o f prenatal alcoho l exposur e o n brai n function wa s assesse d vi a PE T analysi s i n 1 9 youn g adults diagnose d wit h FA S (Clark e t al, 2000) . Glu cose metabolis m i n subcortica l areas , includin g th e thalamic an d caudat e nuclei , i s decreased . I n th e most sever e situations, ethanol cause s gros s structural deficits, wherea s othe r case s sho w subtl e effect s tha t vary b y regio n o r cel l type . These change s ar e remi niscent o f ethanol-induced reduction s i n glucose uti lization amon g specifi c subcortica l structure s i n th e rat brai n (Vinga n e t al. , 1986 ; Mille r an d Dow Edwards, 1993) . Thus , feta l alcoho l exposur e cause s a continuum o f damage to the developin g brain. SPECT has been use d t o evaluate th e brain func tion o f 1 1 childre n (mea n age , 8. 6 years ) wit h FA S (Riikonen e t al. , 1999) . Thes e childre n exhibi t mod erate decrease s i n cerebra l bloo d flo w i n th e lef t parieto-occipital region . Thi s i s consisten t wit h th e abnormalities identified by structural MRI studies. Altered functio n withi n thi s regio n ma y b e relate d t o difficulties tha t childre n wit h FA S hav e wit h arith metic an d speech . Additionally , th e sampl e o f chil dren wit h FA S show s a n asymmetrica l patter n o f frontal lob e perfusion, or the patter n i n which th e radiotracer i s incorporate d b y th e tissue . Specifically , the righ t frontal region showe d sligh t hyperperfusio n

150 ETHANOL-AFFECTE D DEVELOPMEN T compared t o the patter n i n the lef t region . This differ ence ma y relat e t o th e attentio n deficit s commonl y seen i n chidren wit h FAS ? although thi s findin g con trasts wit h th e hypoperfusio n i n th e lef t fronta l an d prefrontal lobe s o f children wit h attention defici t hy peractivity disorde r (Sie g e t al. , 1995 ; Ame n an d Carmichael, 1997 ; Langlebe n e t al. , 2001) . Notably , structural imagin g i n th e childre n wit h FA S showed that the brain s of 6 of the 1 1 subjects ha d gros s struc tural abnormalities , includin g cortica l atroph y an d anomalies of the corpu s callosum and cerebellum . A recen t SPEC T stud y evaluate d cerebra l bloo d flow i n thre e individual s diagnosed wit h FA S (on e child, on e adolescent , an d on e adult ) (Bhatar a et al., 2002). All three case s no t onl y have structura l abnor malities bu t als o reduce d cerebra l bloo d flo w i n th e temporal cortex . Simila r t o th e finding s o f Riikone n and colleague s (1999), the case study report noted lef t hemisphere hyperfusion , whic h i s consistent wit h lef t hemisphere dysfunction.

how structura l deficit s i n th e alcohol-expose d brai n relate t o neurobehaviora l outcome . Accordingly , a recent meetin g o f internationa l collaborator s high lighted th e critica l importanc e o f researc h focusin g on the relationship between brai n structure and func tion i n FAS D (Rile y et al., 2003). In particular, func tional imagin g studies will provide an i n vivo accoun t of ho w prenata l alcoho l exposur e affect s neura l re source allocation , o n a cognitiv e task-by-tas k basis. I t is possibl e tha t futur e researc h wil l sho w tha t a pathognomonic FAS D brai n doe s no t exist . Regard less of the wide-rangin g neura l complication s tha t fetal alcoho l effect s ma y exhibit , th e keyston e o f effective interventio n rests on explicatin g how behav ioral sequelae resul t from teratogeni c alteration o f the brain. Abbreviations ALC prenatall y expose d t o alcohol , bot h wit h and without dysmorphic features CNS centra l nervou s system

SUMMARY AND CONCLUSION S

Teratogenic exposur e t o alcoho l (1 ) cause s perma nent change s i n brai n structur e and (2 ) differentiall y affects specifi c components and region s o f the brain . These structura l change s likel y underli e deficit s i n the cognitiv e abilit y and adaptiv e functioning o f exposed individuals . Structura l brai n deficit s reveale d by quantitative imag e analyse s of alcohol-exposed in dividuals includ e reduction s i n overal l brain volum e and alteration s i n brai n shape , density , an d bilatera l symmetry. Additionally, some structures are especially vulnerable t o th e teratogeni c effect s o f alcohol , no tably the cerebellum , corpu s callosum , an d th e vari ous structures that make up the basal ganglia. From a neurobehaviora l poin t o f view, fetal alco hol effects compris e a continuum, resultin g in a range of devastatin g behaviora l an d cognitiv e disabilities . Targeted researc h definin g the degre e o f insul t t o a structure an d th e continuu m o f neurobehaviora l ef fects wil l be of great clinical utility. Given that individuals with historie s of heavy prenatal alcoho l exposur e face long-lastin g deficits affectin g multipl e aspect s o f cognitive and behaviora l functioning, identification of brain areas likely associated wit h such deficit s will improve diagnostic and treatment strategies. Multidisciplinary researc h i s needed t o fil l i n th e gaps in our current knowledge an d open new light on

CT compute d tomograph y FAS feta l alcohol syndrom e FASD feta l alcohol spectru m disorder s IQ intelligenc e quotien t LV laten t variable MRI magneti c resonanc e imaging PET positro n emission tomograph y PLS partia l least squares analysis SPECT singl e photo n emissio n compute d to mography VBM voxel-base d morphometry

ACKNOWLEDGMENTS Thi s wor k wa s supporte d b y the Nationa l Institutes of Heat h (AA01352 5 an d AA01 0417). References

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Sowell ER, Jernigan TL, Mattson SN, Riley EP, Sobel DF , Jones K L (1996 ) Abnorma l developmen t o f th e cerebellar vermis in childre n prenatall y exposed t o alcohol: siz e reductio n i n lobule s I-V . Alcoho l Clin Exp Res 20:31-34. Sowell ER , Mattso n SN , Thompso n PM , Jerniga n TL , Riley EP, Toga AW (200la) Mapping callosa l mor phology an d cognitiv e correlates : effect s o f heav y prenatal alcoho l exposure . Neurology 57:235—244 . Sowell ER , Thompso n PM , Mattso n SN , Tessne r KD , Jernigan TL , Rile y EP , Tog a A W (200Ib ) Voxel based morphometri c analyse s of the brai n i n chil dren an d adolescents prenatally exposed to alcohol. Neuroreport 12:515-523 . (2002a) Regiona l brai n shape abnormalitie s persist int o adolescenc e afte r heav y prenata l alcoho l exposure. Cereb Cortex 12:856—865 . Sowell ER , Thompso n PM , Peterso n BS , Mattson SN , Welcome SE , Henkeniu s AL , Rile y EP , Jernigan TL, Tog a A W (2002b) Mappin g cortica l gra y matter asymmetr y pattern s i n adolescent s wit h heav y prenatal alcoho l exposure . Neuroimag e 17:1807 1819. Streissguth AP , Conno r P D (2001 ) Feta l alcoho l syn drome an d othe r effec t o f prenatal alcohol : devel opmental cognitiv e neuroscienc e implications . In: Nelson ÇA , Lucian a M (eds) . Handbook o f Developmental Cognitive Neuroscience. MIT Press , Cam bridge, MA, pp 505-518 . Strômland K , Pinazo-Durâ n M D (2002 ) Ophthalmi c involvement i n th e feta l alcoho l syndrome : clini cal and anima l model studies . Alcohol Alcohol 37 : 2-8. Sutherland RJ, Weisend MP , Mumby D, Astur RS, HanIon FM , Koerne r A, Thomas MJ , Wu Y, Moses SN , Cole C , Hamilto n DA , Hoesing JM (2001 ) Retrograde amnesi a afte r hippocampa l damage : recen t vs. remot e memorie s i n tw o tasks . Hippocampu s 11:27-42. Swayze VW 2nd, Johnson VP , Hanson JW , Piven J , Sato Y, Gied d JN , Mosni k D , Andrease n NC . (1997 ) Magnetic resonanc e imaging of brain anomalies in fetal alcoho l syndrome. Pediatrics 99:232-240. Vingan RD , Dow-Edward s DL , Rile y E P (1986 ) Cere bral metaboli c alterations in rats following prenatal alcohol exposure : a deoxyglucos e study . Alcoho l Clin Exp Res 10:22-26. Wass TS, Persutt e WH, Hobbin s JC (2001 ) The impac t of prenatal alcoho l exposur e o n fronta l corte x de velopment i n utero . A m J Obste t Gyneco l 185 : 737-742. Wisniewski K , Dambska M , She r JH , Qaz i Q (1983 ) A clinical neuropathological study of the fetal alcoho l syndrome. Neuropediatrics 14:197-201 .

10

Prenatal Ethanol Exposure and Fetal Programming: Implications for Endocrine and Immune Development and Long-Term Health Joanna H. Sliwowska Xingqi Zhang Joanne Weinberg

Ethanol us e and abuse result in clinical abnormalities of endocrin e functio n an d neuroendocrin e regula tion (Morgan, 1982 ; Adler, 1992) . Direct and indirect effects o f ethanol o n man y hormone systems , including th e adrenal , gonadal, and thyroi d axes, as well as on aldosterone , growt h hormone , parathyroi d hor mone, calcitonin , insulin , and glucagon , hav e bee n reported. Secondar y complication s suc h a s live r disease, malnutrition , and othe r medica l condition s often presen t i n alcoholic s ma y i n an d o f themselves have endocrine consequences an d ca n potentially exacerbate the advers e effects o f ethanol. Whether ethanol-induce d endocrin e imbalance s contribute t o th e etiolog y o f feta l alcoho l spectru m disorders (FASD) is unknown, but i t is certainly a possibility (Anderson , 1981) . Th e effect s o f ethano l o n interactions betwee n th e pregnan t femal e an d fetu s are comple x (Rudee n an d Taylor , 1992 ; Weinberg , 1993a, 1993b , 1994) , and bot h direc t and indirec t effects o f ethanol o n feta l developmen t occur . Ethano l readily crosse s th e placenta , thu s directl y affectin g

developing feta l cell s and tissues , including those re lated t o endocrin e function . I n addition , ethanol induced change s i n endocrin e functio n ca n disrup t the hormona l interaction s betwee n th e pregnan t female an d feta l systems , altering the norma l hormon e balance an d indirectl y affecting th e developmen t o f fetal metabolic , physiological , an d endocrin e func tions. Ethanol-induce d change s i n metaboli c and/o r endocrine functio n can als o affect a female's ability to maintain a successful pregnancy , resulting in miscarriage or , i f the fetu s i s carried to term , possibl e con genital defects. Of particular relevance to the presen t Chapter i s the notio n tha t disturbance s of the recip rocal interconnection s betwee n (a ) th e pregnan t female an d fetu s o r (b ) th e materna l an d neona tal hypothalamic-pituitary-adrena l (HPA ) axes, ma y provide a common pathway by which perinatal exposure t o differen t agent s result s i n feta l programmin g (Angelucci e t al. , 1985) . Fetal o r early programming refers to the concept that earl y environmental or nongenetic factors , includin g pre - o r perinata l exposur e

153

154 ETHANOL-AFFECTE D DEVELOPMEN T to drugs or other toxic agents, ca n permanentl y organize o r imprin t physiologica l and behaviora l systems and increas e vulnerabilit y t o illnesse s o r disorder s later i n lif e (Matthews , 2000 , 2002 ; Bakke r e t al , 2001; Welberg and Seckl , 2001). The presen t Chapte r focuse s o n th e advers e ef fects of prenatal ethanol exposur e on neuroendocrin e and immun e function , wit h particula r emphasi s o n the concep t of fetal programmin g and th e HP A axis, a key playe r i n th e stres s response . Th e HP A axi s i s highly susceptibl e t o programmin g durin g feta l an d neonatal developmen t (Matthews , 2000 , 2002 ; Wel berg an d Seckl , 2001) . Earl y environmenta l experi ences, includin g exposure t o ethanol, ca n reprogra m the HP A axi s suc h tha t HP A ton e i s increase d throughout life . Thi s chapte r present s dat a demon strating tha t gestationa l ethano l exposur e increase s HPA activity in bot h th e pregnan t femal e and th e off spring. Evidenc e suggestin g tha t increase d exposur e to endogenou s glucocorticoid s ove r th e lifespa n ca n alter behavioral and physiologica l responsiveness an d predispose th e organis m t o developmen t o f certai n diseases late r i n lif e i s als o described . Alteration s i n immune functio n may be one o f the consequence s o f fetal HP A programming. Th e chapte r discusse s stud ies demonstratin g tha t ethano l i s a n immunoterato genic agen t an d tha t programmin g o f HP A activity may mediat e som e o f the advers e effect s o f prenata l ethanol exposur e o n immun e competenc e i n late r life.

THE CONCEP T O F STRESS

The ter m stress, used i n the biologica l sense, was popularized b y Hans Selye , who propose d tha t stres s ca n be understoo d withi n th e contex t o f th e "genera l adaptation syndrome. " Thi s concep t gre w ou t o f Selye's observation s tha t a wid e variet y of physically noxious stimuli , suc h a s col d o r hea t exposure , sur gery, muscula r exercise , bacteria, toxins , o r X-irradiation, resulted in essentially the same triad of symptoms: (1) adrena l cortica l enlargement , (2 ) thymi c involu tion o r atrophy, and (3 ) gastrointestinal ulcer s (Selye , 1936, 1950) . The respons e tria d hold s despit e th e fac t tha t a highly specific adaptiv e response exist s for any on e o f these agent s by itself. These symptoms are considere d a nonspecific adaptive response o f the bod y to a physical stresso r and par t o f the "alar m reaction, " th e first

stage o f the genera l adaptatio n syndrome . This stag e is characterized b y activation o f the HP A system, an d the correspondin g change s i n immun e functio n an d gastric ulcération ar e thought to be mediated t o som e extent b y this HPA activation. I f the stressfu l stimulu s persists, the organis m enters the second, o r resistance, phase o f th e genera l adaptatio n syndrome , durin g which adrenocortica l activatio n i s maintained . Afte r repeated o r constan t exposur e t o th e stresso r th e or ganism enter s th e thir d phase , whe n th e capacit y o f the adrena l corte x t o synthesize , store , an d secret e glucocorticoids i s exceede d (Selye , 1946) . Althoug h not all aspects of the general adaptatio n syndrom e ar e supported b y subsequent experimenta l evidence , thi s concept has provided a powerful context fo r stress research fo r many years. The concep t o f nonspecificity (Sely e 1936 , 1950 ) has raise d a critica l question : throug h wha t mecha nisms coul d s o man y divers e agent s transmi t th e common "message " of stress ? Sely e speculate d tha t there mus t b e som e physiologica l "firs t mediator " o f stress, i.e. , som e nonspecifi c chemica l o r byproduc t of biological reaction s tha t produced thes e symptom s indicative o f stres s (Mason , 1975) . Th e searc h fo r physiological firs t mediator s wa s largel y unproduc tive. B y the 1960s , stres s researchers becam e increas ingly awar e tha t (a ) psychologica l an d socia l factor s had effect s simila r to an d perhap s eve n mor e poten t than thos e o f physical stressor s and (b ) that th e HP A axis wa s particularl y sensitive t o thes e psychologica l stimuli (Mason , 1968) . Thi s growin g understandin g of th e rol e o f psychologica l variable s le d Maso n (1975) t o sugges t tha t th e unrecognize d firs t media tors i n man y o f Selye' s experiment s ma y hav e bee n the substrat e i n the centra l nervou s system (CNS ) in volved i n emotional arousal . That HPA activation frequently occurred durin g novel o r aversive stimulation is no t surprisin g because th e HP A axis i s an excellen t indicator o f arousa l (Henness y an d Levine , 1979) . This understandin g altere d th e concep t o f nonspeci ficity, at least in terms of the HP A response. Instea d of viewing hormona l response s a s bein g elicite d b y a great diversit y of stimuli, they coul d b e viewe d as being elicited largel y by the emotiona l arousa l or activation commo n t o mos t i f no t al l nove l an d aversiv e situations. A concept o f stress emphasizing th e "emergency " function o f the sympatheti c nervou s syste m an d th e adrenal medull a an d thei r role s i n maintainin g in ternal homeostasi s come s fro m th e wor k of Cannon

ETHANOL EFFECT S O N ENDOCRIN E AND IMMUNE FUNCTIO N 15 5

FIGURE 10- 1 Schemati c representatio n o f circuitry of the stress system. The tw o major system s central to the stress respons e ar e th e hypothalamic-pituitary-adrena l (HPA) axis and the locus coeruleus noradrenergi c sympathetic adrena l medullary (LC-NE) system , which in teract t o maintain homeostasis . Th e HP A axis consists of a cascade o f responses. Corticotropin-releasin g hor mone (CRH) , secrete d b y th e hypothalamus , stimu lates th e releas e o f adrenocorticotropi c hormon e (ACTH) fro m th e anterio r pituitary , whic h i n tur n stimulates release of the glucocorticoid hormone s fro m the adrena l cortex . Th e glucocorticoid s fee d bac k a t multiple levels of the axi s to inhibit activity. In the LC NE system , norepinephrin e (NE) , release d primarily from sympatheti c nerv e terminals , an d epinephrine , released primaril y from th e adrena l medulla , activat e sympathetic responses. Homeostasis is restored by activation of the parasympatheti c system. The LC-N E system allow s th e organis m t o reac t rapidly , whil e th e HPA hormones act over a longer time frame. There are intimate reciproca l interaction s between th e HP A axis and th e LC-N E system , a s well a s reciproca l neura l connections betwee n th e tw o systems. CR H an d N E stimulate each other , and the two systems are regulated by similar neurotransmitter s and b y mesocortical an d mesolimbic influences . Glucocorticoid s ar e though t to restrai n both system s to preven t th e consequence s of prolonge d o r excessiv e activation . Finally , activit y and sensitivit y of both system s are modulate d b y stress and circadia n influences . AVP, arginin e vasopressin; BZD, benzodiazepine ; GABA , y-aminobutyri c acid ; NPY, neuropeptid e Y ; POMC, proopiomelanocortin ;

and colleague s (Canno n 1914 , 1929 ; Canno n an d de l a Paz, 1911) . Accordingly, homeostasi s i s viewed as th e operatio n o f coordinate d physiologica l pro cesses that maintain the stead y state of the organism . These authors also recognize th e importanc e o f psychological ove r physica l stimul i i n elicitin g a stres s response. In moder n stres s research , i t i s no w widel y ac cepted tha t th e respons e t o stress is mediated b y both the HP A axis and th e locu s coeruleu s noradrenergi c sympathetic adrena l medullar y system (referre d t o as the LC-N E system) and tha t these system s interact to maintain homeostasi s (Fig . 10-1) . Th e LC-N E system i s involve d i n th e "fight-or-flight " respons e an d enables th e organis m t o reac t rapidly . Tw o majo r players i n thi s rapi d respons e ar e norepinephrin e (NE), secreted fro m sympatheti c nerve terminals, and epinephrine, release d fro m th e adrena l medulla . I n contrast, th e HP A axis act s ove r a longe r tim e fram e and help s orchestrat e th e respons e an d adaptatio n of the bod y to the stresso r through variou s physiological and metabolic changes . The ter m stress ha s prevaile d ove r decade s be cause i t attempt s t o addres s a basi c principl e o f na ture: (1 ) the maintenanc e o f balance, equilibrium , or harmony i n th e fac e o f disturbin g stimul i an d (2 ) the counteracting , re-establishin g response s tha t re establish homeostasi s (Chrouso s e t al. , 1988) . Man y definitions an d meaning s hav e bee n an d stil l ar e ascribed t o the term stress, largely because the ter m ha s been use d t o refe r t o the disturbin g stimuli, the stat e of disturbe d equilibrium , and/o r th e result s o f th e counteracting responses . Thi s chapte r use s th e fol lowing definition s (Johnso n e t al. , 1992 ; Mille r an d O'Callaghan, 2002). Stress is a state of threatened in ternal balance o r homeostasis. The threatenin g or disturbing force s ar e define d a s stressors. Thes e ca n range from rea l threats to survival (e.g., immune chal lenges o r physical stressors) to perceived threat s (e.g. , psychological o r socia l stressors) . The counteractin g forces activate d t o neutraliz e th e effect s o f a stressor are adaptive responses, which ca n b e both behaviora l and physica l o r physiological , an d serv e t o re establish homeostasis.

SP, substanc e P . Activatio n i s represente d b y soli d lines, inhibitio n b y dashe d lines . (Source: Reprinte d from Chrouso s (1998) with permission fro m Ne w York Academy of Sciences)

156 ETHANOL-AFFECTE D DEVELOPMENT In recen t years , th e concept s o f allostasis and al lostatic loa d hav e been adde d t o the stres s literatur e and hav e extende d ou r thinkin g about homeostasi s (McEwen, 1998 ; McEwe n an d Wingfield , 2003) . In this framework , th e ter m homeostasis refer s t o th e stability o f physiologica l system s tha t ar e essentia l for lif e an d applie s strictl y to a limite d numbe r o f systems, suc h a s pH , bod y temperature , glucos e contents, an d oxyge n tension , tha t ar e maintaine d within narro w ranges optimal fo r current lif e condi tions. I n contrast , th e ter m allostasis mean s th e achieving o f stability throug h chang e an d refer s t o systems suc h a s th e HP A axis, catecholamines , an d cytokines tha t maintai n homeostati c system s in bal ance. Thes e system s ar e typicall y no t require d fo r immediate surviva l an d hav e muc h broade r bound aries tha n thos e o f homeostatic systems . Further, a s environments o r lif e circumstance s change , th e se t points o r boundaries o f these allostati c systems ma y also change . Allostatic system s enabl e u s t o respon d t o ou r physical state s —for example , awake , asleep , exer cising, etc . —and t o cop e wit h challenge s suc h a s noise, crowding , isolation, extremes o f temperature, danger, an d infectio n (McEwen , 1998) . Th e bod y responds t o challenge s b y turnin g o n allostati c re sponses (mos t commonly , fo r example , activatio n of th e HP A axi s and/o r LC-N E system ) tha t initi ate comple x adaptiv e response s an d the n shuttin g off these response s when th e threa t i s past. The ter m allostatic state refer s t o altere d an d sustaine d ac tivities o f the primar y respons e mediators , th e HP A and catecholamin e hormones , tha t integrat e phys iological an d behaviora l response s t o challenge . Allostatic state s ca n b e sustaine d fo r limite d peri ods, however , i f inactivation i s inefficient o r th e or ganism i s exposed repeatedl y t o intens e challenges . The cumulativ e resul t o f a n allostati c stat e i s allo static load , whic h ca n hav e pathophysiologi c con sequences. The HP A Axis and p-Endorphin Syste m The HP A axi s include s a numbe r o f brai n areas , such a s the paraventricula r nucleus o f the hypothal amus (PVN) , the anterio r pituitary, and adrena l cortex, that act together t o produce a hormonal cascad e in respons e t o stressor s (Fig . 10.1) . Corticotropin releasing hormone (CRH ) an d arginin e vasopressin

(AVP) are synthesized in th e parvocellula r PVN an d released int o th e media n eminence , reachin g th e anterior pituitar y via the hypophysia l portal system. CRH an d AV P act synergisticall y at th e anterio r pituitary t o stimulat e th e synthesi s o f proopiome lanocortin (POMC) , a large precursor hormone tha t when cleave d result s i n th e subsequen t releas e o f adrenocorticotropic hormon e (ACTH ) an d th e en dogenous opioi d p-endorphi n (p-EP ) (Guillemi n et al, 1977 ; Gillie s e t al., 1982) . ACTH the n act s at the adrena l corte x t o stimulat e th e synthesi s an d release o f glucocorticoid s —cortisol i n human s an d corticosterone (CORT ) i n rats . In additio n to a variety o f importan t physiologica l an d metaboli c func tions, th e glucocorticoid s hav e a negativ e feedbac k action, causin g inhibitio n o f HP A activit y b y act ing a t th e anterio r pituitary , th e PVN , an d othe r brain regions , particularl y th e hippocampu s an d prefrontal cortex . Durin g norma l endocrin e adapta tion t o stressors , CRH , ACTH , an d glucocorticoi d concentrations ar e kep t withi n level s tha t promot e health. Whe n copin g fails , a n imbalanc e develop s between driv e an d feedbac k mechanisms , result ing i n th e releas e o f too muc h o r to o littl e o f thes e hormones an d increase d vulnerabilit y t o diseas e (Chrousosetal, 1988) . The Concept of Fetal Programmin g Both epidemiologica l dat a an d experimenta l stud ies sugges t tha t environmenta l factor s operatin g early in lif e markedl y affect developin g systems , per manently alterin g structur e an d functio n through out lif e (Fig . 10-2) . Althoug h th e ide a tha t earl y experiences ca n hav e long-ter m effect s i s no t new , it i s reall y onl y i n th e pas t decad e o r s o tha t th e concept o f fetal o r earl y programming ha s emerge d as a ke y concep t i n development . Thi s concep t arises fro m a larg e bod y o f dat a showin g tha t lo w birth weigh t an d othe r indice s o f feta l growt h ar e associated wit h increase d biologica l ris k fo r coro nary hear t disease , hypertension , an d typ e I I dia betes o r impaire d glucos e toleranc e i n adul t life . Adult lifestyl e factor s suc h a s smoking , ethano l consumption, an d exercis e appea r t o b e additiv e to earl y lif e influences , thu s th e earl y lif e effect s may hav e distinc t role s an d causes . Thes e find ings hav e le d t o th e hypothesi s tha t commo n adult disease s migh t originat e durin g feta l devel -

ETHANOL EFFECTS ON ENDOCRIN E AND IMMUNE FUNCTIO N 15 7 opment, i.e. , th e "feta l origin s o f adul t disease " hypothesis. The biologica l significanc e o f physiological an d behavioral programmin g i s not known . I t ha s bee n suggested tha t environmenta l factor s actin g o n th e pregnant femal e an d fetu s ca n alte r th e se t point o r responsiveness of physiological systems and thu s prepare th e organis m fo r the environmen t int o whic h it will be bor n an d develop . If , however, this process is initiate d b y advers e factor s durin g pregnanc y (e.g., placenta l insufficienc y o r prenata l ethano l exposure) o r i f th e environmenta l circumstance s later i n lif e diffe r fro m wha t wa s anticipated , the n the physiologica l adaptation s migh t alon e resul t i n maladaptive response s an d ultimatel y predispos e the organis m t o diseas e (Welber g an d Seckl , 2001 ; Matthews, 2002). The underlyin g processe s tha t lin k earl y growth restriction with these long-ter m healt h consequence s are no t full y understood . I t i s generall y accepte d that lo w birt h weigh t pe r s e i s unlikel y t o caus e

these increase d risks for disease. Rather, birth weight likely serve s as a marke r fo r th e effect s o f earl y lif e events, an d commo n factor s probabl y underlie bot h the intrauterin e growth retardation and altered phys iological responsivenes s (Welber g an d Seckl , 2001) . The resettin g of key hormonal system s by early environmental event s ma y be on e mechanis m linkin g early lif e experience s wit h long-ter m healt h conse quences. Studie s have identified the HP A axis as one of th e ke y system s likel y involve d (Fig . 10-2) . Th e HPA axis is highly susceptible t o programming during development (Phillip s e t al. , 1998 , 2000 ; Matthews , 2000, 2002 ) an d studie s demonstrat e stron g correla tions betwee n birt h weight , plasm a cortiso l concen trations, an d th e developmen t o f hypertensio n an d type I I diabetes. Thus , i t has been suggeste d tha t in trauterine programmin g o f the HP A axis i s a mechanism underlyin g th e observe d association s betwee n low birth weigh t an d increase d ris k for disease. I n or der t o understan d feta l programmin g mor e fully , it is important to describe how the HP A axis develops.

FIGURE 10- 2 Route s b y whic h th e feta l an d earl y neonata l environmen t can progra m adul t hypothalamic-pituitary-adrena l (HPA ) functio n an d behavior. Th e developin g linibi c system , hypothalamus , an d pituitar y synthesize hig h level s of corticosteroid receptor s and ar e sensitive t o glucocorti coids (GCs) . Earl y exposure to GC s alter s th e developmen t an d subsequen t activity an d functio n o f bot h th e limbi c syste m an d th e HP A axis . I n th e periphery, th e overal l effec t o f programmin g durin g developmen t i s al tered exposur e t o endogenou s GC s throughou t life . Increase d exposur e ha s long-term effect s o n behavior , cognition , learning , an d memory , an d pre disposes th e individua l t o neurological , metabolic , an d immun e disease s and disorder s i n late r life . Conversely , i f programming reduce s exposur e t o GCs ove r th e lifespan , thi s migh t ac t t o protec t agains t thes e pathologica l changes. (Source: Reprinte d fro m Matthew s (2002 ) wit h permissio n fro m Elsevier)

158 ETHANOL-AFFECTE D DEVELOPMEN T DEVELOPMENT OF TH E HP A AXIS

The HPA Axis in Human Developmen t As a result of the difficult y o f sampling an d measurin g ACTH, few data exis t on regulatio n o f ACTH secre tion i n the huma n fetus . By 9 weeks of gestation, th e human feta l pituitar y contain s measurabl e corti cotropic activit y and the content and concentration o f ACTH increase s wit h gestationa l ag e (Kasti n e t al. , 1968; Pavlov a et al, 1968) . B y 12 weeks of gestation, ACTH is detectable b y radioimmunoassay in huma n placenta. ACT H concentratio n i s hig h prio r t o 3 4 weeks an d the n fall s significantl y i n lat e gestatio n (Winters e t al, 1974) . Infusio n o f ACTH to the preg nant female at term doe s not affect the concentration s of plasma ACTH in the fetus, providing evidence tha t this hormone doe s no t cros s the placent a (Miyakaw a et al, 1976) . In contras t t o ACTH , concentration s o f cortiso l are hig h i n th e bloo d o f th e pregnan t woma n an d there i s transplacental transfe r o f cortisol (Pasqualini et al. , 1970) . Feta l exposur e t o cortiso l i s minimized , however, because cortiso l is rapidly inactivated by the placental enzym e 1 1 p-hydroxysteroid dehydrogenas e type 2 (Seckl , 2004a , 2004b) . Abou t 80 % o f cortisol derived fro m th e pregnan t femal e i s converte d t o CORT (Beitin s e t al. , 1973 ; Murph y e t al , 1974) . The remainin g fractio n make s a n importan t contri bution t o feta l plasm a cortisol . Childre n expose d t o dexamethasone ( a syntheti c glucocorticoid ) durin g early pregnancy becaus e o f a ris k of congenital adre nal hyperplasia show increased emotionality , unsociability, avoidance, and behavioral problem s (Trautma n et al. , 1995) . Th e rol e o f prenatal glucocorticoid s i n animal model s o f FAS D i s discusse d i n th e sectio n Prenatal Ethano l Exposur e Alters th e Immun e Sys tem o f Offspring (below) . The HP A Axis in Rodent s Studies on rat s show that CORT secretion i n respons e to stressor s i s ver y hig h durin g th e lat e feta l perio d (Milkovic and Milkovic , 1963) . I n contrast, beginnin g on abou t postnata l da y (P) 2 and continuin g int o th e second wee k of life, the HP A response i s markedly suppressed i n ra t pups. The pioneerin g wor k of Schapir o (1962) identifie s thi s developmenta l stag e an d call s it th e "stres s nonresponsiv e period " (SNRP) . Late r

studies sho w tha t HP A responsivenes s i s no t actuall y abolished durin g thi s perio d bu t simpl y suppressed , and thi s perio d ha s bee n rename d th e "stres s hypo responsive period " (SHRP) . Th e SHR P i s character ized b y blunte d adrena l COR T secretion , despit e normal pituitar y ACTH response s to a variety of stressors. Hypothalamic CRH conten t is decreased t o about 20% o f that foun d i n 21-day-ol d animals , and ther e i s also a decrease i n functionally active glucocorticoid re ceptors t o about 20 % of those foun d i n adult s (Levine et al, 1967; Meane y e t al, 1985a, 1985b ; Walker et al, 1986a, 1991 ; Witek-Janusek, 1988) . This blunted basal and stimulate d COR T secretion i s believed t o be du e in par t t o a decreas e i n adrena l sensitivit y to ACTH (Levine et al., 1967 ) an d increas e in sensitivity to HPA feedback (Walke r e t al, 1986b) . Th e lo w circulatin g glucocorticoid concentration s durin g th e SHR P ap pear to have a protective functio n and are essential for normal brai n and behaviora l development (Rosenfel d et al , 1992 ; Walke r e t al , 2002) . Fo r example , rat s treated wit h glucocorticoid s durin g th e firs t wee k o f life hav e reduce d brai n weights , alteration s i n neu rons (e.g. , reduced number s o f dendritic spines), glia, and myelin , an d behaviora l change s (Sapolsk y an d Meaney, 1986) . Similarly, CORT treatment durin g the first postnata l wee k result s i n lowere d adul t basa l CORT concentrations (Turne r an d Taylor , 1976 ) an d neonatal treatmen t wit h ACTH o n P7-P9 an d P I 7P19 suppresses th e COR T circadia n rhyth m i n adul t rats (Taylo r e t al. , 1976) . Th e presenc e o f th e da m appears t o b e critica l fo r maintaining th e SHRP , an d separation fro m th e mothe r "releases " th e pu p HP A axis from inhibition . I n 10-day-ol d pups , fo r example , maternal separatio n (removin g activ e sensory stimula tion, food , an d passiv e contact ) enhance d basa l an d stimulated ACTH and CORT secretion afte r exposur e to ethe r vapor s an d insulin-induce d hypoglycemi a (Walker, 1995) . Thu s i t appear s tha t th e firs t 2 post natal weeks represent a critical period correspondin g to specific stages in the normal developmen t o f hypothalamic an d forebrai n structure s tha t modulat e ACT H and COR T secretio n a s wel l a s CN S structure s in volved in circadian regulation. By weaning age (P21-P25), the plasma ACTH and CORT responses t o stressors appear to be at essentially adult concentrations. Measures o f CRH hav e shown a similar developmenta l pattern , with adul t responsive ness bein g reache d b y weanin g ag e (Hiroshig e an d Sato, 1970) . I n contrast , negativ e feedbac k mecha nisms continu e t o increas e i n effectivenes s betwee n

ETHANOL EFFECTS ON ENDOCRINE AND IMMUNE FUNCTIO N 15 9 weaning ag e and adulthood . Whe n expose d t o ethe r or shock, for example, the plasm a CORT response of the weanling shows a later peak and delaye d return t o resting concentration s compare d with th e patter n i n adulthood (Goldma n et al, 1973). In summary, in the rat , the postnatal perio d i s critical fo r the developmen t an d integratio n of the CN S and peripheral nervous system mechanisms necessary to maintai n homeostasis. I n contrast , man y o f thes e systems reach a greater degree o f organization at birth in guine a pigs , sheep, nonhuma n primates , and hu mans (Weaver et al., 2004).

GESTATIONAL ETHANOL

EXPOSURE AND HP A ACTIVITY

Human Studies Compared t o the fairl y larg e literature o n th e effect s of ethanol consumption on th e HP A axis of adults following acut e o r chroni c ethano l intake , onl y a fe w human studie s have examined the effect s o f drinking during pregnanc y o n th e HP A axi s o f th e develop ing child . I n cas e studie s b y Roo t an d colleague s (1975), plasma cortisol concentrations are within normal rang e i n childre n wit h FASD . B y contrast, dat a from Jacobso n and colleague s (1999 ) sho w that heavy 7 drinking during pregnancy and a t conception ar e associated with higher basal and post-stress (blood draw) cortisol concentrations i n 13-month-ol d infants . Basal cortisol concentrations are also higher in 2-month-old infants expose d i n uter o t o ethano l o r cigarette s (Ramsay et al., 1996) . Animal studies strongly support this finding that ethano l exposur e i n uter o ca n hav e major effect s o n developmen t an d functio n o f th e offspring HP A axis. Not surprisingly , HPA activity in the pregnan t femal e i s als o altere d b y ethano l consumption. Animal Studie s Effects of Ethanol Consumption on HPA Activity of the Pregnant Female Data fro m roden t model s indicat e tha t gestationa l ethanol consumptio n increases adrenal weights, basal CORT concentrations , th e COR T stres s response , and the CORT stress increment i n the pregnant dam (Weinberg and Bezio , 1987) . Thi s occur s a s early as

gestational da y (G) 11 , persists throughout gestation , may increase as gestation progresses, and occur s even with low concentrations o f ethanol i n the die t (Wein berg and Bezio , 1987 ; Weinber g and Gallo , 1982) . Importantly, thi s activatio n appear s t o b e relate d t o ethanol rathe r tha n t o nutritional factor s a s materna l nutritional status has no major impac t on these effect s (Weinberg and Bezio , 1987) . The stimulator y effect s of ethanol o n basa l hormone concentration s and th e corticoid response to stressors also can extend throug h parturition, eve n whe n ethano l administratio n i s discontinued prio r t o parturitio n (Weinberg , 1989) . Thus regula r consumptio n o f high dose s o f ethano l during pregnancy no t only raises the se t point of HPA function i n th e pregnan t femal e b y increasin g bot h basal an d stres s concentration s of CORT but als o results in HPA hyperresponsiveness to stressors. The ad ditional finding that binding capacity of corticosterone binding globuli n (CBG ) i n ethanol-consumin g fe males is similar to or less than tha t in control females, both durin g pregnanc y an d a t parturition , indicate s that the elevate d CORT concentrations are functionally importan t an d support s th e conclusio n tha t pre natal ethano l exposur e results in both hypersécrétio n and hyperresponsivenes s of the HP A axi s (Weinber g and Bezio , 1987 ; Weinberg, 1989) . The pregnan t femal e an d th e fetu s constitut e a n interrelated functiona l unit. Hence , ethanol-induced alterations i n HP A activit y i n th e pregnan t femal e have implication s for fetal HPA development. COR T crosses the placenta , a t least to some extent (Eguchi , 1969), resultin g i n suppressio n o f endogenou s feta l HPA activity. At the sam e time, ethano l ca n cros s the placenta an d directl y activate the feta l HP A axis. Together thes e influence s may have permanen t organi zational effect s o n neura l structure s tha t regulat e HPA activit y throughou t lif e (Levin e an d Mullins , 1966). Whether th e increased plasma CORT concen tration i n pregnan t female s plays a role i n mediatin g HPA hyperresponsivenes s i n feta l ethanol-expose d (FEE) offsprin g i s as yet unresolved . Adrenalectom y (ADX) o f the pregnan t da m ha s n o effec t o n th e in creased CORT responses to restraint stress in FEE fe male offsprin g (Slon e an d Redei , 2002) . Similarly , CORT treatmen t o f ADX dams doe s no t mimi c th e effect o f prenatal ethano l exposure on HPA activity of offspring (Le e and Rivier , 1992 ) On th e basi s of these data, i t appear s tha t th e increase d stres s responsive ness of FEE offsprin g i s not mediate d primaril y by increased COR T concentration s i n pregnan t females .

160 ETHANOL-AFFECTE D DEVELOPMENT In contrast , ADX of the pregnan t femal e can reverse the effect s o f prenatal ethanol exposur e o n pituitar y POMC mRN A concentrations in FE E offsprin g (Re dei e t al. , 1993) . Furthermore , Trit t an d colleague s (1993) have suggested that removal of the adrena l cortex, but no t the medulla , in the pregnan t femal e prevents th e growth-retardin g effect s o f prenatal ethano l and ma y reverse the dela y in postpartu m weight gain in FE E offspring . Thu s the advers e effect s o f ethano l on birth weight are mediated at least partially through the adrenal gland. Further studie s must be done to resolve the rol e of corticosteroids derive d fro m th e preg nant female in mediating the adverse effects o f ethanol on thei r offspring. I t is likely, however, that a comple x interaction betwee n direc t an d indirec t effect s o f ethanol mediates the adverse consequences o f ethanol consumption durin g pregnancy on fetal developmen t and programmin g of fetal HP A activity. Effects of Prenatal Ethanol Exposure on HPA Activity in Offspring The Preweanin g Period . Th e comple x interactio n of direct and indirec t effect s o f ethanol i s apparent i n the offsprin g followin g parturition. FE E fetuse s exhibit decrease d COR T concentration s compare d t o control fetuse s on G19 (Revsko y et al., 1997) . I n con trast, a t birth , FE E neonate s hav e elevate d plasm a and brai n content s o f CORT, decrease d CB G bind ing capacity, elevated plasma and pituitary concentrations of p-EP, and reduce d pituitary concentrations of p-EP (Kakihan a et al, 1980 ; Taylor et al, 1983; Weinberg et al., 1986; Angelogianni and Gianoulakis, 1989; Weinberg, 1989). Throughout the preweaning period, FEE offsprin g exhibi t blunte d HP A an d p-E P re sponses t o a wid e range o f stressors, including ether , novelty, saline injection, and col d stres s (Taylo r et al., 1986; Weinber g e t al , 1986 ; Angelogiann i an d Gi anoulakis, 1989 ; Weinberg , 1989) . In addition, prenatal ethano l exposur e alters the ontogeneti c expressio n of mRNA s fo r CRH an d POMC , delayin g and exag gerating the rise in CRH expressio n in female (but not male) pups, and suppresses POMC mRNA concentrations i n mal e (bu t no t female ) pup s throughou t th e preweaning period (Aird et al, 1997) . The implicatio n is that sexually dimorphic effects o f prenatal ethanol exposure on these two important glucocorticoid-regulated genes contribute to both the immediate an d the longterm effect s o f prenatal ethano l o n stres s responsive ness of the offspring .

The significanc e o f the reduce d hormonal responsiveness described i n FEE pup s during early development remain s to b e determined . Evidenc e indicate s that i n additio n t o th e reduce d adrenocortica l re sponses t o stressors , plasma CBG bindin g capacity is also reduced i n FE E compare d to that in controls, a t least durin g th e firs t wee k o f lif e (Weinberg , 1989) . Thus i t is possible that althoug h th e COR T stress response i s reduced i n FEE pups , the rati o of bound-to free steroid s ma y no t b e altered . Th e reductio n i n CBG bindin g capacity may represent a compensatory response t o maintai n norma l neuroendocrin e func tion i n FE E offspring . Thi s possibilit y remains to b e tested. Long-Term Effect s o f Prenata l Ethano l Exposure . Importantly, th e reduce d HP A an d p-E P respon siveness i n FE E ra t pup s earl y i n lif e i s a transien t phenomenon. Followin g weaning , FE E rat s are typically hyperresponsive to stressors and t o drugs such as ethanol and morphin e (Taylor et al., 1988 ; Weinberg, 1993a, 1993b ; Ki m e t al , 1996 ; Le e e t al , 1990 , 2000). Simila r result s hav e bee n describe d i n non human primate s (Schneide r e t al. , 2004) . I n rhesu s monkeys, prenatal exposur e to moderat e amount s of ethanol induce d highe r plasm a ACT H an d margin ally highe r plasm a cortiso l response s t o th e stres s of maternal separation. An interestin g findin g fro m roden t model s i s that the sexua l dimorphis m i n th e effect s o f prenata l ethanol exposur e see n durin g th e ontogen y o f th e HPA axis (Aird et al, 1997 ) extend s int o adulthood . Sex differences i n response s of male an d femal e offspring compare d t o thos e o f thei r contro l counter parts ar e ofte n observe d an d ma y var y depending o n the natur e o f th e stresso r an d th e tim e cours e an d hormonal en d poin t measure d (Weinberg , 1988 , 1992a, 1995 ; Halas z e t al , 1993 ; Le e an d Rivier , 1996). Fo r example , i n adulthood , FE E male s an d females exhibi t increase d CORT , ACTH, and/o r p EP response s t o stressors , such a s repeated restraint , footshock, an d immun e challenge s (Taylo r e t al. , 1988; Weinberg, 1993b ; Ki m et al, 1996 , 1999b ; Lee and Rivier , 1996 ; Weinber g e t al, 1996 ; Le e e t al. , 2000). Bot h male s an d female s sho w increase d ex pression o f mRNA s fo r immediat e earl y genes an d CRH followin g stressors (Le e e t al. , 2000 ) a s well as deficits i n habituatio n t o repeate d restrain t (Wein berg e t al , 1996) . I n contrast , i n respons e t o pro longed restrain t o r col d stress , HP A hyperactivit y is

ETHANOL EFFECTS O N ENDOCRIN E AND IMMUNE FUNCTIO N 16 1 seen primarily in FEE male s (Weinberg , 1992a ; Kim et al. , 1999a) , wherea s i n respons e t o acute restrain t or acut e ethano l o r morphin e challenge , increase d CORT and ACTH concentrations occur primarily in FEE female s (Taylo r e t al , 1982 , 1988 ; Weinber g et al. , 1986 ; Weinberg , 1988) . Similarly , altered re sponses i n a consummator y tas k (Weinberg , 1988 ) and t o predictable an d unpredictabl e restrain t stres s (Weinberg, 1992b ) sugges t deficit s i n th e abilit y of FEE female s but no t males to use or respond to environmental cues. Possible Mechanisms Mediating Ethanol-Induced HPA Hyperresponsiveness The mechanism s underlyin g HP A hyperrespon siveness i n FE E offsprin g ar e unknow n a t presen t but coul d reflec t change s a t all levels of the axis . In creased HP A activit y coul d resul t fro m increase d secretion o f secretagogues (e.g. , POMC, CRH , an d AVP), increase d pituitar y and/o r adrena l respon siveness t o thes e secretagogues , increase d driv e t o the hypothalamus , deficit s in feedbac k regulation of HPA activity , and/o r alteration s i n centra l neuro transmitters regulatin g th e HP A axis . Indeed , i t i s likely tha t multipl e mechanism s pla y a role . Fur thermore, the finding that prenatal ethano l exposur e differentially alter s HP A responsivenes s i n male s and females compare d t o that in their contro l coun terparts suggest s tha t th e gonada l hormone s o r al tered adrenal-gonadal interaction s play a significan t role i n mediatin g prenata l ethano l effect s o n HP A activity. This chapter focuses on fou r possibl e mechanisms considere d importan t i n mediatin g HP A hyperresponsiveness in FE E animals : (1 ) alterations i n HPA drive , (2 ) alteration s i n HP A feedback regula tion, (3 ) altered neurotransmitte r regulatio n o f HPA activity, an d (4 ) possibl e interaction s betwee n th e HPA an d hypothalamic-pituitary-gonada l (HPG )

Altered HPA Drive and/or Feedback A numbe r o f studie s sho w tha t stimulator y inputs o r drive to the PVN of the hypothalamus are enhanced by prenatal ethano l exposure . Weanling FE E mal e an d female pup s exhibi t increased basal concentrations of CRH mRN A in the PV N (Lee et al., 1990) , and adul t FEE male s hav e increase d basa l concentration s o f both hypothalami c CRH mRN A and pituitary POMC

mRNA (Redei et al., 1993 ) compare d t o those of controls. Not all studies, however, show these alterations i n central HP A regulation o f basal activity. Kim an d col leagues (1996) reported no differences i n basal CRH o r AVP mRN A concentration s amon g adul t FE E an d control animals. Lee and colleagues (2000) showed n o differences i n basa l CR H heteronuclea r (hn ) RN A or in basal CRH an d AVP median eminenc e protein con centrations i n FE E rat s compared t o controls. Impor tantly, however, the latter investigators also showed that in respons e to bot h footshoc k and lipopolysaccharid e (LPS; an endotoxin used to mimic infection or inflam mation) challenge, FE E animal s exhibit enhanced hypothalamic neurona l activit y compare d t o tha t i n controls, reflecte d i n increase d mRN A concentration s of two immediate earl y genes (c-fos an d NGFI-B) , as well as significantly increase d CRH hnRN A content. Thus, although ther e is some controvers y in the literatur e as to whethe r HP A regulatio n i s altere d i n FE E ani mals unde r basal conditions, the dat a o f Lee an d col leagues (2000 ) provide the first evidence o f a selective stress- and cytokine-induce d increas e i n activit y of hypothalamic CR H neuron s in FEE animals , indicating a potentia l hypothalami c mechanis m throug h whic h ethanol up-regulates the HP A axis. Weinberg an d colleague s (Glava s e t al. , 2001 ; Zhang e t al. , 2005b ) undertoo k studie s t o resolv e conflicting finding s o n whethe r FE E animal s sho w changes in centra l HPA regulatio n unde r basa l or nonstressed conditions . Steady-stat e HP A functio n and th e rol e o f CORT in mediatin g changes i n HPA regulation wer e examine d i n AD X rat s with o r without COR T replacement . I n additio n t o hormona l concentrations, thes e investigator s assesse d th e con centrations o f mRNA for (a) hypothalamic CR H an d AVP a s measures o f hypothalamic activity , (b ) CR H type 1 receptor (CRH-R ^ an d POM C a s indice s of pituitary activity, and (c ) hippocampal mineralocorticoid receptor (MR ) and glucocorticoid receptor (GR) mRNA concentration s a s measures of feedback regulation. As expected , followin g ADX, animal s exhibi t a n increase i n basal plasma ACTH concentration s com pared t o thos e i n sham-operate d animal s (Glava s et al, 2001, Zhang et al., 2005b). Importantly, ACTH elevations ar e greater in FE E male s but no t females, compared t o thos e i n thei r contro l counterparts . These dat a sugges t tha t a CORT-independen t alter ation i n HP A regulation i s unmasked b y remova l o f the COR T feedbac k signal , a t leas t i n males . ADX

162 ETHANOL-AFFECTE D DEVELOPMEN T also reveal s significant alterations in gen e expressio n in FEE animals . Followin g ADX, hypothalamie CRH mRNA concentrations ar e significantly higher i n FE E males an d female s than i n thei r contro l counterparts , despite th e finding that plasm a ACTH concentration s are elevate d onl y in FEE males . FEE male s also have blunted AVP mRNA responses to ADX and lower pituitary CRH-R j mRN A expression overall compared t o controls, but there are no significant differences amon g groups i n pituitar y POMC mRN A levels. In addition , CORT replacemen t i s les s effectiv e i n normalizin g ADX-induced alteration s i n FEE tha n in control ani mals a t several level s of the HP A axis, including hip pocampal M R mRN A concentration s i n bot h male s and females , hippocampa l G R an d hypothalami e AVP mRNA in males , and anterio r pituitary CRH-R! mRNA i n females . The alteration s i n M R an d G R mRNA concentrations ar e particularly noteworthy, as previous studie s have found n o ethanol-induce d differences i n M R an d G R recepto r concentration s o r binding affinit y i n th e hippocampu s an d othe r brai n regions amon g FE E an d contro l animal s (Weinber g and Petersen , 1991 ; Ki m et al, 1999c) . Thes e dat a are th e firs t t o provid e direc t evidenc e fo r possibl e deficits i n sensitivit y t o COR T regulatio n o f HP A feedback i n FE E animals . They suggest an alteratio n in th e balanc e betwee n HP A drive and COR T feedback and exten d previous work supporting alterations in feedbac k sensitivit y in th e intermediat e (Osbor n et al, 1996) , bu t no t the fas t (Hofman n e t al, 1999 ) feedback time domain. An altere d balanc e betwee n HP A drive an d feed back i n FEE compare d t o control animals i s also suggested b y dat a o n HP A responsivenes s followin g glucocorticoid recepto r blockade (Zhan g et al, 2005). Adult female offspring fro m FE E an d contro l group s were injected with the MR antagonist spironolactone, the G R antagonis t RU38486 , o r vehicle . Followin g collection o f basal blood samples, rats were subjected to a 1 hr restrain t stress, and additiona l samples were collected durin g an d followin g stress. Both M R an d GR blockades increase d basa l ACTH concentration s in FE E bu t no t contro l animals , compare d t o vehi cle injection . Furthermore , M R blockad e increase d ACTH concentration s i n contro l bu t no t FE E fe males during restraint, whereas GR blockade increased ACTH concentration s i n FE E female s durin g re straint an d i n contro l female s durin g restrain t an d recovery, compared t o their vehicle-injected counterparts. This differential patter n of responses under both

basal an d stres s condition s suggest s tha t prenata l ethanol exposure enhance s HP A drive and alter s sen sitivity to feedback regulatio n b y MR an d GR , possi bly reflectin g an altere d balanc e betwee n HP A drive and feedback. In summary, ethanol-exposed animals exhibit HPA dysregulation unde r basa l conditions , eve n i n th e face o f similar basal hormone levels , and difference s are furthe r unmaske d followin g perturbation s o f th e system by stress, ADX, or receptor blockade . Dysregulation occur s a t multipl e sites , includin g th e hip pocampus, hypothalamus , an d pituitary , and reflect s changes both i n HP A drive and COR T feedback reg ulation and/or i n the balance betwee n driv e and feedback. Althoug h FE E animal s initiat e compensator y mechanisms to maintain norma l basal hormone con centrations unde r mos t circumstances , perturbation s of the syste m revea l that toni c o r basa l HPA tone in creases an d likel y play s a ke y rol e i n raisin g the se t point o f responsivenes s following stress. The altere d CRH-Rj mRN A responses and th e lac k of difference s in POM C mRN A concentration s i n FE E animal s suggest compensator y mechanisms , probabl y a t th e level o f th e pituitary , suc h tha t th e enhance d ac tivation i n th e hypothalamu s doe s no t translat e di rectly int o enhance d pituitar y activity. This ma y b e protective fo r FE E animals , minimizin g enhance d basal pituitar y activity in th e fac e o f enhanced hypo thalamie drive. Finally, prenatal ethanol exposur e has sexually dimorphi c effect s o n HP A regulation . Thi s finding implies a role for the gonadal steroids or possibly an alteratio n i n adrenal-gonadal interactions me diating thes e effect s o f ethanol o n HP A activity an d regulation. Ethanol Effects on Neurotransmitters Regulating HPA Activity Fetal ethano l exposur e may alter HPA responsiveness through effect s o n centra l neurotransmitte r system s that regulat e HP A function. Data indicat e tha t CR H release i s stimulated b y acetylocholine an d epineph rine in a dose-dependent manner, b y low doses of NE, and b y serotonin (5-HT) , an d i s inhibited b y |3-EP, yaminobutyric acid (GABA) , dynorphin, and substanc e P (Assenmache r e t al, 1987 ; Plotsky , 1987) . Prenata l ethanol exposur e alter s th e developmen t o f centra l neurotransmitter system s i n offsprin g (Druse , 1992) . Of particular relevance to this review are data demon strating long-term change s i n NE , 5-HT , nitri c oxide

ETHANOL EFFECTS ON ENDOCRIN E AND IMMUNE FUNCTIO N 16 3 (NO), glutamate, and GAB A in FEE rat s in brain regions critical for HPA regulation. Norepinephrine. Ethanol-induce d change s i n steady-state concentration s o f N E appea r t o var y b y brain regio n a s wel l a s th e timin g o f th e exposure . Basal concentrations of hippocampal NE wer e not altered b y prenata l ethano l exposur e (Rudee n an d Weinberg, 1993), whereas exposure through both prenatal an d earl y postnatal development (equivalen t of all thre e trimesters ) increase d hippocampa l N E content in males and females i n adulthood (Tra n and Kelly, 1999) . Whethe r prenata l ethanol exposur e alters hypothalamic N E concentration s i s not ye t resolved. N o difference s i n hypothalami c N E concen trations wer e detected betwee n FE E an d control s of either se x (Rudee n an d Weinberg , 1993) . O n th e other hand , Deterin g an d colleague s (1980 , 1981 ) showed that hypothalamic NE concentration s ar e decreased i n bot h youn g an d adul t rat s expose d t o ethanol eithe r pre - o r postnatally . Uniqu e t o th e three-trimester mode l (Tra n an d Kelly , 1999) , however, ethanol-induce d increase s i n basa l hypothala mic NE are found in females, but not males. Exposure t o stressor s differentiall y alter s NE con centrations i n FE E compare d t o contro l animals . Ethanol exposur e increase s hippocampa l N E i n females (Kelly , 1996b ) an d decrease s cortica l an d hypothalamic N E concentrations , bu t i t increase s hippocampal N E conten t in both male s and female s (Rudeen an d Weinberg , 1993) . Thes e dat a sugges t that the NEergi c syste m in the hippocampu s and hypothalamus i s particularly vulnerable t o ethano l an d that alteration s i n N E regulatio n i n thes e area s may contribute to the ethanol-induce d alterations in HPA activity observed in FEE animals . Serotonin. Th e effect s o f prenata l ethano l expo sure o n th e 5-HTergi c syste m hav e receive d special attention becaus e 5-H T als o acts a s a neurotrophic factor an d earl y insul t t o thi s syste m ma y affec t it s own development as well as that of other neurotransmitter systems . Prenatal ethano l exposur e result s in decreased concentration s of 5-H T o r it s metabolites during feta l lif e an d i n weanlin g animal s (Drus e et al , 1991 ; Rathbu n an d Druse , 1985) . Impor tantly, prenata l administratio n o f buspiron e o r ip sapirone, partia l 5-HT 1A agonists , i n conjunctio n with ethanol , ameliorate d som e o f thes e deficit s (Tajuddin an d Druse , 1993) . O n th e othe r hand ,

postnatal ethano l exposur e increase d hypothalami c 5-HT content s i n adul t rat s o f bot h sexes , with fe males showin g greate r overal l concentration s tha n males (Kelly, 1996a). Exposure during pre- and postnatal lif e di d no t differentiall y alte r hypothalamic 5HT concentration s (Tra n an d Kelly , 1999) . Feta l ethanol exposure also increased the numbe r of binding sites for the serotoni n transporter in some areas of the developin g ra t brai n bu t decrease d transporte r binding site numbers in other areas. This finding indicates that ethano l exposur e i n uter o permanentl y al ters 5-H T transmission in discrete brain regions (Zafar et al., 2000). In addition, Sari and Zhou (2004) show that prenatal ethano l exposur e results in long-lasting 5-HT deficit s i n mic e an d propos e tha t ethano l o r its metabolite s caus e los s o f serotonergi c neuron s through apoptosis. Animals prenatally exposed to ethanol exhibit physiological an d behaviora l abnormalitie s consisten t with altered 5-HT function, includin g lack of response inhibition an d increase d anxiet y an d aggression . FEE animal s have altere d hypothermi e response s to 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) , a 5-HT 1A receptor agonist, and FE E females , but no t males, show an increase d rate o f "wet dog shakes" to l-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropan e hydrochloride (DOI), a ^-HT^c recepto r agonist. These effects ar e consisten t wit h ethanol-induce d alter ations i n 5-H T recepto r functio n (Hofman n e t al. , 2002). Furthermore , FE E female s exhibi t blunte d ACTH responses to 8-OH-DPAT bu t increased ACTH responses t o DO I compare d t o controls , which sug gests an altered interaction between the HP A axis and the 5-HTergi c syste m i n FE E animal s (Hofman n et al, 2001). Nitric Oxide . Alteration s in NO concentration s induced by prenatal ethano l exposur e may pla y a rol e in mediatin g HP A hyperresponsiveness i n FE E ani mals. N O i s an intercellula r gaseous signaling molecule that has multiple biological functions, including regulation o f vascula r tone , modulatio n o f neuro transmission, involvemen t i n cell—cel l communica tion an d killin g of pathogens in nonspecifi c immune responses (Lama s e t al. , 1998 ; Moncada , 1997a , 1997b). NO i s generated fro m L-arginin e by NO syn thase (NOS) , a n enzym e foun d i n magnocellula r neurons o f the PV N an d t o som e exten t als o i n th e parvocellular divisio n (Le e an d Rivier , 1993 ; Siau d et al, 1994 ; Hatakeyam a et al., 1996) . NO act s within

164 ETHANOL-AFFECTE D DEVELOPMEN T the hypothalamu s to activat e the HP A axis , with specific stimulator y effect s exerte d o n CRH-producin g neurons (Rivier, 1999). Ethanol-induced brai n damag e i s closel y re lated t o glutamate-induce d exitotoxicity , mediate d through N-methyl-D-aspartat e (NMDA ) receptors . Chronic ethano l exposur e increase s NMD A re ceptors an d Ca 2+ channe l activity , event s associated with seizure s durin g alcoho l withdrawa l (Lancaster , 1992). It has also been postulated that chronic prenatal ethano l exposur e change s glutamate-NMD A receptor-NOS signal transduction i n the developing guinea pi g brai n (Kimur a et al. , 2000) . Kimur a an d coworkers (Kimur a e t al. , 1996 , 1999 ; Kimur a an d Brien, 1998 ) reporte d decrease d feta l hippocampa l NOS activit y and , consequently , decrease d forma tion o f NO, bu t thos e change s are not accompanie d by a los s o f NOS-containin g neuron s i n th e hip pocampal CA I o r CA3 areas or change s i n localization o f hippocampal NO S protei n i n th e near-ter m guinea pig . The critica l period for prenatal ethanol induced los s o f hippocampa l CA I neuron s i n th e guinea pig brain appears to be between G6 2 and PI2 (McGoey e t al, 2003). Glutamate an d NMDA binding site s decrease i n th e hippocampu s o f near-term fetal guine a pigs chronically exposed t o ethanol (Abdollah an d Brien , 1995) . Thus i t has been propose d that chronic prenata l exposure to ethanol suppresses the function o f the glutamate-NMDA receptor-NO signal transductio n syste m i n th e developin g hip pocampus. Furthermore , i t has been postulate d tha t persistent suppressio n o f thi s signalin g syste m dis rupts or delays normal neuronal development, which temporally precede s th e los s o f hippocampa l CA I pyramidal cells in postnatal lif e (Gibso n e t al., 2000; Kimura et al, 2000; McGoey e t al, 2003). In view of the importan t role of the hippocampu s i n HPA feedback, suc h alteration s coul d underli e th e ethanol induced change s i n HP A feedbac k regulatio n tha t have been observed. Changes i n N O synthesi s in FE E animal s ma y mediate th e altere d HP A responses t o immun e sig nals reporte d i n FE E rat s compare d t o thos e i n controls. Gottesfel d (1998 ) repor t increased N O for mation i n respons e t o LP S i n FE E rats . Further more, blockade of NO synthesi s prevents the blunte d sympathetic respons e to LPS or interleukin (IL)- l i n FEE rat s bu t no t i n contro l rat s (Gottesfeld , 1998) . Similarly, changes i n hypothalami c responsivenes s to NO ma y mediate the increased HPA responsiveness of

FEE animal s to stressors . Intracerebroventricula r in jection o f th e N O dono r 3-morpholinosydnonimin e (SIN)-l increase s the ACT H respons e and th e con centration o f mRN A fo r th e immediat e earl y gen e NGFI-B i n FE E male s (Lee e t al., 2003). Moreover, blockade o f NO formatio n abolishe s difference s be tween FE E an d contro l animal s i n respons e t o IL- 1 (Rivier, 1995) . I n norma l (no ethano l exposure ) rats, SIN-1 als o up-regulate s CRH , AVP , an d CRH-R L gene expressio n in th e PVN . Th e functiona l impor tance o f the increase d concentration s o f CR H an d AVP transcript s is evidence d b y immunoneutraliza tion o f endogenous CR H an d AVP , which abolishe s or blunts, respectively, the ACTH respons e to SIN-1 (Lee e t al. , 1999) . I t woul d b e fascinatin g to stud y these relationship s i n animal s prenatall y expose d t o ethanol. Altered HPA-HPG Interactions There ar e bidirectiona l interaction s betwee n th e stress syste m and the HPG axi s (Fig . 10-3) . Hor mones o f th e HP A axi s hav e inhibitor y effects o n HPG activity . In turn, the gonadal steroids feed back to activat e (estradiol ) or inhibi t (testosterone ) HP A activity. Recen t studie s hav e investigate d a possibl e role o f ethanol-induce d alteration s i n HPA-HP G interactions i n mediatin g th e sexuall y dimorphi c effects o f ethano l o n mal e an d femal e offspring . Studies hav e examine d th e effect s o f gonadectom y (GDX), wit h o r withou t hormon e replacement , o n concentrations o f the HP A hormones i n bot h sexe s under basal condition s and followin g stress. Preliminary data sho w that FE E male s have higher plasma ACTH concentration s tha n control s followin g re straint stress , an d tha t GD X eliminate s th e dif ferences amon g group s (La n e t al. , 2004) . Thus , HPA hyperresponsivenes s i n FE E male s appear s t o be mediated , a t leas t i n part , b y ethanol-induce d changes i n testosteron e regulatio n o r i n HP A sensitivity to testosterone . That is , the suppressiv e effect s of testosterone and/o r th e stimulator y effects o f GDX on stress-induce d ACTH releas e are altered i n FE E compared t o contro l males . I n contrast , th e rol e of the gonada l hormone s i n HP A regulatio n i n FE E females i s mor e complex . Preliminar y dat a (Ya mashita et al., 2004) show that overall, FEE female s are les s responsiv e tha n control s t o th e effect s o f estradiol o n bot h reproductiv e an d nonreproduc tive measures . Furthermore, FE E female s appear to

ETHANOL EFFECTS ON ENDOCRIN E AND IMMUNE FUNCTIO N 16 5 PRENATAL ETHANOL EXPOSURE ALTERS

THE IMMUN E SYSTE M OF THE OFFSPRING

Impairments in immune competenc e o f children with FAS have bee n demonstrate d broadl y in bot h innat e and adaptiv e immunity. Innate immunit y is not majo r histocompatibility comple x (MHC ) restricted . I t provides a firs t lin e o f defens e agains t man y commo n pathogens throug h phagocyte s such a s monocytes , macrophages, polymorphonuclear leukocytes, natural killer cells , an d solubl e mediator s suc h a s comple ment an d C-reactiv e protein . Adaptiv e immunit y is MHC restricte d and classifie d a s cellular or humoral, mediated b y T o r B lymphocytes , respectively . Im mune response s agains t microorganisms ar e mediate d by multipl e cel l types , an d interaction s betwee n th e innate an d adaptiv e immun e system s are necessary to launch a n effective immun e response . An understanding of the timeline an d events in normal developmen t of the immune system, a complicated an d multisystem process, is important to an understandin g o f the effec t of ethanol o n the developing immune system. FIGURE 10- 3 Interaction s betwee n th e stres s system and reproductiv e axis . Th e stres s syste m consist s o f the hypothalamic-pituitary-adrena l (HPA ) axi s an d the locu s coeruleu s noradrenergi c sympatheti c ad renal medullar y (LC-NE ) syste m (describe d i n de tail i n Fig . 10.1) . Th e reproductiv e axi s consist s o f gonadotropin-releasing hormon e (GnRH ) neuron s which stimulat e secretio n o f luteinizin g hormon e (LH) an d follicl e stimulatin g hormon e (FSH ) fro m the pituitary , which i n tur n stimulat e secretion o f estrogens (mainl y estradiol ) an d androgen s (mainl y testosterone) fro m th e gonads . Th e reproductiv e axi s is stimulated b y the LC-NE system, primarily through oc-noradrenergic (oc rNE) receptors , an d i s inhibite d at al l level s by component s o f th e HP A axis . Activa tion is represented by solid lines, inhibition by dashed lines. ACTH , adrenocorticotropi c hormone ; CRH , corticotropin releasin g hormone ; E ?, estradiol ; T , testosterone. (Source: Reprinte d fro m Strataki s an d Chrousos (1995 ) wit h permissio n fro m Ne w Yor k Academy of Sciences )

have decreased tissu e responsiveness to estradiol and altered HP G sensitivit y to acute stress . Further stud ies ar e neede d t o elucidat e th e effect s o f prenata l ethanol exposur e o n interaction s betwee n th e HP A and HP G axes .

Normal Timing of the Ontogeny of the Immune Syste m Although developmen t o f the immun e syste m take s place throughou t life , th e basi c framewor k of immunity develop s befor e birth . Th e misconceptio n tha t the newbor n i s immunologically naiv e ha s been cor rected with data demonstrating that neonatal T and B cells ar e mature d t o a stag e tha t the y ca n moun t a n antigen-specific immun e respons e t o antigen s en countered i n utero , suc h a s the parasit e Ascaris an d tetanus toxoi d vaccinatio n (Gil l e t al. , 1983 ; Kin g étal, 1998) . Hematopoietic ste m cell s for m an d develo p i n the yolk sack until 4 weeks of gestation, migrat e to the liver 3 to 4 weeks later, and then further migrat e to the thymus and spleen. Ste m cell s are in the bone marrow at 11-1 2 week s of gestation (Migliacci o e t al, 1986 ; Holt and Jones , 2000). Susceptibility of early lympho hematopoiesis an d cel l migratio n t o environmenta l influences ca n occu r a s early as 7-10 week s postcon ception (West , 2002) . As early a s 1 4 weeks o f gesta tion, differentiate d T an d B cells can be identifie d in fetal bloo d (Goldblatt , 1998 ) an d splee n (Peakma n et al., 1992) . The splee n i s considered completel y im munocompetent b y 1 8 week s o f gestation . A t thi s

166 ETHANOL-AFFECTE D DEVELOPMEN T time, splenic T cells have adult levels of expression of CD3, CD4 , an d CDS and are able to respond t o mitogens (Ka y et al., 1970) , an d spleni c accessor y cell s are full y functiona l i n deliverin g co-stimulatory sig nals (Hol t an d Jones , 2000) . Fetuse s stil l hav e fewe r memory T cell s than adult s do at this time (Hol t and Jones, 2000). Yolk sack-derived pro- B cells develop in the liver and acquire surface immunoglobulin (Ig ) M, IgD, and CD2 0 expressio n b y 10-1 3 weeks of gestation (Hofma n et al. , 1984) . B cells ar e detectabl e i n lymph node s fro m 16-1 7 weeks , in splee n a t 16-2 1 weeks, an d ar e abundan t i n bon e marro w at 16-2 0 weeks of gestation (West , 2002). The structur e of the thymu s develops as a result of epitheliomesenchymal interactions. Endoderm of the third pharyngea l pouch differentiate s int o thymic epithelium. Neural crest cells then migrat e through th e branchial arche s an d becom e th e mesenchym e tha t will form the layers around the epithelial primordium of the thymus and the connective tissu e framework. T cell progenitor s arriv e i n th e thymu s fro m th e live r during the nint h wee k of gestation, an d th e structur e of th e thymu s differentiate s int o a corte x an d a medulla at 10-1 2 week s (West, 2002). Prothymocytes express CD 7 i n th e feta l live r at 7 week s and CD 3 at 8- 9 week s o f gestation, whe n thes e cell s migrat e to the thymus (Holt and Jones, 2000). Gene rearrangement start s a t aroun d wee k 11 , an d expressio n o f the T cel l receptor s (TCR) , CD4 , an d CD 8 fol lows. Thymi c "education " o f immature CD4 +/CD8+ (double-positive) T cell s take s plac e i n th e secon d trimester b y positiv e selection , wit h th e abilit y o f TCR t o bin d t o polymorphi c part s o f nonagonis t MHC-encoded molecules on nonlymphoid cells, and by negativ e selectio n of TCR , wit h hig h affinit y to self-antigens t o avoi d autoimmunit y (vo n Boehme r et al, 2003). Export of mature CD4+ or CD8+ (single positive) cell s begin s afte r wee k 13 , an d a rapi d ex pansion o f th e T cel l poo l happen s durin g 14-1 6 weeks (Ka y et al., 1970 ; Berr y et al, 1992) . At 13-14 weeks, thymocytes acquire proliferative ability to most mitogens. During th e second trimester , susceptibility to environmenta l factor s cause s disruptio n o f th e thymic educatio n processe s an d cel l proliferation , leading to a defective T cell repertoire. As roden t gestatio n (~ 3 weeks ) i s substantiall y shorter tha n tha t o f human s (~4 0 weeks) , immun e system developmen t o f newbor n ra t an d mous e pups occurs at a time equivalent to the end of the sec ond trimeste r i n human s (Zaja c an d Abel , 1992) .

Therefore, studie s i n roden t model s o f prenata l ethanol exposur e have focused primarily on th e early events in ontogeny of the immun e system, i.e., durin g the late gestational period an d early postnatal life . Impaired Immunit y following Prenatal Exposure t o Ethanol Children prenatall y expose d t o alcoho l hav e a n in creased incidenc e o f bacteria l infections , suc h a s meningitis, pneumonia , recurren t otiti s media , gas troenteritis, sepsis , urinar y trac t infections , an d fre quent uppe r respirator y trac t infection s (Johnso n et al, 1981 ; Churc h an d Gerkin, 1988) . These chil dren als o have lowe r cell count s o f eosinophil s an d neutrophils, decrease d circulatin g E-rosette-formin g lymphocytes, reduce d mitogen-stimulate d prolifera tive response s b y periphera l bloo d leukocytes , an d hypo-y-globulinemia (Johnson et al., 1981). Research usin g anima l model s t o investigat e im mune function of FEE offsprin g ha s (a) substantiated the clinica l evidenc e o f impaire d immunit y associ ated wit h FASD and (b ) greatly increased ou r under standing of these immune deficits in terms of both th e spectrum of effects i n differen t orga n systems and th e mechanisms mediatin g th e immunoteratogeni c ef fects o f ethanol . Deficit s i n innat e immunit y hav e typically no t bee n observe d i n anima l studies . Fo r example, on e larg e stud y o n nonhuma n primate s (Macaco, nemestrina) show s that i n uter o ethano l ex posure doe s no t resul t i n significant differences i n total numbers of white blood cells, leukocyte subsets, or monocyte phagocyti c activit y compare d t o tha t i n control subjects (Grossmann et al., 1993). In contrast, marked deficit s in adaptiv e immunity involving both cell-mediated an d humora l immunit y ar e eviden t in FE E animals . Furthermore , a s describe d i n th e discussion below , a marke d sexua l dimorphis m i n ethanol effect s ha s bee n observed , wit h th e majorit y of deficits occurring in male offspring . Deficits i n Adaptive Immunit y Inborn error s of immunocompetent cells in childre n with FASD result in immunodefienc y disorders or increased susceptibilit y to infections . Recurrent oppor tunistic infectio n and infectio n caused b y ubiquitous microorganisms, suc h a s bacteria, viruses , and fungi , typically occu r wit h deficit s in cell-mediate d immu nity, wherea s deficit s i n B cells , immunoglobulins ,

ETHANOL EFFECTS ON ENDOCRIN E AND IMMUNE FUNCTIO N 16 7 complement, an d phagocyte s usuall y lea d t o infec tion b y encapsulated and pyrogeni c bacteria, such as Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus. I n childre n wit h FAS D both types of recurrent infections have been reported, suggesting tha t bot h T an d B cell-mediated immu nity are compromised (Johnso n et al., 1981) . Work with animal models confirm s the findings in human subjects . For example, th e immun e respons e of FEE neonate s t o the intestina l parasit e Trichinella spiralis indicate s a diminished capacit y to respond t o the pathogen , demonstrate d b y a n increase d intes tinal wor m coun t (Steve n e t al. , 1992 ; Seeli g e t al. , 1996). Th e abnormalitie s involve depressed T an d B cell-mediated response s suc h a s lowe r seru m IL- 2 and tumor necrosis factor (TNF ) contents , an d lower IgM an d Ig G antibod y titers. Interestingly , th e detri mental effect s o f ethano l appea r t o increas e acros s generations. Tha t is , FEE pup s (secon d generation ) mothered b y FE E adul t offsprin g (firs t generation ) who themselves consumed ethanol during pregnancy show reduce d proliferativ e response s t o T . spiralis antigen and stimulation with concanavalin A (Con A), lower titer s o f seru m Ig M an d Ig G anti-T . spiralis, and lower percentages of T cells and cytotoxic T cells, compared t o th e first-generatio n FE E an d pair-fe d groups (Seelig et al., 1999) . The nex t two sections reviewr studie s tha t describ e specifi c deficit s i n eithe r cell-mediated o r humoral immunity. Deficits in Cell-Mediated Immunity Prenatal ethano l exposur e alter s development o f the thymus i n rat s an d mice . Delaye d thymi c ontogen y (Ewald an d Walden, 1988) , decreased tota l number s of thymocytes, and diminishe d mitogen-induce d cel l proliferation hav e been reporte d i n near-term fetuse s (Ewald an d Frost , 1987) . Decrease d thymu s weight, size, and cel l count s have also been observe d at birth (Redei et al., 1989) . These changes persisted through the preweanin g perio d an d eve n int o adolescenc e (Ewald an d Frost , 1987 ; Ewald , 1989 ; Giberso n an d Blakley, 1994 ; Taylo r e t al , 1999a) , althoug h on e study o f mic e foun d tha t tota l thymocyt e number s could retur n t o contro l level s as early as P6 (Ewald , 1989). Similarly , mitogen-induce d proliferativ e re sponses o f thymic cell s was suppressed i n FEE male s at weaning (Redei et al, 1989) , but may be increased in adolescen t (44-day-old ) rat s (Chiappell i e t al , 1992; Wong et al., 1992) . This increase i n thymocyte

proliferation durin g adolescenc e doe s no t appea r t o be mediate d b y change s i n numbe r o f GR s o n th e thymocytes; furthe r studie s ar e neede d t o elucidat e the mechanism s underlyin g these varyin g effect s o f ethanol. The advers e effects o f ethanol on thymic develop ment hav e bee n confirme d by in vitr o studies usin g organotypic cultures . Tota l cel l number s an d per centages of immature fetal thymocytes (CD4+/CD8+) decrease i n a concentration-responsiv e manne r i n ethanol-treated orga n culture s (Bra y e t al , 1993) . This decrease is a consequence o f accelerated apopto sis, which the n result s in a n increase d percentag e o f more mature thymocytes expressing CD4+/CD8~ and Y/8 TC R (Ewal d an d Walden , 1988 ; Ewal d e t al , 1991; Bra y et al., 1993 ; Ewald and Shao , 1993) . Similar outcome s ar e observe d i n 20 - to- 40-day-old FE E animals. That is , thymic cel l count s an d tota l num bers of CD4+ and CD8 + cells are decreased through out thi s period , an d immatur e CD8 +/TCR+ an d CD8+/CD4SRC+ thymocyte s ar e reduce d b y P3 5 (Taylor e t al. , 1999a) , finding s suggestin g tha t pre natal ethano l treatmen t alter s the late r stage s of thymocyte maturation , suc h a s afte r double-positiv e (CD4+/CD8+) thymocytes acquire TCR expression. Prenatal ethano l exposur e ha s long-ter m advers e effects o n the immune syste m that last well into adulthood. FE E animal s hav e decrease d number s o f Thyl.2+, CD4+, CD8+, and IgG+ splenocytes (Ewald and Huang, 1990 ; Giberson and Blakley, 1994; Giber son e t al. , 1997) . Decrease d percentage s o f Thyl.2+ splenocytes ar e also evident i n pup s born t o mother s consuming ethano l durin g pregnanc y an d lactatio n or durin g lactation alone , indicatin g direct effect s of ethanol o n postnatal developmen t o f the immun e system (Giberso n an d Blakley , 1994) . Similarly , roden t and primat e studie s indicat e tha t spleni c lympho cytes from FE E male s have decreased proliferative responses to mitogens from adolescenc e throug h youn g adulthood (Norma n et al, 1989 ; Rede i e t al, 1989 ; Gottesfeld e t al , 1990 ; Weinber g an d Jerrells , 1991 ; Grossmann et al, 1993 ; Jerrells and Weinberg, 1998 ) and tha t th e respons e normalizes by 8 months o f age (Gottesfeld an d Ullrich , 1995 ; Norman et al, 1991) . Furthermore, deficit s no t onl y i n th e respons e o f freshly isolate d splenic T cells but also in the response of T blas t cell s (obtaine d followin g treatment wit h Con A ) to IL- 2 o r furthe r Co n A stimulatio n (Got tesfeld e t al , 1990 ; Norma n e t al , 1991 ; Weinber g and Jerrells , 1991; Jerrell s and Weinberg , 1998 ) hav e

168 ETHANOL-AFFECTE D DEVELOPMEN T been observed . I n contrast , th e change s i n mitogen induced proliferation of thymocytes observed in FE E animals normalized i n young adulthoo d (Chiappell i et al, 1992). Deficits in Humoral Immunity Humoral immunit y i s les s affecte d b y feta l ethano l exposure than is cell-mediated immunity . Prepubertal FEE rat s d o no t diffe r fro m control s i n seru m im munoglobulin conten t afte r primar y an d secondar y immunization (Gottesfel d an d Ullrich , 1995) . Simi lar result s ar e eviden t i n nonhuma n primate s afte r immunization wit h tetanu s toxi n (Grossman n e t al. , 1993). On th e other hand, deficits i n humoral immu nity do occur . Abnormal development o f B cell line ages in murine hernatopoietic organs, including bone marrow, spleen , an d liver , occu r fro m feta l age s through adolescence . The tota l numbe r of splenic B cells an d thei r proliferativ e respons e t o LP S i s de creased a t birt h an d throughou t th e preweanin g period (Wolcott et al, 1995). In addition, B cell matu ration i n fetal live r is delayed (Bibe r et al., 1998 ) an d the numbe r o f B220-positiv e hernatopoietic cell s i n liver is decreased i n rat fetuses at the en d o f gestation (Robinson and Seelig , 2002). At birth, the numbers of both immatur e (IgM+/IgD~) and mature (IgM +/IgD+) B cells in spleen and bone marro w are decreased, bu t most recove r to norma l level s by 3 to 4 week s afte r birth, excep t fo r pre- B cell s (B220 +/IgM~) i n bon e marrow, which remai n a t low levels through 5 weeks of life (Moscatell o e t al, 1999) . Ethanol Exposure an d Vulnerability to Stress-Induced Suppression of Immune Function The nervous , endocrine, an d immun e system s exist within a comple x regulator y network in whic h bidi rectional communicatio n occurs via shared receptors and hormona l mediator s t o maintai n homeostasi s (Fig. 10-4) . Th e CM S ca n modulat e bot h th e en docrine an d immun e system s through releas e of neurotransmitters an d throug h direc t innervatio n o f organs. Feedbac k t o th e CN S occur s throug h hor mones an d cytokines , many of which ar e simila r o r even identica l in structure . Immune signal s can acti vate the HP A axis and th e glucocorticoid s play a ma jor rol e in th e stress-induce d suppression of immune and inflammator y reactions.

FIGURE 10- 4 Interaction s betwee n th e stres s system and immun e system. The long-ter m stres s response is maintained vi a secretio n o f stres s hormone s (corti cotropin releasin g hormon e [CRH] , adrenocorti cotropic hormon e [ACTH] , an d glucocorticoids) . The locu s coeruleu s noradrengergi c (LC-NE ) sys tem participate s i n th e effect s o f stres s o n th e im mune respons e through bot h it s interaction wit h th e hypothalamic-pituitary-adrenal (HPA ) axi s an d it s ability t o transmi t neura l signal s from th e peripher y to th e immun e system . Activatio n o f th e HP A axi s occurs durin g th e stres s o f infection , inflammation , or autoimmun e processes , a s wel l a s accidenta l o r operative trauma . Durin g thi s activation , cytokines , neuropeptides, platelet-activatin g facto r (PAF)/prosta noids ar e secrete d an d caus e stimulatio n o f the HP A axis. Th e glucocorticoids , en d hormone s o f the HP A axis, play a major role in the stress-induced suppression of immun e an d inflammator y reactions. Activatio n is represented b y solid lines , inhibition b y dashed lines . (Source: Reprinted from Strataki s and Chrousos (1995) with permission from Ne w York Academy of Sciences)

Chronic stress in adulthood provide s a challenge to the immun e system that differentially affect s FE E an d control animals. Similar to the data demonstrating that basal hormon e concentration s are typically normal i n FEE animals , specific deficit s in the immun e syste m

ETHANOL EFFECTS ON ENDOCRIN E AND IMMUNE FUNCTIO N 16 9 may no t b e observe d unde r nonstresse d conditions , and become apparen t only when FEE animal s are exposed t o stressors . In addition , sexually dimorphic ef fects of prenatal ethano l exposure may be observed, In FE E males , exposur e t o chroni c intermitten t stressors selectivel y down-regulates th e number s o f thymic and periphera l bloo d CD43 + cell s as well as peripheral bloo d CD4 + T cells , an d marginall y decreases th e numbe r o f peripheral bloo d MH C Clas s II Ia + (antige n presenting ) cells . I n contrast , CD4 3 antigen expressio n o n periphera l bloo d T cell s i s selectively up-regulate d (Giberso n an d Weinberg , 1995). Stres s doe s no t differentiall y alte r thes e im mune measure s i n FE E females . Th e findin g tha t FEE male s exhibit greater stress-induced immun e abnormalities tha n FE E females , despit e showin g less adrenal hypertrophy , suggest s tha t male s ar e mor e sensitive t o smal l change s i n glucocorticoi d con centrations tha n females . Role s fo r estroge n a s a n immunoprotective hormon e an d testosteron e a s a n immunosuppresive hormone i n these sexually dimorphic immun e responses remain to be determined . One o f the first demonstrations tha t stres s differen tially alters immune functio n i n FE E an d contro l females is the finding of an interaction between prenata l ethanol an d chroni c col d stres s i n femal e offsprin g (Giberson et al., 1997) . After 1 day of cold stress, FE E females exhibi t increase d lymphocyt e proliferatio n in response t o pokewee d mitoge n (PWM ; a T cell dependent B cell mitogen) an d Con A challenge. No ethanol-induced difference s i n immun e responsive ness ar e detecte d amon g males . I n contrast , FE E males expose d t o 1 or 3 days o f col d stres s have in creased basa l COR T concentration s compare d t o those o f FEE male s no t expose d t o cold. These findings ar e consisten t wit h dat a fro m Halas z an d col leagues (1993) , wh o foun d tha t th e challeng e o f chronic ethano l exposur e i n adulthoo d selectivel y in creased Co n A-induce d lymphocyt e proliferatio n i n FEE females but not in males. These data have important implication s fo r understandin g th e mechanism s underlying immune deficit s induce d by prenatal exposure to ethanol, because defective interactions between T an d B cells woul d significantl y hinder th e develop ment of a normal immune response . Cytokines and the HP A Axis Prenatal ethano l exposur e blunt s th e LPS-induce d febrile respons e i n mal e rat s (Taylo r e t al. , 1999b ,

2002a). Thi s effec t o f ethano l ma y b e mediate d b y a decrease d respons e o f centra l thermoregulator y systems t o IL-1(3 , whic h coul d b e mediate d b y a de creased hypothalami c IL-1( 3 respons e t o LP S ad ministration, possibl y throug h a n impaire d releas e of endogenou s pyrogen s (Ylikorkala e t al. , 1988 ; Yirmiya e t al, 1993 , 1996) . Interestingly , both ADX and sham surger y i n th e pregnan t femal e abrogat e th e effect o f ethanol o n th e febril e respons e o f femal e offspring t o IL-1(3 , bu t onl y AD X ha s a n effec t o n male offspring . Thi s observatio n implie s tha t mater nal adrena l mediator s play a n importan t rol e i n th e blunted febrile response of FEE male s and that nonadrenal mediator s participat e in modulatio n of ther moregulatory system s in FE E female s (Taylo r et al. , 2002a, 2002b). Parallel to blunted hormona l responses to stressors observed i n FEE animal s during th e preweanin g period, FE E preweanling s exhibi t reduce d ACTH , (3EP, an d TNF-o c response s t o immun e challenge s (IL-1(3, LPS) , Thi s reductio n persist s int o adoles cence i n FEE male s but no t in females (Lee and Rivier, 1993 , 1996 ; Chiappell i e t al. , 1997 ; Ki m et al, 1999b). A n altere d abilit y of IL-1 t o stimulat e secre tion o f POMC-relate d peptide s ma y underli e thi s reduced responsiveness (Lee and Rivier , 1993). Interestingly, ovariectom y prio r t o puberty eliminates th e difference i n ACTH response between FE E an d con trol females (Lee and Rivier , 1996), a finding suggesting tha t ethano l an d femal e se x hormones regulat e HPA activity through a common pathway. In contras t to the preweanin g period, ACTH an d CORT response s t o immun e signal s such a s LPS o r IL-1 (3 increase i n FE E animal s b y adulthoo d (Ki m et al. , 1999b) . Thi s respons e parallel s thei r HP A hyperresponsiveness to stressors. The finding that prenatal ethano l exposur e doe s no t alte r cytokin e re sponses to immune challenge s suggest s that cytokine s probably d o no t mediat e th e ethanol-induce d in crease i n HPA responsiveness to immune signals . O n the othe r hand , FE E male s exhibit increase d plasma concentrations o f pro-inflammatory cytokine s to LP S challenge followin g repeate d restrain t stres s (Eske s and Weinberg , 2003 ; Eske s e t al , 2004) . Wherea s CORT response s t o LP S ar e comparabl e amon g groups, FE E animal s hav e greate r an d mor e sus tained elevation s o f IL-1(3 , TNF-ot , an d IL- 6 tha n those i n controls. These data support an d exten d pre vious studie s suggestin g tha t although FEE animal s may no t diffe r i n cytokin e response s unde r basa l o r

170 ETHANOL-AFFECTE D DEVELOPMEN T nonstressed conditions, the y may in fac t be more vulnerable t o th e advers e effect s o f stres s o n immun e function (Eske s et al., 2004). Mechanisms Underlyin g the Effects of Ethanol on the Developing Immune Syste m Direct and Indirect Effects of Ethanol on the Fetus Ethanol ca n directl y disrup t the developmen t o f th e fetal thymus . The highl y specified microenvironment of the thymu s provide d by epithelial an d mesenchy mal cell s i s pivotal i n attractin g immatur e lymphoi d precursors to enable them t o be selected and differen tiated int o matur e T cells . Exces s ethano l exposur e during th e developmen t o f the thymu s inhibit s th e ontogeny o f the thymi c epitheliu m an d disrupt s th e microenvironment fo r maturatio n o f T cells , whic h leads to impaired T cel l immunit y (Bockman, 1997) . These data are interesting in light of the share d clinical characteristic s o f FASD and DiGeorg e syndrome (Ammann et al., 1982) . The latte r is a congenital im mune deficienc y syndrome involvin g mainly T cell s and cause d by an abnormalit y in the developmen t of neural crest-derive d component s o f the thymu s an d parathyroid gland s (Kirb y and Bockman , 1984) . Tar geted effect s o f ethanol o n neura l cres t ar e describe d in Chapter 17 . Selective effect s o f ethano l o n thymi c CR H an d POMC gene expression in male fetuses have been reported. Significan t increase s i n thymi c CR H an d a decrease i n thymi c POM C gen e expressio n hav e been observe d o n G1 9 (Revsko y et al., 1997) . Thes e changes d o no t appea r t o b e relate d t o COR T con centrations i n the fetu s o r pregnant female, but possibly ar e induce d b y th e feta l testosteron e surge . Similarly, the ontogen y o f GR site s per thymocyt e i n FEE animal s i n the first 2 months o f life differ s fro m that in control animal s (Chiappelli e t al., 1992) . This indicates a rol e fo r th e glucocorticoi d hormone s i n the differentia l thymi c developmen t describe d i n FEE an d control animals. Altered IL-2/IL- 2 receptor interaction s ma y play a role in the developmen t of altered immun e function in FE E animal s (Weinberg and Jerrells , 1991; Taylor et al., 1993 ; Chang et al, 1994). For example, despite showing a reduced proliferativ e response to mitogens,

FEE animal s hav e norma l amount s o f IL- 2 produc tion, IL- 2 recepto r expressio n an d distribution , cal cium influ x i n T cells , an d bindin g o f IL- 2 t o it s receptor. Th e internalizatio n and/o r utilization of IL2 by lymphoblasts, however, is reduced, an d th e half time fo r dissociatio n o f IL- 2 fro m it s recepto r i s increased i n T cell s fro m FE E animals . Therefore , impaired intracellula r signalin g events , mediate d b y IL-2/IL-2R interactions , ma y underli e th e immun e deficits observe d i n FE E animals . In contrast , direc t treatment o f lymphocyte s wit h acetaldehyde-seru m protein, whic h form s i n viv o a s a metabolit e o f ethanol consumption , result s i n decrease d IL- 2 pro duction bu t no t IL- 2 recepto r expressio n (Brau n et al. , 1995) . This resul t suggest s that, a t least unde r some conditions , decrease d IL- 2 productio n con tributes to impaired proliferative responses. Altered neurotransmitte r regulatio n o f immun e function ma y play a role i n altered immun e function of FE E animals . Prenata l exposur e t o ethano l de creases concentration s o f NE an d (3-adrenoreceptor s in the lymphoid organs and diminishes synaptic transmission i n th e splee n an d thymus , but no t th e hear t (Gottesfeld e t al. , 1990) . Altere d NEergi c synapti c transmission, includin g a higher rat e o f NE turnove r leading to reduced NE concentratio n and reduce d (3adrenoreceptor density , could affec t immun e capacit y in terms of NE-mediated IL-2 secretion an d cytotoxi c T cell responses . Immunity at the Fetoplacental Interface Immunity a t th e fetal-placenta l interfac e i s biase d toward humoral immunity ; cell-mediated immunit y is suppressed t o preven t feta l rejection . Cell-mediate d immunity i s skewe d towar d Th 2 response s an d pro duction o f cytokine s suc h a s IL-3 , IL-4 , an d IL-5 , rather tha n towar d Thl response s and productio n of cytokines such a s IL-2 and IFN-y (Lin et al, 1993) . It has been suggested tha t progesterone an d CD4 + regulatory T cell s are the key factors enablin g the fetu s to evade immune rejectio n by the pregnant female , thus allowing th e femal e t o maintai n pregnancy . Proges terone suppresse s cytotoxi c activit y o f lymphocyte s from pregnan t wome n vi a progesteron e receptor s (Szekeres-Bartho e t al, 1990) . A timely increas e i n CD4+/CD25+ regulator y T cells , systematicall y and locally at the interface between the fetus and pregnan t

ETHANOL EFFECTS ON ENDOCRIN E AND IMMUNE FUNCTIO N 17 1 female, play s a n importan t rol e i n maintainin g tolerance vi a suppressio n o f a n allogenei c respons e directed agains t th e fetu s (Szekeres-Barth o e t al. , 1990; Aluvihar e e t al. , 2004) . I n uter o exposur e t o ethanol coul d alte r th e balanc e betwee n regulator y T cells and T effecto r cells , an d thu s contribute s to the increase d incidenc e o f spontaneou s abortion s and prematur e births. This conclusio n i s supported by observation s of elevated cor d bloo d Ig E concen trations i n ethanol-expose d infants , indicatin g in creased activit y o f Th2-typ e response s (Somerse t et al. , 2004) . Long-ter m alteration s i n CD4 + regu latory T cell s in FEE male s may play a role in medi ating deficit s i n T cel l functio n (Zhan g e t al. , 2005a). Dexamethasone-induce d apoptosi s an d res cue b y IL-2 of CD4+/CD25+ T regulator y cells fro m FEE animal s an d control s hav e bee n examined . CD4+/CD25+ regulator y T cell s fro m FE E male s were more resistant to dexamethasone-induced apoptosis i n th e presenc e o f IL-2 , resulting i n increase d survival o f T regulator y cells . A s T regulator y cell s are suppressiv e i n nature , th e increase d number s of surviving T regulator y cell s coul d pla y a rol e i n ethanol-induced immun e deficits . Neuroendocrine-Immune Interaction s The CN S i s critica l fo r th e developmen t an d mat uration o f th e feta l immun e syste m throug h bot h sympathetic activit y an d neuroendocrin e mediators , such as growth hormone an d the HP A and HPG hor mones (Fig . 10.4) . Th e sympatheti c nervou s system innervates lymphoid organs, and lymphocyte s express receptors fo r CRH , ACTH , cortisol , NE , an d epi nephrine. Th e glucocorticoi d hormone s ca n exer t profound influence s on T cel l function throug h their interaction with GRs on T cells, which modulate traf ficking an d homing , proliferation , activation , an d apoptosis (Gonzal o e t al , 1994 ; Dhabha r e t al , 1996). Fo r example, transgeni c mic e wit h decrease d GR binding capacity but normal basa l concentration s of ACT H an d COR T sho w a partia l blockag e o f T cell differentiatio n and decrease d apoptosi s in the fetal period but no t i n adult lif e (Sacedo n e t al. 7 1999) . In addition, glucocorticoids cause a shift fro m Thl t o Th2 responses and a change from a pro-inflammatory cytokine patter n (e.g. , IL-1 an d TNF-ot ) t o a n anti inflammatory cytokin e patter n (e.g. , IL-1 0 an d IL-4 ) (DeRijk e t al. , 1997 ; Elenko v an d Chrousos , 1999) .

Growth hormon e increase s IFN- y productio n an d MHC clas s I an d I I expressio n b y thymic epithelia l cells, macrophages , dendriti c cells , fibroblasts , an d extracellular matri x (Savin o an d Dardenne , 2000) . These results suggest that growth hormone facilitate s MHC-mediated influence s o n thymocyt e differentia tion. Thymic epithelia l cells and lymphocyte s in rat s express estroge n an d androge n receptor s fro m G1 6 (Tanriverdi et al., 2003) and thus are influenced by circulating sex steroid hormones. Furthermore, adminis tration o f gonadotropin releasin g hormon e (GnRH ) restores fetal thyrnic and liver-derived T cell proliferative response s afte r surgica l ablation o f the forebrain or th e entir e brai n includin g th e hypothalamu s an d pituitary in 18-day-ol d fetuses (Zakharova et al., 2000). Thus, GnRH appear s to be involve d in regulatio n of T cel l developmen t eve n durin g prenata l ontogene sis. These examples show how closely development of the thymus is regulated by the neuroendocrine system. Cytokines secreted by immune cells , such a s IL-1, IL-2, IL-4 , an d IL-6 , influenc e th e functio n o f hypothalamic neurosecretor y an d thermoregulator y neu rons and pituitary cells (Cunningha m an d De Souza, 1993; Rivier , 1994; Zalcma n e t al., 1994 ; Dun n an d Wang, 1995) , resultin g i n activatio n o f the HP A axis and inducing "sickness behavior" (Watkins and Maier, 2000; Dantzer, 2001). IL-1, IL-6, and TNF-oc are also produced i n th e hypothalamu s b y microgli a an d macrophages (Hetie r et al., 1988 ; Sebir e e t al, 1993 ) and thus can directly influence neuroendocrine func tion. For example, IL-1 stimulates the releas e of CRH and AV P from th e hypothalamu s (Sud a e t al, 1990 ; Chover-Gonzalez e t al. , 1994) , an d IL-1 , IL-6 , an d TNF-oc stimulate ACT H secretio n fro m th e anterio r pituitary (Kehre r e t al. , 1988 ; Shar p e t al , 1989 ; Lyson and McCann, 1991) . Given th e intimat e interaction s between th e neu roendocrine an d immun e system s (Fig. 10.4 ) durin g development and in adulthood , feta l ethanol-relate d developmental change s i n neuroendocrin e activit y could affec t immun e competency . I n turn , feta l ethanol-induced change s i n cytokine secretion b y immune cell s can affec t neuroendocrin e activity . Therefore, th e altere d HP A response s t o immun e signal s (Lee and Rivier , 1993 ) and differentia l vulnerabilit y to stress-induced immun e suppressio n (Giberso n an d Weinberg, 1995; Giberson et al, 1997) shown by FEE rats compared to that of controls could be mediated by the altered neuroendocrine-immune interactions .

172 ETHANOL-AFFECTE D DEVELOPMEN T PRENATAL ETHANOL EXPOSUR E REPROGRAMS FETA L HP A

AND IMMUNE FUNCTIO N

The route s b y which the feta l an d earl y neonatal en vironment ca n progra m adul t HP A function an d be havior ar e describe d eloquentl y b y Matthews (2002 ) (Fig. 10.2 ) and Welberg and Seck l (2001). The devel oping limbic system, primarily the hippocampus , hypothalamus, an d anterio r pituitary , synthesiz e hig h amounts o f GRs and ar e highly sensitive to glucocorticoids. Exposure to high concentration s o f glucocorticoids durin g earl y lif e ca n alte r th e developmen t and subsequen t functio n o f bot h th e limbi c syste m and the HPA axis. The limbi c system, particularly the hippocampus, regulates HPA activity, and in turn, endogenous glucocorticoid s modif y numerou s limbi c system functions . Mechanisms underlyin g these mu tual effects involv e modification of developing neuro transmitter systems , thei r transporte r mechanism s in th e brain stem, th e developmen t o f GR expressio n in th e hippocampus , an d th e developmen t an d re sponsiveness o f the hypothalami c PVN , whic h regu lates glucocorticoid secretion. Prenata l events that can program HP A functio n includ e stres s durin g preg nancy, exposure to synthetic glucocorticoids, and nutrient restriction . Postnata l event s tha t ca n progra m HPA function includ e earl y handling, alteration s i n maternal behavior, and exposur e to exogenous glucocorticoids and infection. The overal l effec t o f earl y developmenta l pro gramming i s altered exposur e t o endogenou s gluco corticoids throughout life (Li u et al., 2001; Matthews, 2002). This altered exposur e i n tur n modifie s behavior, cognition , learning , memory, an d emotion , an d predisposes th e individua l t o cardiovascular , meta bolic, and immune disorders. Although environmental factors pla y an importan t rol e i n feta l programming , it i s likely tha t perinata l environmenta l an d geneti c factors mutuall y influence each othe r i n determinin g HPA activity and behavio r later i n life . Moreover , al though th e effect s o f programmin g ar e ofte n long lasting, postnatal an d late r environmental events can modulate the effect s o f prenatal programming . What is the mechanism underlying fetal o r neona tal programmin g b y earl y lif e experiences ? Earl y experiences resul t i n long-ter m consequence s fro m the behaviora l to the molecula r level through epige netic processes (Weaver et al., 2004). In the mode l of Weaver and colleagues , naturally occurring variations

in materna l behavio r ar e associate d wit h th e devel opment o f individua l differences i n behaviora l an d HPA response s to stressors . Thus, offsprin g of moth ers showin g hig h level s o f maternal behavio r (larg e amounts of licking, grooming, an d arched-back nurs ing o f their young ) ar e les s fearfu l an d sho w better modulated HP A responses t o stressor s than offsprin g of mothers showin g low levels of maternal behavior . This outcome i s not due t o a change i n the underly ing geneti c profile ; i f pup s ar e cross-fostere d fro m low- t o high-materna l behavio r mother s a t birth , the offsprin g profil e i s associated wit h th e adoptiv e rather tha n th e biologica l mother . Therefore , varia tions in maternal behavior can serv e as a mechanism for nongenomi c transmissio n o f individua l differ ences i n stres s reactivity across generations. At least two majo r epigenomi c mechanism s ar e though t t o be involved : demethylatio n o f on e ke y sit e i n th e NGFI-A bindin g sequenc e o f th e firs t exo n o f th e GR gene , an d increase d acetylatio n o f the histone s surrounding th e G R gen e (Weave r e t al. , 2004) . These tw o alterations result in increase d expression of GRs an d increase d acces s o f transcription factor s to GRs , thu s increasin g HP A feedbac k regulatio n and alterin g th e physiologica l an d behaviora l pro files o f the offspring . In ou r model , prenata l ethano l exposur e i s an en vironmental variable that causes long-term alterations in th e physiologica l and behaviora l profile o f the offspring (e.g. , Weinber g an d Jerrells , 1991 ; Giberso n and Weinberg , 1995 ; Ki m et al, 1996 ; Glava s e t al , 2001; Hofmann e t al, 2002 ; Lan et al, 2004 ; Zhan g et al, 2005b) . We hypothesize tha t prenata l ethano l exposure reprogram s th e feta l HP A axis, resultin g i n long-term alteration s in HP A regulation an d respon siveness under basal and stress conditions and increasing HPA tone throughout life. In view of the evidenc e (Phillips e t al , 1998 , 2000 ) tha t feta l programmin g of the HP A axis, at least in part, underlies the connection betwee n th e earl y environment an d adul t stressrelated an d behaviora l disorder s i n humans , furthe r studies are essential to elucidate the mechanism s underlying prenatal ethano l effect s o n programmin g of HPA activity.

SUMMARY AND CONCLUSION S

The presen t chapte r discusse s the advers e effect s o f prenatal exposur e to ethanol o n neuroendocrine an d

ETHANOL EFFECTS ON ENDOCRIN E AND IMMUNE FUNCTIO N 17 3 immune functio n i n th e offspring , wit h particula r emphasis o n th e HP A axi s an d th e concep t o f feta l programming. Ethanol-induce d disturbance s o f th e reciprocal interconnection s betwee n th e pregnan t female an d th e fetu s ma y provide a common pathway by which perinatal exposure to different agent s results in feta l programming . Th e feta l HP A axi s i s repro grammed by ethanol exposur e such tha t HP A tone i s increased throughou t life . HP A activit y is increased and HP A regulation i s altered unde r bot h basa l an d stress conditions . Increase d exposur e t o endogenou s glucocorticoids over the lifespa n ca n alte r behavioral and physiological responsiveness and increas e vulnerability t o illnesse s late r i n life . Deficit s i n immun e competence ma y b e on e o f th e long-ter m conse quences of fetal HP A programming. Prenatal ethanol exposure compromise s offsprin g immun e functio n and increases vulnerability to the immunosuppressiv e effects o f stress. It i s now essential t o explor e possibl e epigenomi c mechanisms mediatin g altere d physiologica l an d be havioral functio n followin g prenata l ethano l expo sure. Th e fac t tha t ethano l cause s alteration s i n th e methylation stat e withi n cell s support s th e possibil ity tha t mechanism s simila r t o thos e describe d b y Weaver an d colleague s (2004 ) mediat e th e change s induced b y ethano l exposur e i n utero . Dat a fro m such investigation s will hav e importan t implication s for th e developmen t o f therapeutic intervention s focused o n reversin g th e long-ter m advers e effect s o f prenatal alcohol exposure .

8-OH-DPAT 8-hydroxy-2-(di-n-propylamino) tetralin FASD feta l alcoho l spectrum disorders FEE feta l ethanol-exposed S-HT serotoni n G gestationa l day GABA y-aminobutyri c acid GDX gonadectom y GnRH gonadotropin-releasin g hormone GR glucocorticoi d receptor hn heteronuclea r HPA hypothalamic-pituitary-adrena l HPG hypothalamic-pituitary-gonada l Ig immunoglobuli n IL interleuki n LC-NE locu s coeruleu s noradrenergi c (sympa thetic adrenal medullary) LPS lipopolysaccharid e MHC majo r histocompatibility comple x MR mineralocorticoi d receptor NE norepinephrin

e

NMDA N-methyl-D-aspartat e NO nitri c oxide NOS nitri c oxide synthase P postnata l day POMC pro-opiomelanocorti n

Abbreviations ACTH adrenocorticotropi c hormon e ADX adrenalectom y AVP arginin e vasopressin J3-EP p-endorphi n CBG corticosterone-bindin g globulin CNS centra l nervou s system

PVN paraventricula r nucleus PWM pokewee d mitogen SHRP stres s hyporesponsive period SIN-1 3-morpholinosydnonimin e SNRP stres s nonresponsive period TCR T cell receptor s TNF tumo r necrosis factor

CON A concanavali n A CORT corticosteron e CRH corticotropin-releasin g hormone CRH-R! corticotropin-releasin g hormone type 1 receptor DO! l-(2 , 5-dimethoxy-4-iodophenyl)-2-amino propane hydrochloride

ACKNOWLEDGMENTS Th e researc h from ou r laboratory tha t i s reporte d i n thi s revie w wa s supporte d by grants fro m th e Nationa l Institut e o f Alcoho l Abus e and Alcoholis m and fro m th e Universit y o f British Co lumbia Huma n Earl y Learnin g Partnershi p t o J.W . J.H.S. wa s supported by a Blum a Tischle r postdoctoral fellowship.

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11 Early Exposure t o Ethanol Affects the Proliferation o f Neuronal Precursor s Michael W. Miller

The numbe r o f neurons constitutin g the brai n is a direct reflection o f the numbe r of cells produced during early development . Th e presen t chapte r explore s th e effects o f ethano l o n spatiotempora l pattern s an d systems regulatin g cell proliferation . During develop ment mor e neuron s ar e generate d tha n th e numbe r comprising th e matur e brain . A "correction " occur s through the natural death of many neurons (se e Chapter 5) . Thi s proces s i s als o affecte d b y exposur e t o ethanol. Th e consequen t effect s ar e addresse d i n Chapters 15 , 16, and 17 .

SPATIOTEMPORAL PATTERN S

The proliferatio n o f neuronal population s occur s i n defined site s ove r restricte d period s o f time . Thes e spatiotemporal pattern s ar e affected b y early exposure to ethanol. Variou s structures involved have been examined, includin g segment s o f th e telencephalo n

(the neocorte x an d hippocampa l formation ) an d brain stem (cerebellu m and pons). Telencephalic Structures Neocortex Patterns o f Neurona l Generation . Neocortica l neurons ar e largel y generated ove r a protracted, bu t defined prenata l period. It has also been argue d tha t neocortical neuron s continu e t o b e generate d a t a low rate i n the matur e corte x (see Gould an d Gross , 2002; Rakic , 2002) . For the purpose s o f this review , any potential contributio n fro m adul t neuronogene sis in neocortex can be considere d to be too low as to have a n impac t o n neurona l number . A possibl e model o f postnatal neurona l generatio n ma y be th e proliferative activit y in the intrahila r zone (IHZ ; also known a s hippocampa l fiel d CA4 ) o f hippocampa l formation wherei n the generation of granule neurons

182

ETHANOL AND PROLIFERATION O F NEURONA L PRECURSORS 18 3 continues throughou t lif e (se e Hippocampal Forma tion, below). In th e rat , afte r productio n o f the pallia i preplate cortical neuronogenesis begins on gestational day (G) 11 and ends on G21 (Bruckner et al, 1976; Lund and Mustari, 1977 ; Miller , 1985 , 1988 ; Al-Ghou l an d Miller, 1989) . Thi s proces s i s orderly and follow s a well-defined inside-ou t sequence . Accordingly , th e first neurons generate d becom e laye r Vi a neurons ; later generations of neurons ar e destine d for progressively more superficial laminae. Ethanol exposur e alter s th e numbe r an d se quence o f neuronogenesi s (Miller , 1986 , 1988) , delaying and extending the period of neuronal generation b y 1 day eac h (Fig . 11-1) . O n eac h day , th e number of neurons with a particular time of origin is reduced. The exceptio n to this pattern is the substantial increas e i n neurona l generatio n a s cortical neu ronogenesis i s winding down (afte r G19) . Th e earl y depression i n neurona l generatio n i s followed b y a late surg e i n term s o f a s neurona l density ; o n G2 0 and succeedin g days, neuronal generation is signifi cantly greate r i n th e cortice s o f ethanol-treate d rat s than i n controls. Interpretatio n o f these dat a leads to four conclusions. The firs t i s that the delaye d onset of cortical neuronal generation suggests an ethanol-related retardin g effect o n th e acquisitio n of critical number s of proliferating cells required to initiate neuronal generation. That is, the population o f proliferating neural precursors has to reach a threshold siz e before neuronal precursors begin t o pas s through thei r fina l cell divisio n and th e youn g neurons begi n t o leav e th e prolifera tive population . Thi s patter n i s consisten t wit h th e concept of founder cell s (see Chapter 2) . The secon d conclusio n i s that ethano l ha s com plex effects o n cell proliferation. Initially, the prolifer ation o f neuronal precursor s is depressed an d late r it is promoted. This conclusion is supported by various studies discusse d belo w (se e Site s o f Proliferation , below). The thir d observation , fro m th e comple x time dependent effect s o f ethanol o n th e numbe r o f neu rons being generated, i s that the lat e surge may be an effort o f the proliferativ e population to compensate for the earl y depression . Indeed , th e densit y of neurons generated ove r the entir e perio d o f neuronogenesis is unaffected b y ethanol exposure . This is an interestin g finding in that the total number of cortical neurons, at

FIGURE 11- 1 Neurona l generatio n i n moto r cortex . These dark-fiel d micrograph s sho w tha t i n contro l rats, neurons generated on gestational day (G) 1 6 and on G20 are distributed in deep and superficia l cortex, respectively. I n ethanol-treated rats, neurons born o n G16 ar e als o distribute d i n dee p cortex , althoug h their number s ar e fewe r an d the y ten d t o b e mor e deeply disposed. Neurons generated o n G20 that are properly distribute d i n superficia l cortex ar e als o of fewer number . Man y late-generate d neurons , how ever, ar e ectopicall y distribute d i n dee p cortex . (Source: Image s taken, with permission, from Mille r (1986), Scienc e 233:1308-1311.) least in somatosensory cortex, is significantly lower in ethanol-treated rats (Miller and Potempa, 1990) . Th e implication i s that ethano l doe s no t affec t th e num ber o f neuron s produce d fro m a n ontogeneti c col umn; rather , it reduces the numbe r o f columns. An

184 ETHANOL-AFFECTE D DEVELOPMENT ontogenetic column i s a hypothetical uni t of developing cortex that includes the proliferative cell s and th e radial arra y of the progen y (e.g. , Rakic , 1978, 2000). This conclusion i s supported b y data showing that the density o f radia l glia l fiber s (th e guides commonl y used t o coordinate neurona l migration ) is unaffected by prenata l exposur e t o ethano l (Mille r an d Robert son, 1993). Finally, th e inside-ou t sequenc e i s largely maintained unti l the en d o f cortical neurona l generation . At thi s time , man y neuron s destine d fo r superficial cortex en d u p i n dee p corte x because o f a defec t i n neuronal migratio n (se e Chapter 13) . Th e effect s o f ethanol o n neuronal generation ma y be best appreci ated b y tracing the ontogen y o f select populations of cortical neurons , fo r example, corticospina l (Miller , 1987) an d callosa l projectio n (Miller , 1997 ) neu rons. I n general , they are distributed in lamina e that are norma l for their phenotype , with the notabl e exception o f the late-generate d callosa l neurons. Man y of thes e neuron s resid e i n infragranula r laminae . Thus, in addition to the conclusions described above, it appears that (a) ethanol doe s not affect th e connectional phenotype of a particular neuron (regardles s of a migratio n defect ) and (b ) th e decisio n abou t neuronal lineag e occur s a t the tim e o f neuronal origin . Similar cortica l disorganizatio n o f neurona l pheno types occur s i n cortice s in whic h cell s i n th e proli ferative zone s ar e heterochronicall y transplante d (McConnell, 1988) . Sites o f Proliferation . Th e proliferatio n o f neuronal precursor s occur s i n specifi c site s generall y located proxima l t o th e ventricula r syste m (se e Chapter 2) . For neocortex, cell proliferation occurs in three sites (Fig. 11-2). The ventricula r zone (VZ) is a pseudostratified epitheliu m linin g th e latera l ventri cles (Sauer , 1936 ; Watterso n e t al. , 1956 ; Saue r an d Chittenden, 1959 ; Jacobson, 1991) . The subventricu lar zone (SZ ) is a stratified epitheliu m externa l to th e neocortical V Z (Ramo n y Cajal , 1909-1911 ; Rakic et al, 1974 ; Shoukimas and Hinds , 1978) . Th e ganglionic eminenc e (GE ) generates som e loca l circui t neurons (Tamamaki et al, 1997 ; Zhu et al, 1999 ; Anderson étal, 2001; Wichterle étal, 2001). The tw o primar y neocortica l proliferatio n zone s are th e V Z an d SZ , an d ther e i s som e controvers y about the contributio n of the VZ an d S Z in the generation o f neocortica l neurons . Som e investigator s

FIGURE 11- 2 Effect s o f ethanol o n the thre e sources of neocortica l neurons . Neuron s tha t populat e th e cortical plat e (CP ) are derive d from th e ventricular zone (VZ) , subventricular zone (SZ) , and ganglioni c eminence (GE) . In norma l rats , V Z an d S Z cell s principally giv e ris e to infra - an d supragranula r neurons, respectively (soli d lines). The G E generate s lo cal circui t neuron s tha t ar e distribute d throughou t neocortex. Followin g prenata l exposur e t o ethanol , many late-generate d neuron s (originatin g largely i n the SZ ) complet e thei r migration s i n dee p corte x (dashed line) . IZ , intermediat e zone ; MZ , margina l

argue tha t th e V Z i s the sit e o f neuronal origi n an d that th e S Z generate s gli a an d glia l precursor s (Gomes e t al, 2003 ; Samuelson et al, 2003) . In contrast, other s argu e tha t al l neuron s ar e derive d fro m the S Z and that the VZ is merely a self-replacing population (Doetsc h e t al , 1997 , 1999; Alvarez-Buylla and Garcia-Verdugo , 2002 ; Marten s e t al , 2000 , 2002). Other studies support a different mode l o f cortical development, that both th e VZ and S Z give rise to cortica l neuron s (Miller , 1992 ; Luo an d Miller , 1998). Studies o n th e consequence s o f prenatal exposure to moderate amounts of ethanol (sufficien t t o produce

ETHANOL AND PROLIFERATIO N O F NEURONA L PRECURSOR S 18 5 peak blood ethano l concentrations o f 15 0 mg/dl) provide unique insigh t into the controversy over the con tribution o f th e V Z an d S Z t o th e populatio n o f neocortical neurons . Dail y neuronal production dur ing th e firs t hal f o f the perio d o f neuronongenesis is depressed b y prenata l exposur e t o ethano l (Miller , 1986, 1988) . Durin g thi s time , th e V Z i s the promi nent neocortica l proliferativ e zone . Change s i n cell proliferation withi n th e V Z paralle l the earl y reduc tions i n neurona l generation . Prenata l exposur e t o ethanol reduce s th e tota l numbe r o f cell s an d th e number of cycling cells in the VZ (Miller , 1989) an d it increase s the tim e cell s need t o transit through th e cell cycle (Miller and Nowkowski , 1991). The ethanol-induce d late surge in neuronal generation occur s when th e S Z becomes th e predominant proliferative zone . Th e S Z i s affected b y ethanol i n a way opposit e tha t o f th e VZ . Th e tota l numbe r o f cells i n th e S Z an d th e numbe r o f cycling cells ar e increased b y ethano l (Miller , 1989 ; Mille r an d Nowakowski, 1991) . Taken together , th e correlativ e dat a o n th e birt h dates o f cortica l neuron s an d characteristic s o f V Z and S Z cells sho w that bot h the VZ and S Z produc e neurons. Thi s thesi s wa s tested i n a n experimen t i n which fetuses were exposed t o ethanol for only 4 days during th e perio d o f (a ) S Z prominenc e (betwee n G18 an d G21) , (b ) VZ prominenc e (betwee n G1 2 and G15) , o r (c ) ste m cel l productio n (betwee n G 6 and G9) (Miller, 1996a). SZ cell proliferation and th e production o f late-generate d neuron s ar e onl y af fected whe n th e ethano l exposur e occur s betwee n G18 an d G2 1 (Fig . 11-3) . Moreover , this lat e exposure t o ethano l increase s both cel l proliferatio n an d production. VZ cel l proliferation and early-generated neurons, b y contrast , ar e maximall y affected b y ex posure t o ethano l tha t occur s betwee n G1 2 an d Gl 5, i.e., when the VZ is most prominent. This exposure t o ethano l inhibit s VZ cel l proliferatio n an d re duces th e numbe r o f neuron s generate d o n G15 . Exposure to ethanol between G6 an d G 9 doe s not affect th e proliferatio n o f cell s i n eithe r proliferativ e zone o r th e numbe r o f early- or late-generate d neu rons. Furthermore , base d o n timing , i t appear s tha t the VZ gives rise to neurons distributed in infragranu lar corte x an d th e S Z generate s supragranula r neurons. This conclusion i s supported b y the tracin g of derivative s of cell s tha t expres s transcript s fo r th e genes Svetl (Tarabyki n e t al. , 2001 ) an d eux (Niet o

et al. , 2004 ) fro m th e S Z t o th e supragranula r laminae. Radial Glia. Radia l glia, thought to serve as guides for neurona l migratio n (Rakic , 1971 , 1978) , ar e among th e first cortical cell s t o be generated . Tradi tionally, radial glia have been considere d transitiona l astrocytes (e.g. , Schmeche l an d Rakic , 1979 ; L u et al, 1980 ; Voigt , 1989 ; d e Lima e t al, 1997) , how ever, mor e recen t evidenc e show s tha t som e radia l glia ca n differentiat e int o neuron s (Malatest a e t al. , 2000; Hartfuss e t al, 2001; Levers et al, 2001; Noctor et al , 2001 , 2002 ; Alvarez-Buyll a an d Garcia Verdugo, 2002 ; Gôtz e t al., 2002; Sanai et al, 2004). This dual lineage is consistent with glial expression of nestin, a cytoskeleta l protei n associate d wit h neura l stem cell s (Hockfiel d an d McKay , 1985 ; Mille r an d Robertson, 1993) . Althoug h n o studie s have directly assessed th e effect s o f ethanol o n th e proliferatio n of radial glia , i t appear s tha t th e periodicit y of the net work o f radia l glia l fiber s i s unaffecte d b y ethano l (Miller an d Robertson , 1993 ; Vallè s e t al , 1996 ; Minana e t al., 2000) . This finding implies that thes e cells proliferat e t o maintai n a consisten t densit y within the expandin g telencephalic vesicle. A discussion o f the effect s o f ethanol o n radia l glia an d thei r role in neuronal migratio n is provided in Chapter 3. Hippocampal Formation Neurons in the hippoeampal formation ar e generated over a protracted period of time: i n the rat , from G1 5 to adulthoo d (Altman , 1962 ; Angevine , 1965 ; Sch lessinger e t al , 1978 ; Bayer , 1980 ; Miller , 1995a) . Most neuron s i n th e thre e hippoeampa l fields , CA1-CA3, an d th e dentat e gyru s ar e generate d pre natally. The sit e o f origin of these neuron s is the VZ . A notable exception is that most (-85%) granule neu rons i n th e dentat e gyru s ar e generate d postnatally . Postnatal granule cell generation occurs in the IHZ. Hippocampal cel l number s ar e substantiall y af fected b y ethanol exposure. These effects ar e spatially and temporall y defined. Total DN A and protei n con tent i n hippoeampa l formatio n i s lowe r i n animal s exposed to ethanol prenatally (Miller, 1996b). I n con trast, postnatal exposur e increases both DN A and protein content . Anatomical studie s hav e provided mor e detailed informatio n o n thes e ethanol-induce d effects. Animal s expose d t o ethano l prenatall y hav e

186 ETHANOL-AFFECTE D DEVELOPMEN T

FIGURE 11- 3 Time-dependen t effects o f ethanol on the tw o neocortical prolif erative zones . A . Exposure t o ethano l durin g three prenata l tim e period s was examined: durin g th e perio d o f neocortical ste m cel l generatio n (gestationa l day [G ] 6 to G9), prominenc e o f the ventricula r zone (VZ ) (G12-G15) , an d prominence o f the subventricula r zone (SZ ) (G18-G21). B. Exposure during the period o f stem cel l generation doe s not affec t th e proportio n o f cells prolif erating o n G1 5 an d G2 1 (whe n th e V Z an d S Z predominate , respectively) . Later exposures , by contrast, hav e specific effects o n th e zon e most active during the tim e o f exposure, i.e. , the V Z betwee n G1 2 an d G1 5 and th e S Z between G1 8 an d G21 . (Source: Image s taken , wit h permission , fro m Mille r (1996a), Alcohol Clin Ex p Res 20:139-143.)

fewer neuron s in the pyramida l cell layer of CAI an d granule cel l laye r of the dentat e gyru s than d o con trols (Miller , 1995a) . Interestingly , whereas postnata l exposure generall y has littl e effec t o n th e numbe r of these neurons , it can increas e the numbe r of granule neuron s i n th e dentat e gyru s (Miller , 1995b ; Pawlak et al., 2002). This effec t o n granule neurons is concentration-dependent Moderat e amount s o f ethanol ar e stimulator y and hig h concentration s of ethanol are depressive. The ethanol-induced changes in neuronal number directly reflect alterations in neuronal generation . Fo r example, the generatio n o f dentate granul e neurons is reduced b y prenatal ethano l exposur e an d promote d by postnatal exposure . Thus, ethano l affect s develop ing hippocampal formatio n in th e sam e manne r tha t it affect s th e generatio n o f neocortica l neurons . As postnatal generation of granule neurons occurs in th e IHZ, i t appear s that ethano l affect s al l telencephali c auxiliary (derived) proliferative zone s similarly.

Brain Ste m The effec t o f chronic gestationa l exposur e t o ethano l on th e principa l sensor y nucleu s o f th e trigemina l nerve (PSN) and cerebellum has been examined . The PSN i s a simple structure. All of its neurons are derived from th e pontin e VZ. Prenatal exposure to ethanol reduces the numbe r o f neuons i n th e PS N (Mille r and Muller, 1989 ; Miller , 1995b) . This reductio n i s partially (two-thirds ) du e t o decreased cell generation an d partially (one-third ) to exacerbatio n of mechanisms of naturally occurring neuronal death (Miller, 1999a). The reduction in cell generation appears to be equivalent for neurons and gli a and neurona l generation i s delayed by about 1 day (Miller and Muller, 1989) . The cerebella r corte x is also vulnerable to ethano l toxicity. A gross indication that ethanol affect s cel l generation i n the cerebellu m i s the effec t o f ethanol o n its size (Bauer-Moffett an d Altman, 1975; Kornguth et al., 1979; Dia z an d Samson , 1980 ; Pierc e an d West ,

ETHANOL AND PROLIFERATION O F NEURONA L PRECURSOR S 18 7 1986; Bonthiu s and West, 1988) . Exposur e t o moder ate amounts of ethanol (bloo d ethano l concentration s of-150 mg/dl ) causes reductions (15%-20%) in cerebellar siz e simila r to tha t eviden t fo r cerebra l corte x (Miller, 1996b) . Followin g exposur e t o high amount s (sufficient t o produc e bloo d ethano l concentration s of >250 mg/dl), the siz e of the cerebellu m i s more adversely affected . Cerebellar neuron s ar e generate d i n tw o site s — the VZ and a derived proliferative zone, th e externa l granular layer (EGL). Attention ha s been directed to ward the EG L becaus e i t is most prominent i n the rat postnatally an d i t give s birt h t o a populou s neuro n type i n th e centra l nervou s syste m (CNS) , th e cere bellar granul e neuron . Postnata l exposur e t o hig h amounts o f ethano l prolong s th e existenc e o f th e EGL an d delay s its depletion (Bauer-Moffet t an d Altman, 1977 ; Korngut h e t al. , 1979) . Moreover , cel l proliferation i n th e EG L i s reduced. These dat a suggest that the EG L i s different fro m othe r derive d proliferative zone s i n tha t it s proliferative activity is no t stimulated by ethanol. Th e EG L differ s fro m th e S Z and IH Z insofar as it is the sol e source of granule neurons, whereas the S Z and IH Z produc e neuron s tha t can be generated i n the VZ. Nonetheless, thi s conclu sion remain s unconfirme d unti l dose-respons e dat a analysis of the EG L i s performed.

CELL CYCLE KINETICS

In vivo and In Situ Studies As cells proliferat e the y pass through a defined se t of steps, cumulativel y known a s the cell cycle. The cel l cycle ha s four phases : Gl, S, G2, and M. During th e Gl phas e th e cell s eithe r prepar e fo r anothe r pass through the cell cycle or exit from th e cell cycle. Durings, cells replicat e their nuclear DNA . G 2 i s a short period whe n preparatio n fo r mitosi s take s place , which occur s durin g M . Dat a o n th e cel l cycl e ar e best appreciated for VZ cells . The somat a o f VZ cell s move rhythmicall y as the cell s pass through th e fou r phases (Sauer , 1936 ; Watterso n e t al. , 1956 ; Saue r and Chittenden , 1959 ; Atla s an d Bond , 1965) . Through thi s process , calle d interkinetic nuclear migration, mitoti c cell s tha t ar e a t th e ventricula r surface reconstitute and mov e their nuclei to positions in the external third of the VZ where they replicate their DNA during S.

Ethanol doe s no t affec t th e patter n o f interkinetic nuclear migration in the VZ per se (Kennedy and El liott, 1985 ; Miller, 1989) . On th e other hand, ethano l does affec t th e tim e a VZ cel l take s t o pas s throug h the cell cycle. For example, i n the neocortical V Z of a 21-day-old rat fetus (Miller and Nowakowski, 1991) or a slic e o f corte x explante d fro m a 17-day-ol d fetu s (Siegenthaler an d Miller , 2005a) , th e tota l lengt h o f the cel l cycl e (T c) i s increased 26%-29% . Thi s in crease ha s a direc t negativ e effec t o n th e numbe r o f cells produce d i n th e VZ . An increase i n th e T c is also eviden t i n th e EG L o f the cerebellu m (Borge s and Lewis , 1983) . Ethanol-induce d increase s in Tc primarily resul t from a lengthening o f Gl. Thes e effects o f ethanol ar e no t th e sam e fo r all proliferative populations. Fo r example, th e Tc of the neocortica l SZ i s not affecte d b y moderat e exposure s t o ethano l (Miller and Nowkowski , 1991). In addition to cell cycle kinetics, the production of new cells i s defined by three othe r feature s of proliferating populations: th e growt h fractio n (GF), th e pro portion of cells that exit the cell cycle, and the leaving fraction (LF) . The G F i s the proportio n o f total cel l population tha t i s activel y cycling. Overall , th e G F for th e cerebra l wal l extend s ove r th e perio d o f neo cortical neuronongenesi s (Mille r an d Kuhn , 1995 ; Takahashi e t al, 1995 , 1996) . Althoug h ther e i s no ethanol-induced differenc e i n th e G F a t th e begin ning of this period, as neuronal generation proceeds a significant differenc e takes place . Ethano l cause s a n increase i n the GF. The effec t o f ethanol on the over all G F i s largely due t o ethanol-induce d increase s in the SZ . Both th e specifi c G F fo r the S Z (Mille r and Nowkowski, 1991 ) and th e numbe r o f cells in the S Z (Miller, 1989 ) ar e increase d b y ethanol. Th e G F fo r the VZ i s either unaffecte d by ethanol in vivo (Mille r and Nowkowski , 1991) or subtly, but significantly , reduced i n th e V Z o f an organotypi c slic e preparation (Siegenthaler an d Miller , 2005a) . The effec t o f th e increases i n GF i n the S Z is accentuated wit h time as the S Z become s th e majo r neocortica l proliferativ e zone. After cells pass through M, the daughter cells either re-enter the cycle or exit the cell cycle. The numbe r of cells tha t exi t th e cel l cycl e ca n b e determine d b y tracing the proportio n o f cells tha t hav e incorporate d a DN A precursor during S relative to the tota l popu lation o f cycling cells, for example , cell s tha t express Ki-67 (Chen n an d Walsh , 2002 , 2003 ; Siegenthale r and Miller , 2005a ; 2005b) . Accordingly , a n i n sit u

188 ETHANOL-AFFECTE D DEVELOPMEN T study show s tha t ethano l increase s th e numbe r o f cells that exit the cel l cycl e (Siegenthale r an d Miller , 2005a). A thir d featur e contributin g to neurona l produc tion i s th e LF . Thi s differ s fro m th e exi t fractio n i n that i t encompasses cel l cycl e exi t and th e proces s of moving fro m th e proliferativ e zones . Th e L F rise s steadily over the period of neuronal generation (Miller, 1993, 1999b ; Takahashi et al, 1996) . Ethanol does not affect th e L F durin g this period (Miller , 1993) . Inasmuch a s ethano l doe s affec t th e numbe r o f exitin g cells, thi s lac k o f increas e i n th e L F implie s tha t ethanol induce s postmitoti c cell s t o remai n i n th e proliferative zones for a longer time before commenc ing their migration. Various cell cycle-relate d protein s ar e affecte d b y ethanol: th e expressio n o f cycli n A ( a regulato r o f S and G2) , cycli n Dl, and cdk 2 (proteins that regulate the progressio n of cells fro m G l t o S ) i s reduced b y ethanol exposur e (Li et al, 2001, 2002; Siegenthale r and Miller , 2005a) . Cerebella r expressio n o f cycli n D2, cdk4, and cdk 6 (regulator s of Gl) ar e unaffected by ethano l (L i et al, 2002) . By contrast, p2 7 ( a protein involve d in movin g cells from G l ou t o f the cycle) i s depresse d b y ethano l i n th e V Z o f cortica l expiants (Siegenthale r an d Miller , 2005a ) an d i n developing rat cerebellum (L i et al., 2002). Thus, the effects of ethanol o n cell cycle-related protein s not only corroborate, bu t provid e a mechanis m fo r ethanol induced increase s in the Tc and cell cycle exit and a decrease i n the GF. Cell Culture Studies Studies i n cel l cultur e model s replicat e th e i n viv o data an d provid e systems in which t o explore cellular and molecula r mechanism s underlyin g th e ethanol induced changes . Variou s cell s hav e bee n use d a s models o f proliferatin g neura l precursors—primar y cultures of neurons (Jacobs and Miller , 2001; Miller, 2003a) an d astrocyte s (Kenned y and Mukerji , 1985 ; Guerri e t al. , 1990 ; Snyde r e t al, 1992 ; Kòtte r an d Klein, 1999 ; Lu o an d Miller , 1999a ; Cost a an d Guizzetti, 2002 ; Mille r an d Luo , 2002a ) an d neu ronal (Lu o an d Miller , 1997a , 1999b ; Mille r an d Luo, 2002b ) o r glia l (Wazir i e t al , 1981; Isenber g et al. , 1992 ; Resnicof f et al, 1994 , 1996b ; Lu o an d Miller, 1996 ) cance r cel l lines . Ther e i s consensu s that ethanol increase s the time these neural cells take to double the siz e of their populations.

Doubling tim e i s a comple x yardstic k reflectin g the su m o f simpler parameters. On e o f these parame ters is an increase in the Tc, and mos t of this increase is from a lengthened G l (Guerr i et al., 1990 ; Luo an d Miller, 1997a ; Jacobs and Miller , 2001). Remarkably, the effect s o f ethano l o n th e proliferatio n o f dissoci ated neuron s an d B10 4 neuroblastom a cell s strongl y parallel thos e o f V Z cell s i n viv o (cf . Mille r an d Nowakowski, 1991 ; Lu o an d Miller , 1997a ; Jacob s and Miller , 2001). Another factor i s the GF . Alteration in the GF has an invers e effect on doubling time . For example, a s i n th e cas e o f B10 4 cells , a n ethanol induced decreas e i n th e numbe r of cycling cells con tributes t o a n increas e i n doublin g tim e (Lu o an d Miller, 1997a) . A thir d facto r definin g th e doublin g time i s the incidenc e o f cel l death . Deat h normall y occurs amon g proliferatin g population s (Blaschk e et al., 1996; Thomaidou et al, 1997; Jacobs and Miller, 2000) (se e Chapter 5) . Ethanol affect s th e balanc e between cel l proliferation and deat h (Jacob s and Miller , 2001; Miller , 2003a) . Fo r example , ethano l cause s a steady decreas e i n th e numbe r o f primar y culture d cortical neurons . Mos t (two-thirds ) of the decreas e i s due t o compromised cell proliferation, whereas the remainder i s from ethanol-induce d cel l death . Ethano l affects neurona l generatio n an d deat h i n th e imma ture PSN in a similar manner (Miller , 1999a).

LIGAND-MEDIATED SYSTEM S

Many factors may contribute t o the differentia l effec t of ethano l o n specifi c population s o f proliferating cells. A majo r playe r is the myria d of growth factor s that tightly regulate cell proliferation. This sectio n focuses o n a smal l numbe r o f growt h factor s tha t ha s been studie d i n relatio n t o th e effect s o f ethanol . These factor s includ e platelet-derive d growt h facto r (PDGF), basi c fibroblas t growt h facto r (bFG F o r FGF-2), insulin-lik e growth factor (IGF)- l an d IGF 2, an d transformin g growth facto r (TGF ) pi . The y can b e classifie d a s mitogeni c (FGF-2 , IGF-1 , an d PDGF) or anti-mitogenic (TGFp). Mitogenic Factors Platelet-Derived Growth Factor The cyclin g activit y o f neura l cell s i s potentl y pro moted by PDGF. It is noteworthy that PDGF receptors

ETHANOL AND PROLIFERATION O F NEURONA L PRECURSOR S 18 9 are expresse d i n th e S Z o f normal rodent s (Sasahara et al, 1991 ; Yeh et al, 1991) . Th e growth-promoting effect o f PDGF has also been described fo r astrocytes (Luo an d Miller , 1999a ) an d B10 4 neuroblastom a cells (Luo and Miller, 1997a) . PDGF i s active as a dimer , forme d as a combina tion o f th e tw o isoforms , PDGF- A an d PDGF-B . PDGF-BB i s the mos t potent ligand , doubling or trebling the growt h o f B104 cells an d astrocytes , respec tively, ove r 3 days (Lu o and Miller , 1997a , 1999a) . In contrast, PDGF-A A onl y increase s cel l growt h b y -50% (B104 neuroblastoma cells ) or -70% (astrocytes) over the sam e period. Although the tota l length o f the cell cycl e i s reduced, mos t o f the growt h result s fro m the recruitmen t o f cells int o the cyclin g population— i.e., throug h a n increas e i n th e GF . Thi s patter n i s reminiscent o f th e respons e o f S Z cell s t o ethano l (Miller an d Nowakowski , 1991 ; Mille r an d Kuhn , 1995). The PDGF-mediate d increase in cell proliferation is mediated throug h tw o intracellular signal transduc tion cascades : the ras-raf pathwa y and th e protei n kinase C (PKC ) pathwa y (Fig . 11-4 ) (Lu o an d Miller , 1999a). Th e ultimat e effect s o f PDGF ar e th e tran sient (phasic ) activation o f mitogen-activate d protei n kinase and extracellula r signal regulated kinase (ERK) and th e transcriptio n of proliferation-associated genes . PDGF-mediated growt h i s compromise d b y ethanol, th e growt h amon g B10 4 cell s (Lu o an d Miller, 1997a) and astrocytes (Luo and Miller, 1999a ) being reduced i n a concentration-dependent manner . Interestingly, the effect s o f ethanol o n th e tw o ligand isoforms are cell type-specific . The actio n of PDGFBB on B10 4 cells i s twice as sensitive to ethano l a s it is to PDGF-AA (Lu o and Miller , 1997a) , an d th e in verse situatio n i s eviden t fo r neocortica l astrocyte s (Luo and Miller, 1999a) . This may mean tha t PDGF differentially affect s cel l lineage s i n vivo . Moreover , the variatio n i n effect s ma y underli e th e differentia l activity o f SZ cells , whic h i n th e ra t ar e largel y neu ronogenetic prenatally and gliogenetic postnatally. The effec t o f ethanol o n ligand-mediate d activity apparently reflects ethanol-induced changes in recep tor expression and activation . Two receptors can bind PDGF ligands : PDGF-a r an d PDGF-pr . I n B10 4 cells, ethanol target s the PDGF-ar, which ultimately leads to a change in ERK activation (Luo and Miller, 1999a). Ethano l doe s not sto p ERK phosphorylation; rather, i t induce s a sustained ER K activation, which in turn can lead to changes in gene transcription.

Fibroblast Growth Factor-2 FGF-2 i s an importan t regulato r o f cell proliferatio n in th e V Z an d derive d zone s o f developing rodents . An immunohistochemical study shows that FGF-2 receptors ar e predominantl y expresse d i n V Z durin g normal, cortical development (Weis e et al., 1993) . SZ cells in the neonata l rat brain are also FGF-2 responsive (Bartlet t et al, 1994 ; Gritt i e t al, 1999 ; Wagne r et al , 1999 ; Decke r e t al. , 2002) . Fo r example , in traperitoneal injection of FGF-2 stimulates the prolif eration o f neuronal precursor s in the S Z (Lachapell e et al. , 2002) . Similarly , intraventricula r administra tion of FGF-2 expands the populatio n of SZ neuronal progenitors (Kuh n e t al., 1997 ) an d increase s mitoti c activity in the SZ and IH Z in adul t rat s (Wagne r et al, 1999) .

FIGURE 11- 4 Signa l transductio n triggere d b y ethanol-sensitive growt h factors . Cel l proliferatio n is regulated b y platelet-derive d growt h facto r (PDGF ) and transforming growth factor (TGF ) pi . PDGF acts as a dime r o f A o r B isoforms . Thes e growt h factor s bind t o specifi c trans-membran e receptor s tha t au tophosphorylate. Thi s proces s trigger s a cascad e o f phosphorylation event s throug h variou s intermedi aries. Interestingly , although PDG F an d TGFp l in duce opposit e effect s (mitogeni c an d anti-mitogeni c actions, respectively) , they bot h stimulat e extracellular signal-related kinase (ERK). The circle d plu s and minus sign s identif y th e action s o f ethanol . Briefly , ethanol ha s a specific effec t o n a single PDGF receptor isofor m (PDGF-ar ) and , throug h bot h pro - an d antimitogenic factors , ethanol stimulates ER K activ ity. This i n turn ca n alte r nuclear gene transcription. MEK, methylethylketone ; nCAM , nerv e cel l adhe sion molecule; PKC , protein kinase C.

190 ETHANOL-AFFECTE D DEVELOPMENT Dissociated cultures provide model systems to illuminate th e effect s o f FGF-2 o n proliferatin g populations. Cell cycle kinetics among B104 cells is affected by FGF-2 (Lu o and Miller, 1997a) . The Tc and G F are equally affected b y FGF-2. These data are consis tent with the mixe d expression of FGF-2 response by VZ and S Z cells. Other preparation s that presumably model V Z an d S Z cells impl y that FGF- 2 promote s the proliferation o f VZ and S Z derivatives (Martens et al., 2000, 2002). The effect s o f ethano l o n FGF-2-mediate d cel l proliferation ar e no t well understood. Studie s explor ing thi s interactio n hav e examine d C 6 gliom a cell s (Luo an d Miller , 1996 ; Mille r an d Luo , 2002a ) an d telencephalic neuroepithelial cell s (M a et al., 2003). They sho w tha t ethano l eliminate s th e FGF-2 promoted increas e i n cell proliferatio n such tha t th e effects o f FGF-2 and ethano l nullif y eac h other . The depressiv e effect s o f ethano l ar e mediate d through th e FGF- 2 receptor , fl g (Lu o an d Miller , 1996b, 1997b) . ER K phosphorylation, a downstream event o f flg activation, increase s i n cortica l astrocyte s (Smith an d Navratilova , 2003 ) an d ste m cell s (M a et al. , 2003 ) treate d with ethanol . Althoug h ethano l does not alter protein expressio n of FGF-2 receptors , ERK activity remains elevated, implying that ethano l affects recepto r phosphorylation . Insulin-like Growth Factor-1 Prenatal exposure to ethanol inhibition of intrauterine and earl y postnatal growth correlates with the effec t of ethanol o n the IGF system (Breese et al, 1993 ; Sing h et al., 1994) . Ethanol halves plasma IGF-1 content i n the pregnant dam (Singh et al., 1994; Breese and Son ntag, 1995 ) and in the fetal brai n (Singh et al, 1996) . Data o n the effect s o f prenatal exposur e to ethanol o n IGF-2 are mixed. One repor t states that the amount of IGF-2 is doubled (Brees e and Sontag , 1995) , whereas others sho w tha t IGF- 2 expressio n i s halve d (Sing h et al., 1994 , 1996) . Likewise , the effec t o f ethanol o n binding protei n (IGF-BP ) i s als o mixed . Som e evi dence show s tha t IGF-B P expressio n i s reduce d (Breese an d Sontag , 1995) , an d othe r studie s sho w that it is increased (Singh et al, 1994, 1996) . Although the reason s for thes e disparat e data ar e unclear , i t is agreed tha t (1 ) ethano l affect s circulatin g plasma an d fetal expressio n of IGF-2 an d IGF-B P an d (2 ) ethano l causes opposing effects o n the expressio n of these two proteins. The reductio n in IGF-1 persist s in the young

offspring (u p t o 3 weeks old), but i t abates by 40 day s of ag e (Brees e e t al , 1993) . I n contrast , IGF- 2 an d IGF-BP ar e unaffected in preweanling rats. The effec t o f IGF-1 ha s been studie d in C6 gliom a cells (e.g. , Resnicof f e t al , 1994 , 1996a , 1996b) . These cells have the advantage of being able to proliferate i n a serum-free medium an d i n th e absenc e o f any priming growth factors (e.g. , Isenberg et al., 1992 ; Resnicoff e t al, 1994 ; Lu o and Miller , 1996) . IGF- 1 is a potent mitoge n fo r proliferating C6 cell s (Resni coff e t al., 1994 , 1996a , 1996b) . Thi s effec t i s at least partially growt h factor-specific , a s proliferatio n o f these cell s i s unaffected b y PDGF (Resnicof f et al. , 1994). It is worth noting that this differential respons e to growth factors may be a quality of the cell s becaus e primary cultur e astrocyte s and B10 4 neuroblastom a cells are highly regulated b y PDGF (Luo and Miller , 1997a, 1999a ) (se e Platelet-Derive d Growt h Factor , above). Ethanol inhibit s th e proliferatio n o f C 6 cell s (Waziri e t al, 1981 ; Isenber g e t al, 1992 ; Resnicof f et al, 1994 , 1996a , 1996b ; Luo and Miller, 1996) , and it has been suggested that this inhibition i s mediated by ethanol-induced disruption s of IGF-1 recepto r (IGF lr; Resnicof f e t al , 1994 , 1996a , 1996b) . Ethano l blocks th e mitogeni c effec t o f the ligan d (IGF-1 ) an d interferes wit h receptor (IGF- 1 r) activation. Autophos phorylation o f the IGF- 1 r is inhibited b y low concentrations o f ethanol . Also , ethano l inhibit s th e anti-apoptotic effect s o f IGF-1 (Gu i étal, 1997) . Anti-mitogenie Facto r TGFp ligand s ar e intracortica l factor s tha t tur n of f neural cel l growt h (Camero n e t al. , 1998 ; Bôttne r et al, 2000) . Exogenou s TGFpl inhibit s the growth of variou s cells , includin g C 6 cells , astrocyte s (Ro zovsky e t al , 1998 ; Ric h e t al. , 1999 ; Rob e e t al , 2000; Mille r an d Luo , 2002a) , B104 neuroblastom a cells (Luo and Miller, 1999b) , an d neural progenitor s (Miller an d Luo , 2002b) . TGFp l doe s thi s b y removing cell s fro m th e proliferativ e population, i.e. , reducing th e G F (Siegenthale r an d Miller , 2005b) . Interestingly, it does not affec t cel l cycle kinetics. Transforming Growth FactorMediated Signal Transduction The effec t o f TGFpl o n cel l proliferatio n is medi ated throug h ER K activatio n (Fig . 11-4) . TGFp l

ETHANOL AND PROLIFERATION O F NEURONA L PRECURSORS 19 1 induces sustaine d (>9 0 min ) activatio n (Lu o an d Miller, 1999b) , a n anti-intuitiv e resul t i n that ER K is generally associate d with mitogenic activity . A recent study o f proliferating cells i n organotypi c culture s of fetal corte x shows that cycling cells in the VZ respond to TGFpl (Siegenthale r an d Miller , ZOOSb) . As with the dissociated cells, TGFpl inhibit s VZ cell prolifer ation in situ by increasing the exi t of cells from th e cycling populatio n an d concomitantl y decreasin g the GF. Combined treatmen t o f cortica l slice s wit h ethanol and TGFp 1 affects the expressio n of cell cycle-related protein s (Siegenthale r an d Miller , 20 0 5b) . For example , tota l cycli n D l expressio n i s reduce d in th e VZ , an d becaus e ethano l an d TGFpl d o no t affect th e frequenc y o f cyclin Dl-positiv e cells , i t ap pears tha t th e amoun t o f cycli n D l pe r cel l i s reduced. A simila r decremen t i n p2 7 expressio n ha s been detected . A s for p21, co-treatment wit h ethano l and TGFp l eliminate d th e increas e i n p2 1 expres sion promoted b y TGFpl alone . Thus, three proteins involved in the transition o f cells from Gl (t o either S or GO ) ar e depresse d b y combine d exposur e t o ethanol an d TGFpl. It appears that ethanol interferes with TGFpl regulatio n o f cell cycle activity. In Vivo Transforming Growth Factor Systems Various immunohistochemica l analyse s hav e show n that th e developin g nervou s system contain s a n en dogenous TGFp system (Flanders e t al., 1991 ; Pelto n et al , 1991 ; Miller , 2003b) . TGF p ligand s ar e expressed i n th e V Z an d S Z o f th e cerebra l vesicl e in vivo (Fig . 11-5) . TGFp l i s expresse d i n th e V Z pre- an d postnatall y and TGFp 2 i s expressed during the firs t postnata l week . Potentiall y mor e importan t than ligan d expressio n i s the distributio n o f the tw o chief TGF P receptors , TGFpI r an d TGFpIIr . Th e TGFpIr i s expressed in bot h neocortica l proliferativ e zones throughou t life , wherea s th e TGFpII r i s only expressed i n th e VZ . Moreover , bot h TGFp 2 an d TGFpIr are expressed by radial glia. Prenatal exposur e t o ethano l affect s endogenou s TGFP syste m expressio n i n viv o (Miller , 2003b) . TGFpl expressio n falls i n immature an d mature rats, whereas TGFp 2 expressio n rise s onl y i n perinates . Changes i n recepto r expressio n ar e mos t eviden t for the TGFpIr ; it s expressio n i s significantly highe r i n ethanol-treated rats , regardless o f age. These ethanol -

FIGURE 11- 5 Transformin g growth facto r (TGF ) P ligands an d receptor s i n th e developin g ventricula r and subventricula r zones . Pair s of images depict th e distribution o f immunolabeling for TGFpl (to p left) , TGFp2 (to p right) , TGFpIr (botto m left) , an d TGF pIIr (bottom right ) in control (Ct ) and ethanol-treate d (Et) perinates . Ethano l markedl y affect s ligan d an d TGFpIIr expressio n in the subventricula r zone (SZ) . Open and solid arrows identify immunolabelin g i n the ventricular zone (VZ ) and SZ , respectively. Curved arrows labe l radia l gli a i n th e intermediate zon e (IZ) . Scale bars = 10 0 jlm. (Source; Images taken, with permission, fro m Mille r (2003b) , J Com p Neuro l 460:410-424.)

induced change s ar e associate d wit h alteration s i n the distributio n o f cell s tha t ar e TGFpl , TGFp2 , TGFpIr, an d TGFpII r immunoreactive . Th e mos t marked change s ar e increase s i n S Z expressio n o f TGFp ligands an d especiall y receptors . Thi s finding may underli e th e increas e i n gliosi s tha t occur s fol lowing early postnatal ethanol exposure (Fletcher an d Shain, 1993 ; Goodlett et al, 1993) . Lineage Specificity Studies o f th e effect s o f exogenou s TGFp l impl y that the endogenous TGFp system in the developin g and/or adul t brai n i s affected b y ethanol. I n general ,

192 ETHANOL-AFFECTE D DEVELOPMEN T ethanol block s TGFpl inhibition , however , thi s ef fect is lineage-dependent Fo r astrocytes and C 6 cells , the combine d effec t o f ethanol an d TGFp l i s competitive (Mille r and Luo , 2002a) , that is , in th e pres ence o f ethano l an d TGFpl , th e proliferatio n o f either of these cel l types is the sam e as that occurring in untreate d cultures . I n contrast , th e individua l inhibitory effect s o f ethanol an d TGFp l i n B10 4 cells (Luo an d Miller , 1999b ) an d cell s determined t o b e neurons (Mille r and Luo , 2002b ) are additive. Treatment o f B10 4 cell s wit h a n agen t tha t block s ER K activity largely eliminates the inhibition of [3H]thymidine incorporation . Thi s resul t implie s tha t bot h ethanol an d TGFpl-mediated inhibitio n of neuronal progenitors is transduced through ERK. Interestingly, th e T c fo r in situ V Z cell s treate d with TGFp l an d ethano l i s the sam e a s the T c for untreated preparations . The sam e effect i s evident for the GF: TGFpl an d ethanol cancel the inhibitory action o f th e othe r anti-mitogen . Thus , V Z cell s i n organotypic cultures behave the same way as cultured cortical astrocytes . This findin g i s particularly interesting in light of data showing that radial glia, at least in cortex, are stem cells that can give rise to neurons or astrocytes (see Radial Glia, above). This phenomeno n begs th e questio n o f ho w astrocyte s are identifie d — i.e., is the commonl y use d marker , glia l fibrillary acidic protein, astrocyte-specific (se e Chapters 1 3 and 18)? Equally , i f no t mor e important , i s th e findin g that afte r a cell i s determined a s becoming a neuron , even though i t may retain mitotic ability (Jacobs and Miller, 2000), its reaction to its environment, such as combined TGFp l an d ethanol , ha s changed . Th e implications o f thi s shif t i n responsivit y ma y fram e our understandin g of stem cell s an d progenitor s an d their future utilit y in transplantation. Interactions amon g Growth Factor s Growth factor s d o no t ac t i n vivo i n isolation . They are multiply expressed, and cell s express multiple re ceptors. Th e effect s o f PDGF , FGF-2 , IGF-1 , an d TGFpl ar e affecte d b y th e mediu m i n whic h th e cells ar e raised . The actio n o f each facto r i s amplified, or onl y expressed , when th e mediu m contain s fetal seru m (Lu o an d Miller , 1997a , 1999b ; Mille r and Luo , 2002a) . (Seru m i s ric h i n a plethor a o f growth factors. ) A specific exampl e o f an interactio n among growt h factor s i s tha t betwee n FGF- 2 an d TGFpl (Mille r an d Luo , 2002a) . I n culture s o f

neocortical astrocyte s and C 6 cells , these two factors effectively cance l ou t th e actio n o f each other . Like wise, ethanol inhibits the primar y action of each factor. Th e amoun t o f cel l growt h tha t occur s amon g cells treate d wit h FGF- 2 an d TGFpl, wit h o r without ethanol , i s the sam e as that eviden t in untreate d cultures. The multipl e effect s o f ethano l and/o r growt h factors o n cel l proliferatio n appea r t o depen d o n a single gatekeeper , ERK . Wit h ou r curren t under standing, however , it is difficul t to deciphe r the action o f this transduction funnel . Ethano l ca n inhibi t cell proliferatio n by altering ER K activit y (Lu o an d Miller, 1999a , 1999b) . Furthermore , bot h mitogeni c (e.g., PDGF and IGF-1) an d antimitogenic (TGFpl) growth factors activate ERK. It seems parsimonious to conclude tha t ERK is not a simpl e switc h tha t is either o n o r off ; ERK i s activate d b y a mitoge n an d turned of f by an anti-mitogen . Instead, ERK i s a complex switc h tha t ca n b e activate d b y substances with opposing effects , an d th e characte r o f that activatio n (acute o r sustained) defines th e effec t o n subsequen t transcription. The effect s o f ethanol ar e not ubiquitous , they are quite specific. For example, ethanol affect s th e expres sion and activit y of the PDGF-oer , but no t the PDGF pr. Potentially , thi s differentia l effec t o f ethano l depends o n the distribution of the receptors. Could it be tha t on e recepto r i s associate d wit h a lipi d raft , whereas another is not?

WHAT CONTROLS CELL NUMBER?

Ethanol studie s sugges t that , a t leas t i n som e telen cephalic structures, there is a dynamic interaction between th e tw o proliferativ e zones . A t lo w an d moderate exposure s t o ethanol , cel l proliferatio n in the V Z i s reduced , bu t proliferatio n i n th e S Z an d IHZ i s increase d (Miller , 1989 , 1995a) . Hence , th e implication is that there i s a set point, an implicit goal for cel l number , and tha t the stimulate d proliferatio n in th e S Z an d IH Z i s compensating for the earl y depression of VZ proliferation. The numbe r o f neurons i n a define d cylinder of neocortex i s unaffecte d b y moderat e ethano l expo sure (Miller and Potempa, 1990) . Nevertheless, cortex is smaller (Miller , 1987 , 1996b) . Thi s decrease i n size likely results from th e ethanol-induce d eliminatio n of ontogenetic columns . Presumably , this alteration i s a

ETHANOL AND PROLIFERATION O F NEURONA L PRECURSOR S 19 3 result of the goa l of the developin g nervous system to maintain th e integrit y of ontogenetic columns , even if th e numbe r o f column s i s reduced . Exposure s t o high concentration s of ethanol overwhel m the com pensatory response. The change s i n th e telencephalo n contras t with those i n region s o f the brai n derive d from onl y th e VZ. In the absence of a derived proliferative zone, no compensatory respons e i s possible an d reductio n i n cell numbe r occur s regardless of ethanol concentra tion. Th e depressio n o f proliferativ e activit y i n th e VZ ca n occu r throug h tw o nonmutuall y exclusiv e mechanisms. Ethanol ca n inhibi t the actio n of mitogenie growt h factor s and/o r ethano l potentiate s the actio n o f anti-mitogeni c factors . Empirica l evidence fro m cel l cultur e studie s support s both o f these theses. Derived proliferatin g populations , fo r example , the SZ , are affected b y ethanol in a complex, bimodal fashion. This provides a mechanism fo r the S Z to off set primary damage to the VZ in an attempt to generate a structure with "proper" cell density . The abilit y of the S Z t o compensate fo r reductions i n VZ prolif eration ma y resul t from th e differentia l sensitivit y o f the tw o cell populations to ethanol. A s such, i t would reflect differentia l responsivit y t o particula r growth factors. Ke y support for this conclusion i s that the tw o zones respon d t o differen t combination s o f growt h factors and they express different receptors . By pulling together al l o f the dat a o n th e effect s o f ethanol o n growth facto r activity , w e postulat e tha t ethano l af fects S Z cells in defined ways. Take th e exampl e o f cell proliferatio n i n th e SZ . The S Z contain s receptor s fo r PDG F an d TGFp . Ethanol block s PDG F bindin g wit h a n ED 50 o f 100-200 mg/dl, dependin g o n th e recepto r isofor m (Luo an d Miller , 1999a) . Thi s result s i n a reduce d mitogenic effec t o f PDGF . Ethano l interfere s with TGFp-induce d inhibitio n wit h a n ED 50 o f <50 mg/dl (Lu o and Miller , 1999b) . The interactio n of ethanol an d TGF p result s in a los s of the inhibi tion. Thus , th e combine d effect s o f ethanol, PDGF , and TGF p are a s follows. First , at relatively low con centrations, ethano l inhibit s TGFp-mediated inhibi tion i n th e relativ e absenc e o f ethanol-induce d interference wit h PDGF-promote d cel l proliferation . Second, at high concentrations, ethanol eliminate s the mitogenic effect s o f PDGF that supersed e the los s of TGFp-mediated inhibition . Finally , a t intermediat e concentrations, ethano l produce s a mixe d response .

Thus, the concentration-dependen t effect s o f ethanol on two growth factor system s can explai n the bimodal response of ethanol o n S Z cells in vivo. Abbreviations bFGF (FGF-2) basi c fibroblast growth factor EGL externa l granular layer of the cerebellum ERK extracellula r signal regulated kinase G gestationa l day GE ganglioni c eminence GF growt h fraction IGF insulin-lik e growth facto r IGF-BP insulin-lik e growth factor binding protein IGF-lr insulin-lik e growth factor- 1 receptor IHZ intrahila r zone, or hippocampal field CA4 LF leavin g fraction PDGF platelet-derive d growt h factor PKC protei n kinase C PSN principa l sensory nucleus of the trigemina l nerve SZ subventricula r zone Tc tota l length of the cell cycle TGF transformin

g growth factor

TGFpIr transformin g growth factor p i receptor TGFpIIr transformin g growth factor pi i recepto r VZ ventricula r zone

ACKNOWLEDGMENTS Thi s wor k wa s supporte d b y the National Institute on Alcohol Abuse and Alcoholism and th e Departmen t of Veterans Affairs .

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12

Effects of Ethanol on the Regulatio n of Cell Cycle i n Neural Ste m Cells W. Michael Zawada Mita Das

The effect s o f early exposure to ethanol o n th e pro liferation o f neurona l precursor s ar e describe d i n Chapter 11 . Th e presen t chapte r discusse s the ef fects o f ethanol on th e cel l cycl e with the particular reference t o neura l ste m cell s (NSCs ) i n th e devel oping brain . Relativel y littl e i s know n abou t th e mechanism b y which ethano l affect s th e cel l cycl e machinery i n NSC s becaus e mos t studie s focu s on the effect s o f ethanol on other cel l types . Understanding ho w ethano l affect s th e cel l cycl e o f NSCs migh t be valuable i n the contex t of treatment of ethanol-induce d developmenta l defects , suc h as those observed in fetal alcohol spectru m disorders (FASD). Befor e delvin g int o th e effect s o f ethano l on th e progressio n o f th e cel l cycle , NSC s an d the majo r molecula r component s o f cel l cycl e ar e defined.

DEFINITION O F NEURAL STEM CELLS

Origin of Neural Ste m Cells Stem cell s mak e the developmen t an d maintenanc e of mammalia n tissue s an d henc e organ s possible. Embryonic stem (ES) cells reside in the inne r mass of a blastocyst, are pluripotent, and are capable of generating cells o f all three primar y germ lineages (endo derm, mesoderm , an d ectoderm. ) A subdivisio n of the ectoderm , neuroectoderm , give s rise to th e cen tral nervous system (CNS). In the developing CNS, a subset of stem cells known as NSCs shape s the struc tural and functional layout of the brain. Neural stem cells ar e define d a s cells with a high proliferative capacit y tha t allow s fo r self-renew 7al, retention o f multilineag e potentia l (multipotency) ,

199

200 ETHANOL-AFFECTE D DEVELOPMEN T and clonality , which permit s a single NS C t o gener ate progeny with the sam e multi-lineag e potentia l a s its own. In contrast, neurona l precursors are probably best define d a s a type of stem cel l tha t i s oligopotent, that is, capable o f generating onl y a few selected type s of cells , i n thi s cas e neurons . Althoug h adul t brai n NSCs ar e less abundant an d generall y characterize d by a reduce d proliferativ e potential (se e Cel l Cycl e of Ste m Cells) , the y maintai n th e homeostasi s o f cell pool s wit h hig h turnove r rates , suc h a s granule cell neuron s o f th e hippocampu s an d granul e an d periglomerular interneurons of the olfactory bulb. Characteristics of Neural Stem Cells During development o f the CNS, tw o distinct populations o f NSC s exist . Th e firs t populatio n consist s o f relatively homogenous populatio n tha t participate s i n neural tub e formation . Thes e cell s ar e primaril y located in the ventricular zone (VZ) and lack expression of markers typical of differentiated neura l cells . Their proliferation an d surviva l are controlled b y transmissible protein s suc h a s basi c fibroblas t growt h facto r (bFGF or FGF-2) (Kilpatrick and Harriett , 1995) . Mitotically activ e cell s derive d fro m th e V Z con tribute t o derive d proliferativ e zones suc h a s the sub ventricular zone (SZ ; also known as the subependymal zone) i n th e cas e o f th e telencephalon . Thi s zon e grows as the VZ diminishes in thickness (Miller, 1989) . With th e ris e of the SZ , a second identifiabl e population o f NSC s appear s (Reynold s an d Weiss , 1992 , 1996). These stem cells proliferate in response to both bFGF an d epiderma l growt h facto r (Vescov i e t al. , 1993). Thus , during the secon d hal f o f telencephali c generation, tw o populations o f NSCs exist, a principal population i n th e V Z an d a growin g populatio n i n the SZ. Despite differences i n the VZ and SZ, both popu lations shar e similaritie s in protei n expression . Cell s in thes e zone s expres s nestin , ATP-bindin g cassett e protein (ABCG2) , telomeras e revers e transcriptas e (TERT), telomerase-repeat-bindin g facto r (TRF ) 1 , and TRF 2 (Klappe r e t al, 2001) . Moreover , telom erase activit y is detectable i n bot h th e V Z and SZ . A fuller descriptio n o f telomerase i s provided i n the sec tions Stem Cells , and Telomerase . In addition t o the VZ and SZ , NSCs, some radial glia, and cell s expressing glial fibrillary acidic protein (GFAP) hav e bee n postulate d t o b e ste m cell s (Doetsch e t al. , 1997 ; Garcia-Verdug o e t al. , 1998 ;

Hartfuss e t al. , 2001 ; Alvarez-Buyll a an d Garcia Verdugo, 2002) . I t shoul d b e note d tha t expressin g GFAP doe s no t necessaril y mean tha t a cell i s an astrocyte. Rather , it means that a cell expresses an inter mediate filamen t als o expresse d b y astrocytes . Fo r example, radia l gli a ca n expres s GFAP , bu t the y d o not appea r t o b e astrocytes . Instead , the y hav e trait s associated wit h NSCs, such a s the abilit y to generat e neurons an d glia . Consequently , th e effect s o f ethanol exposur e o n GFAP-positiv e cell s an d radia l glia during development also have wide-ranging consequences fo r NSCs (see Chapters 1 3 and 18) . In adul t rodents, four distinc t cell type s have bee n identified in the SZ (Doetsch e t al., 1997). They can be discriminated on th e basis of the lengt h o f their cell cycle and ultrastructural characteristics. These are (1) proliferating migratory neuroblasts (type A cells), (2) slowly dividing astrocyte s (typ e B cells) , (3 ) rapidl y dividing progenitors (typ e C cells) , and (4 ) ependyma (typ e D cells) tha t lin e th e ventricula r wall. Whether ependy mal cells themselves ar e stem cells is contested (Chias son e t al. , 1999 ; Johansson et al. , 1999 ; Spassky et al. , 2005). Furthermore , parenchyma l cell s positiv e fo r neural cel l adhesio n molecul e an d transdifferentiate d cells are candidates for NSCs (Pevny and Rao, 2003). Symmetric and Asymmetric Cell Division among Neural Stem Cells Like othe r type s o f stem cells , NSC s underg o eithe r symmetric o r asymmetri c cel l division s (Zhong , 2003). During a symmetric cel l divisio n both daugh ter cell s shar e th e sam e fate . Accordingly , bot h cell s remain in the proliferative population o r both exi t the cell cycle . I n contrast , a n asymmetri c divisio n pro duces tw o cells wit h different fates . For example, on e remains a stem cel l an d th e othe r assumes a defined phenotype such as that of a neuronal progenitor. Ho w is this fate determination possible ? Although mitoti c cel l divisio n produce s tw o daughter cell s containin g identica l geneti c material , the epigeneti c molecule s withi n th e cytoplas m ca n be distribute d unequall y betwee n th e tw o daughte r cells. Such differentia l distributio n of cytoplasrnic determinants i s postulate d t o affec t cel l fat e decision s (Zhong, 2003) . Asymmetric cel l divisio n is indeed a fundamental attribut e o f ste m cells , becaus e fo r a stem cell both t o self-renew and to produce differenti ated progeny , th e cel l mus t hav e an abilit y to divide asymmetrically. A specifi c exampl e o f asymmetri c

EFFECT OF ETHANO L ON NEURA L STE M CELL S 20 1 cell divisio n come s fro m th e Drosophila CN S i n which a neuroblas t give s ris e t o a smalle r daughte r cell know n a s th e ganglio n mothe r cel l (GMC) . A GMC i n turn produces two neurons or glial/neuronal siblings (Doe et al., 1998), Among severa l componen t protein s tha t partici pate i n asymmetri c division s in Drosophila, prosper o and Num b are retained in the basa l region of a dividing neuroblast at metaphase and preferentiall y segregate int o th e ganglio n mothe r cel l (Knoblic h e t al. , 1995). Although tracing cell lineage trees is currently impossible in vertebrates in vivo, it is feasible to trace the progen y of single cells labeled retrovirally . Other methodologies fo r tracing stem cell s i n vivo , suc h a s transplantation o f labeled cells , ar e discusse d belo w in th e sectio n Us e of Neural Ster n Cel l Transplanta tion to Examine the Effect s o f Ethanol o n Cell Regulation In Vivo . A discussion on the asymmetri c cel l division i s particularly relevan t here , becaus e asym metric distributio n o f cytoplasmi c determinant s i s cell-cycle dependent . Althoug h i t i s no t know n whether ethanol ca n disrupt this distribution, the possibility exists because th e asymmetri c cell divisio n occurs early in the proces s of fate determination . Thus , even small changes during this period might result in substantial abnormalities later in development. Markers of NSC Populations No discussio n o f NSC s i s complet e withou t a n at tempt t o defin e the m b y mean s o f unique ste m cel l markers. Although n o absolut e marke r fo r NSCs exists, th e presenc e o f a hematopoieti c ste m cel l marker, C D (cel l determinant ) 13 3 (know n a s prominin i n rodents) , i s require d fo r th e clonogeni c NSCs tha t self-rene w and retai n niultilineage differ entiation propertie s (Uchid a e t al. , 2000) . Suc h clonogenic NSC s ca n b e derive d fro m huma n feta l brain tissue s by fluorescence-activate d cel l sortin g for cells that are GDI33% CD34-, CD45~, and CD23-710. These cell s appea r t o be th e onl y huma n feta l brain cells that display clonogenic characteristics , retain th e ability t o differentiat e int o bot h neuron s an d glia , and participat e i n a neurogeneti c progra m following transplantation int o th e roden t CN S (Uchid a e t al. , 2000; Tamaki et al., 2002). Evidence o f the importanc e o f CD 133 as an indi cator o f "sternness" (i.e., their proliferative an d differ entiative potential ) wa s discovered whe n testin g th e cancer ste m cel l hypothesis . A s few as 10 0 CD133 +

cells isolate d fro m huma n brai n tumor s giv e ris e t o phenotypically identica l tumor s whe n transplante d into brain s o f immunodeficien t mic e (Sing h e t al. , 2004). B y contrast, th e CD133 " cells fai l t o produc e tumors in these transplants, even when engrafte d as a bolus of 10 5 cells. These findings do not imply that all NSCs are tumorigenic, but that stem cells introduce d into th e brai n ectopicall y and/o r a t a n inappropriat e time hav e a capacity for tumor generation . Althoug h GDI33 labelin g ha s been use d mostl y fo r detectio n of human NSCs , expressio n of other potentiall y useful marker s fo r NSC s suc h a s Sox-1 , Sox-2 , nestin , musashi, Hu , neuralstemnin , Le X (trisaccharid e 3 frucosyl-N-acetyllactosamine)/stage-specific embryoni c antigen 1 , an d ABCG 2 ha s bee n use d t o identif y NSCs i n a variet y o f specie s (Capel a an d Temple , 2002; Pevny and Rao , 2003).

CELL CYCLE

The mos t fundamenta l property of living organisms is their abilit y to reproduc e themselves . This property is based upo n th e featur e that cell s ar e able t o duplicat e by a process known as the cel l o r mitotic cycle. Mammalian brai n developmen t depend s o n a precis e sequence o f cell division s controlle d b y th e cel l cycle . Sculpting of the CN S an d othe r organ s in the embryo is greatly affected b y the kinetic s o f the cel l cycl e an d the qualit y o f chromosoma l replicatio n tha t occur s during this process. Alterations in the amount s of necessary trophic factor s (e.g., glucose and growth factors), mutations in the genes that control th e cell cycle (e.g., cyclins and cyclin-dependent kinases [Cdks]), and various biophysica l stresse s (e.g. , excessiv e hypoxi a an d acidemia) occur naturally . These alterations ca n affec t the cel l cycl e and resul t in stunted o r abnormal development, an d they cannot b e easily controlled. Cel l cycle regulation , an d hence , developmen t ca n b e als o affected b y unnatural causes such a s excessive ethanol consumption. Befor e discussin g the specifi c effects o f ethanol o n the cel l cycle , it is important to review the components o f cel l cycl e machiner y an d th e differ ences between th e cell cycle s of somatic and ES cells. Cell Cycle of Somatic Cells As mentione d above , th e cel l cycl e i s th e mean s b y which organism s reproduce themselves . I n virtually all cells, the cel l cycle is composed o f four discrete phases :

202 ETHANOL-AFFECTE D DEVELOPMEN T (1) the DNA synthesis phase (the S phase), (2) the cel l division phase (the M phase), and tw o gap phases, (3 ) the Gl phas e between M and S and (4) the G2 phase between S and M (Fig. 12-1). During Gl, the cell prepares for DNA replication that results in duplication of the chromosome s durin g S . The secon d ga p period , G2, i s the post-DNA synthetic phase during which the cell prepares for mitosis. The ter m cell cycle checkpoint refers t o a mechanism b y which th e cel l activel y halts the progression of the cell cycle until it can ensure that an earlier process, such as DNA replication o r mitosis, is complete. Suc h cell cycle checkpoints are mediate d by p53 at the end of Gl an d during G2 (Tabl e 12-1) . P53 is also known as a tumor suppressor and whe n inactivated permits oncogenes to promote transformation and tumorigenesis (Lowe et al., 2004). Cyclins and their partners, the Cdks, constitute th e basis o f the molecula r mechanis m tha t control s cel l cycle regulation (Fig. 12-1) (Murray , 2004). Cdks are serine/threonine protei n kinase s that requir e bindin g of a cycli n t o becom e activated . Mammalia n cell s contain multipl e Cdk s tha t ar e activate d b y multiple cyclins. Much o f the differenc e betwee n the behavior of different cyclin s reflects their location an d timin g of expression rathe r than thei r direct effects o n substrate specificity. Th e activit y of Cdks i s highly regulated b y a number of mechanisms: (1 ) the leve l of their respec tive partne r proteins , the cyclins , (2) the amount s of inhibitory proteins of the Cip/Ki p (e.g., p21, p27, and p57) and Ink4 (e.g., pi5, p!6, p!8, and p!9) familie s (Cdk inhibitors or CKIs), and (3 ) inhibitory and stim ulatory phosphorylatio n o f variou s Cd k residue s (Table 12-1 ) (Cheng , 2004 ; Siegenthale r an d Miller , 2005). High concentrations of cyclins therefore generally stimulat e cell-cycl e progressio n an d proliferatio n through activatio n of Cdks, wherea s high amount s of CKIs antagonize these processes. Cyclins ar e activatin g subunit s tha t interac t wit h specific Cdk s t o regulat e their activit y and substrat e specificity. I n mammals , thes e complexe s include : (1) the D-type cyclin s (cyclins Dl, D2, an d D3 ) tha t activate Cdk4 and Cdk6 to execute critica l regulatory events i n Gl ; (2 ) the E - and A-typ e cyclin s that activate Cdk2 to affec t event s in S including DNA replication; and (3 ) the A-typ e cyclins and B-typ e cyclins that activate Cdk l t o direct structura l and regulator y events in M (Fig . 12-1) . Inactivation o f Cdkl i n late M contributes to progression into G L In somati c cells , movemen t throug h G l an d int o S is driven by the activ e forms o f the cycli n Dl, D2 ,

or D 3 complexin g with Cdk 4 or Cdk6. This triggers an intricat e cascad e o f events. Formatio n o f a cycli n D-Cdk comple x induce s th e phosphorylatio n o f retinoblastoma (Rb ) protein (Fig . 12-2 ) (Classo n an d Harlow, 2002). Before a cell prepares to ente r a ne w cell cycl e durin g the Gl/ S transition, a critica l tran scription factor , E 2F-1 i s inactive and boun d t o Rb . Once the new cell cycle begins, Rb is phosphorylated and E 2F-1 i s partiall y release d fro m R b inhibition . This activates cell cycle regulators including cyclin A and cycli n E tha t comple x wit h Cdk 2 an d Cdc25 A phosphatase. Cdc25 A phosphatas e remove s phos phates tha t inhibi t Cdk 2 an d th e resultan t cyclin E/Cdk2 comple x furthe r phosphorylate s Rb . The re sult is a complete release of E2F and the transcriptio n of a series of genes essential for progression into S and DNA synthesis . Parallelin g thi s activit y is the direc t contribution o f the c-myc pathway to the Gl/ S transi tion b y elevatin g the transcriptio n fo r cycli n E an d Cdc25A(Fig. 12-2). In additio n t o th e positiv e regulatio n b y cyclins, Cdks ar e regulate d (1 ) b y multipl e phosphorylatio n and dephosphorylation events and (2 ) by several CKIs that physicall y associat e wit h th e cyclin-Cd k com plexes to inhibit their activities and promote cell cycle arrest or delay. Since phosphorylatio n i s fundamental to the regulation of cell cycle progression, a large subset ofDrosophila protei n kinases (kinome) studied for their necessity in the norma l cell cycle was examined by silencin g genes wit h RNA-mediate d interferenc e (Bettencourt-Dias e t al. , 2004) . O f 22 8 protei n ki nases that are down-regulated by this silencing, downregulation o f 8 0 o f the m ca n induc e cel l cycl e dysfunction. Man y o f thes e enzyme s wer e alread y known to phosphorylate microtubules , actin , an d associated proteins , but a s a consequence o f these studies, th e protei n kinase s hav e bee n assigne d ne w functions i n cel l cycle . Thes e finding s underscor e the complexit y of cell cycle regulation and the multi tude o f potentia l target s tha t ethano l migh t affec t during development. Cell cycle progression is regulated by growth factors (see Chapter 11) , the extracellular matrix, and cell-cel l contacts. Upo n deprivatio n o f cells fro m a n essentia l component (nutrient s o r growt h factors ) th e cell s become quiescen t an d ente r th e so-calle d GO-phase . This exit from th e cel l cycle can be reversible in astrocytes (L i e t al. , 1996 ; MacFarlan e an d Sontheimer , 2000) or irreversible, as evident in man y types of postmitotic neurons (Zawada et al., 1996) . In mammalian

SOMATIC CELLS

ES CELLS

FIGURE 12- 1 Cel l cycle o f embryonic ste m (ES ) an d somati c cells . Not e tha t ES cell s hav e greatl y shortene d ga p phases, exhibi t constan t expressio n o f cyclins A, E , and D , and lac k p53 checkpoints tha t allow for rapid cel l division . bFGF, basi c fibrobals t growt h factor ; IGF, instilin-lik e growt h factor ; PDGF , platelet-derived growth factor; TGFpl, transforming growth factor (31 .

203

204 ETHANOL-AFFECTE D DEVELOPMENT TABLE 12- 1 Endogenou s cel l cycl e inhibitor s slo w dow n cel l cycl e at differen t stage s by actin g on variou s cyclin-CDK complexes . Ink4 Family Cell Cycle Stage

Cyclin-Cdk Complexes plS

Gl

cyclinD-Cdk4/6 + cyclinE-Cdk2 cyclinA-Cdk2 cyclinB-Cdk2 -

Gl/S S

G2/M

pi

+

cells, th e cel l cycl e machiner y tha t determine s whether cell s continu e proliferatin g or ceas e dividing and differentiate appear s to operate mainl y in the Gl. Cell Cycle of Stem Cells Embryonic Cells In stem cells , there i s a distinct cell cycle control tha t operates i n orde r t o maintai n thei r sternness . Th e need for higher rate s of proliferation i s met b y minimizing the lengt h o f time tha t ES cell s spend i n G l and G 2 (Fig . 12-1) . Mouse E S cells have a defective Rb pathwa y an d a nonresponsiv e p5 3 pathwa y

6 pl8

+

Cip /Kip Family

p!9 p2l ++ -+

+ +

P27

pS7

+1+ -

+/+ + -

pS3 — + +

(Savatier and Malashicheva, 2004). Furthermore , ES cells hav e constitutiv e phosphatidyl-inositol-3-kinas e activity an d ectopi c cycli n E/Cdk 2 kinas e activity , features ofte n eviden t i n tumor s (Sherr , 1996) . Th e uncoupling o f G l fro m th e influenc e o f extrinsi c stimuli migh t explai n i n par t th e rapi d proliferation rate o f E S cells . Anothe r metho d throug h whic h stem cells ar e abl e t o maintai n a hig h rat e o f self renewal i s reduction i n expressio n o f CKIs, particu larly p2 1 an d p2 7 (Chen g an d Scadden , 2004 ; Siegenthaler an d Miller , 2005) . I t i s likely tha t th e distinct cell cycle profil e i n ste m cell s is mediated either b y distinc t upstrea m intracellula r mediator s o r by uniqu e combinatoria l relationship s o f commo n

FIGURE 12- 2 Majo r regulator s of cell cycl e initiatio n i n Gl . Se e tex t fo r details. Cdk, cyclin-dependent kinase , RB, retinoblastoma.

EFFECT OF ETHANO L ON NEURA L STE M CELL S 20 5 biochemical mediator s that limi t the intensit y of signals to exit the cell cycle. Stem cell s are not only key building blocks in normal developmen t bu t the y ar e als o candidates as the cell o f origin for cancer becaus e o f their pre-existing capacity o f self-renewa l an d unlimite d replicatio n (Beachy e t al. , 2004) . Cance r mos t ofte n emerge s i n those tissues undergoing constant renewal by the proliferation o f ste m cells . B y retaining o r re-acquirin g stem cell behavior, malignant cell s may be able to recreate a tumor durin g metastasis o r after therapeuti c elimination o f th e bul k o f th e tumo r populatio n (Beachy et al., 2004). Neural Stem Cells During normal developmen t o f the CNS, th e division rates o f NSC s withi n th e germina l zone s var y wit h brain regio n an d wit h tim e (se e Chapter s 2 an d 11) . The principl e of the cel l cycl e o f ES cell s (described above) composed solely of S and M no longer applies. By contrast, as the developmen t o f CNS progresses , a multitude a cel l cycl e "types " emerge s (Fig . 12-1) . These cell cycle s differ i n length , whic h i s most com monly determined by the period of time that the cel l is in Gl and/o r G2. Studies o f murine cortical neurogenesi s detail th e cell cycle over the 6-da y long process (Caviness et al., 1999). Founde r cell s an d thei r progen y underg o 1 1 cell cycles . The proportio n o f the cells becoming post mitotic increases with eac h successiv e cel l cycl e an d the length o f the cycl e increase s due to a gradual prolongation o f Gl. Gl i s characterized by two main enzymatic activities : Cdk4 i n mid-Gl an d Cdk 2 i n lat e Gl jus t prio r t o initiatio n o f S (Fig . 12-2) . A s described abov e i n Origi n o f Neura l Ste m Cells , th e SZ cells ca n b e subdivide d int o slowl y dividin g GFAP-positive (typ e B) cells , rapidly dividing transitamplifying progenitors (type C cells), and proliferating migratory neuroblast s (typ e A cells) . On e propose d model o f neurogenic program defines th e flowin g lineage progression : typ e B t o typ e C t o typ e A cell s (DoetschetaL, 1997) . The CK I p27 is an inhibito r of Cdk2 tha t can pro long th e entr y o f a proliferatin g cel l int o S . Durin g neurogenesis, loss of p27 ha s n o effec t o n th e numbe r of stem cells (type B cells), but selectivel y increases the number o f transit-amplifying typ e C cells , whil e con comitantly reducing the numbe r o f type A neuroblasts (Doetsch e t al., 2002). By contrast, NSC s i n transgenic

mice over-expressin g p27 have longer Gl (Mitsuhash i et al., 2001). The lengt h o f cell cycle (Tc) in germinal zones is under temporal and spatia l control. Fo r example, i n th e mouse , divisio n rate s ca n b e a s rapi d a s 7-10 hr i n earl y gestation an d u p t o 1 8 hr long at th e end o f cortica l neuronogenesi s (Takahash i e t al. , 1995). Likewise , the lengt h o f the cel l cycl e increases in rat cortex from 1 1 to 1 7 hr acros s the perio d o f neuronogenesis (Miller and Kuhn, 1995) . Cell cycle varies by location, too. The Tc for the rat neocortical VZ on G21 i s 1 9 hr an d 1 6 hr fo r S Z cell s (Mille r an d Nowakowski, 1991) . In the adul t brain , th e frequency of cel l proliferatio n can b e a s infrequen t a s day s o r months (Garcia-Verdug o et al., 1998 ; Morshea d e t al., 1998; Doetsch e t al., 1999) . In addition to the apparen t differences i n cell cycle lengths, the details of molecular control s dictatin g characteristics o f various cell cy cles of NSCs are speculative .

EFFECTS O F ETHANOL ON CEL L CYCLE

Somatic Cell s Reports on the effect s o f ethanol o n cell cycl e regulatory molecule s sugges t tha t th e ethanol-mediate d effects o n ke y regulator s o f cel l cycl e ar e tissue specific an d cell type dependent. Fo r example, in hu man lymphocytes , ethanol-induce d inhibitio n o f mitosis i s mediate d throug h G2/ M blockad e (Ahluwalia e t al. , 1995) . I n huma n epithelia l cells , ethanol delays cell cycle progression by up-regulating the expressio n of transcripts for p21 ( a key CKI) an d inhibiting Cdk 2 kinas e activit y i n G l (Gu o e t al. , 1997). I n epithelia l cells , thes e ethanol-mediate d ef fects on the cell cycl e are independent o f the cellula r status of a checkpoint mediator , p53. Other studies in a variet y o f tissues corroborat e th e finding s tha t spe cific CKI s serve as targets o f ethanol actions . Fo r example, a n increas e i n p2 l gen e expressio n upo n ethanol infusio n ha s bee n reporte d i n th e isolate d perfused hear t (Jankal a et al., 2001) . Hepatocyte proliferation afte r partia l hepatectom y i s als o inhibite d by ethano l throug h amplificatio n o f Gl checkpoin t mechanism involvin g inductio n o f p21 gen e an d in hibition o f cyclin D (Koteis h et al., 2002) . Yet the ef fects o f ethano l migh t no t influenc e onl y a singl e phase o f the cel l cycle . Transient blockade o f cell cycle a t G2/ M an d preventio n o f the cel l fro m exitin g the cycl e b y ethanol exposur e hav e been show n i n a

206 ETHANOL-AFFECTE D DEVELOPMENT fibroblastic cel l lin e derive d fro m activel y dividing mouse connectiv e tissu e (Mikami etal., 1997) . Ethanol intak e by pregnant rat s inhibits growth of the intestina l epitheli a (Pere s e t al. , 2004) . Ethano l substantially attenuates cryp t cell proliferatio n in bot h the jejunu m an d ileu m an d prolong s cel l cycl e tim e among crypt cells. In ethanol-fe d rabbits , neo-intima l hyperplasia i n coronar y arteries following balloo n an gioplasty is diminished (Merrit t et al., 1997) . Similarly , local deliver y o f ethano l b y balloo n catheter s i n balloon-injured coronar y arterie s inhibit s intima l hy perplasia (Liu et al., 1997). In cultured vascular smooth muscle cells , ethano l inhibit s the progressio n o f cells from th e Gl t o S by modulating the expression/activity of key regulatory molecules of the cycl e (Sayeed et al., 2002). I n particular , ethano l induce s th e expressio n of p21 , inhibit s Cdk 2 activity , and halt s th e induc tion o f cycli n A , resultin g i n a decreas e o f hyper phosphorylation of Rb and failure to initiate a new cell cycle. Cumulatively , report s o n ethanol-mediate d ef fects on the cell cycle of somatic cells suggest that p21, the ke y negative regulator of mammalian cell cycle, is an important target of ethanol. Despite a n overwhelmin g number o f reports sup porting th e anti-proliferativ e rol e o f ethanol, instea d of perturbin g th e cel l cycl e progression , it s action s can actually induce the cell cycle in certain cell types under particula r conditions . I n suc h cases , reduce d expression o f cel l cycl e inhibitor s upo n exposur e t o ethanol accelerate s progressio n from th e G l t o S. Indeed, ethanol-mediate d inhibitio n o f pi8, pi9 , an d p21 expression, increased R b phosphorylation and in creased proliferation have been reported i n squamous cell carcinoma cell lines of the head and nec k (Hager et al., 2001; Kornfehl e t al., 2002). Increased hepato cyte proliferation in rat s after chroni c ethanol feedin g has als o bee n demonstrate d b y severa l investigator s (Chung e t al., 2001). Immortalized NSCs , grown under proliferation-encouragin g conditions , als o re spond t o ethano l b y increasin g th e rat e o f thei r division (Morgan et al., 2003) (described i n mor e detail below) . Likewise, following prenata l exposur e to moderate amount s o f ethanol , S Z cel l proliferatio n increased (Mille r and Nowakowski , 1991) becaus e of recruitment o f cells int o th e cyclin g population, no t because o f a decrease i n the Tc or the lengt h o f the Gl. Take n together , thes e findings indicate tha t th e effects o f ethanol o n cel l cycle progressio n are highly variable an d depen d o n th e molecular , cellular , and system contexts under which they occur .

Stem Cells Neural Stem Cells Mechanisms o f neuronogenesis durin g developmen t (Miller, 1986 ) an d adulthoo d (Herrer a e t al. , 2003 ) are affecte d b y ethano l (se e Chapter s 1 1 an d 13) . Much of the followin g discussion is based on findings from adul t NSCs, as studies examining molecular de tails of cell cycl e modulatio n b y ethanol durin g brain development ar e few . Nonetheless , approache s an d findings presented her e should b e usefu l i n studying effects o f ethanol o n th e characteristic s o f cell cycl e in the cours e of neurogenesis. Both bing e (Nixo n an d Crews , 2002) an d chroni c (Miller, 1995; Rice etal., 2004; He etal, 2005) ethanol consumption reduc e the postnatal proliferation of neural progenito r cell s i n th e S Z o f the latera l ventricl e and th e intrahila r zone (IHZ ; also known a s the sub granular zone) o f the dentat e gyrus. Adult rats receiving liquid diet containing ethanol (6.5 % v/v) for 3 days reduced thei r neura l progenito r proliferatio n i n th e IHZ a s evidenced b y a decrease i n the numbe r of cells that incorporat e bromodeoxyuridin e (BrdU ) (Ric e et al., 2004). I n contrast, animal s receiving an ethano l diet fo r 1 0 or 3 0 days n o longe r displa y reduced pro genitor proliferation , indicating activation o f a com pensatory respons e t o th e actio n o f ethanol . A t leas t during the early postnatal period, the effect s o f ethanol are concentration-dependen t (Miller , 1995) ; hig h doses inhibi t cel l proliferatio n an d lowe r dose s ar e stimulatory. Durin g feta l development , ethano l migh t also stimulate proliferatio n of neural progenitor s a s illustrated by enhanced proliferatio n of cerebral cortical neuroepithelial precursor s upo n exposur e t o alcoho l (Miller an d Nowakowski , 1991 ; Miller , 1995 ; Santil lano e t al. , 2005) . Treatment wit h ethano l fo r 4 days increases th e size , variation , an d numbe r o f cortica l precursor neurospheres , induces S , and increase s pro gression through G2/ M (Santillano et al., 2005). Analysis of the effect s o f ethanol o n postnata l neu rogenesis shoul d b e conducte d i n term s o f the fina l numbers of cells generated an d retained. This measure is affected b y not only the rat e of cell proliferation, but also b y the rat e o f cell deat h (Miller , 2003) . The im portance o f thi s approac h ha s been documente d b y studies reportin g tha t ethano l actuall y increase s cel l proliferation an d concomitantl y increases cell death as determined wit h terminal deoxynucleotid e transferasemediated deoxyuridin e triphosphate nick-en d labelin g

EFFECT OF ETHANO L ON NEURAL STEM CELLS 20 7 (TUNEL) (Pawlak et al., 2002; Miller, 2003). TUNEL identifies nucleosome-sized products of DNA degradation. These finding s underscor e the complexit y of the effects o f ethanol o n cel l cycl e and cel l survival . On e study explores the effect s o f a 24 hr exposure to ethanol on C17.2 cells, a line of NSCs derived from a postnatal mouse cerebellu m (Morga n e t al. , 2003) . Exposure to ethano l double d th e numbe r o f survivin g NSCs when grown under proliferation-favoring culture conditions (10% serum/uncoated culture plate). In contrast, ethanol reduces the number of NSCs grown under dif ferentiating condition s (1.0 % serum/poly-L-lysine coated cultur e plate) . A s thi s NS C lin e ha s bee n immortalized with v-myc, it is conceivable tha t the in crease i n proliferatio n of C17. 2 durin g exposur e t o ethanol i s related t o the effect s o f ethanol o n the activity of the oncogene , v-myc. Oncogenes ar e known activators of telomerase, which in turn maintains telomere length necessar y for unabated proliferatio n (see Ste m Cells, and Telomerase, below). Mechanisms Underlying Cell Cycle Despite th e abundance o f literature describing the effects o f ethano l o n neura l progenito r proliferatio n during neurogenesis , onl y a fe w studie s hav e exam ined th e molecula r mechanism s mediatin g thes e changes. One o f the few models used to date is that of cerebellar neurogenesis . Cerebella r granul e neuron s represent a majo r neurona l typ e i n th e cerebellu m and aris e fro m th e proliferatin g externa l germina l layer durin g th e firs t 2- 3 postnata l week s i n th e ra t (Miale an d Sidman , 1961 ; Altman , 1972) . Cycli n A and Cdk2 , tw o positiv e regulator s o f th e cel l cycle , exert contro l ove r neurogenesi s i n th e developin g cerebellum (L i et al., 2002) . Cerebellar granul e pro genitors expres s predominantly Cdk 2 (Courtne y an d Coffey, 1999 ; L i e t al. , 2001) . L i an d colleague s (2002) repor t tha t i n cerebella r granul e progenitor s derived from 3-day-ol d rat pups, ethanol exposure decreases th e expressio n o f Cdk 2 an d cycli n A . Thi s ethanol-mediated reduction o f Cdk2/cyclin A expression ma y induce cell cycl e arrest in neuronal precursors by increasing the duration of Gl an d S. P27, a negative regulator of the cell cycle, plays an important role during neurogenesis of the developin g cerebellum. I n th e neonata l cerebellum , p2 7 i s severely down-regulated after earl y postnatal exposure to ethanol (L i et al., 2002). P27 induces cell cycle arrest in neuronal precursors and accelerate s differentiatio n

of neurons (Perez-Juste and Aranda, 1999; Farah et al., 2000; Miyazaw a et al. , 2000) . Repressio n o f p2 7 b y ethanol exposur e may reactivat e postmitotic neurons and evok e apoptoti c cel l death . Collectively , report s on the effect s o f ethanol o n neurogenesis suggest that p27, Cdk2 , an d cycli n A ar e primar y targets o f th e ethanol i n the developing cerebellum. Unfortunately, such detailed analysis of cell cycle proteins during prenatal exposure to ethanol i s unavailable. Different neurona l population s fro m divers e locations in the developing brain exhibit different degrees of susceptibilit y t o ethano l toxicit y (Maie r e t al. , 1999). Ethano l als o inhibits carbachol-induced DN A synthesis o f astrogli a (Guizzett i e t al. , 2003) . Mus carinic agonist-drive n progression from th e G l t o S also ca n b e affecte d b y ethano l i n thes e cell s (Guizzetti an d Costa , 1996 ; Guizzett i e t al. , 2003). Taken together , thes e report s o n ethanol-induce d ef fects o n th e cel l cycl e o f differen t neurona l an d as troglial population s sugges t tha t S an d G l ar e th e primary targets of ethanol-induced effects . Not onl y ca n cel l cycl e pathway s b e studie d i n vitro and i n vivo (see Use of Neural Stem Cell Transplantation t o Examine the Effect s o f Ethanol on Cell Cycle Regulation In Vivo), but gene array technology makes i t possibl e t o scree n ste m cell s fo r ethanol niediated gene expressio n patterns in a n effor t t o discover ne w ethanol-responsiv e genes . A repor t b y Treadwell and Sing h (2004), who used microarrays of adult mous e brai n gen e expressio n followin g acut e ethanol exposure , suggest s that a significan t proportion of ethanol-responsive gene s have roles in cell cycle transitio n an d arrest . Th e genes , p2 l an d th e growth arres t an d DN A damage-inducibl e gen e (Gadd45r), ar e up-regulate d i n respons e t o ethanol . P21 an d Gadd45 r ar e implicate d i n bot h p53 mediated an d p53-independen t inductio n o f G l growth arres t i n respons e t o ethano l an d oxidativ e stress (Guo et al., 1997; Kitamur a et al., 1999). The molecula r mechanism s o f ethanol-induce d effects o n cel l cycl e involv e ethanol-responsiv e ele ments o n promoter s o f targe t genes . Ethano l alter s the expressio n an d DN A bindin g activit y of various transcription factors , suc h a s activatin g protei n 1 (AP-1), nuclear facto r K-B , an d earl y growth response gene (Beckman n e t al. , 1997 ; Ceber s e t al. , 1999 ; Fried e t al, , 2001 ; Acquaah-Mensa h e t al. , 2002) . These transcriptio n factors regulat e the transcriptio n of genes encoding cyclins , Cdks, and CKI s (Sylvester et al., 1998 ; Beie r et al., 2000; Milde-Langosch et al.,

208 ETHANOL-AFFECTE D DEVELOPMENT 2000; Passegu e an d Wagner, 2000 ; Henniga n an d Stambrook, 2001) . AP- 1 bind s t o th e cAMP responsive elemen t i n th e promote r o f cyclin A gene (Sylvester e t al. , 1998 ; Beie r e t al. , 2000 ) an d en hances transcription of cyclin A. Suppression of AP-1 activity b y prenata l ethano l exposur e (Acquaah Mensah e t al. , 2002 ) inhibits the transcriptio n of cyclin A in developin g cerebellum . Give n th e critica l role of Cdks and CKI s in neurogenesis , disruption of the Cdk/CKI system may be the mechanis m underly ing ethanol-induce d damag e o f man y developin g CNS nuclei, includin g the cerebellum . Telomerase A potential opportunit y for ethanol t o influence stem cell proliferatio n i s t o affec t th e chromosoma l ter mini, the telomeres . In all vertebrates, telomeres con sist o f non-coding G-rich DN A repeat s (TTAGGG) . These specialized chromati n structure s are necessar y for successfu l cel l divisio n an d ar e shortene d wit h each roun d o f DN A replication . The y ca n als o b e preserved b y elongatio n mediate d b y a n enzym e called telomerase. I n norma l huma n somati c cells , consecutive round s o f telomer e shortenin g induc e replicative senescence, particularl y in the absenc e o f telomerase. Los s o f TTAGG G sequence s lead s t o telomere dysfunctio n characterize d b y end-to-en d chromosome fusio n tha t result s i n genomi c instabil ity. Telomere s ar e protecte d b y a collectio n o f pro teins that , i n additio n t o telomerase , includ e several other protein s regulatin g telomer e length , includin g protection o f telomeres 1 (Lei et al., 2004) and other s (Bekaert et al., 2004). Telomerase activit y requires (1 ) the revers e transcriptase TERT and (2 ) an RN A component tha t add s TTAGG G repeat s t o th e end s o f chromosomes to prevent chromosomal shortening . Telomerase activit y in the developing rodent brain is high on gestational day (G) 1 3 but declines by G18 , remains low until postnata l da y (P) 3 , and finall y be comes undetectabl e b y P1 0 (Klappe r e t al. , 2001) . These observations are consistent with a role of telomerase i n proliferatio n o f NSCs. Interestingly, accelerated shortenin g o f telomere s i n mic e deficien t i n telomerase abrogate s proliferation of adult NSCs isolated fro m th e SZ , however , it does no t hav e that effect i n th e NSCs isolated from th e feta l brai n despite severe telomere erosio n resulting in chromosomal ab normalities and nuclea r accumulation of p53 (Ferro n et al. , 2004) . Thi s observatio n parallel s th e findin g

that mous e E S cells lac k p53-dependent checkpoint s (Savatier an d Malashicheva , 2004) . Whethe r feta l NSCs a t variou s stage s o f developmen t shar e inde pendence i s not clear , bu t variou s "types" o f cell cycles diffe r i n lengt h an d differin g expressio n pattern s of cell cycl e regulator y proteins hav e been identifie d both i n somatic cells and ES cells (Fig. 12-1) . At least som e NSC s display cell cycl e characteris tics like those o f somatic cells. This is evident in adult NSCs, whic h ca n hav e a prolonge d G O (Takahashi et al. , 1995 ; Garcia-Verdug o e t al. , 1998 ; Morshea d et al., 1998; Doetsc h etal., 1999) and those dependent on the p53 checkpoints (Meleti s et al., 2005). In neurospheres derive d fro m th e S Z o f p53-null mice , th e number of BrdU-positive cells is increased as much as 20%, whereas p2 1 conten t i s reduced (Meleti s e t al. , 2005). Concomitan t wit h thi s increase d capacit y fo r self-renewal i s a reduction i n cell death, evidence d by reduced caspase 3 activity and annexin V staining. When examining neurogenesis, it is important no t to presume tha t the protein s involve d share the sam e function a t all stages of development. Suc h i s the cas e for caspas e 3 , a pro-apoptotic protease , i n late r stages of brai n organogenesi s an d adulthood . Caspas e 3 is necessary for normal growth of morula and blastocys t embryos, a s it s inhibitio n stall s th e growt h o f suc h embryos (Zwaka etal., 2005). NSCs undergoing telomere shortening, invariabl y enter an irreversible growth arrest, an d eventuall y senesce . Developmen t o f per petual NS C line s fo r studies o n huma n feta l NSC s has bee n successfull y attempte d usin g transfectio n with a proto-oncogene, v-myc (Vill a e t al, 2004) . I n such immortalize d NSCs , v-myc maintain s hig h telomerase activit y and allow s culturing of NSCs for up to 4 years with preservation of short, but stable and homogenous telomeres . Ethanol affect s th e expression of a proto-oncogene , c-myc, but i t fails to change th e amounts of p53 in th e skeletal muscl e o f rat s expose d t o chroni c ethano l (Nakahara e t al., 2003). Prenatal exposur e to ethanol increases gestational expression of p53 in the cerebra l cortex (Kuh n an d Miller , 1998) . Thi s findin g paral lels th e effect s o f ethano l o n S Z cells , bu t whethe r ethanol alter s th e expressio n an d activit y o f onco genes and p53 in NSCs remains to be examined. Ionic an d Metabolic Homeostasis Calcium an d magnesiu m ar e bot h believe d t o pla y an importan t rol e i n cel l proliferatio n (Wol f an d

EFFECT OF ETHANO L ON NEURA L STE M CELL S 20 9 Cittadini, 1999 ; Ziesch e e t al. , 2004) . Ethano l ca n cause a decrease i n cytosolie-fre e Ca 2+ (Zhang e t al., 1992) and a rapi d depletion of intracellular Mg 2+ in vascular smooth muscl e cell s (Altur a et al., 1995) . In addition, ethano l ca n adversel y affec t mitochondria l cytochrome oxidas e (Kennedy, 1998 ) an d AT P synthesis (Dev i e t al. , 1994) . Therefore , th e effect s o f ethanol o n io n distributio n and/o r cellula r bioener getic metabolis m should no t b e overlooke d as possible modulators of cell cycle regulatory molecules an d cellular proliferation.

vivo. I n suc h labeled , transplante d ste m cell s an d their progeny , one ca n asses s the effect s o f ethanol in take o n cel l cycl e regulator y proteins . A suitabl e model o f neurogenesi s tha t i s as clos e a s possibl e t o human neurogenesi s (i.e. , transplantatio n o f huma n NSCs into a pregnant primate that i s available for examination o f effects o f ethanol o n cel l cycl e o f stem cells) has been develope d (Ouredni k e t al., 2001) (for details see section below).

USE O F NEURA L STE M CELL

It has been hypothesize d that multipl e bonafid e ste m cell pool s exis t an d represen t descendant s o f a com mon NS C (Ouredni k e t al. , 2001) . Thes e cell s emerge, b y design, in the contex t of a single developmental proces s o f allocatio n an d segregatio n during the earliest stages of eerebrogenesis and then establis h a lifelong reservoir, presumably for use i n homeostasis and repair . This phenomeno n coul d represen t a de velopmental strateg y i n whic h plasticit y i s "pro grammed" into the CN S a t the singl e cell leve l fro m the earlies t stage s o f embryogenesis . Thi s progra m depends o n both progenito r proliferatio n and differen tiation. A clon e o f NSCs o f huma n derivatio n (hNSCs ) was grafte d int o developin g brain s o f feta l bonne t monkeys (Macaca radiatd), a n Ol d Worl d monke y species (a s close a s feasible experimentally to the hu man condition ) (Ouredni k e t al., 2001) . Unilaterally injected hNSC s distribut e themselve s evenl y throughout bot h cerebra l hemisphere s an d a t all levels of the neura l axis, settling in diverse widespread regions o f the telencephalon , principally at th e fronta l and frontoparietal levels. Although individua l hNSCs are clonally related, they appear to segregate into two subpopulations. Cells i n subpopulatio n 1 traverse great distance s through th e developin g cerebru m b y migrating from the periventricula r germina l zone s alon g hos t radial glia t o terminate a t developmentally an d temporall y appropriate cortica l lamina e an d differentiat e ac cordingly int o severa l neurona l an d glia l cel l types . Those hNSC s tha t migrate d to the mor e distan t superficial neurogeni c lamina become neurons , identified b y dua l immunoreactivit y t o antibodie s agains t neuron-specific nuclea r protein , calbindin , and neu rofilament, intermixe d seamlessly with the monkey' s own neurons .

TRANSPLANTATION TO EXAMINE

THE EFFECTS O F ETHANOL ON CELL CYCLE REGULATION I N VIVO

Labeling Neura l Ste m Cell s for In Vivo Studie s Cultured cell s are much more amenabl e t o the study of cel l cycl e regulator s than ar e cell s i n situ . Therefore, mos t studie s hav e examine d NSC s tha t hav e been derive d fro m th e CN S an d experimentall y manipulated i n culture . Bot h th e remova l o f cell s from thei r natural surroundin g in the brain and artifi cial cultur e condition s contribut e t o a degre e o f un certainty a s t o whethe r observation s fro m tissu e culture truly reflect the natural responses of stem cell s in th e intac t brain. This issu e can onl y be addresse d through examinatio n of NSCs i n liv e animals. Many novel technique s ar e currentl y availabl e t o stud y NSCs i n situ. The initia l ste p o f any suc h stud y is to develo p a method fo r identifying the cell s o f interest i n vivo . A review o f these method s i s beyond th e scop e o f this chapter. Nonetheless , one approac h i s to use genetic techniques tha t allo w creatio n o f transgeni c mic e with expressio n of marker genes under NS C promot ers such a s nestin o r CD 133. Viral vectors delivering a labe l suc h a s green fluorescen t protei n o r Lac Z (3galactosidase, or the exhaustivel y used BrdU-labelin g can b e use d a s well (Zawada et al. , 1998 ; Ouredni k et al. , 2001) . Another approac h i s transplantation o f NSCs int o th e brain , following thei r e x vivo labeling and/or geneti c modification . Potentia l advantage s of this approach includ e th e abilit y to control th e num ber o f cell s t o b e studied , thei r placement , an d th e ability to follow their movement an d differentiation i n

Transplanted Huma n Neural Ste m Cells Participate i n Brain Development

210 ETHANOL-AFFECTE D DEVELOPMEN T Most hNSC-derive d neuron s are found i n superfi cial cortica l layer s II/III (which , a t th e tim e o f trans plant, profite d fro m a n intensiv e suppl y o f newl y formed neurons ) (Frant z an d McConnell , 1996) . hNSC-derived cells , that stop and integrate within the cortical layer s IV-VI, differentiat e appropriatel y int o glia, a s identifie d b y immunoreactivit y t o GFA P (fo r astrocytes) o r t o 2 /,3/-cyclic-nucleotide phosphodi esterase (fo r oligodendrocytes). Gli a o f dono r origi n are als o observe d i n th e margina l zon e (MZ ; laye r I) and i n subcortical regions. Som e donor cell s also con tribute to the populatio n o f radial glia. Cells i n subpopulatio n 2 are small , non-processbearing, undifferentiate d BrdU-positiv e cell s dis persed throughou t th e S Z a s single cell s o r a s small clusters intermingle d wit h th e germina l cell s o f th e host. When double-staine d for cell type-specifi c antigens, thes e cell s expres s vimenti n (a n immatur e progenitor/stem cel l marker) , but ar e negativ e fo r all other marker s o f differentiation . A smal l numbe r o f subpopulation 2 cells , however , ar e presen t withi n the striatu m an d cortex , intermixe d wit h the differen tiated cell s appropriate t o the specific lamina. The transplantatio n experimen t answers a numbe r of important questions concerning NSC s an d provides a model fo r studying individual NSCs during neurogenesis. (1) The dat a provid e a plausible explanatio n fo r the generatio n o f multiple, disparate stem cel l populations as part of a unitary strategy of NSC allocation . (2) The clona l progen y o f a given NSC segregat e t o yield differentiated cell s fo r organogenesis (e.g. , subpopula tion 1) , whereas others (e.g. , subpopulation 2) are de posited i n germina l zones (e.g. , the SZ ) as a reservoir. (3) Grafte d hNSC s ca n integrat e int o th e morphoge netic program of the developing primate host brain. FIGURE 12- 3 Transplante d neura l ste m cell s (NSC) . Neural ste m cell s ca n b e grow n i n vitr o and success fully transplanted int o mammalian brain for studies examining thei r survival , proliferation , differentiation , and migration. A. C17.2 murine NSCs cultured in low serum differentiate int o astrocytes (large GFAP-positive cells), whereas some remain undifferentiated an d positive fo r nesti n (fou r cell s abov e th e asterisks) . B. En largement o f the tw o nestin-positive C17. 2 ste m cell s from pane l A . C . Te n day s afte r transplantatio n int o striatum an d nucleu s accumben s o f adul t C57BL/ 6 mice, C17. 2 NSC s surviv e an d expres s activ e p galactosidase as evidenced by staining this 40 jam-thick coronal sectio n fo r 5-bromo-4-chloro-3-indoly l p d-galactopyranoside (X-gal) . D. Enlargemen t o f X-galpositive cell s i n boxe d are a i n pane l C . E . Fou r markers of various stages of cell cycle. Shown here are:

Examination o f Cell Cycle Machinery I n Vivo Although th e mode l o f neurogenesis described abov e is instructive, it is very labor intensive and costly . Simpler model s hav e bee n developed . A prototypica l NSC-line, C17.2 , ha s bee n derive d fro m postnata l murine cerebellum . Thes e cell s ar e nestin-positiv e and differentiat e int o neurons, astrocyte s (Fig. 12-3) , and oligodendrocyte s i n culture . C17. 2 cell s ar e proliferating cel l nuclea r antige n (PCNA ) tha t label s the entir e cel l cycle , Ki-6 7 (nearl y entire cycle) , bro modeoxyuridine (BrdU) (a marker for DNA synthesis), and phosphorylated histone H 3 (pHisH3, for G2/M).

EFFECT OF ETHANO L ON NEURA L STEM CELL S 21 1 highly engraftabl e i n th e brai n and ca n b e identified by their expression of (3-galactosidase (Fig. 12-3) . Immunocytochemical determinatio n o f th e particula r phase i n whic h NSC s ar e presen t i s als o possible . Four commo n cel l cycl e markers , proliferatin g cell nuclear antigen that labels the entir e cell cycle , Ki-67 (nearly entir e cycle) , Brd U (DN A synthesis) , an d phosphorylated histone H3 (pHisH3, for G2/M), have been commonl y use d (Fig . 12-3) . I n on e elegan t study, these markers were used to determine that morphine induce s premature mitosi s in proliferating cells in the adult mouse S Z (Mandyam et al., 2004). Commercially availabl e antibodie s agains t cyclins , Cdks , and CKI s ar e usefu l fo r exploratio n o f expressio n of cell cycle mediators in even greater detail .

CKI cyclin-dependen t kinas e inhibitor CNS centra l nervous system ES embryoni c stem FASD feta l alcoho l spectru m disorders G gestationa l day Gadd45 growt h arres t an d DN A damag e in ducible GFAP glia l fibrillary acidic protein GMC ganglio n mothe r cel l hNSC huma n neura l stem cel l IHZ intrahila r zone Ki-67 proliferation-associate d nuclear antigen MZ margina l zone NSC neura l stem cel l

SUMMARY AND CONCLUSION S Prenatal exposur e t o ethano l ca n lea d t o FASD . Ethanol in the developin g brain not only triggers cell death, bu t als o negativel y affect s cel l division . Sinc e the lengt h o f the cel l cycle differs fo r ES cells , NSCs , and somati c cells , th e effec t o f ethanol o n cel l cycl e progression varies in these cel l populations and i s developmentally controlled . Take n together , ethano l generally reduces proliferatio n of NSCs during brain development b y affectin g th e expressio n of Cdks, cyclins, and CKIs such a s p21 and p27 . Although thes e complex response s are cell-typ e dependent , an d spa tially and temporarily controlled, th e net result generally i s prolongatio n o f ga p phase s o f th e cel l cycle , particularly Gl . Othe r potentia l cel l cycle-alterin g targets o f ethano l i n NSC s migh t b e telomerases , transcription factors , and protein s regulatin g cellula r bioenergetics. With th e adven t of novel technologies, many ne w cel l cycl e regulator s are bein g discovere d and wil l become importan t player s i n futur e studie s examining how ethanol affect s NS C proliferatio n and differentiation i n vivo. Abbreviations ABCC2 ATP-bindin g cassett e protei n AP-1 activatin g protein-1 bFGF basi c fibroblast growth factor (als o known as FGF-2 ) BrdU bromodeoxyuridin e CD 13 3 cel l determinan t Cdk cyclin-dependen t kinase

P postnata l day Rb retinoblastom a SZ subventricula r zon e Tc tota l length o f the cell cycl e TERT telomeras e revers e transcriptase TRF telomerase-repeat-bindin g factor TUNEL termina l deoxynucleotid e transferase mediated deoxyuridin e triphosphat e nick-end labeling VZ ventricula r zone

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13

Mechanisms of Ethanol-Induced Alterations i n Neuronal Migratio n Julie A. Siegenthale r Michael W . Miller

Neuronal migration i s the proces s b y which postmi totic neuron s translocat e fro m thei r birthplace i n proliferative zone s t o th e appropriat e target structur e where th e neuron s integrat e int o th e emergin g net work. I n th e cerebra l cortex , fo r example , neuron s generated i n one o f two germinal zone s adjacen t to a lateral ventricle , th e ventricula r an d subventricula r zones (se e Chapters 2 and 11) , migrate i n a n orderl y fashion t o a defined laminar residence (Angevine an d Sidman, 1961 ; Berr y and Rogers , 1965 ; Rakic , 1972 , 1974;Rakicetal., 1994) . The inabilit y of neurons to properly migrate is one cause of neurological disorders. The cerebra l cortex is the brai n region most frequently described as exhibiting malformation s designated a s neuronal migratio n disorders (NMDs). Tw o likely reasons fo r this are (1 ) its immense siz e and th e larg e numbe r o f constituen t neurons and (2 ) its highly laminated structure. These features mak e morphologica l disruption s more read ily identifiable.

Structural Malformation s i n Human Brain Two type s o f cortica l malformation s are eviden t i n humans wit h NMDs : lissencephal y o r heterotopias . Both malformation s are ofte n associate d wit h debili tating cognitiv e defect s i n individual s who ar e men tally retarde d and/o r epilepti c (Anderrnann , 2000 ; Pilzetal.,2002). The ter m lissencephaly, meanin g "smoot h brain, " is derived from the eve n appearance o f the surfac e of the brai n cause d b y a reductio n (pachygyria ) or absence (agyria ) of cortica l gyri . Othe r defect s associ ated wit h lissencephal y includ e increase d cortica l thickness an d reduce d o r poorl y define d cortica l lamination (Crome , 1956 ; Friede , 1989) . Heterotopia refer s to a cluster of neurons tha t is abnormally distribute d relativ e to neuron s wit h a com mon phenotype , suc h a s birthdat e o r connectivity . Heterotopias ca n b e detecte d a t fou r sites . Cluster s

216

ETHANOL AFFECTS NEURONAL MIGRATION 21 7 of ectopic neuron s may be distributed withi n the cor tical gra y (Miller, 1986 ; Anto n e t al. , 1999) . Hetero topias can als o be distribute d in the subcortica l white matter. A common exampl e i s a subcortical band het erotopia, a thick strip of ectopic cells beneath the cor tex. A ban d heterotopi a i s ofte n associate d wit h lissencephaly (Chevassus-au-Loui s and Represa , 1999 ; Kato and Dobyns , 2003) . Heterotopias are also found outside th e corte x beyon d th e ventricula r (Eksiogl u et al , 1996 ) o r pia l surfac e (William s e t al, 1984 ; Saito et al., 1999 ; Taked a e t al, 2003). Ethanol-Induced Malformation s Neuronal migratio n disorder s in human s hav e gene tic and/o r epigeneti c roots . Mutation s i n gene s tha t transcribe protein s regulatin g neurona l migratio n lead t o a spectru m o f defects , includin g cortica l lissencephaly an d heterotopias (Mochid a an d Walsh , 2004). Exposur e t o drug s o f abuse , i n particula r ethanol, ca n lea d t o NMDs. Feta l brain s expose d t o ethanol i n uter o ma y exhibi t a diminutio n o f gyr i and sulc i an d disruption s i n cortica l laminatio n (Fig. 13-1) (Clarre n e t al. , 1978 ; Wisniewsk i e t al. , 1983; Konovalo v et al, 1997 ; Roebuc k e t al, 1998) . Morphological defect s cause d b y gestational ethano l exposure likely contribute t o the cognitiv e defects and mental retardatio n associate d wit h feta l alcoho l syn drome (FAS) . Several brai n region s i n anima l model s o f FA S display morphologica l defects , includin g hetero topias an d structura l disorganizatio n (Fig . 13-2A) , similar t o thos e reporte d i n humans . Thes e region s include the cerebra l corte x (Miller , 1986 ; Kotkoski e and Norton , 1988 ; Komats u e t al. , 2001 ; Moone y e t al, 2004) , striatu m (Heato n e t al, 1996) , midbrai n (Sari et al., 2001; Zhou e t al, 2001), and cerebellu m (Kornguth e t al , 1979 ; Borge s an d Lewis , 1983 ; Quesada e t al, 1990a , 1990b) . Th e similaritie s between human s an d laborator y animal s establis h th e utility o f anima l model s fo r examinin g th e effec t o f ethanol o n neurona l migration . Rigorou s measure s of all aspect s of cell migratio n ar e require d to evaluate the effec t o f ethanol o n neurona l migration . Du e to th e obviou s limitation s o f human studies , anima l models o f FA S have bee n use d (a ) t o furthe r docu ment th e phenomenon of ethanol-induced defects in neuronal migratio n an d (b ) t o stud y underlyin g mechanisms.

FIGURE 13- 1 Brai n malformations in humans exposed to ethanol in utero. The gyra l pattern of the normal human infan t brain A. contrasts with the smoother cortical surface i n the brain of an infan t with FAS B. The brai n is marke d b y it s smal l size , whic h i s likely caused b y ethanol-induced defect s in both neurona l proliferatio n and cel l death. The brai n also exhibits a paucity of gyri, and these gyri tend to be broad. These features are signs of lissencephaly . Moreover , th e brai n i s covere d b y sheets of heterotopic cells over the surfac e o f the brain. The lissencephal y and heterotopia s are consistent with defects i n neurona l migration . C . Ethano l exposur e can also induce heterotopias t o form i n other regions of the brain , includin g th e brai n stem . Arrow s depict th e breach i n pia l surfac e throug h whic h th e migratin g neurons presumabl y streame d t o form th e heterotopia . D. Heterotopia s ar e als o collection s o f cells tha t pre sumably terminate thei r migrations i n inappropriate locations. Suc h neuron s ar e ofte n foun d i n th e cerebra l cortex. Cells with large (arrow ) and smal l (arrowhead) somata form a heterotopia withi n a portion o f layer I, a region of the cortex that is normally cell-sparse. (Source: Images provided courtesy of Sterling Clarren.)

MIGRATION DEFECT S I N ANIMAL MODELS OF FETAL ALCOHOL SYNDROM E

A metho d commonl y use d t o stud y neurona l migra tion i s mappin g o f spatiotempora l change s o f cell s with [ 3 H]thymidine autoradiography . Throug h thi s

218 ETHANOL-AFFECTE D DEVELOPMEN T method, proliferating cells can b e permanently radio labeled an d th e migratio n o f tagged cell s traced fro m their "birth " through th e completio n o f their migra tion. Durin g norma l development , mos t neuron s la beled with pHjthymidin e earl y i n corticogenesi s eventually ar e distribute d i n dee p layers , wherea s neurons labele d late r in development occup y superficial layer s (Angevin e and Sidman , 1961 ; Berr y an d Rogers, 1965 ; Rakic , 1974 ; Mille r an d Nowakowski , 1988). Thus, cortica l layer s are forme d in a n orderl y inside-out manner , durin g which migratin g neuron s move pas t those tha t have already completed cel l mi gration. Migration ca n b e divide d int o thre e phases . Th e first is the onset of migration, which occurs in the proliferative zones . Second , youn g neurons activel y migrate throug h th e intermediat e zon e (IZ ; th e anläg e of the whit e matter) to the superficia l limi t of the cor tical plate, the predecessor of the cortica l gray matter. The fina l ste p i s the terminatio n of neuronal migra tion in the developing structure . Although th e basi c inside-ou t developmen t o f th e cortex i s largel y preserve d i n roden t model s o f FAS , each phas e o f migratio n i s affecte d b y ethanol . Th e birth of cells that will occupy analogous site s in corte x is 1 to 2 days later i n ethanol-treate d rat s than i n con trols (se e Fig. 11-1 , Chapte r 11 ) (Miller , 1986 , 1987 , 1993). Par t o f this dela y (as much a s 3 1 hr) i s due t o postmitotic cell s lingerin g longe r i n th e proliferativ e zones befor e initiatin g thei r migrations . Thi s likel y contributes to the increase in size of the subventricular zone, which contain s many young neurons beginning their migrations (Miller, 1989). On the other hand, the number o f neuron s beginnin g migratio n a t an y on e time, the leavin g fraction, i s not affected b y ethanol. Ethanol als o reduce s th e rat e o f migratio n throughout development (Fig. 13-2B ) (Miller , 1993). For example, in the cortice s of 17-day-old fetuses, the mean rat e o f migratio n i s 138u,m/day , wherea s i n ethanol-treated fetuse s th e mea n rat e i s 8 2 jilm/day. Studies usin g slic e culture s o f corte x sho w tha t ethanol ha s a simila r effec t i n vitr o (Siegenthale r and Miller, 2004 ) and the rat e of migration is reduced in a concentration-dependen t manne r (Hira i e t al. , 1999b). The findin g tha t th e rat e o f migration i n situ is th e sam e a s tha t i n vivo suggest s tha t neurona l migration i s regulated b y loca l microenvironment — i.e., tha t extracortica l signal s (monoamines) ma y no t be critical regulators.

G13

G17

G20

TIME OF ORIGIN FIGURE 13- 2 Anima l model s o f feta l alcoho l syn drome (FAS) . A. Malformations incidental t o prena tal alcoho l exposure , includin g cortica l heterotopia s (box indicates area magnified in B), can be replicate d in anima l model s o f FAS. B. Thi s particula r hetero topia appear s to be caused by cells streaming through layer 1 at multiple sites (arrows), forming a layer of ectopic cell s outsid e th e cortex . C . Th e rat e o f migration throughou t corticogenesi s i s particularl y vulnerable to ethanol. Pulse-chas e experiment s usin g [ 3 H] thymidin e sho w tha t ethano l cause s significant decreases i n th e rat e o f migration o f cells generate d on gestational da y (6) 13 , G17, and G20 . Th e migra tion o f cell s generate d o n G1 7 i s mos t effecte d b y ethanol. O n Gl 3 and G17, the cells that begin migra tion first (outermost 10 ) and the total migratin g population (mea n population ) ar e similarl y slowe d b y ethanol. I n contrast , migratio n o f th e outermos t 1 0 cells generate d o n G2 0 i s les s sensitiv e t o alcoho l than tha t o f th e tota l population . (Source: Image s A and B provided courtesy of William Shoemake r an d Gordon Sherman. ) During late r stage s o f cortica l development , ethanol cause s migratin g neuron s t o finis h thei r migration i n inappropriat e locations . Fo r instance , neurons i n the corte x o f mature, norma l rat s that ar e generated o n gestationa l da y (G ) 2 0 are destine d fo r

ETHANOL AFFECTS NEURONA L MIGRATIO N 21 9

layer II/III. Followin g prenata l exposur e t o ethanol , however, most of these neuron s eithe r terminat e thei r migrations i n dee p laminae (layer s IV, V, and VI ) o r they overmigrat e int o laye r I (Miller , 1986 , 1993) . Other studie s sho w tha t ethano l ca n caus e th e production o f suprapial heterotopias , o r warts (Kotkoskie and Norton , 1988 ; Komatsu e t al , 2001 ; Mooney et al. , 2004) . The implicatio n i s that ethano l disrupts mechanisms involved in stopping neuronal migration.

MIGRATION MACHINERY : PROTEIN TARGETS O F ETHANOL TOXICIT Y

The thre e phases of neuronal migratio n are regulated by a numbe r o f differen t protei n complexe s an d signaling pathways that wor k in concer t t o mov e young neurons fro m th e proliferativ e zone s t o thei r targets. Each phase of neuronal migration is vulnerable to the effects o f ethanol . Therefore , protein s tha t regulat e migration, includin g cel l adhesio n protein s (CAPs) , cytoskeletal proteins, extracellula r matri x (ECM) proteins, an d growt h factors , ar e likel y candidates a s targets of ethanol toxicity .

cytoskeleton reorganizatio n (Panicker et al., 2003). Integrin receptor s bin d integrin s and ECM component s (e.g., laminin , fibronectin , an d glycoproteins) . Inte grin bindin g trigger s cytoskeleta l change s throug h a cascade of phosphorylation events (Juliano, 2002). Up-regulating expression of CAPs i s an importan t part o f initiating and propellin g neurona l migration. CAPs ar e richl y expresse d i n region s of the cerebra l wall, th e IZ , an d th e cortica l plat e (CP) , containin g migrating neurons (Edmondso n e t al., 1988 ; Seki and Arai, 1991; Anton e t al, 1999 ; Siegenthaler an d Miller , 2004). Disruptio n o f normal cel l adhesio n slow s th e progression o f glial-guided migratio n an d ca n lea d t o detachment o f neurons fro m th e glia l fibe r (Chuon g etal, 1987 ; Antonetal, 1996, 1999). Ethanol-lnduced Changes in Cell Adhesion Proteins

Ethanol exposur e reduce s th e rat e o f migration an d causes neurons t o be marooned i n ectopic cortical locales (Miller , 1986 , 1987 ; Mille r an d Robertson , 1993). A likel y mechanis m underlyin g ethanol induced defect s i n neurona l migratio n i s disrupte d cell adhesion . Fo r example , ethano l inhibit s LI-L I mediated cel l adhesio n by physically blocking LI-LI Cell Adhesion Proteins homophilic bindin g (Charnes s e t al , 1994 ; Ra The migratio n o f most cortica l neuron s begin s soo n manathan et al, 1996 ; Wilkemeyer et al, 1999 , 2000, after thei r birth i n a zone proxima l to the latera l ven- 2002a, 2002b) . Ethano l treatmen t als o inhibit s Li tricle (see Chapter 3) . The neuro n migrate s out of the mediated neurit e outgrowt h b y culture d cerebella r germinal are a usin g radia l fibers that spa n th e widt h granule cells ; however , ethanol apparentl y doe s no t of the cerebra l wal l and remain s apposed t o the guid - affect LI-L I bindin g per se (Bearer et al, 1999) . The ing fiber throughou t its migratory tre k (Rakic , 1971, implications o f this findin g ar e tha t ethano l ca n dis1972; Edmondso n an d Hatten , 1987) . Appropriat e rupt LI-mediated cell adhesio n throug h mechanism s adherence t o the glia l guide is regulated by cell adhe - other than th e LI protein-protei n interaction. sion receptor s tha t lin k th e neura l cytoskeleto n t o Unlike LI , th e effect s o f ethano l o n nCA M ap CAPs, component s o f th e ECM , an d membrane - pear to be a t the leve l of protein expression . Ethano l treatment increase s nCAM protei n expression in pribound moitié s of the glia l fiber. mary cortical neurons and i n slice s from feta l corte x (Fig. 13-3A ) (Mille r an d Luo , 2002a ; Siegenthale r Cell Adhesion Proteins Regulate Neuronal and Miller , 2004) . Specifically , ethanol-induce d Migration in Normal Animals increases i n nCAM expressio n ar e note d o n th e surAdhesion protein s fro m bot h th e cel l adhesio n mole - face o f migratin g neuron s i n cortica l expiant s (Fig. cule (CAM ) and integri n families are particularly im- 13-3B, 3C ) (Hira i e t al , 1999b ; Siegenthale r an d portant fo r neurona l migratio n (Ron n e t al. , 1998; Miller, 2004) . Th e expressio n and post-translationa l Clegg et al, 2003; Schmid an d Anton, 2003). Neural processing o f th e thre e isoform s o f nCAM i n th e CAM (nCAM ) an d L I mediat e neurona l migration postnatal brain are differentiall y affecte d b y prenatal through homophili c binding and consequen t activa- ethanol exposure , however , ther e ar e n o significan t tion o f intracellula r signalin g cascade s centra l t o changes i n nCA M niRN A expressio n wit h ethanol ,

220 ETHANOL-AFFECTE D DEVELOPMENT

FIGURE 13- 3 Expressio n o f cel l adhesio n protein s i s effecte d b y ethanol . A. Expressio n o f three isoform s (120 , 140 , an d ISOkD a isoforms ) o f neura l cell adhesio n molecul e (nCAM ) i s increase d i n primar y cortica l culture s (top) and organotypi c slice cultures (18 0 kDa only ) (bottom). B . Cells i n th e intermediate zon e (IZ ) fro m untreate d organotypi c slices immunolabel with nCAM antibody. nCAM expression is highest proximal to the cel l membrane and appear s to localize to patches (arrows) . C. Patche s o f nCAM immunore activity appear to be larger , denser, and mor e frequen t i n I Z cell s exposed to ethanol. Ethano l exposur e also increases the expressio n of integrin subunits a?, oc v, and ß j i n organotypi c cortical cultures D. Larg e increase s in nCA M and integri n receptors, like those observed with ethanol exposure , may cause a migrating cell to adhere too tightly to the surrounding environ and thus slow motility. (Source: Fro m Siegenthaler an d Miller , 2004 , wit h permission).

suggesting a defec t i n nCA M translatio n (Minan a et al., 2000). Ethanol-induced changes i n integri n expressio n and function are only beginning to be investigated. A compelling reaso n t o explore integrin s is the similarities betwee n cortica l migratio n defects i n mic e lack ing specifi c integri n subunit s an d anima l model s o f FAS. I n mic e lackin g integrin oc 3 an d i n animal s exposed prenatally to ethanol, cortica l neurons destine d for superficia l layer s li e i n ectopic , deep-laye r posi tions (Miller, 1986 , 1993 ; Anton etal, 1999). Neural specific integri n ß } knockou t mic e als o displa y abnormal neuronal migration . Ectopi c cell s are com mon i n the margina l zone an d th e pia l surfac e i s disrupted, analogou s t o th e heterotopia s see n wit h prenatal ethanol exposur e (Graus-Porta etal., 2001). Despite th e similarities between anima l models of FAS and integri n knockou t animals , i n vitro studies indicate that ethanol potentiate s integri n protein con tent (Fig . 13-3D ) (Siegenthale r an d Miller , 2004) . Interestingly, thi s findin g i s similar t o th e effec t ob served wit h nCA M expressio n (Hira i e t al. , 1999b ; Luo and Miller, 1999 ; Miller and Luo, 2002a; Siegen thaler an d Miller , 2004) . Ethano l increase s th e ex pression of integrin subunit s OC 3, ot v, and ß j i n cortica l

expiants (Siegenthaler and Miller , 2004) . In culture d neurophils (Bautista , 1995 ) an d hepatocyte s (Schaf fert et al., 2001), integrin oCj , oc 5, and ß ! are increase d following treatmen t wit h ethanol , settin g u p th e possibility tha t to o muc h integrin , lik e to o muc h nCAM, is detrimental t o neuronal migration . Indeed , neurons tha t overexpres s integri n receptor s hav e im paired migratio n (Palece k e t al. , 1997) . Presumably , excess CA P cause s increase d cel l adhesio n an d pre vents migratin g neuron s fro m breakin g adhesion s a t the rea r o f the cell . Essentially , th e adhesio n i s too sticky. Alterations i n integri n expressio n ma y als o con tribute t o ethanol-induce d defect s i n th e radia l glia l scaffold. Radia l glial fibers traverse the migratio n path way in th e developin g corte x and provid e a guide for neurons durin g thei r migratio n t o their ultimat e resi dence. Afte r neurona l migratio n i s complete , radia l glia transfor m into stellate astrocyte s (Schmeche l an d Rakic, 1979 ; Voigt , 1989 ; Culica n et al, 1990 ; Mille r and Robertson , 1993) . Ethano l exposur e i n uter o leads t o th e prematur e differentiatio n o f radia l gli a into astrocytes in both th e corte x (Mille r and Robert son, 1993 ; Miller , 2003) and cerebellu m (Shett y and Phillips, 1992) . Neuron s activel y migrating o n thes e

ETHANOL AFFECTS NEURONAL MIGRATIO N 22 1 transforming radia l glia ar e strande d i n dee p cortica l layers, unable t o complete thei r migration. This finding i s further supporte d b y [ 3H]thymidine studies in which neuron s intende d fo r superficia l layer s ar e found i n dee p corte x followin g ethano l exposur e (Miller, 1986 , 1993) . Incidentally, absence of integrin OC3 also causes premature transformation of radial glial (Anton e t al , 1999 ) an d attachmen t o f radia l glia l endfeet i s disrupte d i n integrin-ß } knockou t mic e (Graus-Porta e t al., 2001) . Thus, ethanol-induced disruptions i n integri n functio n o r expressio n may pre cede the prematur e withdrawal of radial glia. Disruptions in Ca2+ channel flux during neuronal migration ma y contribute t o the ethanol-induce d re duction i n the rat e of migration. Ca2+ currents in migrating neurons are mediated by N-methyl-D-aspartate (NMDA) subtype of glutamate receptors . Cel l migration decrease s followin g treatment wit h NMD A an tagonists (Komur o an d Rakic , 1992 , 1993 ; Hira i e t al., 1999a) . Ethano l affect s bot h th e expressio n an d function o f NMDA receptors . Chronic ethano l expo sure up-regulate s specifi c subunit s of the NMD A re ceptor i n feta l cortica l neuron s (Kumari , 2001) . I n contrast, acut e treatmen t wit h ethano l block s NMDA-dependent Ca 2+ current s i n culture d hip pocampal neuron s an d slice s (Lovinger e t al. , 1989 , 1990). Ca2+ currents ar e particularly important at the trailing edg e o f migratin g cells . Los s o f integrin dependent cel l adhesio n a t th e rea r edg e o f motil e cells depend s o n increase s i n intracellula r Ca 2+ (Marks an d Maxfield , 1990 ; Lawso n an d Maxfield , 1995). Thus , ethanol-dependen t change s i n norma l Ca2+ currents might slo w neuronal migratio n by preventing normal separation at the rea r edge of the cell . Extracellular Matrix The pia l membran e (PM ) (o r pia l basemen t mem brane) is an ECM-rich region. The EC M contribute s to the rol e of the P M in the norma l developing cortex as a barrier (chemical and physical ) around develop ing neura l tissu e and a n attachmen t poin t fo r radial glia (Siever s e t al, 1994) . Breache s in th e P M allo w neurons to migrat e beyond th e pia l surface an d for m heterotopias (Marin-Padilla, 1996) . Prenatal ethano l exposur e ca n induc e formation of heterotopia s i n human s expose d prenatall y t o ethanol (Clarre n e t al., 1978) . Roden t model s o f prenatal ethanol exposure also show heterotopias i n both the cerebellu m an d corte x (Kotkoski e an d Norton ,

FIGURE 13- 4 Ethanol-induce d heterotopia s ar e associated wit h disruption s i n th e margina l zon e (MZ ) o f organotypic cortica l cultures . A . Severa l cel l som a oc cupy a breach (ope n arrows) in calretinin-positive fibers within th e MZ . Th e cell s appea r t o b e streamin g through th e brea k and contributin g t o the heterotopia , or "wart, " formin g outsid e th e cerebra l wall . Cajal Retzius cel l bodie s (close d arro w wit h line ) an d pro cesses (closed arrows) that occupy the MZ immunolabel with calretini n antibodie s (close d arrows). CP , cortica l plate. B. Breaches in the MZ (* ) contain nestin-positive radial glial fibers (arrows). These fibers normally terminate a t the pia l membran e (PM ) adjacen t to the MZ . Ethanol-induced disruption s i n th e M Z and/o r P M likely allow the fibers to course inappropriately into the heterotopia. (Source: Fro m Moone y e t al. , 2004 , wit h permission).

222 ETHANOL-AFFECTE D DEVELOPMENT 1988; Komats u e t al, 2001; Sakata-Haga et al, 2001 ; Mooney e t al., 2004). In rodents, breaches in the glial limitans an d margina l zone , a superficia l cortica l region adjacen t t o th e PM , ar e ofte n foun d a t th e base o f ethanol-induce d heterotopia s (Fig . 13-4 ) (Komatsu e t al , 2001 ; Sakata-Hag a e t al , 2001 ; Mooney e t al, 2004). Despite repeated report s of heterotopias associate d with prenata l ethano l exposure , a clea r lin k betwee n defects i n th e EC M constituent s o f th e P M an d ethanol ha s not been made . Nevertheless, the appear ance o f heterotopia s i n animal s i n whic h specifi c ECM protein s ar e dysfunctiona l strongl y indicate s that these protein s are disrupted b y ethanol. Deletio n or dysfunction of PM protein s lamini n (Halfte r et al., 2002), perlecan (Costel l e t al., 1999) , an d chondroitin sulfate proteoglyeans (Blackshear et al, 1997 ) leads to overmigration o f neuron s an d formatio n o f ectopia s reminiscent of ethanol-induced heterotopias . Integri n receptors bin d t o an d organiz e EC M proteins . Mic e lacking the integrin a6 or integrin ßj feature defect s in the cortica l pia l membran e tha t lea d t o th e appear ance o f ectopic cel l bodie s (Georges-Labouess e e t al, 1998; Graus-Porta et al, 2001). As noted above, ethanol does affec t th e expressio n o f integrin subunits , includ ing ß j (Siegenthale r an d Miller , 2004) . Conceivably , ethanol targetin g of these integrins is key to the gener ation o f heterotopia s and , b y deduction , thes e inte grins are key to the integrit y of the PM . Reelin, a larg e glycoprotei n componen t o f th e marginal zon e ECM , i s produce d an d secrete d b y Cajal-Retzius cell s (D'Arcangel o e t al, 1995 ; Hirot sune e t al , 1995 ; Ogaw a e t al , 1995 ; Sod a e t al , 2003). Reeli n i s believed to ac t a s a sto p signa l causing migratin g neuron s t o en d migratio n an d detac h from thei r glia l guide s (Pearlma n an d Sheppard , 1996; Marin-Padilla , 1998 ; Dulabo n e t al , 2000 ; Hack e t al. , 2002) . Immunohistocheniistr y studie s show tha t reeli n i s abnormally distributed i n ethano l induced-heterotopias (Moone y e t al. , 2004) . I t i s absent directly below heterotopias, but i t is distributed toward th e perimete r o f a heterotopia . Conceivably , the periphera l displacemen t o f reelin underlie s con tinued migration of neurons into a heterotopia . Cytoskeleton Successful cel l adhesion an d releas e during neurona l migration depends o n th e rapi d reorganization of the cytoskeleton. Thi s reorganizatio n i s essentia l fo r th e

initiation an d maintenanc e o f neurona l migratio n (Lambert de Rouvroi t and Goffinet , 2001 ; Marin an d Rubenstein, 2003) . Th e tw o mai n cytoskeleta l ele ments in neurons are actin and microtubules, proteins that ca n polymeriz e int o lon g chain s o f individua l subunits tha t compris e th e intracellula r scaffold . Actin filament s ar e enriche d a t th e leadin g edg e o f migrating neurons . Microtubules , in contrast , for m a cage-like structur e withi n th e cel l somat a an d ar e aligned longitudinall y i n th e leadin g proces s (Riva s and Hatten , 1995) . The importanc e of both actin and microtubule assembl y is emphasized b y finding s tha t several geneti c NMD s ar e cause d b y defect s i n pro teins tha t bin d an d organiz e cytoskeleta l protein s (Sapir et al, 1997; Gleeso n et al., 1999) . Ethanol negativel y affects th e cytoskeleton i n neu ral and non-neura l cells . Actin assembly is aberrant i n cultured neura l cres t cell s treate d wit h ethano l out growth (Hassle r and Moran , 1986a , 1986b) . Ethanol induces th e formatio n of thick bundles o f actin fibers that prevent the norma l process . The acti n cytoskele ton i n astrocyte s i s als o sensitiv e t o treatmen t wit h ethanol (se e Chapter 18) . The norma l organization of actin filament s i n culture d astrocyte s i s severely disrupted b y ethanol i n a concentration-dependent man ner (Allansso n e t al. , 2001 ; Guasc h e t al. , 2003 ; Tomas e t al, 2003) . Ethano l promote s th e transfor mation o f actin stres s fiber s int o dysfunctiona l acti n rings. Interestingly , the effec t o f ethanol o n th e acti n cytoskeleton i s alleviated b y overexpressio n o f RhoA, a smal l GTPase that induce s actin stres s fiber assembly (Guasc h e t al , 2003) . This effec t suggest s tha t ethanol exposur e may disrupt signaling pathways regulating cytoskeleta l reorganization . Microtubul e polymerization i s also affected b y ethanol exposur e i n astrocytes an d hepatocyte s (Yoo n et al. , 1998 ; Toma s et al. , 2003) . Afte r treatmen t wit h th e microtubule disrupting agen t nocodazole , th e spee d o f micro tubule repolymerizatio n is reduced i n the presenc e of ethanol. Growth Factors Role in Neuronal Migration Growth factors are proteins tha t regulate cel l ontogen y via extracellula r mechanisms . Growt h factor s tha t affect neurona l migratio n includ e member s o f th e transforming growt h facto r (TGF ) ß superfamil y (TGFßl an d bon e morphogeni c protei n [BMP ] 4 )

ETHANOL AFFECTS NEURONA L MIGRATIO N 22 3 and brain-derive d neurotrophi c facto r (BDNF) . Several studies imply that these factor s are active endogenously. Eac h ligan d an d th e relevan t receptor s are expresse d i n proliferativ e zone s durin g cortica l development (Flander s et al, 1991 ; Fukumitsu et al, 1998; L i et al, 1998; Miller, 2003). Functional studie s relyin g o n exogenou s growt h factor furthe r indicat e tha t TGFßl , BMP-4 , an d BDNF ar e involve d i n th e initiatio n and/o r mainte nance o f neurona l migration . Bot h TGFß l an d BMP-4 decrease cel l proliferatio n and promot e neuronal migratio n (L i e t al. , 1998 ; Lu o an d Miller , 1999; Siegenthale r an d Miller , 2004 , 2005a) . Complimentary line s o f evidenc e indicat e tha t BDNF, though no t classicall y associate d wit h neurona l mi gration, promote s th e initiatio n o f neurona l migra tion. First , studie s sho w tha t cerebella r granul e cell s from BDNF~ f~ mic e ar e abl e t o attac h t o glia l fiber s but fai l t o begi n migratin g (Borghesan i et al. , 2002). Also, addition of exogenous BDN F enables BDNF~ /granule cells to migrate properly . Finally, injectio n o f BDNF int o th e latera l ventricle s o f embryoni c animals induces the prematur e initiatio n of migration of neurons destine d fo r dee p corte x (Ohmiy a e t al. , 2002). Effect ofEthanol on Growth Factor-Mediated Neuronal Migration Ethanol effect s bot h th e expressio n o f th e endoge nous TGFß system and th e functio n of exogenously applied TGFß l i n vitro . Rat s prenatall y exposed t o ethanol exhibi t altere d TGF ß ligan d an d recepto r expression (Miller , 2003 ) Specifically , ethano l in creases TGFßl an d TGFß2 content i n the prolifer ative zone s prenatally , bu t i t diminishe s perinata l TGFß2 expressio n i n th e interna l segment s o f ra dial glia. In contrast, ethanol increase s TGFß recep tor typ e 1 expressio n i n radia l glia l fibers . Th e presence o f an endogenous TGF ß system in the proliferative an d intermediat e zone s suggest s tha t TGFß i s importan t fo r a t leas t th e initiatio n an d propagation o f neurona l migratio n throug h th e in ner half of the cerebral wall. Further, th e vulnerability o f thi s portio n o f th e TGF ß syste m t o ethano l may contribut e t o ethanol-induce d defect s i n neuronal migration. Ethanol disrupts TGFßl-mediated neuronal migration. I n slic e culture s o f cerebra l cortex , ethano l im pedes th e effect s o f exogenousl y applie d TGFß l o n

both cel l migratio n and CA P expression (Siegenthaler and Miller , 2004 , 2005b) . In vitro studies o f dissociated cortica l cell s an d neura l tumorgeni c cell s de scribe a simila r effec t o f TGFß l an d ethano l o n a specific CAP , nCAM. nCAM expressio n is increased with TGFß l treatmen t alon e bu t i s reduced t o control level s b y combine d treatmen t wit h ethano l (Luo an d Miller , 1999 ; Mille r an d Luo , 2002a , 2002b). Collectively , thes e studie s sho w tha t (1 ) ethanol interfere s with TGFßl-mediated promotion of cell migratio n an d (2 ) i t ma y d o s o b y blockin g TGFßl-dependent increase s i n expressio n o f nCAM and othe r CAPs . BMP signalin g is involved in the initiatio n of cell migration i n th e developin g cerebra l corte x (L i et al., 1998) . BMP- 4 specificall y is expressed by cells about to begin migratio n (e.g., at the ventricula r surface). Althoug h th e effec t o f ethano l o n BMP mediated initiatio n o f migratio n i s no t establishe d per se , ethano l doe s bloc k BMP-4-induce d cel l ad hesion amon g culture d neuroglioblastom a cell s (Wilkemeyer e t al. , 1999) . Presumably , suc h inhibi tion ma y be involved in delay of the initiatio n of neuronal migration. BDNF promotes th e initiatio n o f glial-guided mi gration (Borghesan i e t al. , 2002) . Incidentally , th e endogenous BDN F syste m i s perturbed b y ethanol . Perinatal ethano l exposur e down-regulate s BDN F mRNA and it s receptor, trkB , in the developin g cere bellum (Heato n e t al, 2000 ; Ligh t e t al, 2001 ) and olfactory bul b (Maie r e t al. , 1999) . A similar downregulation o f BDN F protei n accompanie d b y a ethanol-induced decreas e i n BDN F signalin g i s evident i n prenata l corte x (Climen t e t al. , 2002) . An ethanol-induced reductio n i n BDN F activit y i n the developin g brai n likel y affect s BDNF-regulate d processes includin g th e initiatio n o f neurona l migration.

SUMMARY AND CONCLUSION S

Neuronal migratio n i s a uniqu e an d critica l compo nent o f neural development . I t provides a bridg e be tween th e birt h o f a cel l an d it s prope r integratio n into a n evolvin g structure . NMD s ar e cause d b y breakdowns i n th e commencement , completion , and/or cessatio n o f neuronal migration . Th e NMD s detected in case s of FAS are likel y caused by ethanol interfering with each phas e o f neuronal migration .

224 ETHANOL-AFFECTE D DEVELOPMEN T

FIGURE 13- 5 Ethano l disrupt s cortical migratio n a t many levels. A. Ethanol reduce s the rat e of neuronal migration i n th e developin g cerebra l cortex . Migrat ing neuron s depen d o n prope r cell adhesio n (e.g. , cell adhesio n molecule s an d integrins ) and cytoskeletal reorganizatio n (e.g. , microtubule s an d actin ) fo r motility alon g glia l guides . Ethanol-dependen t in creases in cell adhesion protein expression and abnor mal cytoskeleta l organizatio n ar e a likel y caus e o f reduced cortica l cel l motility . B. Heterotopi a forma tion ha s been linke d t o disruptions i n both the extra cellular matri x component s o f th e pia l membran e (PM) an d thei r organizatio n b y integri n receptors . The resultin g "holes" i n th e P M an d adjacen t M Z permit overmigratio n o f neuron s tha t woul d nor mally sto p an d incorporat e int o th e cortica l plate . In particular , absenc e o f th e stop-signa l reeli n within th e breache s likel y contribute s t o hetero topia formation . Evidenc e o f radia l glia l fiber s coursing into the heterotopia ma y mean tha t migrating neuron s migrat e int o th e war t on overextende d glial fibers . C . Ethano l exposur e cause s prematur e transformation o f radial glia int o astrocytes. Neurons still migratin g o n thes e retractin g fiber s woul d b e stranded inappropriatel y i n deepe r cortica l layers . This phenomeno n ma y explai n th e appearanc e o f layer II/III neurons are foun d i n layers IV, V, and V I in ethanol-expose d brains . D. Variou s growth factor s (e.g., TGFßl , BMP-4 , an d BDNF ) ar e involve d i n

Ethanol-induced defect s i n neurona l migratio n have seriou s consequence s fo r downstrea m develop mental events . For example, exposure to ethanol dur ing th e perio d o f neurona l migratio n ca n caus e neurons t o lan d i n th e wron g cortical layer s (Miller, 1986, 1987) . Despite this, the neurons may retain their destined phenotype , fo r example , a s corticospina l (Miller, 1987 ) o r callosa l (Miller , 1997 ) projectio n neurons. As the cell bodies are distributed in inappropriate locations , the y integrat e int o th e synapti c web incorrectly (Al-Rabia i an d Miller , 1989) . Conse quently, abnormalitie s i n cortica l metabolis m an d activity occu r (Vinga n e t al, 1986 ; Mille r an d Dow Edwards, 1988 , 1993) . Flawe d synapti c connection s due t o heterotopia s ca n caus e a myria d of problems , the foremos t being epileps y (Chevassus-au-Louis an d Represa, 1999) , a neurological symptom prevalen t in children with FAS (Burd et al, 2003). Over th e las t two decades, studie s of neuronal mi gration hav e identifie d a number o f possible mecha nisms fo r ethanol-induce d defect s i n migration . Ethanol alter s bot h th e microenvironmen t encoun tered by the migratin g neuron (e.g. , radial glial fibers, CAPs, EC M proteins , an d growt h factors ) an d th e motility o f neuron s withi n tha t environmen t (e.g. , CAPs an d cytoskeleta l proteins ) (Fig . 13-5) . The di versity of cellular pathways affected b y ethanol speak s to the complexit y of ethanol-induced disturbance s i n neuronal migration. Abbreviations BDNF brain-derive d neurotrophic facto r BMP bon e morphogenic protei n CAM cel l adhesion molecul e CAP cel l adhesio n protei n CP cortica l plat e ECM extracellula

r matrix

FAS feta l alcohol syndrome G gestatio n day IZ intermediat e zon e

the initiatio n o f neuronal migration . Ethano l disrupts the norma l function of these growth factors. The dela y in initiatio n o f migration see n wit h ethano l exposur e likely result s fro m inhibitio n o f growt h factor-medi ated activities.

ETHANOL AFFECTS NEURONAL MIGRATIO N 22 5 nCAM neura l cell adhesion molecule NMD neurona l migration disorde r NMDA N-methyl-D-aspartat e PM pia l membrane TGF transformin

g growt h facto r

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14

Effects of Ethanol on Mechanisms Regulating Neuronal Proces s Outgrowth Tara A. Lindsley

Establishment o f normal neura l circuitr y in the cen tral nervou s syste m (CNS ) require s spatia l an d tem poral contro l o f th e outgrowt h o f neuntes . Thi s outgrowth an d th e specificatio n of axon s an d den drites ar e amon g th e earlies t event s i n th e develop ment of postmigratory neurons. Three decade s o f researc h sho w tha t exposur e t o ethanol disrupt s th e developmen t o f axon s an d den drites an d alter s neura l circuitr y i n divers e brai n re gions. Th e primar y actions o f ethanol o n developin g neurons are still largely unknown, however, in part, because th e endogenou s mechanism s an d extracellular signaling pathway s regulating proces s outgrowt h an d guidance during normal development ar e not fully un derstood (se e Chapter 4) . Fo r comprehensive descriptions of effects o f ethanol o n th e microscopi c structure of the developing brain, the reader is referred to reviews by Pentney and Miller (1992) and Berma n and Hanni gan (2000), respectively. The presen t chapte r summarize s th e effect s o f ethanol on the outgrowth and maturation o f dendrites

and axons , focusin g o n CN S neuron s developin g in vivo or in vitro. The selecte d observation s highlight both well-establishe d an d emergin g insight s regarding th e effect s o f ethano l o n axona l an d dendriti c growth. Althoug h not exhaustive , one sectio n describes how recent advances in developmental neural cell biology are informing work aimed at understanding th e mechanism s underlyin g disruptio n o f neu ronal development b y ethanol.

EFFECTS O F ETHANOL O N DEVELOPMENT OF DENDRITES In Vivo Studies The comple x an d characteristi c shap e o f a dendriti c arbor, where neurons receive and integrate inpu t fro m multiple synapti c partners , i s a critica l determinan t of th e electrophysiologica l propertie s o f a neuron . Ethanol-induced change s i n dendriti c morpholog y

230

ETHANOL AND PROCESS OUTGROWTH 23 1 could severel y affec t neurona l function . Result s o f morphometric studie s in rodent s expose d t o ethano l during development sugges t that regions of the brai n are no t equivalentl y affected b y ethanol. Eve n withi n a brain region vulnerable to ethanol-induced change s in dendriti c shape , th e spécifi e parameter s o f th e dendritic arbo r (e.g. , lengt h o f dendriti c segments , number o f primary dendrites, o r branching ) ma y b e differentially affected . The expans e o f dendritic arbor s i s determined b y the numbe r an d lengt h o f it s primar y dendrite s (which exten d directl y fro m th e cel l body ) an d den dritic branches . I n youn g mic e expose d t o ethano l pre- an d perinatally , pyramida l neuron s i n th e CA 1 region o f th e hippocampu s hav e shorte r basa l den drites, but th e lengt h o f apical dendrites is unaffecte d (Davies an d Smith , 1981 ; Smit h an d Davies , 1990) . Parallel results have been reporte d for the dendrite s of pyramidal neuron s i n the sensorimoto r corte x (Ham mer an d Scheibel , 1981 ; Fabregue s et al., 1985) . The number o f primary dendrites an d th e exten t o f their branching are also decreased in other areas including chick spina l cor d serotonergi c neuron s (Mendelso n and Driskill , 1996) , ra t dopaminergi c neuron s o f the substantia nigr a (Shett y et al., 1993) , an d ra t cerebel lar granul e neuron s (Smit h an d Davies , 1986) . Tha t ethanol may differentially affec t distinc t parameters of dendritic morpholog y i s a potentiall y importan t ob servation give n evidenc e tha t th e number , length , and branchin g o f dendrite s ma y b e independentl y regulated (Scott and Luo, 2001). Although man y report s emphasiz e th e inhibitor y effects o f prenata l ethano l exposur e o n th e siz e o f dendritic arbor s i n variou s region s o f immatur e ro dent brain, studies indicat e tha t ethano l ca n increase the siz e o f dendritic arbors. For example, ethanol in creases th e numbe r an d lengt h o f apical an d basila r dendritic branche s o f callosa l projection neuron s i n the ra t (Qiang et al., 2002) . The stimulator y effec t o f ethanol o n dendrite s appear s t o involv e N-methyl-Daspartate (NMDA ) receptors , sinc e dendrite s are nor mal i n ethanol-expose d transgeni c mice wit h deletio n of the NR 1 subtype of NMDA recepto r (Den g and El berger, 2003). Notably, th e dat a cite d abov e exemplif y effect s o f developmental exposur e t o ethano l observe d whe n neonatal or immature animals are examined. In some cases, however , whe n prenata l ethanol-expose d ani mals ar e examine d a t othe r youn g ages, o r a s adults, areas that had stunted dendrite s are found to be eithe r

normal (Pentne y e t al , 1984 ) o r hav e significantl y larger an d mor e comple x dendrite s tha n control s (Miller e t al. , 1990) . Thes e dat a ar e generall y be lieved t o indicat e tha t ethanol-induce d reductio n i n dendritic arbo r siz e reflect s delayed dendriti c devel opment o r that initial inhibition o f growth is followed by an abnorma l compensatory growth response when the anima l is no longe r expose d to ethanol . Anothe r possibility i s tha t ethano l interfere s wit h dendriti c pruning, a proces s associate d wit h a glutamatergic NMDA recepto r mechanism (Kozlowsk i et al., 1997) . This concep t i s supported b y evidenc e tha t ethano l can caus e axona l hypertroph y as well (se e Effect s o f Ethanol on Development of Axons). That dendriti c morpholog y coul d chang e s o significantly i s not surprising , given that dendrit e development i s a highl y dynami c process . Dendriti c growth i n viv o i s characterized by extensive remodeling, as exemplified by the postnata l retractio n o f th e apical dendrite s o f some laye r V callosa l projection neurons, fro m cortica l laye r I to layer IV (Koester and O'Leary, 1992) . Thus, assessment of prenatal ethano l effects o n dendrite s ove r extende d development , an d after withdrawa l fro m ethanol , i s important . Mor e studies are neede d to explor e the possibilit y tha t ethanol affects th e cellula r event s whereby exuberan t early growth is followed b y partial pruning, or initially stunted developmen t i s later reverse d b y compensa tory growth. Dendritic remodelin g ca n b e directl y visualize d by time-lapse imaging , as performed on neuron s fro m frog opti c tectum , in which rapi d addition an d retrac tion o f ne w branche s an d remodelin g o f existin g branches, a s well as the differentia l regulatio n o f these events during maturation, has been documente d (W u et al. , 1999) . I t i s now possibl e t o imag e neuron s i n complex environment s ove r extende d tim e period s (Danuser an d Waterman-Storer , 2003) . Suc h time lapse studie s coul d hel p distinguis h whethe r ethano l delays o r alter s on e o r mor e feature s o f dendriti c growth in the intac t developing brai n (e.g., outgrowth, elongation, branching, or remodeling). In Vitro Studies Several neuron cultur e systems have been used t o examine th e effect s o f ethanol o n developin g neurons . Some cell cultur e models ar e poorly suited to exploring effect s o f ethano l specificall y o n dendrite s o r axons. Fo r example , i n som e dissociate d cultures ,

232 ETHANOL-AFFECTE D DEVELOPMEN T (1) the neuron s are too dense or there are various cell types present , limitin g bot h morphometri c an d mo lecular analyses ; (2) th e neuron s d o no t surviv e o r develop long enoug h fo r researchers to investigate th e persistence o f ethano l effect s o r th e capacit y o f th e neuron to recover normal morphology, or (3) the neu rons are no t th e type s sensitive to ethanol damag e i n vivo. No t surprisingly , a n inconsisten t pictur e o f ethanol effect s o n neuromorphogenesis has emerged. Some report s sho w that ethano l inhibit s proces s out growth (Do w an d Riopelle , 1985 ; Kentrot i an d Ver nadakis, 1991 ; Heato n e t al. , 1994 ; Saunder s e t al. , 1995; Beare r e t al. , 1999) , wherea s other s repor t a stimulatory effec t (Messin g e t al. , 1991 ; Woote n an d Ewald, 1991 ; Roivaine n e t al , 1993 ; Zo u e t al , 1993). The followin g discussion focuses o n studies for which ther e is reasonable certaint y that th e processe s being studied are either dendrites or axons. Unique characteristic s o f low-densit y culture s o f fetal ra t hippocampal pyramida l neurons mak e the m a valuable model fo r examining the molecula r mech anism^) b y whic h ethano l interfere s wit h dendriti c and axona l growth. These cultures, detailed in Chapter 4, are th e mos t extensivel y characterized primar y culture o f mammalian CNS neurons , and numerou s studies hav e been conducte d comparin g the m t o pyramidal neuron s developin g i n sit u (Crai g an d Banker, 1994 ; Gosli n e t al, 1998) . Neuron s i n thes e cultures ar e nearl y homogeneous ; typicall y 94% ar e readily distinguished as pyramidal neurons. They live for u p t o 4 weeks , undergoin g stereotypical , nearl y synchronous development , each neuron havin g a single axo n an d severa l dendrites of well-defined shap e and characteristi c molecula r constituents . Th e cul tured neuron s for m synapti c relationship s tha t ar e representative o f thos e normall y present i n th e hip pocampus (e.g. , Schaffe r collateral s interconnectin g pyramidal neurons). This pattern parallels hippocampal developmen t i n situ, which i s largely complete by the en d o f th e thir d postnata l wee k (Minkwit z an d Holz, 1975). Moreover, the developmental progression that occurs during the elaboratio n o f axons, dendrites, and synapse s can b e readil y quantified by time-laps e microscopy o f individua l neurons (Dott i e t al. , 1988 ; Goslin an d Banker , 1989). Some of the inhibitory effects o f ethanol on dendrite development ar e supporte d b y results from studie s using these cultures, in which definitive dendrite s can b e identified b y their characteristi c proximal-dista l tape r and b y immunolocalization o f microtubule-associated

protein (MAP ) 2, a protein distribute d in th e somato dendritic compartmen t (Gosli n e t al. , 1998) . I n hip pocampal cultures , quantitativ e morphometri c analyses o f MAP2-stained, pyramidal neuron s 6 days in vitro (DIV) , expose d continuousl y t o 43-8 7 mM ethanol, sho w concentration-dependent decrease s i n total dendriti c length , in the numbe r o f primary den drites and dendritic branches, and in the length o f individual dendrite s (Yann i an d Lindsley , 2000 ; Yanni et al. , 2002) . Ethano l als o decrease s th e numbe r o f synapses forme d by these neuron s (Yann i an d Linds ley, 2000) . Nevertheless , synapti c densit y i s no t af fected. Hence , i t i s likel y secondar y t o effect s o n dendrites an d no t a direct effec t o n synapse formation per se . Delaying th e additio n o f ethanol unti l 1 DIV, when mos t neuron s hav e develope d a n axon , results in significantl y shorter, less-branche d dendrite s a t 6 DIV, relative to the neuron s exposed to ethanol a t the time of plating (Lindsley et al., 2002). Thus the matu rity of hippocampal neuron s influences their vulnerability to morphoregulatory effects o f ethanol. The duratio n o f ethanol exposur e an d th e timin g of withdrawal have variable effects o n dendrit e devel opment i n hippocampa l culture s (Lindsle y an d Clarke, 2004). When ethanol i s added to the mediu m shortly after plating , continuous exposure for up t o 14 DIV decrease s th e lengt h an d numbe r o f dendrite s formed, but i t has no effect o n neuron survival . When ethanol exposur e is limited to the first day post-plating and withdrawa l occurs befor e dendrites elongate , in dividual dendrite s achieve normal length b y 1 4 DIV. The arbors , however , ar e stil l smalle r tha n control s because o f a persisten t reductio n i n th e numbe r o f primary dendrites. Thes e findings are consistent wit h in vivo observations that inhibitor y effects o f prenatal ethanol o n dendriti c lengt h ma y no t endur e afte r withdrawal from ethanol (Pentne y et al., 1984; LopezTejero e t al. , 1986) , but th e effec t o n th e numbe r o f primary dendrites may persist even after limite d expo sure to ethanol (Duran d et al, 1989). The additio n of ethanol a t any time point prior to 6 DIV does no t affec t neuro n survival . In contrast , addi tion of ethanol on Day 6 (after rapid growth of dendrites and synapses has begun) triggers cell death, despite th e shorter duratio n o f ethano l exposur e (Lindsle y e t al. , 2002). Developmenta l stage-dependen t sensitivit y to ethanol-induced cel l deat h ha s also been reporte d fo r cerebellar neuron s i n vivo an d i n vitro (Goodlet t an d Johnson, 1999 ) and may involve the nitric oxide-cyclic guanosine 5 /-nionophosphate-dependent protei n

ETHANOL AND PROCESS OUTGROWTH 23 3 kinase pathwa y (Bonthiu s e t al, 2004) . Elucidatin g the mechanisms b y which the timing of ethanol exposure influences the natur e and severit y of its effects o n dendrites and o n neurona l surviva l is a goal o f ongo ing research.

EFFECTS O F ETHANOL O N DEVELOPMENT O F AXONS

In Vivo Studies The effec t o f prenata l ethano l o n axona l growt h i n various brain regions is less well characterized than its effects o n dendrites . I t i s mos t ofte n reporte d a s a n overall hypertroph y o f axon s fro m projectio n neu rons, such a s those comprisin g the corticospina l trac t and th e moss y fiber tract i n th e hippocampus . Thi s hypertrophie effec t o f ethano l i s no t a s consistentl y observed i n axo n tract s that cros s the midlin e i n th e brain, such a s the corpu s callosum an d anterior com-

Axons of Projection Neurons Prenatal exposur e t o ethano l markedl y alter s th e number an d distributio n of axons of projection neu rons in various brain regions. I n mos t cases , change s in the numbe r of axons cannot be attributed to corresponding change s i n th e numbe r o f neurons i n th e brain regio n fro m whic h th e trac t extends . Fo r example, prenatal exposur e to ethanol increase s the num ber o f neuron s projectin g fro m ra t somatosensor y cortex t o the spina l cor d (Miller , 1987) , th e numbe r of axons in the cauda l pyramida l tract (Miller and Al Rabiai, 1994) , an d th e tota l axoplasmi c volum e i n layer V o f th e somatosensor y corte x (Al-Rabia i an d Miller, 1989) . Although the absolut e number of corticospinal neuron s i s no t affected , becaus e th e tota l number of neurons in layer V is reduced by one-third (Miller an d Potempa , 1990) , th e relativ e numbe r i s substantially increased . I t ha s bee n suggeste d tha t these exuberan t projections are persistent , immatur e connections an d ma y indicat e tha t ethano l inhibit s the prunin g of axons that normall y refines ove r time (Miller, 1987) . A hypertrophi e axona l projectio n ha s bee n ob served i n axon s extendin g fro m granule cell s i n th e dentate gym s (moss y fibers ) t o apica l dendrite s o f the pyramida l neuron s i n th e CA 3 regio n o f th e

hippocampus. Adul t rats prenatally exposed to ethano l have a conspicuous abnorma l ban d of infrapyramida l mossy fibers in hippocampal subfiel d CA3 a a t middl e and temporal levels (West et al., 1981). In addition, th e band o f suprapyamidal moss y fibers in the stratu m lucidum is disorganized. This exposure period coincide s with th e birt h o f pyramidal neurons i n CA3 , bu t i t is before mos t granule cell s or their axons appear. Therefore, it has been postulated that alterations in the terminal field induce hypertrophie growt h of mossy fibers. As with the corticospina l tract, moss y fibers in th e hippocampus underg o stereotype d prunin g o f axonal branches tha t sculpts the connection s int o the matur e pattern. Althoug h th e mechanism s controllin g moss y fiber axon pruning are not fully understood, recen t evidence points to a role for the repulsiv e guidance mole cule Sem a 3 F (Bagri et al., 2003). There are intriguing similarities between the abnorma l mossy fiber trajectories of prenatal ethanol-exposed rats and mice deficient in the axon guidance molecule semaphorin 3 F (Sahay et al. , 2003 ) o r i n member s o f th e holorecepto r fo r semaphorins, neuropilin- 2 (Che n e t al. , 2000 ) an d plexin A3 (Cheng et al., 2001). It is appealing to speculate that ethanol interferenc e with mossy fiber pruning is mediated by altered semaphorin signaling. Axons That Cross the Midline During brai n development , axon s fro m specifi c re gions within each cerebral hemispher e ar e guided by extracellular signal s t o gro w acros s th e midlin e t o synapse on neurons in the opposit e hemisphere . Th e largest axo n trac t crossin g th e midlin e i s the corpu s callosum. I t consist s of axon s projecting to th e con tralateral cortex , thereb y allowin g sensory and moto r integration betwee n th e lef t an d righ t side s o f th e brain. A higher tha n normal incidenc e o f corpus cal losum abnormalities has been reporte d for individuals with feta l alcoho l syndrom e (FAS ) (Rile y e t al , 1995). Magnetic resonanc e imagin g an d autops y stud ies i n human s expose d prenatall y t o ethano l sho w that ethano l decrease s the cross-sectiona l area of the corpus callosum, especiall y the posterio r region (sple nium), an d displace s i t relativ e to othe r brai n struc tures (e.g. , Clarre n e t al , 1978 ; Peife r e t al, 1979 ; Pratt an d Doshi , 1984 ; Schaefe r e t al , 1991 ; Rile y et al, 1995; Roebuck et al, 1998 ; Swayz e et al., 1997 ; Sowell e t al., 2001). In contrast, prenatal ethano l ca n increase th e siz e o f the corpu s callosu m i n childre n

234 ETHANOL-AFFECTE D DEVELOPMENT prenatally exposed t o ethanol (Bookstei n et al, 2002) and i n a nonhuma n primat e (Macaca nemestrina) (Miller et al, 1999) . Indeed, th e tota l numbe r o f axons i s increased. The mos t labile segment i s the ros tral portion (genii) , without affecting th e thicknes s of axons o r their myelin sheaths . The difference s i n th e primates ma y reflect concentration-dependent effect s of ethanol: a t lower exposures, ethanol leads to hypertrophic effects , wherea s a t highe r exposure s i t i s full y depressive. Result s in roden t model s ar e similarl y in consistent, with some studies showing ethanol-induced decreases in variou s measures of corpus callosum size (Chernoff, 1977 ; Zimmerber g an d Mickus , 1990 ; Moreland e t al. , 2002 ) and other s showin g no effec t (Livy and Elberger , 2001 ) or an increas e i n the num ber of callosal projection neurons (Miller, 1997). Noncallosal axo n tract s tha t cros s the midlin e are vulnerable t o disruption by ethanol. Prolonged expo sure to relatively high dose s of alcohol ca n reduc e th e size o f th e hippocampa l commissur e (Liv y an d El berger, 2001) . Th e midsagitta l are a o f th e anterio r commissure i s reduced b y prenatal ethanol exposur e in som e inbre d mic e (Cassell s et al., 1987) , however , this effec t ha s no t bee n observe d i n studie s o f rat s (Zimmerberg an d Mickus , 1990 ; Liv y and Elberger , 2001). Granat o e t al. (1995 ) reported reduce d termi nal axona l arbor s o f corticothalami c projections , i n which th e mos t sever e damage i s in th e selectiv e absence o f corticothalami c projection s tha t cros s th e midline, a characteristi c o f ra t corticothalami c sys tems that is not seen i n humans . Some o f thes e inconsistencie s ma y reflec t differ ences in species, strain differences i n the developmen tal sensitivit y of commissural axons, differences i n th e dose o r timin g o f ethano l administration , o r othe r méthodologie factors (Mille r et al, 1999). In any case, the susceptibilit y o f crossin g fiber s t o disruptio n b y ethanol has some important mechanistic implications because the midlin e is a uniqu e developmenta l field where the respons e of axons to attractive and repulsive cues i s finely regulated b y precise molecula r mecha nisms no w bein g identifie d (Tessier-Lavign e an d Goodman, 1996 ; Hube r et al, 2003). In Vitro Studies Partial t o complet e agenesi s o f the corpu s callosum , as seen i n FAS , is also common i n people wit h mutations in LI, the so-called CRAS H syndrom e (Franse n

et al , 1995 ; Bearer , 2001) . Thi s findin g le d t o th e hypothesis tha t disruptio n o f L I functio n ma y con tribute t o neuropathologi c effect s o f prenatal ethano l exposure (Charnes s e t al. , 1994) . L I i s a neura l cell adhesion molecul e expresse d o n axon s that facilitates axon growt h throug h bot h heterophili c an d ho mophilic binding (Brummendor f and Rathjen, 1996) . Some i n vitr o evidenc e is consistent with ethano l disruption o f Ll-mediated axon outgrowth fro m CN S neurons. Ra t cerebella r granule neuro n culture s extend neunte s whe n culture d o n substrate s coate d with LI . Ethano l inhibit s Ll-mediated outgrowt h o f the longes t neurit e forme d 1 2 hr afte r platin g (pre sumably the axon ) in thes e cultures , even a t concen trations a s lo w a s 3- 5 m M (Beare r e t al. , 1999) . Apparently, thi s i s no t du e t o inhibitio n o f Ll mediated adhesio n pe r se, because th e sam e effec t o f ethanol on axon growth is observed when the cell s are plated o n poly-lysine substrates and treate d wit h a soluble for m o f the extracellula r domain o f LI. This re sult wa s take n t o indicat e tha t ethano l disrupt s signaling downstream of LI tha t influences organization of the growth cone cytoskeleton. There i s growin g evidenc e fro m i n vitro experi ments tha t second messenge r cascade s an d cytoskele tal reorganizatio n are triggere d b y LI, eithe r directly or vi a it s interaction s wit h co-receptor s (Burden Gulley et al., 1997 ; Castellan i et al, 2000; Watanabe et al, 2004). Other subsets of axons in the developin g CNS expres s LI i n hig h amounts , includin g pyramidal trac t axon s (Jooste n an d Gribnau , 1989) , whic h are sensitiv e t o ethano l disruptio n durin g develop ment (se e Axon s o f Projectio n Neurons , above) . Thus, elucidating the mechanism s by which ethano l disrupts LI regulate d axo n growth i s an importan t focus o f future studies . Various studies have shown that LI i s but on e o f many cell adhesio n protein s affecte d by ethano l (Lu o an d Miller , 1999 ; Mille r an d Luo , 2002; Siegenthaler an d Miller , 2004). These protein s are regulate d b y factor s suc h a s transformin g growth factor ßl , whic h i s know n t o promot e neurit e out growth (Ishihar a et al., 1994) and i s a target of ethanol (e.g., Lu o an d Miller , 1999 ; Mille r an d Luo , 2002 ; Siegenthaler and Miller , 2004). Although studie s i n vivo have no t focuse d o n th e effects o f ethanol on early stages of process outgrowth, some i n vitro studies point to effects o f ethanol o n th e timing of initial axon outgrowt h (axo n specification) . In culture s o f hippocampa l pyramida l neurons , th e

ETHANOL AND PROCESS OUTGROWTH 23 5 proportion o f neuron s tha t reac h Stag e 2 (extende d neuntes, bu t befor e axo n specification ) an d th e proportion that reac h Stag e 3 (axon specification) are both increase d when ethanol is added to the medium, beginning shortl y after platin g (Clam p an d Lindsley , 1998). Subsequen t time-laps e studies , which includ e only cells that achieved Stag e 3 , show that ethanol de lays axonal outgrowth (Lindsley et al., 2003). Both observations indicate that the increas e in the proportion of cell s wit h undifferentiate d neunte s i s responsibl e for th e fina l increas e i n th e proportio n o f polarized cells at 1 DIV, and that ethanol ma y increase the likelihood of forming an axon even as it decreases the rate at which this occurs. Some caution is required when interpreting studies of ethanol effect s o n proces s outgrowt h tha t compar e neurite morpholog y a t a selecte d tim e afte r plating . Differences i n th e lengt h o f processes measure d a t a single tim e poin t d o no t tak e int o accoun t effect s o n the time it takes for a neuron to initiate axon extension and th e rat e of elongation onc e the axo n i s formed. A time cours e o f ethanol effect s durin g initial stage s of neurite outgrowth, or time-lapse analyse s on live cells, is needed to make this distinction. An eve n higher-resolution tim e cours e i s required to asses s th e effect s o f ethano l o n th e rat e o f axo n growth afte r a n axo n emerges . Thi s i s because onc e axons ar e formed , the y gro w i n a saltator y manne r characterized b y cyclin g betwee n period s o f growth (when length increases ) an d period s of relative quiescence ("non-growth" ; whe n shor t retractio n occurs ) (Fig. 14-1) . Molecula r an d pharmacologi e treat ments ca n selectivel y alter specific aspects of process outgrowth, suc h a s the relativ e duration o f periods of growth, the timin g of axon establishment, o r the mag nitude o f retraction durin g pauses (Esc h e t al., 1999 ; Ruthel an d Banker , 1999 ; She a an d Beerman , 1999 ; Walker et al, 2001; Lindsley et al., 2003). These findings sugges t tha t these feature s of process growt h dynamics ar e independentl y regulate d unde r norma l conditions an d coul d b e differentiall y sensitiv e t o ethanol. Time-lapse analysi s of living neuron s ha s demon strated saltator y axonal growt h i n dissociate d culture s (Katz et al., 1984; Aletta and Greene, 1988; Ruthe l and Banker, 1999 ; Lindsle y et al., 2003), tissue slices (Halloran an d Kalil , 1994) , an d th e intac t brain (Kaethner and Stuermer , 1992) . I n som e axons , suc h a s thos e comprising th e corpu s callosum , thi s ca n b e quit e

FIGURE 14- 1 Axona l growt h o f a hippocampa l py ramidal neuron . Phase-contras t image s o f a ra t hip pocampal pyramida l neuron , capture d a t variou s times postplatin g (show n i n hr). The dista l ti p o f the axon advance s an d retract s durin g elongation . Scal e bar= I S |im.

dramatic, with extensive lengths o f axon retracting or sprouting rapidl y (Den t e t al. , 1999) . Dendrite s als o undergo significan t retraction s durin g outgrowth an d remodeling, a s demonstrate d b y withdrawa l o f th e apical dendrit e fro m subset s of pyramidal neurons i n the developin g corte x (Koeste r an d O'Leary , 1992) . Aside from affectin g ne t growt h rate , saltator y growth is associate d with othe r importan t feature s o f neura l development, includin g branc h formatio n (Szebeny i et al. , 2001) , branc h growt h alternatio n (Futerma n and Banker , 1996) , an d growt h con e response s t o attractive an d repulsiv e guidance cue s (Son g an d Poo , 1999; Min g et al, 2002). Time-lapse analysi s of the effect s o f ethanol o n th e saltatory growt h dynamic s i n hippocampa l culture s shows that ethano l increase s th e overal l rat e o f axon elongation b y decreasin g th e exten t o f retraction s

236 ETHANOL-AFFECTE D DEVELOPMENT during pause s i n growth . Ethano l doe s no t increas e the rat e o f extension durin g growth spurts or the rela tive time axon s spend growin g and pausin g (Lindsley et al., 2003).

MECHANISMS UNDERLYING ETHANOL-INDUCED CHANGE S IN DENDRITI C AN D AXONAL MORPHOLOGY

As described above, in vivo and i n vitr o data indicat e that specifi c morphologi c feature s tha t mak e u p th e overall shape o f dendrites an d specifi c features of axonal growth dynamics may not be equall y affected b y ethanol. The y als o indicat e tha t th e morphoregula tory effects o f ethanol ma y be correlated wit h the ear liest stage s o f proces s outgrowth . Mor e studie s ar e needed t o identif y th e molecula r basi s fo r such spe cific vulnerabilit y t o ethanol-induce d damage . Ad vances i n understandin g ke y cellula r regulator s o f growth con e motilit y provid e ne w opportunitie s t o speculate o n ho w ethano l exposur e migh t modulat e morphogenesis o f axons an d dendrite s durin g devel opment. Tw o o f these emergin g idea s ar e discusse d below. Small Rho Guanosine Triphosphatases Although regulator y mechanism s underlyin g den dritic remodeling ar e not full y understood , the mole cules an d signal s involve d ar e though t t o influenc e the acti n an d microtubul e cytoskeleton, sinc e signifi cant cytoskeleta l rearrangemen t occurs . Th e Rh o family, smal l guanosin e triphosphatase s (GTPases) , Rac, Cdc42, and RhoA , ar e essentia l component s o f signaling pathways that transduce a variety of extracellular signal s (e.g. , neurotrophin s an d integrins ) int o morphological changes . Rh o guanosin e S'-triphos phatases (GTPases) d o this by regulating downstream kinases tha t alte r th e activit y o f acti n an d tubuli n binding protein s involve d i n actin-base d cytoskeleta l remodeling i n growt h cone s (Lu o e t al. , 1997 ; Mackay an d Hall , 1998 ; Luo , 2000 ; Redmon d an d Ghosh, 2001; Lundquist, 2003). Different member s o f Rho-famil y GTPase s regu late distinct features of dendritic development, including initia l outgrowth, rat e o f growth , numbe r o f primary dendrites, and extent of branching (Redmond

and Ghosh , 2001). For example, results of experimentally altere d Rac l o r Cdc4 2 expressio n i n cortica l neurons indicate s tha t thes e protein s ar e importan t for dendritic branching and remodeling, but that they have littl e effec t o n dendriti c growt h rat e pe r s e (Threadgill e t al, 1997 ; L i et al, 2000) . I n contrast , overexpression o r activatio n o f Rho A inhibit s den dritic growt h an d underexpressio n o r inactivatio n o f RhoA lead s t o a n increas e i n dendriti c lengt h (Le e et al, 2000; Li et al, 2000). Studies focuse d o n the cellula r mechanism s regu lating extension and retractio n behavior of axons during growth an d i n respons e t o guidance factor s (e.g., semaphorins) als o poin t t o Rh o famil y GTPase s an d their downstrea m effector s (Lu o e t al. , 1997 ; Küh n et al, 2000; Luo , 2000 ; Redmon d an d Ghosh, 2001). In general , proces s extensio n i s correlate d wit h in creased formation of lamellipodia and filopodia at the leading edg e o f the growt h cone , which require s acti vation o f Cdc42 an d Racl , wherea s retraction i s correlated wit h increase d Rho A signalin g (Hall , 1998 ; Luo, 2000) . Two studies show that ethano l ca n dramaticall y rearrange F-acti n structure s in some non-neurona l cells and that activation of small Rho GTPases may mediate these effects. Ethano l induce s actin reorganization and shape chang e i n astrocyte s in vitro, and i t ha s bee n suggested tha t these effect s ar e mediated b y decreased RhoA activity, since they could b e reverte d by expression o f constitutivel y activ e Rho A (Guasc h e t al. , 2003). In the endothelial cell line, SVEC4-10, ethano l induces actin reorganization and motility. Since thes e effects ca n be blocked b y expression of dominant neg ative Cdc42, it has been suggested tha t they are medi ated b y activation of Cdc42 (Qia n e t al, 2003) . O n the othe r hand , th e effec t o f ethanol o n F-actin distri bution i n the growt h cones of developing neurons has not been reported . Som e preliminar y data are consistent wit h altere d F-acti n organizatio n i n axona l growth cones of hippocampal neurons maintained for 1 DIVwith ethanol (Sha h and Lindsley, unpublished observations). Figur e 14- 2 contain s example s o f confocal image s showing F-actin ric h structures in axonal growth cone s o f contro l an d ethanol-treate d hip pocampal neuron s 1 DIV tha t ar e fixed and staine d with phalloidin-Alex a 568 . Continuou s exposur e t o low doses of ethanol induce s extensive elaboration of lamellipodia an d numerou s thickene d filopodi a i n axonal growt h cones , an d hig h dose s onl y increas e

ETHANOL AND PROCESS OUTGROWTH 237

FIGURE 14- 2 Effec t o f ethanol o n F-acti n distribution i n growth cones . Repre sentative confoca l image s o f F-acti n distributio n i n axona l growt h cone s o f 1-day-old hippocampa l neuron s culture d fo r 1 da y i n vitr o withou t o r wit h ethanol (concentration s shown) . Images were captured a t 0.20 \im intervals using 8-image jump averaging. Each imag e i s the lowes t section fro m th e z-serie s stack. The lo w dose o f ethanol increase d the exten t of lamellipodia an d filopodia compare d t o controls , an d th e hig h dos e increase d filopodi a only . Scal e bar= 5.0 Jim.

filopodia formation . Moreover , th e involvemen t o f small GTPase s i n reorganizatio n o f growt h con e actin i n neuron s expose d t o ethano l i s indicate d by pull-dow n experiment s showin g change s i n Rac l activation i n whole-cel l lysate s o f hippocampa l cultures expose d t o ethano l fo r 1 da y durin g axo n elongation. Important advance s i n understandin g th e effect s of ethano l o n growth-relate d signalin g i n axon s an d dendrites ca n b e expected , give n th e availabilit y o f appropriate mode l system s amenabl e t o measurin g the effect s o f expressio n o f relevan t dominant negative or constitutively active mutant Rh o GTPases on ethanol-induce d disruptio n o f morphogenesis . Interestingly, man y o f the signalin g pathway s known to contro l growt h con e extension-retractio n an d at traction-repulsion behavior s appea r t o b e differen tially regulate d i n axon s an d dendrites . Som e evidence suggest s that Rh o GTPase s ar e differentiall y distributed i n growt h cone s (Renaudi n e t al. , 1999) , and experimenta l manipulations t o over - or underexpress Rho-famil y protein s hav e differentia l effect s o n the number and length o f dendrites compared to their effects o n axona l growth (Threadgil l e t al., 1997 ; Lee et al., 2000 ; Luo, 2000). Thus, if Rho-family GTPase modulation i s indeed involve d in th e morphoregula tory effect s o f ethanol, the n differentia l effect s o f th e various Rho-famil y member s o n axona l vs . dendritic effects ma y help explain the various effects o f ethanol on these distinct process types.

If ethanol-induce d change s i n growt h dynamic s and/or cytoskeleta l reorganizatio n d o indee d involv e altered GTPas e signaling , the n on e coul d as k what upstream modulator s o f Rho GTPases are particularly important i n morphoregulator y effect s o f ethanol. I n NIH 3T 3 cells , growt h factor-induce d Rac l activa tion i s sensitive to protein kinase C (PKC ) inhibition , placing PK C upstrea m o f Ra c i n tha t syste m (Buchanan e t al. , 2000) . Th e relationshi p betwee n Rho GTPas e signalin g and PKC s i s potentially quit e interesting. I n P C 12 cells, exposur e t o 25-20 0 mM ethanol fo r 2-8 day s stimulates neurite growth via upregulation o f PKC e (Messin g e t al , 1991 ; Hundle étal, 1997) . What rol e migh t downstrea m effector s o f Rho GT Pases have i n mediatin g th e effect s o f ethanol o n neuronal proces s growth ? Amon g a numbe r o f know n effectors, severa l are of particular interest. In hippocam pal neuron cultures , axon specification is linked to activation o f Cdc42 and Ra c 1 in a positive feedbac k loo p involving a highly localized comple x o f mPar3/mPar6, PKC, an d phosphoinositid e 3 kinas e (PI3K) , whic h modulates actin and microtubule cytoskeletal dynamics in th e growt h con e o f the nascen t axo n (Sh i et al. , 2003). It is not yet clear, however, whether PI3 K initiates signal s that activat e Racl/Cdc42 downstrea m o r whether Racl/Cdc4 2 are upstream signal s for PI 3K in this cascade . Othe r know n downstrea m effector s o f potential interes t includ e acti n cytoskeleton-regulating proteins, suc h a s ezrin , radixin , an d moesin , an d

238 ETHANOL-AFFECTE D DEVELOPMEN T regulators of mitogen-activated protein kinase pathways (Hall, 1998 ; Redmon d an d Ghosh, 2001). Some insigh t int o th e regulatio n o f neurit e out growth may come from studie s of neuronal migration , as commo n protein s ar e involve d i n bot h processe s (see Chapters 3 and 13) . Reduced F-acti n at the lead ing edg e o f migratin g neuron s i s associate d wit h modulation of RhoA, Racl, and Cdc42 activity (Kholmanskikh e t al., 2003) . Since abnorma l neurona l mi gration i s a ke y neuropathologi c featur e o f prenata l alcohol exposur e (Miller , 1986 , 1993 ; Hira i e t al , 1999; Moone y et al, 2004 ; Siegenthale r an d Miller , 2004), a n understandin g o f the effect s o f ethanol o n Rho GTPas e signaling in neuron s may shed ligh t on cellular mechanisms contributin g to prenatal ethanol induced neuronal migration errors. Intracellular Calcium Results o f numerou s studie s o f th e molecula r path ways linkin g activatio n o f specifi c Rho-famil y pro teins and thei r effector s poin t t o involvement of Ca2+ influx (Küh n e t al , 1998) . Thu s i t i s likely tha t ef fects o f ethano l o n dynami c growt h con e behavio r might ultimatel y b e linke d mechanisticall y throug h Rho-family effector s actin g o n th e growt h con e cytoskeleton, eithe r directl y o r indirectly , t o Ca 2+ sig naling. It ha s lon g bee n appreciate d tha t intracellula r Ca2+ i s a critica l secon d messenge r tha t modulate s growth con e behavior durin g neuronal developmen t by communicatin g growth-relate d signal s to th e cy toskeleton an d vesicula r apparatu s o f th e growt h cone (Kate r an d Mills , 1991 ; Letournea u an d Cypher, 1991 ; Neel y an d Nicholls , 1995) . Ethano l can alte r the functio n and expressio n of L-type Ca 2+ channels i n neura l cells (Walter and Messing , 1999) . The effect s o f ethanol o n Ca 2+ signalin g i n neuron s have focused primarily on mature neurons, i n which Ca2+ channels an d receptor s figur e importantl y int o cellular adaptation s t o ethano l an d t o withdrawal induced neurona l damag e (Fadd a an d Rossetti , 1998). Ethano l affect s Ca 2+ homeostasi s i n matur e neurons b y alterin g th e expressio n o r functio n o f voltage-dependent Ca 2+ channel s (VDCCs) , iono tropic receptors , and Ca 2+-induced Ca 2+ release fro m intracellular stores (Leslie et al., 1990 ; Bergamasch i et al., 1993) . Unfortunately, far fewer studie s hav e pur sued th e ide a tha t ethano l disrupt s Ca 2+ signalin g

regulating neuronal development. Th e potentia l importance o f this distinction i s suggested b y evidenc e that th e effec t o f ethano l o n VDCC s ma y no t b e constant durin g neurona l differentiatio n (Berga mashi et al, 1995 ; Webb e t al., 1997) . The "set-poin t hypothesis " propose s tha t proces s growth occur s withi n a narro w range o f intracellular Ca2+ concentratio n [Ca 2+]j, an d tha t sustaine d in creases o r decrease s i n [Ca 2"1"^ lea d t o growt h con e collapse and/o r cel l damage (Kate r and Mills , 1991) . In contrast , oscillation s o r transien t increase s i n [Ca2+]j are associated with signaling induced b y effec tor pathways , similar to those though t t o be involve d in effect s o f ethanol o n proces s growth. Some o f these transient Ca 2+ signals are highly localized. Fo r example, growt h cone s generat e transien t increase s i n [Ca2+]j as they migrat e i n viv o (Gome z an d Spitzer , 1999) an d i n vitro (Gome z e t al , 1995 ; Ciccolin i et al, 2003 ; Tang e t al, 2003) . The frequencie s and amplitudes o f these [Ca 2+]j transients are inversely related t o th e rat e o f axo n elongatio n an d increas e several-fold whe n axon s pause durin g periods o f nongrowth. Thes e transient s ar e likel y t o alte r growt h cone extension b y reorganizing the cytoskeleton , per haps b y disassemblin g local acti n an d microtubul e networks. I n cortica l neurons , thes e transient s ar e spontaneous an d Ca 2+-dependent an d ac t primaril y through L-typ e VDCCs (Tang et al, 2003). Signaling to the cytoskeleto n appears , at least in part, to involv e the phosphatase calcineri n (Lautermilc h and Spitzer , 2000). The result s o f time-series , confoca l imagin g o f Ca2+ flux in axon s o f hippocampal neuron s expose d to ethano l unde r condition s tha t alte r axona l growth dynamics hav e bee n recentl y reporte d (Gilli s an d Lindsley, 2004) . I n thes e studies , th e frequenc y an d kinetics o f spontaneou s [Ca 2+]j transient s i n axona l growth cone s o f hippocampa l neuron s a t 1 DIV i n control mediu m wer e compared t o thos e o f neuron s maintained i n mediu m t o whic h ethano l (22 , 43, or 87 mM) was added a t the time of plating. Figure 14- 3 illustrates typica l time-series image s o f axonal growt h cones from contro l and ethanol-expose d cultures, and corresponding plot s o f relative fluorescenc e intensit y in th e subscribe d regio n o f th e growt h con e ove r a 10 min recordin g period . A s describe d b y Gome z and colleague s (1995) , a transient i s define d b y a n increase in [Ca 2+]i of >200% of baseline, slow kinetics (~20 sec to peak), and retur n t o baseline.

ETHANOL AND PROCESS OUTGROWTH 23 9

FIGURE 14- 3 Time-serie s confoca l image s of axona l growt h cones . The growth cone s o f 1-day-ol d neuron s wer e loade d wit h Fluo-3 . A growth con e from a contro l cultur e (to p row ) and one fro m a cultur e expose d to 87 mM ethanol (botto m row ) are shown . Fo r each growt h con e tw o images from th e time serie s are presented, on e depictin g baselin e fluorescenc e (left) , an d th e other, peak fluorescenc e durin g a spontaneous calcium transient (right). Peak calcium elevation s are image d i n white. The plot s on th e righ t show the rela tive fluorescenc e intensit y as a percent o f baseline for the growt h cones i n th e panels (left). Scal e bar =5.0 iim.

Results of recording more than 10 0 neurons fro m 10 cultures showed that only 18 % of axons in contro l cultures exhibite d one o r more transient s during the 10 min recordin g period . Exposur e t o lo w dose s o f ethanol reduce d th e numbe r o f neurons with one o r more transients to 8% (p < 0.005), but exposur e to th e highest dos e o f ethano l increase d th e proportio n o f growth cone s wit h transient s t o 39 % (p < 0.005) . Ethanol, at any concentration tested , had no effect o n the peak amplitude, time to peak, or total duration of the transients . Thes e finding s sugges t tha t ethano l modulates th e frequenc y of spontaneou s Ca 2+ tran sients i n axona l growt h cones . I t i s yet t o b e deter mined whethe r dendriti c growth dynamics and Ca 2+ transients ar e similarl y altere d b y ethanol . I n hip pocampal cultures , a globa l increas e i n [Ca 2+]j ca n inhibit dendritic growth but promotes or has no effec t on axona l growt h (Mill s an d Kater , 1990 ; Mattso n et al, 1988 ; Mattso n an d Kater , 1987) . Thi s finding indicates potentia l difference s i n Ca 2+-dependent, growth-related signalin g in axon s and dendrites . Th e modulation o f transient Ca2+ flux in the growt h cone could b e a n importan t cellula r target of ethanol tha t mediates it s differentia l effect s o n axon s an d den drites, considerin g the sensitivit y o f growth cone s t o elevated fre e Ca 2+ and th e numbe r o f Ca2+-activated

proteins expresse d differentiall y b y axon s an d den drites.

SUMMARY AND CONCLUSIONS

Descriptive studie s detailin g specifi c morphologica l features o f neuronal development that are sensitive to disruption by ethanol in a variety of neuronal cell cultures are essential in laying the groundwork for identifying cellula r mechanism s underlyin g th e effect s o f ethanol o n neurona l morphogenesis . Man y ethanol sensitive developmenta l phenomen a discovere d s o far—the altere d growt h dynamic s an d stage-specifi c effects o f ethanol — could no t hav e been so readily derived fro m fixed sections taken fro m animal s expose d to ethanol i n utero. Neuronal development in vivo involves a diversity of interactions between neuron s and their environmen t tha t experimenta l condition s i n vitro cannot fully reproduce . Therefore, a n importan t goal for the futur e shoul d be to test specific mechanis tic hypothese s arisin g from cel l cultur e experiment s in mode l system s tha t preserv e th e endogenou s spatiotemporal expressio n o f extracellula r growth regulating molecules. Such model systems might provide opportunitie s t o manipulat e th e expressio n o f

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15 Neuronal Survival Is Compromised by Ethanol: Extracellular Mediator s Michael W. Miller Maria B. Bruns Paula L. Hoffma n

A salient characteristi c o f fetal alcoho l spectru m dis order (FASD ) is microcephaly an d th e associate d mi croencephaly (Lemoin e e t al. , 1968 ; Jone s an d Smith, 1973 ; Stratton et al. , 1996 ; se e Chapter s 1 , 8, and 9). Major contributors to this microencephaly are ethanol-induced reductions i n the additive ontogenetic processe s (cel l proliferatio n an d neurona l migration) and increase s in the amoun t o f neuronal death. The effect s o f ethano l o n th e additiv e processe s ar e addressed i n Chapter s 11 , 12 , an d 13 . The presen t and followin g chapte r (Chapte r 16 ) describe th e ef fects o f ethano l o n neurona l deat h and , conversely, neuronal survival . This chapter deal s with the effect s of ethano l o n th e incidenc e o f neurona l deat h an d the extracellular mediators that define this death, and the next chapter covers intracellular mechanisms that underlie this death. Neuronal deat h i s a natura l proces s i n th e devel oping nervous system (Jacobson, 1991; Martin , 2001; Lossi an d Merighi , 2003). It affect s proliferatin g an d postmitotic, postmigrator y neuron s (se e Chapte r 5) .

The amoun t o f neuronal deat h i n the nervous system can var y from 20 % to 80% . Factors that contribut e t o the amount of death ar e the location and the maturational stat e of the vulnerabl e neurons. One exampl e is the cerebra l cortex , in which the incidenc e o f neuronal deat h i s lamina- an d neuro n type-dependen t (Miller, 1995a) . Two- to threefol d mor e neuron s die in superficia l laye r VI (o r laye r Via) than i n laye r IV and local circuit neurons (which, in part, differentiate on a later tim e line ) ar e twic e as likely to di e a s projection neurons in the same layer.

245

EFFECTS O F ETHANOL O N BRAIN STRUCTURE S I N VIVO

In viv o studie s sho w tha t ethano l affect s the spa tiotemporal patter n of neuronal deat h throughout th e nervous system. Most studies have focused on the outcome, i.e. , th e effec t o f ethano l o n tota l neurona l number. Fe w have teased ou t the effect s o n neurona l

246 ETHANOL-AFFECTE D DEVELOPMEN T death fro m th e effect s o f ethano l o n othe r develop mental events . The presen t review describes the limitations an d advance s i n ou r understandin g o f suc h effects o n fou r structures : th e principa l sensor y nu cleus of the trigeminal nerve (PSN), cerebellum, hip pocampal formation , and neocortex . Eac h structur e has a differen t developmenta l histor y that provide s a distinctive platfor m fo r understandin g th e mecha nism of ethanol toxicity. Principal Sensor y Nucleus of the Trigeminal Nerv e Documentation o f neuronal death in vivo has consisted of two challenges: difficulties i n identifyin g dyin g neurons an d distinguishin g the effect s o f ethano l o n cel l proliferation from thos e on neuronal death. The PS N is an idea l structure for separating these issue s because i t is a small, pontine nucleus that lends itself to quantitative studies . Th e PS N i s largel y surrounded b y fibe r bundles, facilitatin g th e delineatio n o f border s an d comprises a relatively homogeneous population of neurons (Miller and Muller , 1989) . Furthermore , th e rol e of the PS N i n neurona l developmen t implicate s i t in FASD because it is a key site for the passag e of information fro m somatosensor y afférents arisin g i n facia l ski n to highe r centers— a primar y characteristi c o f FAS D is craniofacia l malformation s (Lemoine e t aL , 1968 ; Smith and Jones, 1973; Sulik et al., 1988 ; Johnston and Bronsky, 1995; Astley etal, 1999) . The PS N i n th e norma l ra t has 28,00 0 neurons . Following prenatal exposure to ethanol , the nucleu s has onl y 19,60 0 neuron s (Mille r and Muller , 1989) . This exposur e occur s durin g th e period s whe n PS N neurons proliferate and migrat e (Nornes and Morita , 1979; Miller and Muller, 1989 ; Al-Ghoul and Miller, 1993a). A longitudinal study of the tempora l effect s of prenatal exposure to ethanol showed that one-third of this differenc e i s a laten t effec t o n neurona l surviva l (Miller, 1999 ) (Fig . 15-1) . Thi s findin g wa s docu mented b y two changes in the neonate: (1) a decreas e in th e tota l numbe r o f neurons an d (2 ) increase s in the numbe r o f pyknotic and silver-staine d cells. PS N neurons als o di e whe n exposur e occur s durin g th e periods o f primar y synapse formation an d naturall y occurring neuronal death (NOND) (Miller , 1995b) — i.e., postnatall y before postnatal da y (P ) 1 0 (Ashwell and Waite, 1991 ; Al-Ghoul and Miller, 1993b ; Miller and Al-Ghoul , 1993) . I n contrast , ethano l exposur e during adolescenc e doe s no t hav e a significant effect

on neurona l number (Miller, 1995b) . Thus, the criti cal window for ethanol-induced neuronal death i n th e PSN i s framed by the period s o f neuronal generatio n and synaptogenesis. A prim e afferen t o f th e PS N i s th e infraorbita l nerve. A neonatal lesio n of this nerve causes massive neuronal death, confine d to the ventral portion o f the PSN wher e th e infraorbita l nerv e terminates (Miller et al., 1991) . Exposure to ethanol doubles the amoun t of thi s frans-synapti c deat h (Miller , 1999) . Further more, thi s death is rapid, as the expressio n of Fos (a marker o f cellular activity) an d ALZ-5 0 ( a marke r of dying neurons) increases within 2 hr of placing the lesion (Miller and Kühn , 1997) . Cerebellum The cerebellu m i s susceptibl e t o ethanol-induce d damage in a dose-dependent manner ; ethanol-exposed rats hav e smalle r cerebell a tha n control s (Dia z an d Samson, 1981 ; Wes t e t al, 1989) . A key factor con tributing t o th e degre e o f damage i s the pea k bloo d ethanol concentratio n (BEG ; Pierce an d West, 1986 ; Bonthitis et al., 1988) . Ra t pups expose d t o the sam e amount o f ethano l dail y d o no t hav e th e sam e amount of damage if the schedul e of ethanol delivery differs. Exposure s t o relativel y large bol i o f ethano l are significantl y mor e deleterious than a constant de livery of low doses of ethanol. The stud y of neuron number s i n th e cerebellu m has centere d o n Purkinj e neuron s (Goodlet t an d Horn, 2001 ; Light et al, 2002) . Adult rats exposed to ethanol durin g the perio d fro m P 4 to P10 have signif icantly fewe r neuron s tha n controls . Th e amoun t o f Purkinje cel l loss can var y from 10 % to 70 % depend ing on the developmental history and cerebellar compartment (Fig . 15-2) . Fo r example, lobule s I-V , IX, and X are mor e vulnerabl e to ethano l exposur e than lobules V I an d VI I (Pierc e e t al. , 1989 ; Goodlet t et al., 1990) . Th e developmen t o f neurons i n lobule s I, II , IX, and X precedes that o f neurons i n th e inter vening lobules VI and VII (Miale and Sidnian , 1961 ; Altman and Das , 1966 ; Altaian and Bayer , 1978). Exposure to ethanol during the restricted period of P4 or P5 cause s a s muc h o f a differenc e a s does exposure between P4 and P1 0 (Light et al, 2002). This effec t is concentration-dependent. Thus , exposur e t o a hig h BEG, eve n fo r a shor t time, ca n resul t i n th e los s of vulnerable neuron s i n th e cerebellu m (Wes t e t al. , 1989; Miller, 1996b ; Thomas et al, 1996) .

EXTRACELLULAR MEDIATOR S OF NEURONA L DEAT H 24 7

FIGURE 15- 1 Neurona l deat h i n th e developin g principa l sensor y nucleus o f the trigemina l nerv e (PSN). A. The number s o f neurons i n the PS N rises prenatally an d fall s durin g th e firs t 2 postnatal week s (left) . Thi s decreas e coin cides wit h a n increas e i n th e frequenc y o f pyknoti c cell s (right) . Prenata l exposure to ethanol delays the period o f neuronal death . B . Presumptive dyin g neurons (arrows ) can be identified in a silver-stained section fro m th e pon s of a 3-day-old rat . They are distributed throughou t th e PSN . C . Mor e argyrophili c neurons are evident in the PS N o f an ethanol-treate d 3-day-ol d pup. D . An argyrophilic cel l (arrow ) ha s a dens e cor e an d ofte n i s surrounded b y granules. These are presumed t o be blebs and release d nucleosomes . E. A pyknotic cell is characterized b y a condensed nucleu s tha t ofte n is broken int o small bodies . Scale bar s = 10 0 ujn ( B and C ) an d 1 0 Jim ( D an d E) . (Source: Dat a i n A are taken with permission fro m Miller , 1999. )

Ethanol-induced difference s i n cel l numbe r ma y result from reduction s i n neuronal number and/o r increases i n neurona l death . Th e cerebell a o f rat s ex posed to ethanol durin g gestation alone o r during the pre- and earl y postnatal period weigh less and have no fewer Purkinj e neurons tha n rat s expose d durin g th e postnatal perio d alon e (Wes t e t al. , 1994 ; Miller ,

1996b; Light e t al, 2002) . These dat a hav e bee n in terpreted a s evidenc e tha t ethano l doe s no t affec t neuronal generation . Thi s ma y i n par t b e true , a s ethanol ma y not affect th e generation of Purkinje cell s which occur s prenatall y (Mial e an d Sidman , 1961 ; Altman an d Bayer , 1978) . Th e possibilit y stil l exists , however, tha t ethano l doe s reduc e th e generatio n o f

248 ETHANOL-AFFECTE D DEVELOPMEN T

FIGURE 15- 2 Effec t o f ethano l exposur e o n number s o f Purkinj e neurons . Rats were exposed t o ethanol b y gavage on postnata l da y 4. Th e number s of Purkinje neuron s in various cerebellar lobule s were determined. Th e dat a for two pairs of lobules ar e presented: lobule s I and IX , which ar e earl y developing lobules, and lobules VI and VII, which develo p relativel y late. Each poin t describes th e chang e i n the numbe r o f Purkinje neurons relativ e to the num ber identified in controls on P4. No significant changes were detected for latedeveloping lobules . Bot h o f th e early-developin g lobules , however , wer e vulnerable to ethanol toxicity. Significant differences ( p < 0.05; noted b y asterisks) were detected onl y 24 hr o r 1 0 days after exposure . (Source: Dat a take n from Ligh t and colleagues , 2002. )

granule neurons produced postnatally, which ca n secondarily lead to the degeneration o f Purkinje neuron s dependent o n thei r afférent s (Reza i an d Yoon , 1972 ; Herrup and Trenkner, 1987) . Recent evidence, from studie s of restricted ethano l exposure, supports the notio n tha t ethanol cause s th e death of Purkinje neurons. It is unlikely that a 1-da y exposure to ethanol affect s granul e cel l generatio n s o profoundly an d rapidl y a s t o caus e th e short-ter m death o f Purkinje neurons . Moreover, various studies show that ethanol increase s th e expressio n o f two indices of apoptotic deat h b y Purkinje neurons (Ligh t et al., 2002)—th e expressio n of activate d caspase-3 , a n enzyme involved in the clippin g of DNA into packets during apoptoti c death , an d evidenc e o f termina l uridylated nick-en d labelin g (TUNEL) , a n inde x of DNA fragments . Moreover, exposur e t o ethano l o n P4 lead s t o a n increas e i n whol e cerebella r catalas e and reactiv e oxygen species (Horn , personal comm.) .

Activated caspase- 3 expressio n is markedly increased in man y Purkinj e cell s an d i n a smatterin g o f small cells in the interna l granul e cel l layer. The sensitivity of th e granul e neuron s pe r s e t o ethanol-induce d death remain s a question , a s does th e mechanis m o f ethanol-induced los s of Purkinje neuron s (West et al., 1994; Goodlet t and Horn , 2001) . Suffice i t to say that ethanol ca n compromis e neurona l survival . Intracel lular event s underlyin g thi s deat h ar e reviewe d i n Chapters 6 an d 16 . Th e molecula r mechanis m o f Purkinje cel l deat h deserve s mor e detaile d analysis, given recen t evidence that Purkinj e neurons ma y die by a proces s o f autophag y followin g remova l o f trophic support (Florez-McClure e t al., 2004). Hippocampal Formatio n The hippocampa l formatio n i s a comple x structur e containing a t leas t tw o majo r subdivisions , th e

EXTRACELLULAR MEDIATOR S OF NEURONA L DEATH 24 9 hippocampus an d th e dentat e gyms . Th e axon s of granule cells in the dentat e gyru s synapse with pyramidal neuron s i n th e hippocampus . Thes e neurona l populations develo p differently . Fo r example , hip pocampal pyramida l neuron s ar e generate d prena tally i n th e ventricula r zone , wherea s mos t (85% ) granule neuron s ar e generate d postnatall y in th e in trahilar zon e (Schlessinge r e t al. , 1978 ; Nowakowski andRakic, 1981). Prenatal an d postnata l exposur e t o ethano l ha s differential effect s o n th e number s o f cells i n th e hip pocampal formation . Th e amoun t o f DNA i n the hip pocampal formation is not affecte d followin g prenatal exposure; however, postnatal exposur e leads to an in crease in DNA content (Miller, 1996a) . Although thi s result suggest s tha t ethano l doe s no t affec t neurona l survival, these biochemical measure s do not discriminate betwee n neuron s an d gli a o r differentiat e th e specific effect s o f ethano l o n th e hippocampu s o r dentate gyrus. Careful anatomica l studies have shown that prenatal exposure to ethanol reduce s the numbe r of neurons in th e hippocampu s (Miller , 1995c) . I t is likely tha t som e o f this reductio n result s fro m a de crease i n cel l proliferation . Earl y postnata l exposur e does no t affec t neurona l numbe r unles s th e ethano l exposure i s high (BE G >30 0 mg/dl; Miller , 1995c) . Thus, i t appear s tha t ethano l doe s no t induc e th e death o f neuron s i n th e hippocampa l formatio n be yond that occurring naturally. Neocortex In Vivo Studies Cortex contrast s wit h th e cerebellu m an d hippocam pus i n tha t th e generatio n o f it s neuronal subpopula tions, suc h a s projectio n an d loca l circui t neurons , occurs concurrently (Miller, 1985) . Likewise, the periods of NOND and synaptogenesi s are largel y synchronized for all cortical neurons (Rakic et al., 1986; Miller, 1988). In these aspects, neocortex is more like the PS N than the cerebellum and hippocampal formation . Pre- an d postnata l exposure s to ethano l induc e a difference i n the numbe r o f cortical cells . Overall , total DN A conten t i s 29 % lowe r i n th e cortice s o f ethanol-treated rat s tha n tha t o f control s (Miller , 1996a). If the same mechanisms are at work in the de veloping corte x a s i n th e PS N (Miller , 1999) , one third o f the differenc e cause d b y prenatal exposure to ethanol i s due t o laten t death . Prenata l exposur e t o

ethanol cause s a -33 % differenc e i n th e numbe r o f neurons in somatosensory cortex (Miller and Potempa , 1990) and n o differenc e i n the numbe r o f neurons i n visual cortex (Moone y e t al., 1996 ; Mooney and Nap per, 2005). Apparently, not all parts of the pallium are equally affecte d b y ethanol . Postnata l exposur e t o ethanol als o cause s a significan t differenc e i n DN A content, althoug h thi s difference i s only 13 % (Miller, 1996b), a differenc e expecte d t o have equal effec t o n neurons an d gli a (Mille r and Potempa , 1990) . Inter estingly, the amoun t o f neuronal deat h (estimate d a t 29% x 1/ 3 = 10% ) caused b y exposur e during the pe riod o f neuronogenesis (betwee n gestationa l da y [G ] 11 and G2 1 ) is similar to that caused by exposure during early synaptogenesis (betwee n P 4 and P10). Does this mea n tha t ethano l cause s an equivalen t amount of cel l deat h regardles s of the tim e o f exposure, i.e., that ther e i s n o critica l perio d fo r ethano l toxicity ? No. Exposur e to ethanol afte r th e en d o f the period s of NON D an d synaptogenesi s doe s no t affec t th e number o f cortica l neuron s (Miller , unpublishe d results). Changes i n cel l numbe r ar e parallele d b y alter ations i n th e expressio n o f protein s associate d wit h neuronal death . Thes e includ e ALZ-5 0 (Küh n an d Miller, 1998) , be l proteins , an d activate d caspase- 3 (Mooney an d Miller , 2000) . Th e expressio n o f eac h of these markers rises during the first and second post natal weeks . Ethano l significantl y affect s eac h marker, mostl y by delayin g the perio d o f expression (Kühn and Miller, 1996; Moone y and Miller, 200la). A prim e sourc e o f cortical afférent s an d targe t of cortical efferent s i s the thalamus . I t is noteworthy that although ethanol-induce d neurona l deat h occur s i n the neocortex , apparentl y n o such deat h occur s in at least some thalamic nuclei (Liv y et al., 2001), includ ing th e ventrobasa l thalamu s (VB ; Moone y an d Miller, 1999a) . No t onl y i s the numbe r o f VB neu rons the sam e i n matur e ethanol-treate d an d contro l rats, but ther e appear s to be n o differenc e i n the tem poral chang e i n number s durin g th e period s o f NOND an d synaptogenesi s (Moone y an d Miller , 200 Ib). Thi s phenomeno n i s particularly interesting in that the differential effec t o f ethanol sets up a potential mismatc h i n thalamocortica l afférent s an d thei r target an d wit h corticothalami c afférent s an d thei r target. A series of studies b y Olne y an d colleague s (e.g. , Ikonomidou et al., 1999 , 2000) describe the effect s of early postnata l exposur e t o ethano l o n th e deat h o f

250 ETHANOL-AFFECTE D DEVELOPMEN T cortical cells . Th e author s clai m tha t earl y develop mental exposur e t o ethano l cause s massiv e neurona l death whe n th e exposur e selectivel y occur s durin g synaptogenesis. I n cortex , thi s proces s begin s o n P 3 and peak s afte r P3 0 (Miller , 1988) . Allegedly , tran sient exposur e t o ethano l cause s a wav e o f neurona l death tha t i s initiall y greates t i n dee p lamina e an d progresses superficially . Thei r wor k suggests tha t th e vast majority of cortical neurons di e as a consequenc e of earl y postnata l exposur e t o ethanol . Thes e dat a are i n conflic t with a wealt h o f data (som e o f whic h are reference d above ) an d mus t b e interprete d wit h caution. As th e dat a o f Olne y an d colleague s hav e re ceived considerabl e attention, the y deserve comment. The dat a ar e base d o n a silver-stainin g method (d e Olmos an d Ingram , 1971 ; Friedma n an d Price , 1986) that purportedly identifie s dyin g neurons. I t is difficult t o assess the applicabilit y of this method be cause section s fro m th e brain s o f contro l animal s have virtuall y n o label . Thi s lac k o f labelin g i s disturbing becaus e th e consensu s i s that th e firs t post natal wee k i s par t o f th e perio d o f NON D i n th e normal ra t brai n (e.g. , Finla y an d Slattery , 1983 ; Ferrer e t al, 1990 ; Miller , 1995a ; Spreafic o e t al, 1995). Th e dat a fro m th e ethanol-treate d rat s ar e equally disturbing . Massiv e number s o f neuron s i n the cortice s o f ethanol-treate d rat s ar e argyrophili c (Ikonomidou e t al. , 1999) . Recen t evidenc e show s that neuron s tha t exhibi t a "marker " o f cel l deat h (e.g., TUNEL o r caspase immunoreactivity ) ar e no t obliged t o di e (Gordo n e t al. , 2002 ; Cheng and Zo chodne, 2003 ; Ishid a e t al, 2004 ; Oomma n e t al. , 2004). Furthermore , i f al l o f th e cortica l neuron s that exhibi t ethanol-induce d silve r stainin g indee d do die , the n earl y postnata l exposur e t o ethano l would caus e a collaps e o f the cerebra l wall . Careful studies show, however, that less than one-sixt h o f all cortical neuron s di e a s a resul t o f ethano l exposur e (Miller, 1996a ; Moone y e t al , 1996 ; Moone y an d Napper, 2005). According to Olney an d colleagues , th e ravage s of ethanol are not confined to cerebral cortex. For example, variou s thalamic nucle i als o exhibi t widesprea d neuronal degeneratio n followin g earl y postnata l ex posure t o ethanol. I t appears that most neuron s in th e anterior nucle i an d ventra l thalamus o f 8-day-old rat s are argyrophilic . Studie s o f mature ra t thalami , how ever, sho w tha t rat s expose d t o ethano l pre - o r post natally hav e robus t anterio r nucle i an d stereologica l

data sho w that ethano l doe s no t affec t th e numbe r o f neurons i n thalami c nucle i (Moone y an d Miller , 1999a;Livy et al, 2001,2002). Finally, Olne y an d colleague s conclude , incor rectly, that the tempora l window of ethanol toxicit y is restricted t o the perio d o f synaptogenesis. Indeed , exposure t o ethano l durin g thes e ontogeneti c event s can caus e neurona l deat h durin g the perio d o f synaptogenesis (Miller , 1995b , 1999 ; Küh n an d Miller , 1998; Moone y an d Miller , 1999b). Prenatal exposur e to ethanol ca n als o cause neuronal deat h (se e above). Furthermore, ethano l ha s profoun d effect s o n cel l proliferation (se e Chapters 1 1 an d 12 ) and neurona l migration (se e Chapter 13). Cell Culture Studies Cultured cortica l neuron s obtaine d fro m 17-day-ol d fetuses exhibi t a 25 % los s ove r th e firs t 2 day s post plating (Fig . 15-3 ) (Seabol d e t al. , 1998 ; Jacob s an d Miller, 2001 ; Mille r e t al. , 2003 ; Ramachandra n et al. , 2003) . This los s is equivalent to th e amoun t o f natural deat h occurrin g amon g post-migrator} ' neu rons i n corte x i n vivo (Finlay and Slattery , 1983 ; Fer rer e t al., 1990 ; Miller , 1995a ; Spreafic o et al, 1995) . Ethanol exposur e cause s a n additiona l 25 % loss . In terpretation o f thes e dat a i s confounde d becaus e a t least som e o f th e neuron s i n feta l cortica l culture s have th e abilit y t o proliferat e (Jacob s an d Miller , 1999). Thi s confoundin g variabl e ca n b e addresse d by applyin g a mathematica l mode l tha t determine s the tota l amount s o f cel l proliferatio n an d deat h (Miller, 2003) . A particular strength o f this mode l i s that i t does not rel y on a method fo r identifying dyin g cells, which ca n bia s the results . Accordingly, ethano l causes a 30 % differenc e i n th e numbe r o f culture d cortical neuron s or B104 neuroblastoma cells. Of this difference, two-third s resul t fro m decrease s i n cel l proliferation an d one-thir d fro m ethanol-induce d cel l loss. Thus, the i n vitro studies of cortical neurons con cur wit h the i n vivo data.

SURVIVAL-PROMOTING FACTORS

Evidence tha t neurona l deat h occur s durin g the pe riod o f synaptogenesi s support s th e hypothesi s tha t neuronal deat h i s driven b y neuron s failin g t o com pete fo r a n entit y ( a growt h facto r and/o r synapti c sites) i n limite d supply . Among th e factor s tha t hav e

EXTRACELLULAR MEDIATOR S O F NEURONA L DEAT H 25 1

FIGURE 15- 3 Effec t o f ethano l o n neurotrophin supported neurona l survival . Cortical neuron s derive d from 17-day-ol d fetuse s wer e culture d i n th e presenc e of a neurotrophi n ( 0 o r 1 0 ng/ml) an d ethano l ( 0 o r 400 mg/dl). Over 3 days postplating, one-quarter o f the untreated cortical neurons were lost. Nerve growth factor (NGF ) prevente d thi s loss , wherea s brain-derived neurotrophic facto r (BDNF ) an d neurotrophi n 3

(NT-3) di d not . Ethano l exacerbate d th e "natural " death observe d i n untreate d cultures . Ethano l als o eliminated th e abilit y o f NG F t o maintai n viability . Asterisks an d poun d sign s denot e significan t differ ences relativ e t o the untreate d control s o n da y 0 an d day 3, respectively. A single sign identifies data that are different a t a level oïp < 0.05 and a double sig n designates difference s a t the p < 0.01 level o f significance.

most commonl y bee n implicate d a s promotin g neuronal surviva l ar e nerv e growt h facto r (NGF) , brain-derived neurotrophi c facto r (BDNF) , insulin like growt h factor- 1 (ÏGF-1) , N-methyl-D-aspartat e (NMDA), an d pituitar y adenyly l cyclas e activatin g polypeptide (PACAP).

associated wit h trkA or trkB in certai n populations . A low-affinity receptor , p75, may also bind t o any of the neurotrophins. Thi s recepto r ma y act i n cooperatio n with o r independently o f the trks. The tyrosin e kinase associated with the high-affinit y trks can activat e a t least thre e differen t signalin g path ways to support differentiation , proces s outgrowth, cell survival, an d activity-dependen t plasticit y (Bibel an d Barde, 2000). The p7 5 receptor ha s no kinase activity associated wit h i t per se , but i t is now known t o hav e signaling capability . The mechanism s o f p7 5 signal ing are far less characterized. Depending o n th e extracellula r environmen t an d the compositio n an d abundanc e o f receptors o n th e membrane surface , neurotrophin s ca n hav e benefi cial o r deleteriou s effect s o n neurons . Th e trk s an d p75 for m homo - an d heterodimer s wit h eac h other , and thes e dynamic complexe s ca n cooperat e to exer t varied effect s (Chao , 2003 ; Huan g an d Reichardt , 2003). Receptor interaction s can als o be antagonistic, depending on which ligand s are available. The expressio n o f neurotrophin s change s i n a time-dependent manne r durin g development . Tha t is, certai n population s o f neuron s ar e dependen t o n

Neurotrophins Neurotrophins ar e secreted , diffusibl e signalin g pro teins know n t o exer t neuromodulator y effect s o n adaptive processes such as synaptic plasticity, required for learnin g and memory , and neurona l survival. This family o f protein s act s i n a n autocrin e an d antero grade fashio n simila r t o tha t o f neurotransmitter s (Pitts and Miller , 1995 , 2000; Mille r and Pitts , 2000; Neet an d Campenot , 2001) . A numbe r o f neu rotrophic factor s hav e bee n identified , includin g NGF, BDNF, neurotrophi n (NT)3 , and NT-4/5, each of which supports distinct populations of neurons. To perform thei r functions, the neurotrophin s bind t o tyrosine kinase receptors (trks) . NGF bind s to trkA with high affinity , wherea s BDN F an d NT-4/ 5 bin d t o trkB. NT-3 binds primarily to trkC, bu t ha s also bee n

252 ETHANOL-AFFECTE D DEVELOPMEN T one neurotrophi n fo r a perio d o f time. Then , afte r a critical period , thes e neuron s becom e dependen t o n another neurotrophin(s) . Fo r example, Purkinj e cell s switch from BDN F dependence t o NT-3 dependenc e between P 4 an d P 6 (Davies , 1997) . Coincidentally , this i s the perio d o f highest vulnerabilit y o f Purkinj e cells t o ethano l (Ligh t e t al. , 2002 ; se e above) . Ethanol neurotoxicity may be mediated by changes in neurotrophin systems. The distributio n o f neurotrophi n ligand s i n vari ous brain tissues during norma l developmen t i s critical t o appropriat e signa l transductio n fo r survival . Alterations in the abundanc e o f the neurotrophi n ligands and receptor s ma y result after ethano l exposure . In othe r words , withdrawa l fro m o r a n inadequat e supply o f neurotrophins ma y underlie decrease d cel l survival. Conversely , ethano l ma y down-regulat e re ceptor expressio n below a threshold require d t o initiate signals, interfere wit h ligand-receptor binding , or render neurotrophi n receptor s nonfunctional . Mos t likely, severa l mechanisms ar e a t play , depending, o f course, on the populatio n o f neurons an d the tim e of ethanol exposure . The differentia l effect s o f ethano l on distinc t populations of neurons ca n offe r valuabl e insight t o th e norma l interaction s o f neurotrophin s during development . Nerve Growth Factor NGF prevent s NOND—i.e. , i t i s an anti-thanatopi c factor (e.g. , Majda n an d Miller , 1999 ; Bibe l an d Barde, 2000 ; Morriso n e t al, 2002) . This i s particularly evident among cultures of cortical cells in which NGF uniquel y support s cel l surviva l (Fig . 15-3 ) (Seabold e t al., 1998 ; Mille r e t al, 2003) . NGF ca n also affec t othe r developmenta l phenomena , notabl y the cel l cycl e and th e surviva l of cells that exit the cycling population (Lopez-Sanche z an d Frade , 2002). Ethanol ca n interfer e wit h NGF-mediate d func tion bot h i n vitr o an d i n vivo . I t disrupt s th e NGF mediated surviva l o f primar y culture d cortica l neurons (Seabol d e t al., 1998 ; Mille r e t al, 2003). A neuroprotective rol e o f NGF i s supported b y studies of NG F over-expressio n i n mic e (Heato n e t al , 2000b). Earl y postnata l exposur e t o ethano l kill s fewer Purkinj e cell s i n thes e mic e tha n i n wild-type controls. Altered NGF abundanc e coul d hav e patho logical consequences, fo r example, NGF ca n potenti ate fre e radical-induce d apoptosi s i n vitro (Spea r etal, 1998) .

The effect s o f gestationa l ethano l exposur e o n NGF expressio n hav e als o bee n examine d i n vivo. The amoun t of NGF protei n was measured at PI an d P10 afte r gestationa l ethano l exposure . NG F i s in creased i n the corte x and striatum of ethanol expose d animals a t PI , bu t doe s no t diffe r fro m control s a t P10. No other neurotrophin s are affected i n any brain region a t P I (Heato n e t al. , 2000e) . Expressio n o f NGF protei n i s als o increase d i n th e hippocampa l formation transientl y a t PI5 afte r gestationa l ethano l exposure (Angelucci etal., 1997) . In additio n t o affectin g neurotrophi n ligan d ex pression, ethano l ca n alte r recepto r expression . Ethanol-induced cel l deat h i n vitro i s associated with a selective decreas e i n p75 expression; the expressio n of al l tr k isoform s i s unaffecte d b y ethano l (Seabol d et al. , 1998) . Thi s pattern reverberate s in othe r cells , including septohippocampa l neuron s (Angelucc i et al. , 1997 ) an d P C 12 pheochromocytom a cell s (Luo e t al., 1996 , 1999) . In cerebellar Purkinj e cells, following earl y postnatal ethano l exposure , there i s a significant decreas e i n trk A and p7 5 recepto r expres sion f? 7 vivo, despit e ther e bein g n o chang e i n th e amount o f NGF (Dohrma n e t al , 1997) . Selectiv e down-regulation o f th e neurotrophi n receptor s b y ethanol durin g th e earl y postnata l developmen t pe riod i s fatal t o Purkinje cell s (Light et al, 2002). Th e differential effec t o f ethanol o n receptor s ma y reflec t the distributio n of these receptors in the plasma mem brane. Conceivably , p7 5 i s distribute d i n lipi d raft s (Higuchi et al., 2003), the fluidit y o f which i s affecte d by ethanol (People s e t al., 1996) , whereas trks are relatively protected fro m ethano l becaus e they appear no t to be dispersed within rafts (Muto h et al., 2004). In addition , gli a offe r a critica l suppl y o f neu rotrophins. Unde r norma l homeostati c conditions , glia d o no t expres s NGF, however , they ma y elevat e NGF unde r pathological conditions (Pitts and Miller, 1995; Miller and Pitts , 2000). This glial production of neurotrophins ma y be an attempt a t neuroprotection . For example , astrocyte s increas e NG F expressio n after ethano l exposur e in vitro (Vallès et al. , 1994) . C6 glioma cell s secret e increase d amount s o f NGF an d BDNF, quantitie s tha t ar e sufficien t t o protec t cere bellar granul e neuron s fro m ethano l insul t (Pantazi s et al., 2000). Antibodies specific t o neurotrophins and trk receptors block this ethanol-induced neuroprotec tion o f the granul e cells . Interestingly, trkA receptor s are no t localized i n cerebellar granule cells, and the y express onl y low amounts o f p75. The implicatio n i s

EXTRACELLULAR MEDIATOR S OF NEURONA L DEATH 25 3

that NG F mus t b e actin g through a promiscuous trk isoform. A downstream effec t o f ethanol toxicity in cortica l cultures i s the quashin g o f the expressio n o f a gen e up-regulated b y NGF , ne g (nerv e growt h factor stimulated, ethanol-depresse d gene ) (Mille r e t al, , 2003). The functio n of the gene produc t is unknown, although i t appear s tha t Ne g i s involve d i n NGF mediated cel l survival . The gen e product ha s protein domains suggestin g tha t i t i s a niitochondria l trans membrane protei n (Nagas e et al., 1996) . This finding is intriguin g in ligh t o f evidence tha t ethano l affect s other niitochondria l protein s tha t regulat e cel l sur vival, fo r example , bcl- 2 an d ba x (Moone y an d Miller, ZOOla) (see Chapter 16) . Brain-Derived Neurotrophic Factor BDNF promote s cel l surviva l i n viv o an d i n vitro. During critica l period s o f norma l development , BDNF i s necessar y fo r bot h neurit e outgrowt h an d synaptic pruning . Developin g neurona l processe s compete fo r a limite d suppl y o f BDNF . Thos e neu rons that ar e unabl e t o obtai n a sufficient amoun t o f BDNF o r tha t hav e insufficien t trk B receptor s un dergo cell death . Like NGF, BDN F i s also thought t o play a neuroprotective rol e i n respons e t o ethano l exposure . In deed, BDN F ma y pla y a rol e i n th e tissue - an d age-dependent component s of ethanol-induced neu ronal cel l death . Th e striatu m i s a good example . I n the normal striatum, NOND is more commo n o n P3 than P1 4 (Heato n e t al., 2003) . This sequence i s the inverse o f the patter n o f endogenous BDN F expres sion; striata l BDN F conten t i s highe r o n P1 4 (Fig. 15-3) . Earl y postnata l exposur e to ethano l increases striatal BDNF content (Heato n e t al, 2000e). Cerebellar Purkinj e cell s revea l a contrastin g situa tion: Purkinj e cell s decrease BDN F expressio n afte r early postnata l ethano l exposur e (G e e t al. , 2004) . Within hour s o f ethano l exposure , there i s also de creased expressio n o f cerebella r trkB . Afte r P9 , ethanol exposur e doe s no t affec t BDN F o r trk B expression. Compromised , time-dependen t BDN F support i n cerebellu m ma y underli e hig h rate s o f Purkinje cel l apoptosi s durin g it s perio d o f ethano l sensitivity (P4 to P5). Manipulation o f availabl e BDN F suppor t i n vivo can furthe r demonstrat e neurotrophi n system-specifi c vulnerability t o ethanol . Transgeni c mic e lackin g

BDNF expressio n in the cerebellu m sho w significant losses o f Purkinj e cell s afte r earl y postnata l ethano l exposure (Heato n e t al. , 2002) . There ar e als o con current decreases i n anti-apoptotic factor s (e.g. , bcl-2) in these mice , suggestin g that BDN F signals mediate neuronal survival . The contrastin g results observed in cerebellum an d striatu m illustrat e tha t ethano l ca n exert tissue-specific effects dependen t o n BDNF 1 regulation. In cortica l tissue , the effect s o f ethanol o n BDN F and trk B expression are also specific to the time of exposure. Earl y postnata l exposur e cause s significan t increases i n neocortica l BDN F abundance , simila r to change s observe d i n hippocampu s an d striatu m (Heaton e t al. , 2000e) . I n contrast , gestationa l expo sure result s i n significan t decrease s i n BDN F an d trkB recepto r expressio n i n cortex , whic h correlat e with significant increase s in apoptosis of cortical neu rons (Climent e t al., 2002). Both of these observations support th e hypothesi s tha t a sufficien t amoun t o f BDNF mus t be present to promote cel l surviva l in response t o ethanol . Insulin-Like Growth Factor- 1 IGF-1 i s another survival-promotin g factor tha t i s target of ethanol (Zhan g e t al, 1998) . Activatio n of the IGF-1 recepto r ca n inhibi t cel l deat h i n vitr o (W u et al, 1996 ) an d in vivo (Resnicoff et al., 1995) . An in vivo model i n which transplante d tumo r cell s overex pressed IGF- 1 receptor s show s that IGF- 1 play s a major protectiv e role . I n contrast , a decreas e i n IGF- 1 receptor expression results in heavy amounts of apoptosis i n vivo. The cytotoxi c effects ar e mor e dramati c than when tumor cell s are manipulated i n vitro. Like th e neurotrophins , IGF- 1 expressio n fluctu ates durin g development . Norma l change s o f IGF- 1 receptor distributio n indicat e tha t differen t popula tions of cells have different period s of IGF-1 depend ence durin g the postnata l period o f brain maturation (Breese e t al. , 1991) . Norma l neonate s expres s th e greatest abundanc e o f IGF- 1 protei n a t birth ; IGF lcontentfalls61%byP20. Gestational ethano l exposur e alter s IGF- 1 abundance an d distributio n durin g postnata l brai n development (Brees e e t al. , 1994) . I n offsprin g o f ethanol-exposed mothers, the decrease in IGF-1 expression i s attenuated, fallin g onl y by 25 % during the firs t 20 day s afte r birth . Localizatio n o f IGF-1 an d IGF- 1 receptors, however , i s not affected . Thus , ethanol ma y

254 ETHANOL-AFFECTE D DEVELOPMEN T disrupt th e regulatio n o f IGF-1 availabilit y (i.e., pro duction an d secretion ) rathe r than th e ligand-receptor signals. Ethanol ca n revers e th e anti-apoptoti c effect s o f IGF-1 recepto r signalin g (Resnicof f e t al , 1996) . Fi broblasts lacking IGF-1 receptor s are completely insensitive to ethanol-induced cell death in vitro. Cerebellar granule neurons can normally be rescued from apopto sis i n th e presenc e o f IGF- 1 (D'Mell o e t al. , 1993) . Ethanol ca n inhibi t this neuroprotectio n eve n whe n excess IGF-1 i s available (Zhang et al., 1998) . The amoun t o f IGF- 1 i n materna l plasm a i s reduced afte r ethano l exposur e (Brees e an d Sonntag , 1995). Insufficien t IGF- 1 ligan d o r receptor s ca n cause massiv e amounts o f apoptosis. Thus, the conse quences o f insufficient plasm a IGF- 1 o n metabolis m and hormon e physiolog y ma y contribut e t o bod y growth deficiencie s tha t compoun d th e pathologica l microcephalic brain development associated with FAS. The vulnerabilit y of an individua l IGF-1 recepto r to ethanol ca n var y in that the affinit y o f the recepto r for it s ligan d ma y b e altered . Th e enzym e activit y of the IGF- 1 recepto r ca n als o be disrupted . Several subclones o f 3T 3 cell s generate d t o expres s equa l amounts of IGF-1 recepto r demonstrate d tha t no t all IGF-1 receptor s are created equal (Seiler et al., 2000). IGF-1 receptor s in some subclones resisted the effect s of ethanol; ethano l di d no t inhibi t the autophospho rylation of the IGF- 1 receptor . Like the tissue-specific distribution o f neurotrophi n receptor s an d variabl e expression of trk isoforms, relative abundance of IGF1 receptor s an d thei r variabl e resistanc e t o ethano l may explain the differentia l effect s o f ethanol o n various cell populations . N-Methyl-D-Aspartate Glutamate i s a majo r excitator y neurotransmitte r i n the mammalia n nervous system. Its actions ar e medi ated b y interactio n wit h bot h metabotropi c an d ionotropic receptors (Hollman and Heinemann, 1994 ; Ozawa et al., 1998) . The NMD A subtype of ionotropic glutamate receptor plays an importan t role in synaptic plasticity (e.g., learning and memory) , but hyperactivation of NMDA receptors can lead to excitotoxic, primarily necrotic, death of mature neurons (Choi, 1992). During neurona l development , NMD A recepto r function i s necessary for the activity-dependen t organization of synapses (Collingridge and Lester , 1989; Mc Donald and Johnston, 1990). In addition, activation of

NMDA receptors can hav e a trophic o r protective effect on developing neurons. This protective effect ha s been mos t extensivel y studied wit h cultured cerebel lar granul e neurons. Cerebellar granul e neurons iso lated fro m 7-day-ol d rat s requir e a growt h mediu m containing a depolarizing concentration of K+ (25 mM KC1) fo r surviva l (Gall o e t al , 1987 ; Balâz s e t al , 1988). Th e dependenc e o f cel l surviva l o n depolar ization develop s ove r a narro w time frame . Thi s pe riod include s the tim e when the glutamatergic mossy fibers innervat e postmitoti c granul e neurons . Treat ment of cerebellar granul e neuron s wit h NMDA also can protect thes e postmitotic neuron s fro m cel l death when they are grown in medium containin g a physiological concentratio n o f K + ( 5 mM KC1 ) (Balâz s et al, 1988 ; Burgoyn e e t al, 1993 ; Bhav e an d Hoff man, 1997) . Thi s neuroprotectiv e actio n o f NMD A apparently mimic s th e effec t o f innervatio n o f th e granule neurons by mossy fiber afférents i n vivo. Two model s o f culture d cerebella r granul e cell s have been use d t o explor e the neuroprotectiv e prop erty of NMDA. In th e first model, neuron s from post natal rat s ar e culture d i n a mediu m containin g a physiological concentration o f KC1 (see above). These cells underg o spontaneou s death . I n th e secon d model, th e cell s ar e grow n i n mediu m containin g 25 mM KC 1 an d afte r severa l day s i n culture , th e medium i s replaced wit h mediu m containin g a lo w KC1 concentration and ofte n i s serum-free (Yan et al , 1994). Unde r bot h o f thes e conditions , neuron s di e over a period o f 1 or 2 days, and ca n b e protecte d b y the additio n o f NMDA (Ya n et al , 1994 ; Bhav e an d Hoffman, 1997 ) o r polyaniine s (Harad a an d Sugi moto, 1997) . Polyaniine s modulat e th e functio n o f NMDA receptors (Dod d e t al, 2000 ) and act in part through a n NMD A receptor-dependent mechanis m (Harada an d Sugimoto , 1997) . I n bot h instances , NMDA ha s a neuroprotectiv e effec t o n cerebella r granule neurons, however , the molecular mechanis m of cel l deat h differ s i n th e tw o model s (Kharlamo v et al, 1995 ; Bhave et al, 1999 ; Bonn i et al, 1999) . It i s well-established tha t ethano l acutel y inhibits NMDA recepto r function . Thi s actio n ha s bee n demonstrated i n viv o an d i n vitro , and i n recombi nant systems where the NMD A recepto r i s expressed in heterologou s cell s (Hoffman , 2003) . These find ings led to the proposa l that ethanol ca n promote th e death o f cerebella r granul e neuron s b y interferin g with th e protectiv e effec t o f NMDA . Interestingly , ethanol no t onl y reduce s th e protectiv e effec t o f

EXTRACELLULAR MEDIATOR S O F NEURONA L DEAT H 25 5 NMDA o n cerebella r granul e neuron s grow n i n th e presence o f a lo w concentratio n o f K + bu t als o re duces th e toxi c effec t o f NMDA o n cerebella r gran ule neuron s grow n i n a hig h concentratio n o f K + (Wegelius an d Korpi , 1995) . Neuron s grow n i n lo w K+-containing medium ar e thought t o have the char acteristics o f immatur e neurons , thu s cerebella r granule neurons grown under these condition s represent a mode l i n whic h th e effect s o f ethano l an d NMDA o n neurona l surviva l durin g developmen t can b e assesse d in a mechanistic manner . Cerebellar granul e neuron s grow n i n lo w K +, o r subjected t o remova l o f high KC1 , exhibi t apoptoti c features, suc h a s DN A fragmentation , nuclea r con densation, somati c blebbing , an d caspas e activation (Yan e t al. , 1994 ; Bhav e an d Hoffman , 1997 ; Bhav e et al. , 1999) . Th e deat h o f these neuron s i n cultur e appears to mimic the apoptosi s observed in the developing ra t cerebellu m i n vivo betwee n P 5 an d P l l (Wood e t al., 1993) . Man y factors , such a s activation of variou s caspases , th e proteasom e an d accumula tion o f ubiquinated proteins, mitochondrial function, Bel-family proteins , and oxidativ e stress , hav e bee n implicated i n apoptoti c cel l los s (Atabay et al., 1996 ; Alavez e t al, 2000 ; Can u e t al, 2000 ; Vogel , 2002 ; Konishi an d Bonni , 2003) . Treatment o f cerebella r granule neuron s wit h NMD A prevent s thi s apoptoti c death (Ya n et al. , 1994 ; Bhav e an d Hoffman , 1997 ; Bhave et al, 1999) . Ethanol promote s apoptosi s b y interferin g wit h the protectiv e effec t o f NMDA (Bhav e and Hoffman , 1997). Neurons grown in low KCl-containing medium for 4 days prior to addition o f ethanol and/or NMD A for 2 4 h r experienc e th e sam e amoun t o f apoptoti c death a s neurons treated wit h ethanol alone . Ethano l also inhibit s th e neuroprotectiv e effec t o f NMD A when cerebella r granul e neuron s ar e switche d fro m high- t o low-KC l mediu m (Castold i e t al , 1998) . NMDA protect s cerebella r granul e neuron s fro m apoptosis b y increasin g th e expressio n o f BDNF , which i n tur n activate s phosphatidylinosito l 3 phosphokinase (PI3K ) an d Ak t phosphorylatio n (Bhave e t al., 1999) . Ethano l inhibit s th e first step of this pathway , th e NMDA-stimulate d increas e i n BDNF expression , whic h i s mediate d b y NMDA induced increases in intracellular calcium (Zafra e t al., 1990, 1991 ; Ghosh et al, 1994). This action of ethanol is specific , i.e. , ethano l doe s no t affec t late r step s i n the neuroprotectiv e pathwa y (Bhav e e t al. , 1999) . Thus, ethano l apparentl y doe s no t interfer e wit h th e

protective effec t o f BDN F (Hoffma n e t al , 1994 ; Seabold e t al., 1998) . The sit e of action o f ethanol a t the NMD A recepto r i s further supporte d b y studies of cerebellar granul e neuron s showin g tha t th e in hibitory effec t o f ethano l o n NMD A recepto r func tion (measure d a s a n increas e i n intracellula r calcium) an d tha t o n th e protectiv e rol e o f NMD A are both attenuate d i n the presenc e of a high concen tration o f glycine, the co-agonis t at the NMD A recep tor (Bhave and Hoffman , 1997) . Although ethano l acutel y inhibit s NMD A recep tor function , chroni c exposur e of mature neuron s t o ethanol up-regulate s NMDA recepto r functio n (Hoff man, 2003) . This change , i n vivo, is thought t o con tribute t o ethano l withdrawa l hyperexcitabilit y an d the brai n damag e associate d wit h long-ter m ethano l ingestion. By contrast, chroni c treatmen t o f culture d "immature" cerebella r granul e neuron s (grow n i n 5.0 mM KC1) with ethanol decreases the neuroprotec tive effec t o f NMDA , simila r t o th e effec t o f short term ethano l treatmen t (Bhav e e t al. , 2000) . Th e effect o f chronic ( 3 day) exposure to ethano l persists even afte r ethano l i s removed (Castold i e t al. , 1998 ; Bhave et al., 2000), attributed to decreased expressio n of NMD A recepto r subunit s b y th e ethanol-treate d "immature" neurons , resultin g i n decrease d NMD A receptor-mediated stimulatio n o f BDN F expressio n (Bhave e t al. , 2000) . Similarly , chroni c prenata l ethanol treatmen t i n vivo reduce s th e expressio n of NMDA recepto r subunit s i n th e offsprin g (Hughe s etal, 1998). The ag e o f th e culture d cerebella r granul e neu rons defines the respons e to ethanol an d NMDA . Fo r example, ethano l exposur e induce s th e deat h o f cerebellar granul e neuron s harveste d o n P1 0 an d treate d with ethano l (5 0 o r 20 0 mM) fo r 1 da y (Pantazi s et al., 1995) . This death ca n b e prevente d b y adding NMDA t o the cultures (Pantazis et al, 1995) . In contrast, i f cell s ar e maintaine d i n cultur e (5. 0 mM o r 25 m M KC1 ) fo r 7 days and the n expose d t o ethano l for 2 4 hr, there i s no evidence o f increased cel l death . The underlyin g mechanism o f thi s phenomeno n i s unknown. I t mus t b e considere d tha t th e ethanol induced deat h o f neurons o n th e first day in cultur e could reflec t interferenc e with cellula r adhesio n t o the cultur e dishe s and thu s b e a n artifac t o f the cul ture system . Nevertheless, unde r condition s i n whic h ethanol alon e produce s neuronal death , a number of anti-apoptotic agent s (e.g. , NMDA , neurotrophins , and PACAP ) ca n preven t ethanol-induce d deat h

256 ETHANOL-AFFECTE D DEVELOPMENT (Pantazis e t al , 1995 ; Beato n e t al. , 1999 ; 2000a , ZOOOb; de la Monte et al, 2000; Vaudry et al, 2002). Whether thes e result s ar e indicativ e o f th e mecha nism b y which ethano l itsel f induces neurona l deat h or reflec t algebraicall y additive effects o f ethanol an d protective agents has yet to be determined. Suc h studies of the tempora l vulnerabilit y of cultured cerebel lar granul e neuron s t o ethanol-induce d deat h ar e reminiscent o f the i n vivo studies demonstrating windows o f vulnerabilit y of th e cerebellu m t o ethanol induced neurona l los s durin g developmen t i n viv o (e.g., Hamre and West, 1993) . An i n viv o interactio n o f ethanol an d th e NMD A receptor wit h respect t o neuronal deat h i s not wel l established. Studie s reporte d b y Olne y an d colleague s (2002b) suggest such an interaction. They found tha t a single treatmen t wit h ethano l produce s apoptoti c de generation i n mous e o r ra t brain (Ikonomido u e t al. , 2000). On th e basi s of patterns o f neurodegeneration and comparison s with th e effect s o f NMDA recepto r antagonists, the y propose d tha t th e pro-apoptoti c ac tion o f ethano l i s mediate d throug h NMD A (an d yaminobutyric aci d A ) receptor s (Ikonomido u e t al , 1999, 2000 ; Olne y e t al, 2002a) . In th e cerebellum , the fact that cerebellar granule neurons undergo apoptosis at a specifi c tim e durin g developmen t (Woo d et al., 1993 ) an d th e possibilit y that this apoptosis i s a consequence o f the lac k o f innervatio n b y the gluta matergic mossy fiber afférents (Balâz s et al., 1988) sug gest tha t inhibitio n o f NMD A recepto r functio n b y ethanol a t thi s critical perio d o f development i n vivo would resul t i n inappropriat e cel l loss . Although th e mechanism o f cell deat h propose d b y Olney an d col leagues i s consistent with results from th e i n vitro studies, th e caveat s raised earlie r in thi s chapter , tha t th e massive amoun t o f cell deat h reporte d b y Olney an d colleagues is not consistent with studies of the numbe r of neurons i n adul t animals treate d pre - or postnatally with ethanol, cas t doubt on the interpretatio n o f the in vivo studies. Pituitary Adenylyl Cyclas e Activating Polypeptid e Activation of the transcription factor cycli c adenosine monophosphate (cAMP ) respons e elemen t bindin g protein (CREB ) ma y b e a downstrea m mediato r o f stimulus-dependent neurona l surviva l (Fig . 15-4 ) (Shaywitz an d Greenberg , 1999 ; Finkbeiner , 2000 ; Walton an d Dragunow , 2000) . Persisten t phosphory -

lation of CREB is considered necessary to increase the expression o f gene s neede d fo r cel l survival . Therefore, activation of several signal transduction pathway s that contribut e t o CRE B phosphorylatio n ma y pro vide optima l condition s fo r survival . On e suc h path way i s th e cAMP/protein kinas e A (PKA ) pathway . Activation o f the intracellula r messenge r cAM P ca n increase neurona l proliferation , differentiation , an d survival (Tojim a e t al , 2003 ; Fujiok a e t al , 2004) . Many studie s o f th e rol e o f th e cAMP/PKA/CRE B pathway in neuronal death hav e used cerebellar granule neuron s a s a model. Raisin g cAMP concentratio n in cerebellar granule neurons in many (D'Mello et al., 1993; Chan g e t al, 1996 ; Kienle n Campar d e t al , 1997; Mora n e t al. , 1999 ; L i e t al. , 2000 ) bu t no t al l (Yan et al., 1995 ) instances has prevented apoptosis . PACAP, a cAMP generator, i s a neuropeptide originally isolate d fro m a n ovin e hypothalami c extrac t (Miyata e t al. , 1989) . Thi s peptid e exist s in tw o isoforms (PACAP-2 7 an d PACAP-38 ) tha t ar e member s of th e vasoactiv e intestina l polypeptid e (VIP) / secretin/glucagon family . Bot h isoform s o f PACA P have simila r biochemica l effects . The y interac t wit h high-affinity PAC j receptor s (whic h hav e hig h spe cific affinit y fo r PACAP) and wit h two forms o f VPAC receptors (whic h hav e equa l affinit y fo r PACA P an d VIP; Harma r e t al. , 1998) . Bot h PACA P an d severa l splice variant s of the PAC j recepto r (Spengle r e t al, 1993) ar e found i n th e cerebellu m an d expresse d b y cultured cerebella r granul e neuron s (Basill e e t al. , 1995; Favit et al, 1995 ; Tabuchi et al, 2001) . In these neurons, activation of the PAC j receptor leads both t o cAMP accumulatio n an d polyphosphoinositid e hy drolysis (Basill e et al, 1995 ; Favit et al, 1995) . Treatment o f cerebella r granul e neuron s wit h PACA P attenuates apoptosi s produced eithe r b y growing th e neurons in low KCl-containing medium or by switching them from a high to low KCl-containing mediu m (Cavallaro et al, 1996 ; Campar d e t al, 1997 ; Villalba et al , 1997 ; Bhav e an d Hoffman , 2004) . PACA P i s likely an endogenou s facto r tha t promotes cerebella r granule neuron surviva l because treatmen t of granule neurons grow n i n a hig h KCl-containin g mediu m with a PACA P recepto r antagonis t reduce s neurona l survival (Tabuchi etal, 2001). The mechanis m b y whic h PACA P promote s cerebellar granul e neuro n surviva l ha s bee n evalu ated in severa l studies. Th e cAMP/PK A pathway an d the mitogen-activate d protei n kinas e (MAPK ) path way (Villalb a e t al , 1997 ) hav e bee n implicate d

Casuases

FIGURE 15- 4 Neuroprotectiv e pathways in cerebellar granule neurons. Cerebel lar granul e neuron s fro m postnata l rats , culture d i n mediu m containin g 5 mM KC1, undergo spontaneou s apoptosis . These neuron s ca n b e protecte d b y addition o f n-methyl-D-aspartat e (NMDA) , brain-derive d neurotrophi c facto r (BDNF), insulin-lik e growth factor 1 (IGF-1), or pituitary adenylyl cyclase activating polypeptide (PACAP) to the cultur e medium . Th e anti-apoptoti c effect of NMDA depend s o n NMDA-induced expressio n of BDNF, which i s inhibited b y ethanol. The protectiv e effect s o f NMDA an d BDNF are mediated throug h acti vation o f phosphatidylinositol-3' hydroxy kinase (PBK) , which i n tur n activate s Akt. NMDA ha s also been suggeste d to activate Akt directly via Ca2+/calrnodulindependent protein kinas e kinase (CaM-KK ) (dotte d line) . BDN F can als o promote cerebella r granul e neuron surviva l by activation of microtubule-associated protein kinas e (MAPK ) extracellula r signal-related kinas e (ERK) 1/2 . Activation o f this pathway results in phosphorylation o f the pro-apoptoti c protei n Bad on th e Ser 112, whic h i s mediated b y Rsk-2 , a membe r o f the MAPK-activate d pp90 ribosoma l S6 kinase family. Phosphorylation o f Bad results in dissociation of Bad from th e anti-apoptoti c Bcl- 2 protein, allowin g the surviva l function of Bcl-2 to be expressed. Activation of the PBK/Ak t pathway results in phosphorylation o f Bad o n Ser 136, an d phosphorylatio n o n bot h site s ma y be necessar y t o reduc e apoptosis (Bonni et al., 1999) . Although treatmen t o f cells with NMDA activate s MAPK, a n inhibito r o f thi s pathwa y doe s no t bloc k th e protectiv e effec t o f NMDA. Ther e is also evidence tha t the anti-apoptoti c actions of the MAP K and PI 3-kinas e pathways may converge at the leve l of cyclic AMP respons e elemen t binding protei n (CREB) . CREB activatio n can increas e th e expressio n o f bcl-2 mRNA. The overal l effect o f activation of these pathways is to release Bcl-2 fro m dimerization wit h Ba d an d t o increas e Bcl- 2 levels. The anti-apoptoti c effec t o f PACAP in cerebella r granul e neuron s i s mediated throug h activatio n o f PKA by cAMP and by activation of the PBK pathway via a pertussis toxin-sensitive G protein. Ethanol enhance s bot h o f these PACAP-stimulated pathways. Protein Kinas e A (PKA) can phosphorylate Bad at Ser 112 while Akt phosphorylates Bad at Ser 136, as discusse d above . Activation of PKA and PB K als o leads t o CRE B activation . Therefore, the protective effect o f PACAP may depend o n the convergence of two signaling pathways at the level of phosphorylation of Bad and/or CREB.

257

258 ETHANOL-AFFECTE D DEVELOPMEN T (Campard e t al, 1997 ; Vaudr y et al, 1998) . Ethano l acutely enhance s receptor-stimulate d cAM P produc tion i n variou s neurona l an d non-neurona l system s (Tabakoff an d Hoffman , 1998) . I f ethano l als o en hances PACAP-stimulate d cAM P production , on e could postulat e tha t ethano l ha s a neuroprotective effect when granule neuron s ar e treated simultaneousl y with ethanol and PACA P The signalin g pathway s tha t mediat e th e anti apoptotic effec t o f PACAP on cerebella r granule neu rons hav e bee n examined . I n cell s grow n i n th e absence o r presence o f ethanol i n a medium contain -

ing 5 mM KC1 , th e anti-apoptoti c effec t o f PACA P was reduced b y inhibitors of the cAMP/PK A pathway and th e PB K pathwa y (Bhav e and Hoffman , 2004) . The anti-apoptoti c effec t o f PACAP is evident within 4 hr . Ethano l (lOOmM ) accelerate s thi s proces s a s the anti-apoptoti c effec t o f PACA P i s maxima l afte r only 1 hr o f exposure t o ethanol an d PACAP . The ef fect of ethano l is blocke d by inhibitor s of bot h the cAMP/PKA an d th e PB K pathways , an d ethano l stimulates PACAP-induced productio n o f cAMP an d phosphorylation o f Akt (Bhav e an d Hoffman , 2004) . In addition , ethano l enhance s PACAP-induce d

FIGURE 15- 5 Schemati c o f th e relationshi p o f neurotrophi n expressio n an d neuronal ontogeny . Ther e ar e tw o period s o f cel l deat h durin g neura l on togeny: during the perio d o f cell proliferatio n (in the germina l zone [GZ] ) an d during th e perio d o f neuronal differentiatio n (i n th e gra y matter). At least th e latter i s associated wit h neurotrophi n expression . Neurotrophin expressio n increases durin g the perio d o f neuronal differentiatio n t o suppor t th e growt h o f neuntes an d promot e synaptogenesis . Nevertheless, neurotrophi n expressio n is limited, hence it defines the numbe r o f neurons tha t can successfull y compete for enoug h neurotrophi n t o maintai n thei r survival . Ethanol delay s th e num bers o f neurons tha t are generated an d successfull y migrate t o the CN S struc ture an d reduce s th e amoun t o f available neurotrophin . Th e consequenc e of this reductio n i s tha t fewe r neuron s survive . Soli d an d broke n line s identif y changes for control an d ethanol-treate d subjects, respectively. Thicker line s depict change s i n the number s o f neurons i n a CN S structur e and thinne r line s describe changes i n neurotrophin expression . The graph s at the botto m depic t changes i n the number s of cells involved in a particular ontogenetic event (for a control subject) .

EXTRACELLULAR MEDIATORS OF NEURONA L DEATH 25 9 CREB phosphorylation . These result s indicate d tha t ethanol treatmen t o f cerehellar granul e neuron s ca n increase neuroprotection induce d by PACAP. Although exposur e o f th e developin g brai n t o ethanol i s associate d wit h a n overal l los s o f neurons , and th e cerebellu m i s particularly sensitive to ethanolinduced neurona l los s (Goodlet t e t al. , 1990 ; Wes t et al, 1990 ; Miller , 1992 , 1996b ; Maier e t al, 1999) , the finding s fo r PACA P offe r a possibl e approac h fo r amelioration o f th e overal l deleteriou s effec t o f ethanol. Sinc e PACA P i s endogenousl y presen t i n cerebellar granul e neurons , th e presenc e o f ethanol a t critical time s o f development coul d enhanc e the pro tective effec t o f this endogenous factor . Neurona l survival durin g development depend s o n th e interpla y of various neurotrophi c factors . Ethano l ca n affec t th e balance amon g th e effect s o f thes e factors . On e ap proach t o alleviatin g the damagin g effect s o f ethano l during centra l nervou s syste m developmen t i s t o in crease th e contributio n o f neuroprotectiv e pathway s that are targeted b y ethanol.

the developin g periphera l nervou s syste m (Davies , 1997). Potentially, neurons ar e more susceptible whe n only a singl e neurotrophi n syste m i s active, but the y become more resistant to ethanol toxicit y as additional intercellular communicatio n system s are established . How thi s affect s intracellula r processin g i s currentl y unknown. A n alternativ e bu t no t mutuall y exclusive mechanism i s that ethanol target s receptors (e.g. , p75, but no t tr k isoform content) . Recepto r selectivit y may reflect th e differentia l distributio n o f p75 an d trk s i n the plasm a membrane, for example, in lipid rafts . In summary, ethanol vulnerability is determined by extracellularly initiate d events . Th e effect s o f ethano l on ligand s and receptor s may be additiv e or synergistic. Regardless , th e differentia l effect s ar e instructiv e about th e rol e o f neurotrophin system s during early development. Abbreviations BDNF brain-derive d neurotrophic facto r BEC bloo d ethanol concentratio n

SUMMARY AND CONCLUSION S

Ethanol-induced neurona l deat h occur s durin g defined period s o f th e developmenta l tim e lin e (Fig. 15-5) . These time period s coincid e wit h thos e of neuronogenesi s an d primar y synaptogenesis , which ar e periods of NOND (se e Chapter 5) . Exposure to ethanol a t this time ma y exacerbate processes involved i n NOND . This pattern applie s t o part s of the brai n i n whic h th e developmen t o f th e con stituent neuron s i s relatively synchronized, as in th e PSN an d cerebra l cortex . A n interestin g counter point i s th e cerebellum , i n whic h population s o f neurons follo w nonoverlappin g ontogeneti c tim e lines. Fo r example , th e proliferatio n of granule cel l precursors coincide s wit h th e synaptogenesi s o f Purkinje neurons . I n thi s case , th e windo w o f vulnerability t o ethano l i s narrow; i t i s only 2 days (P 4 and P5) , early during the perio d o f Purkinje neuron synaptogenesis. Ethanol ha s multiple targets , includin g extracellu lar component s o f growth facto r systems , i.e., ligand s and receptors . Ethano l i s no t a promiscuou s toxin ; rather, it affects th e action s of spécifie ligand s (e.g., for fetal cortica l neurons , NGF , bu t no t BDNF or NT-3). Ligands ma y b e temporall y expresse d an d overlai d upon pre-existin g systems. This situation i s evident i n

cAMP cycli c adenosine monophosphat e CREB cAM P response element bindin g protein ERK extracellula r signal-related kinase FASD feta l alcoho l spectru m disorders G gestationa l day IGF-1 insulin-lik e growth factor- 1 MAPK mitogen-activate d protein kinase NGF nerv e growth facto r NMDA N-methyl-D-aspartat e NOND naturall y occurring neuronal death NT neurotrophi n P postnata l day PACAP pituitar y adenyly l cyclas e activatin g polypeptide PI3K phosphatidylinosito l 3'-phosphokinas e PKA protei n kinas e A PSN principa l sensory nucleus of the trigemina l nerve trk tyrosin e kinase receptor TUNEL termina l uridylate d nick-end labelin g VB ventrobasa l nucleus o f the thalamu s VIP vasoactiv e intestinal polypeptid e

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EXTRACELLULAR MEDIATOR S O F NEURONA L DEAT H 26 5 Pantazis NJ, Dohrman DP , Luo J, Thomas JD, Goodlett CR, Wes t J R (1995 ) NMD A prevent s alcohol induced neurona l cel l deat h o f cerebellar granul e cells in culture. Alcoho l Cli n Exp Res 19:846-853. Pantazis NL, Zaheer A, Dai D, Zaheer S, Green SH, Lim R (2000 ) Transfection o f C6 gliom a cell s wit h gli a maturation facto r upregulate s brain-derive d neu rotrophic facto r an d nerv e growth factor: trophi c effects an d protectio n agains t ethano l toxicit y i n cerebellar granule cells. Brain Res 865:59—76. Peoples RW , Li C , Weigh t F F (1996 ) Lipi d v s protein theories o f alcoho l actio n i n th e nervou s system. Annu Rev Pharmacol Toxicol 36:185-201 . Pierce DR , Goodlet t CR , Wes t J R (1989 ) Differentia l neuronal los s following early postnatal alcoho l ex posure. Teratology 40:113-126 . Pierce DR , Wes t J R (1986 ) Alcohol-induce d microen cephaly durin g th e thir d trimeste r equivalent : rela tionship t o dos e an d bloo d alcoho l concentration . Alcohol 3:185-191 . Pitts AF, Miller M W (1995 ) Expression o f nerve growth factor, p75 , and trk i n the somatosensory-moto r cortices o f mature rats: evidence for local cortica l support circuits. Somatosensory Motor Re s 12:329—342. (2000) Expressio n o f nerv e growt h factor , brain derived neurotrophic factor, an d neurotrophin- 3 in the somatosensor y corte x o f th e rat : co-expressio n with hig h affinit y neurotrophi n receptors . J Comp Neurol 418:241-254. Rakic P , Bourgeoi s JP , Eckenhof f MF , Zecevi c N , Goldman-Rakic P S (1986 ) Concurren t overpro duction o f synapse s i n divers e region s o f th e pri mate cerebral cortex. Science 232:232—235 . Ramachandran V , Watt s LT , Maff i SK , Che n J , Schenker S , Henderson G (2003 ) Ethanol-induce d oxidative stres s precedes mitochondriall y mediated apoptotic deat h o f culture d feta l cortica l neurons . JNeurosci Res 74:577-588. Resnicoff M , Abraha m D , Yutanawibooncha i W , Rot man HL , Kajstur a J , Rubin R , Zoltick P , Baserga R (1995) Th e insulin-lik e growt h facto r I recepto r protects tumo r cell s fro m apoptosi s i n vivo. Cancer Res 55:2463-2469 . Resnicoff M , Cu i S , Coppol a D , Hoe k JB , Rubi n R (1996) Ethanol-induced inhibitio n of cell prolifera tion i s modulated b y insulin-like growth factor-I re ceptor levels. Alcohol Clin Ex p Res 20:961-966. Rezai Z , Yoon C H (1972 ) Abnormal rate of granule cell migration i n th e cerebellu m o f "weaver " mutan t mice. DevBiol 29:17-26. SchlessingerAR, Cowa n MW , Swanso n L W (1978) Th e time o f origin of neurons i n Ammon's horn an d th e associated retrohippocampa l fields . Ana t Embryo l 154:153-173.

Seabold G , Lu o J , Miller M W (1998 ) Effec t o f ethano l on neurotrophin-mediate d cel l surviva l an d recep tor expressio n i n cortica l neurona l cultures . De v Brain Res 128:139-145. Seiler AE, Ross BN, Green JS, Rubin R (2000) Differential effect s o f ethanol o n insulin-lik e growth factor-I receptor signaling . Alcoho l Cli n Ex p Re s 24:140 — 148. Shaywitz AJ , Greenberg M E (1999 ) CREB : a stimulus induced transcriptio n facto r activate d b y a divers e array o f extracellula r signals . Annu Re v Bioche m 68:821-861. Spear N , Esteve z AG , Johnso n GV , Bredese n DE , Thompson JA , Beckman J S (1998 ) Enhancemen t of peroxynitrite-induced apoptosi s i n PC 12 cells by fibroblast growt h factor- 1 an d nerv e growt h facto r requires p21Ras activation and i s suppressed b y bcl2. Arch Biochem Biophy s 356:41-45. Spengler D , Waeber C , Pantalon ! C, Holsboer F , Bockaert J , Seebur g PH , Journo t L (1993 ) Differentia l signal transductio n b y fiv e splic e variant s o f th e PACAP receptor. Nature 365:170-175 . Spreafico R , Frassoni C, Arcelli P, Selvaggio M, De Bias i S (1995 ) I n sit u labeling of apoptotic cel l deat h i n the cerebra l corte x and thalamu s o f rats during development. J Comp Neurol 363:281-295 . Stratton K , How e C , Battagli a F (1996 ) Fetal Alcohol Syndrome: Diagnosis, Epidemiology, Prevention and Treatment. Nat l Aca d Se i Press , Washingto n DC. Sulik KK, Cook CS, Webster W S (1988 ) Teratogens and craniofacial malformations : relationship s t o cel l death. Developmen t 103(Suppl):213-231 . Tabakoff B , Hoffma n P L (1998 ) Adenyly l cyclases an d alcohol. Ad v Secon d Messenge r Phosphoprotei n Res 32:173-193. Tabuchi A, Koizumi M, Nakatsubo J, Yaguchi T, Tsuda M (2001 ) Involvement o f endogenous PACA P ex pression in the activity-dependen t survival of mouse cerebellar granul e cells . Neurosc i Re s 39:85-93. Thomas JD , Wasserma n EA , Wes t JR , Goodlet t C R (1996) Behavioral deficits induced b y bingelike ex posure t o alcoho l i n neonata l rats : importanc e o f developmental timin g an d numbe r o f episodes . Dev Psychobiol 29:433-452. Tojima T , Kobayash i S , Ito E (2003 ) Dua l rol e of cyclic AMP-dependent protei n kinas e i n neuritogenesi s and synaptogenesis during neuronal differentiation . J Neurosci Res 74:829-837. Vallès S , Lind o L , Montoli u C , Renau-Piquera s JR , Guerri C (1994 ) Prenata l exposur e t o ethano l in duces change s i n the nerv e growth facto r and it s receptor i n proliferating astrocytes in primary culture. Brain Res 656:281-286.

266 ETHANOL-AFFECTE D DEVELOPMENT Vaudry D, Gonzale z BJ , Basili c M , Anoua r Y, Foumier A, Vaudr y H (1998 ) Pituitar y adenylat e cyclase activating polypeptid e stimulate s bot h c-fo s gen e expression an d cel l surviva l i n ra t cerebella r gran ule neuron s throug h activatio n o f th e protei n ki nase A pathway. Neuroscience 84:801-812. Vaudry D , Rousselle C, Basili c M , Falluel-More l A, Pamantung TF , Fontain e M , Foumie r A , Vaudry H , Gonzalez B J (2002 ) Pituitar y adenylat e cyclase activating polypeptid e protect s ra t cerebella r gran ule neuron s agains t ethanol-induce d apoptoti c cel l death. Pro c Natl Acad US A 99:6398-6403. Villalba M, Bockaert J, Journot L (1997) Pituitary adenylate eyclase-activating polypeptide (PACAP-38) protects cerebella r granul e neuron s fro m apoptosi s by activating th e mitogen-activate d protei n kinas e (MAP kinase) pathway. J Neurosci 17:83-90 . Vogel M W (2002 ) Cell death , Bcl-2 , Bax , and th e cere bellum. Cerebellu m 1:277-287 . Walton MR , Draguno w I (2000) Is CREB a key to neu ronal survival? Trends Neurosc i 23:48-53. Wegelius K , Korpi E R (1995 ) Ethano l inhibit s NMDA induced toxicit y and trophis m i n culture d cerebel lar granule cells. Acta Physiol Scand 154:25—34 . West JR, Chen WJ, Pantazis NJ ( 1994) Fetal alcoho l syndrome: th e vulnerabilit y of th e developin g brai n and possibl e mechanism s o f damage. Meta b Brain Dis 9:291-322. West JR , Goodlett CR , Bonthiu s DJ , Hamr e KM , Mar cussen B L (1990) Cell populatio n depletio n associ ated wit h feta l alcoho l brai n damage : mechanism s of BAC-dependen t cell loss . Alcohol Cli n Ex p Re s 14:813-818. West JR , Goodlett CR , Bonthiu s DJ , Pierc e D R (1989 ) Manipulating pea k blood alcoho l concentration s i n

neonatal rats : revie w o f a n anima l mode l fo r alcohol-related developmenta l effects . Neurotoxi cology 10:347-365 . Wood KA , Dipasquale B, Youle RJ (1993) In situ labeling of granul e cell s fo r apoptosis-associate d DN A frag mentation reveal s differen t mechanism s o f cell los s in developing cerebellum. Neuro n 11:621—632 . Wu Y , Tewari M , Cu i S , Rubi n R (1996 ) Activatio n of the insulin-lik e growth factor- I recepto r inhibit s tu mor necrosi s factor-induced cell death . J Cell Physiol 168:499-509 . Yan GM , Li n SZ , Irwi n RP , Paul S M (1995 ) Activation of G protein s bidirectionall y affect s apoptosi s o f cultured cerebella r granule neurons . J Neurochem 65:2425-2431. Yan GM , N i B , Weller M , Woo d KA , Paul S M (1994 ) Depolarization o r glutamat e recepto r activatio n blocks apoptoti c cel l deat h o f culture d cerebella r granule neurons. Brai n Res 656:43-51. Zafra F , Castre n E , Thoene n H , Lindhol m D (1991 ) Interplay betwee n glutamat e an d gamma aminobutyric acid transmitte r system s i n the physi ological regulatio n o f brain-derive d neurotrophi c factor an d nerv e growt h facto r synthesi s i n hip pocampal neurons . Pro c Nat l Aca d Se i US A 88 : 10037-10041. Zafra F , Hengerer B , Leibrock J , Thoenen H , Lindhol m D (1990 ) Activit y dependent regulatio n o f BDNF and NG F mRNA s i n the ra t hippocampus i s mediated b y non-NMDA glutamate receptors . EMB O J 9:3545-3550. Zhang FX , Rubin R, Rooney TA (1998) Ethanol induce s apoptosis i n cerebella r granul e neuron s b y inhibit ing insulin-lik e growth facto r 1 signaling. J Neurochem 71:196-204.

16

Intracellular Events in Ethanol-Induced Neuronal Death Sandra M. Mooney Michael W . Miller George I . Henderso n

Developmental exposur e to ethanol ca n significantly reduce th e numbe r o f neurons i n specific areas of the brain. This may result fro m a decrease i n the additive processes o f cell generatio n an d migratio n (Chapters 11, 12 , and 13) and/o r a n increas e in th e subtractiv e process o f cel l deat h (se e Chapte r 15) . The presen t Chapter explore s the effect s o f ethanol o n intracellular pathways involved in neuronal death . Before embarkin g on a description o f the various pathways and th e effect s o f ethanol o n them , it is important t o acknowledge caveat s tha t ma y affect inter pretation o f the relevan t data . Exposur e t o ethano l alters expressio n and/o r activatio n o f man y protein s involved i n apoptosis . Evidenc e show s tha t ethano l can trigger intrinsic and extrinsi c apoptotic pathways. For example, ethanol-induced cel l deat h i n viv o ca n be mediate d b y Be l proteins , i.e. , th e intrinsi c pathway (Moone y and Miller , 2001 ; Young et al, 2003). Studies wit h organotypi c cultures , b y contrast , show that Fa s receptor expressio n ca n als o b e affecte d b y ethanol (e.g. , Cheema e t al., 2000 ; de l a Monte an d

Wands, 2002) , thu s implicatin g th e extrinsi c path way. Par t o f th e resolutio n o f thi s riddl e i s tha t ethanol ma y trigge r deat h throug h bot h pathways . The relevan t research is thus based on a variety of (in vivo an d i n vitro ) model s tha t encompas s multipl e variables, such a s the pea k concentration o f ethanol , the duratio n of the exposure , the typ e of cell or tissue being examined, and the time afte r exposur e that th e cells o r tissue s wer e examined . Althoug h thes e different model s ma y provid e complementar y ap proaches, the y ma y als o generat e nonphysiological , model-dependent responses . Thus , i t i s challengin g to reconstruc t the mechanism s of ethanol-induce d apoptosis. When reviewin g th e literatur e o n th e effect s o f ethanol o n neurona l death , i t mus t b e kep t i n min d that developmenta l exposur e t o ethano l interfere s with othe r developmenta l events , notabl y cel l prolif eration an d migratio n (se e Chapter s 11 , 12 , and 13) . Without includin g th e effect s o f ethano l o n thes e early ontogenetic events the data ma y be confounde d

267

268 ETHANOL-AFFECTE D DEVELOPMENT and misinterpretation s ma y result . Fo r example , longitudinal studies of the effect s o f ethanol on neural cell number in vitro (Jacobs and Miller , 2001; Miller, 2003) o r i n viv o (Miller , 1999 ) documen t a signifi cant reductio n i n cel l number . Two-third s o f thi s change i s due t o a depression in cell proliferation ; the remaining thir d result s fro m ethanol-induce d cel l death. Th e consequen t lesso n mus t be heede d b y alcohol investigator s examinin g cel l number : reduc tions i n cel l numbe r ar e no t necessaril y due t o cel l death.

CELL DEAT H PATHWAYS

The effect s o f ethano l o n thre e pathway s of neuronal death ar e discusse d below : (1 ) th e intrinsi c caspase dependent pathway , (2) the extrinsic caspase-dependen t pathway, an d (3 ) th e caspase-independen t pathwa y (Fig. 16-1) . These pathway s are describe d i n greate r detail i n Chapte r 6 , bu t a brie f outlin e i s included below. The intrinsi c pathwa y i s mitochondriall y medi ated, typically in response to an apoptotic signal such as DN A damag e o r reactiv e oxyge n specie s (ROS) . An initia l even t i s releas e o f proapoptoti c protein s from th e niitochondria l intermembran e space . Per meabilization o f the mitochondrial oute r membran e appears t o be mediate d b y Bcl-2 famil y protein s an d may includ e direc t bindin g o f p5 3 t o on e o r mor e anti-apoptotic proteins , suc h a s Bcl-X L o r Bax . Up regulation of Bax may allow insertion of Bax-Bax ho modimers into the mitochondria l membrane, which alters membrane permeabilit y and allows intermembrane substance s t o escape int o the cytoplasm . On e such substance , Cytochrom e C , associate s wit h apoptotic proteas e activatin g facto r 1 and caspase- 9 in th e presenc e o f adenosin e triphosphat e (ATP ) to for m a n apoptosome . Thi s activate s caspase-9 , which i n tur n activate s caspase-3. Activ e caspase- 3 inactivates poly-adenosin e diphosphat e ribos e poly merase (PARP ) 1 , thus repressing DNA repai r mechanisms. I n combinatio n wit h activatio n o f enzymes such a s DN A fragmentatio n factor 40 , th e resul t is DNase-mediated fragmentatio n o f DN A an d cel l death. The extrinsi c pathwa y is activated b y binding of a ligand t o a cel l surfac e receptor . On e exampl e i s the bindin g o f Fa s ligand (FasL ) to it s recepto r Fas . This bindin g cause s recepto r oligomerization , an d

FIGURE 16- 1 Schemati c of three pathway s leading to cell death . Thre e pathway s o f cel l death , outlinin g the flow of information fro m externa l effects t o nuclear response, are depicted : intrinsi c and extrinsi c caspasedependent pathway s an d a caspase-independen t path way. A subse t o f th e molecule s involve d i n thes e pathways is shown; a mor e comprehensiv e figur e de tailing the pathways is shown i n Figure 6- 1 i n Chapter 6 . Factor s know n t o b e affecte d b y ethano l ar e identified. Smal l arrow s sho w th e effec t o f develop mental exposur e t o ethano l o n eac h factor . Th e blunted lin e signifie s a n inhibitor y effect. AIF , apopto sis inducin g factor ; PARP , poly-adenosin e diphos phate ribose polymerase; ROS, reactive oxygen species. See text for details.

recruitment o f th e Fas-associate d deat h domai n (FADD) and procaspase s 8 and 1 0 (Benn and Woolf, 2004). Th e FADD-caspase-8/1 0 comple x form s th e death-inducing signalin g comple x tha t cleave s an d activates caspase-3 . As with the intrinsi c pathway, active caspase-3 inactivates PAR P and allow s DNA frag mentation. I n addition , activ e caspase- 8 cleave s th e Bel family protein, Bid. Truncated Bi d translocates to the mitochondri a wher e i t ca n activat e the intrinsi c pathway b y promotin g insertio n of Bax-Ba x homod imers into the mitochondrial membrane. Like th e intrinsi c pathway , the caspase-indepen dent pathwa y is mitochondria-dependent . Followin g release o f apoptosi s inducin g facto r (AIF ) fro m th e mitochondrial intermembran e space , it can eithe r directly up-regulate DNase activity or cleave and inacti vate PARP, thus repressing DNA repair and promotin g cell degeneration.

INTRACELLULAR EVENT S I N ETHANOL-INDUCE D NEURONAL DEATH 26 9 EFFECT O F ETHANOL ON APOPTOSIS

Intrinsic Pathway s Bel Family Proteins Exposure t o ethano l alter s th e i n viv o expressio n o f members o f the Be l protein family . This is apparent i n changes of the mRNAs and the proteins. Changes ma y be rapid , as in the cas e of the mRN A (Moor e et al. , 1999; Inoii e et al, 2002), or occur later , as detected i n protein expressio n (Mooney an d Miller , 2001) . Corti cal expressio n of Bcl-2 , a prosurviva l membe r o f th e family, i s lowe r i n th e cortice s o f rat s expose d t o ethanol prenatally , most notabl y i n the perio d aroun d postnatal day (P) 6. This finding is particularly interesting i n ligh t of the lac k of change i n th e expressio n of bax in cerebral cortex , the ne t result being a reductio n in th e rati o of Bcl-2 to Ba x on gestationa l day (G) 16 , P6, an d P12 . Thes e times , prenata l an d postnata l week, coincid e with th e period s o f both naturall y occurring and ethanol-induced cell death i n the prolifer ative zone s an d i n th e immatur e corte x (Küh n an d Miller, 1998 ; se e Chapter 5) . It is noteworthy that an other immunoblottin g study of the cortice s of rats prenatally exposed to ethanol showed an increase in Bcl-2 expression on PI an d n o change betwee n P 7 and P3 5 (Climent e t al. , 2002) . Th e difference s i n reporte d Bcl-2 expressio n i n th e tw o studie s ma y reflec t th e feeding paradigms . Although fetuse s were expose d t o ethanol i n bot h studies , i n the stud y by Climent and colleagues ethanol exposur e continued int o the postnatal period. The chang e i n corte x contrast s wit h th e lac k o f a change in the relative amounts o f bcl-2 and bax in the thalamus. The thalamu s i s a structure that exhibit s no neuronal deat h followin g prenata l exposur e t o ethanol (Moone y an d Miller, 1999 ; Liv y et al, 2001). Thus, these dat a indicat e tha t th e chang e i n the bal ance o f bcl- 2 an d ba x i s a ke y determinan t o f neu ronal death an d survival. The expressio n o f Be l protein s i s altere d i n th e murine cerebellu m followin g earl y postnatal exposure to ethanol (Heato n et al, 2002a, 2003a). Expression of the anti-apoptoti c protei n Bcl- 2 decrease s an d o f proapoptotic Ba x an d Bcl-x s increase s i n concomitan t temporal manner s tha t correlat e wit h th e perio d o f Purkinje cel l vulnerability (Heaton e t al., 2003a). That is, during the perio d of vulnerability, on P4, expression of Ba x and Bcl-x s increase s within 2 hr o f exposure to

ethanol. I n contrast, ethanol exposur e on P7, a time of relative invulnerability , causes a n earl y up-regulatio n of th e survival-promotin g proteins, Bcl- 2 an d Bcl-xl , and a down-regulation o f the pro-apoptotic Bcl-xs. The change s i n protei n expressio n apparentl y follow earlie r change s i n transcrip t expression. Fo r ex ample, exposur e t o ethano l induce s a n immediat e change i n th e expressio n o f bd famil y transcript s i n the cerebra l corte x an d cerebellu m (Moor e e t al. , 1999; Inou e et al., 2002). Ethanol up-regulate s the expression o f pro-apoptotic genes , ba x and bcl-xs, in th e cerebellum. Interestingly , however, the up-regulatio n of hax i n th e cerebellu m occur s in th e absenc e o f an effect o n cel l survival . This finding concurs with data from a strain of bax knockout animal s i n which ther e was n o increas e i n cell deat h followin g exposur e to ethano l (Youn g e t al. , 2003) . Overexpressio n o f bcl-2, by contrast , confer s invulnerabilit y t o ethanol induced deat h o f cerebella r Purkinj e cell s (Heato n etal, 1999) . Cytochrome C Release of cytochrome C fro m mitochondri a i s a central even t i n th e intrinsi c apoptoti c pathwa y (Polste r and Fiskum , 2004). This release, combined wit h that of othe r proapoptoti c proteins , elicit s activatio n o f initiator caspase s tha t ultimatel y produce s effecto r caspase activation . Thus , mitochondria l releas e o f Cytochrome C i s a n early , feed-forwar d ste p i n th e apoptotic process , an d combined wit h subsequent ac tivation o f effecto r caspases , i s compellin g evidenc e that apoptotic deat h i s a work in progress. Several laboratorie s hav e use d Cytochrom e C re lease to address ethanol-mediated apoptotic brai n cell death followin g i n viv o ethano l exposure . Appropriately, thes e observation s have bee n linke d t o activation o f caspase-3 . Mitochondri a isolate d fro m feta l brains, that have been expose d to ethanol i n utero (on G17 an d G18) , exhibi t enhanced releas e of both cy tochrome C an d AI F (Ramachandra n e t al. , 2001) . This resul t parallel s th e activatio n o f caspase- 3 i n whole feta l brai n homogenates . Simila r response s have been reporte d followin g ethanol treatmen t o f 4to 10-day-ol d ra t pup s (Ligh t e t al. , 2002 ; Carlon i et al, 2004) or infant mic e (Youn g et al, 2003). The ethanol-induce d i n viv o releas e o f Cyto chrome C ha s bee n replicate d i n vitro. Exposure o f cultured feta l ra t cortical neurons to ethanol generates enhanced releas e o f Cytochrom e C withi n 2 hr o f

270 ETHANOL-AFFECTE D DEVELOPMENT exposure (Ramachandra n e t al. , 2003) . Thi s i s followed b y activation o f caspase-3 withi n 1 2 hr o f treatment. Thus, neurons exposed to ethanol eithe r i n vivo or i n cultur e ca n elici t mitochondria l releas e o f Cytochrome C in some, as-yet, undetermined manner . Caspases The researc h community has focused on the effects of developmental exposur e t o ethano l o n tw o caspases : caspase-3 and caspase-8 . These are of interest because (a) the y ar e fro m tw o differen t molecula r subgroup s and (b ) they ar e involve d i n tw o pathways; caspase- 3 is associated wit h the intrinsi c apoptoti c pathwa y an d caspase-8 with the extrinsi c pathway (Fig. 16-1) . There i s considerable i n vitro (Sait o e t al. , 1999 ; Jacobs an d Mille r 2001 ; Jan g e t al , 2001 ; Sohm a et al, 2002; Vaudry et al, 2002; Ramachandran e t al., 2003) an d i n vivo (Moone y an d Miller , 2001 ; Ra machandran e t al, 2001 ; Climent e t al, 2002; Ligh t et al., 2002; Olney et al, 2002a, 2002b; Mitchell an d Snyder-Kelly, 2003 ; Ragjopa l e t al. , 2003 ; Youn g et al., 2003; Carloni e t al, 2004) evidenc e tha t exposure t o ethano l increase s the activatio n o f caspase-3. This activation ma y be model-dependent . Fo r exam ple, Sohm a an d colleague s (2002 ) repor t tha t al though bot h C 6 ra t gliom a cell s an d A54 9 huma n adenocarcinoma cell s show evidence of apoptosis following exposure to ethanol th e C6 cells increas e thei r caspase activity, whereas the A549 cells do not . Ethanol cause s apoptosi s (a t leas t i n vitro ) whe n trophic factor s ar e i n limite d suppl y (Oberdoerste r and Rabin , 1999) . Cerebellar granul e neuron s grow n in serum-containin g mediu m supplemente d wit h high concentration s o f potassium chlorid e d o not un dergo apoptosi s i n respons e t o ethanol . Remova l o f the seru m result s i n increase d activatio n o f caspase- 3 and apoptosis i n response t o ethanol . To date, ther e ar e few data regardin g the effec t o f ethanol o n othe r caspases . One i n vitr o stud y shows that ethanol-induced deat h of cerebellar granul e neu rons i s accompanied b y a n increas e i n activatio n o f initiator caspase s 2, 8, and 9, and effecto r caspase s 3 and 6 (Vaudry et al, 2002). This deat h i s abrogated by the additio n o f pituitary adenylate cyclas e activat ing polypeptid e (PACAP ; se e Chapte r 15) . Additio n of exogenou s PACA P ameliorate s th e activatio n o f caspases 3 an d 6 , bu t no t caspase s 2 , 8 , o r 9 . Thi s implies that activation of an initiato r caspase does no t oblige a cell to die.

Extrinsic Pathway s Although littl e i s known about th e effec t o f develop mental exposur e to ethanol o n tumor necrosis facto r a or Apo2/TRAIL systems i n the brain , th e Fas L syste m has been studie d i n viv o and i n vitro. Fas and Fas L are transiently expresse d i n developin g corte x durin g th e time o f naturally occurrin g neurona l deat h (Cheem a et al., 2000). Exposure t o ethanol induce s Fa s mRNA expression in vitro, but onl y at subapoptotic concentra tions an d no t a t concentrations tha t induc e apoptosis . Similarly, there i s an increas e i n Fa s receptor an d lig and i n cultures o f cerebellar granul e cell s taken i n th e postnatal perio d fro m rat s expose d t o ethano l prena tally (de la Monte an d Wands, 2002) . Other evidence showing tha t ethano l activate s extrinsi c pathway s i s that th e ethanol-induce d deat h o f cultured cerebella r granule cells is accompanied b y an increase in the ac tivity of caspase-8 (Vaudr y et al., 2002). Caspase-Independent Pathway In a numbe r o f neurona l cel l deat h paradigm s i n which caspase s ar e normall y activated , inhibitio n o f caspase activity delays but doe s not prevent cell deat h from occurrin g (Mille r e t al. , 1997 ; Stefani s e t al. , 1999; D'Mell o e t al, 2000 ; Keramari s e t al, 2000 ; Selznick e t al., 2000) . Suc h dat a impl y that prenata l exposure to ethanol affect s caspase-independen t pathways, bu t fe w dat a directl y addres s thi s possibility . Ethanol exposur e doe s induc e th e releas e o f Cy tochrome C and AIF from mitochondri a (Ramachan dran e t al. , 2001) . Consequently , AI F ca n initiat e a feedback loo p that furthe r induce s Cytochrome C release fro m th e mitochondri a (Porter , 1999) . Thus , ethanol-induced initiatio n o f caspase-independen t death ca n be a self-perpetuating phenomenon .

EFFECTORS OF CEL L DEAT H

p53-Related Factors p53 i s a critica l fulcru m definin g th e fat e o f cells : whether the y di e o r surviv e (Mille r e t al. , 2000 ; Morrison e t al, 2002). The teeter-totter i s pushed on e way or the othe r b y the phosphorylatio n of serines on p53, and particularl y by the position s of the phospho rylated serines . Although a n effec t o f ethanol o n thi s process ha s been surmise d (Küh n an d Miller , 1998) ,

INTRACELLULAR EVENT S IN ETHANOL-INDUCE D NEURONA L DEAT H 27 1 direct data ar e lacking. Nonetheless, i t is known that ethanol ca n affec t kinas e activit y i n othe r system s (e.g., Resnicoff et al, 1994 ; Lu o and Miller, 1999) . In additio n to potential effect s o f ethanol on posttranslational modificatio n of p53, the effectivenes s o f p53 can be affecte d b y an ethanol-induced change i n the amoun t o f p53 available. Indeed, p5 3 expression in vivo is affected b y prenatal exposure to ethanol i n a spatiotemporal manne r (Küh n an d Miller , 1998) . Ethanol increase s p53 expression in the fetu s an d re duces it in the first and second postnatal weeks . Interestingly, followin g chroni c gestationa l exposur e t o ethanol, expression of p53 is increased in the ra t cerebellum durin g th e earl y postnata l perio d (d e l a Monte an d Wands , 2002) . The increas e i n p5 3 oc curs concurren t with increase s in Fa s ligand an d re ceptor, implyin g that the increas e i n p53 is related to cell death. Although many ethanol-susceptible activities occu r coincidentall y i n th e developin g cere bellum, tw o are wort h noting : th e reductio n i n th e population o f proliferatin g granul e cell s (Bauer Moffett an d Altman, 1977; Kornguth et al., 1979 ) and the death o f Purkinje neuron s (e.g., Light et al, 2002; see Chapter 15) . Thus, the ethanol-induce d increas e in cel l mortalit y among th e proliferatin g neurona l precursors i n th e corte x and cerebellu m ma y be signaled by an increase in p53 expression. A phosphorylated form o f p53 may be recognize d by th e antibod y ALZ-5 0 (Küh n an d Miller , 1998) . ALZ-50 immunoreactivit y i s associate d wit h dyin g central nervou s syste m (CNS ) neuron s (Al-Ghou l and Miller , 1989 ; Valverd e et al., 1990 ; Mille r e t al, 1991). Interestingly, the expressio n profile o f ALZ-50 in the developin g corte x parallel s that of p53 expression (Kühn and Miller , 1998) . Moreover, prenatal exposure t o ethano l affect s bot h protein s similarly . I t reduces the amoun t o f peak ALZ-50 and p5 3 expression an d induce s this pea k to occur earlier . The im plication i s that ethano l alter s the timin g of neuronal death i n the developing cortex. Oxidative Stress Basic Findings Over the last four decades, studies have linked ethano l intake to oxidative stress (D i Luzio , 1963 ; Sha w et al., 1983). The earlies t reports connecting maternal drinking t o feta l oxidativ e damage wer e b y Dreost i (1987 ; Dreosti and Patrick, 1987), who reported that maternal

ethanol intak e increased malondialdehyde (MDA ) in fetal ra t live r mitochondria . Thi s increas e ha s als o been describe d i n culture d feta l hepatocyte s (Dev i et al. , 1994 , 1996) . Likewise , ethano l ca n increas e both MD A an d conjugate d diene s i n feta l brain . Chronic prenata l ethano l exposur e ca n reduc e glu tathione (GSH ) conten t o f feta l brai n (Reye s e t al, 1993) an d ethano l produce s fre e radica l damag e i n neural cres t cell s (Che n an d Sulik , 1996) . Thus , ethanol-mediated oxidativ e damag e t o feta l cell s occurs in vivo and this can be reproduced i n culture d fetal cells . These data sets laid the conceptual groundwork fo r numerou s subsequen t report s o n ethanol related oxidativ e damag e t o feta l cells . The y ar e reviewed below in the context of mechanisms underlying ethanol-mediated apoptotic death of neurons. Ethanol induces oxidative stress and activates apoptotic pathways in the developin g brain and i n cultured neurons (d e l a Mont e e t al, 2001 ; d e l a Mont e an d Wands, 2001 ; Ramachandra n e t al. , 2001 , 2003 ; Heaton et al, 2002b, 2003a, 2003b, 2003e). The parallel observation s of these tw o ethanol-related phenom ena are correlative and do not establish oxidative stress as a causative factor for neuronal death. Extensive documentation o f suc h concomitan t events , combine d with the effectivenes s o f anti-oxidant interventions and time lines illustrating that onse t of oxidative stress ca n precede apoptosis , i s convincing evidence tha t oxidative damage is one mechanism underlyin g the ethanol mediated loss of neurons in the developing brain. In utero exposure to ethanol o n G17 and G18 can elicit bot h oxidativ e damage t o brai n mitochondria , such a s 4-hydroxynonenal (HNE ) formation , an d in duction of markers of mitochondria-mediated apopto sis. Th e latte r include s change s o f mitochondria l permeability transition , activation o f caspase-3 , an d mitochondrial releas e of Cytochrome C and AIE (Ramachandran e t al. , 2001) . A mechanism underlying the ethanol-mediate d leakag e o f pro-apoptoti c pro teins from mitochondri a i s activation of mitochondri ally generated HNE . Mitochondri a isolate d from th e brains o f fetuse s treate d wit h HN E mimi c th e re sponse observe d i n th e brain s o f rodent s expose d t o ethanol. Th e abilit y o f HN E t o elici t apoptosi s i n neurons make s it an attractiv e mechanism, however, a clear causative link remains to be established . Ethanol-related increase s in RO S hav e been examined in young rats acutely exposed to ethanol presented as a binge exposure . Increased amount s o f ROS wer e detected in the cortices of these rats on P7 and i n their

272 ETHANOL-AFFECTE D DEVELOPMENT cerebella o n P4 , P7 , o r P1 4 (Heato n e t al , 2002b , 2003c). Thi s ethano l exposur e als o increase d cortica l Bax, pAkt, and pJN K expression. Taken together , thes e data strongl y suppor t th e occurrenc e o f enhance d apoptotic deat h concomitan t wit h oxidativ e stress . Cerebella o f 2-day-old rat s exposed to ethanol i n utero are hypoplastic and exhibit increased amounts ofapop tosis, beyond that occurring naturally (de la Monte and Wands, 2002) . I n culture d granul e neuron s isolate d from thes e cerebella , th e previou s ethano l exposur e generated increase d oxidativ e stress (dihydrorosarnin e fluorescence) an d apoptosi s (evidence d by phosphorylation of GSK-3ß, Akt, and Bad) , along with inhibitio n of insulin-stimulate d viabilit y an d pro-apoptoti c gen e expression (e.g. , increase d ba d expression) . Thus, ges tational ethano l exposur e impair s neurona l surviva l systems and insuli n signaling mechanisms durin g th e early postnatal period . These ethano l effect s appea r t o be mediated b y oxidative stress. The abov e reports document tha t ethano l ca n con comitantly elicit oxidative stress and enhance apoptosi s in the developin g brain . Using in vitro models, several laboratories have shown that oxidative stress can pla y a direct rol e in ethanol-mediated neuro n death . On e ap proach probe d th e causalit y of oxidative stress by determining i f thi s phenomeno n precede d o r followe d evidence o f apoptosis and/o r decrease d neuro n viabil ity (Ramachandra n e t al, 2003) . In culture d feta l ra t cortical neurons, confoca l microscopy of live cells preloaded wit h dichlorofluorescei n diacetat e illustrate d that ethano l ca n stimulat e RO S productio n withi n minutes o f exposure. Thi s initia l RO S burs t persisted for 2 hr an d wa s followed b y increased HN E i n neu ron mitochondria (withi n 2 hr; Fig. 16-2) . By 2 hr, an

FIGURE 16- 2 Ethanol-induce d oxidativ e stress originating in mitochondria. Feta l cor tical neurons , grow n on coverslips , were exposed t o ethano l (4. 0 mg/ml) fo r 2 hr a t 37°C. Afte r ! 3 /4hr, hydroethidiu m (Het ) (400 nM) , whic h detect s generatio n o f Superoxide anio n radicals , an d 5 0 nm Mi totracker Gree n (MG) , whic h label s mito chondria, wer e added t o the medium . Liv e

cell image s were acquired through confoca l microscopy wit h a n argo n lase r t o excit e MG an d an He-Ne543 laser to excite Het. A. This imag e identifie s enhance d generatio n of Superoxide anion radical s in discrete areas in th e cel l bod y an d i n th e processes . Th e Het fluorochrom e freel y crosse s th e plasm a membrane, i t is very specific for the superox ide anio n radical , and , a t nanomola r con centrations, i t preferentiall y localize s i n mitochondria. B . Mitochondri a ar e distrib uted i n th e perinuclea r regio n a s well a s i n the processes . C . Th e exten t o f the neurite s can b e appreciate d i n a differentia l interfer ence contrast imag e o f the neuron .

INTRACELLULAR EVENT S IN ETHANOL-INDUCE D NEURONA L DEATH 27 3 increase in annexi n V binding i s seen, along with increased Cytochrom e C release . Thes e event s wer e followed b y caspase-3 activation. This time course of ethanol responses , wit h increase d RO S conten t oc curring a s a n upstrea m event , suggest s causality . More convincin g evidenc e i s th e mitigatio n o f ethanol-mediated apoptosis by prevention o f oxidative stress vi a normalizatio n o f neuro n glutathion e con tent (Ramachandra n e t al. , 2003 ) and b y other antioxidant interventions (see Anti-oxident Interventions, below). Potential Role for Mitotoxic Effects of Ethanol The specifi c mechanism(s ) underlyin g ethanol induced apoptosi s and oxidativ e stress remains undetermined an d ar e likel y rnultifactoria l an d cel l type-dependent. One consisten t finding from numer ous laboratories , however , i s that ethano l i s a mito toxin, and mitochondrial damage may be an important player i n bot h th e enhance d productio n o f ROS an d apoptotic deat h associate d with ethanol exposur e (Hirano et al., 1992 ; Kukielka et al., 1994 ; Garcia-Rui z et al., 1995 ; Dev i e t al. , 1996 ; Che n e t al. , 1998 , 2002 ; Goodlett and Horn, 2001; Mooney and Miller, 2001 ; Bailey and Cunningham , 2002). With respec t t o th e fetus , there i s abundant evi dence tha t ethano l negativel y alter s mitochondria l function i n liver and brain . Among th e earlies t fetal related studie s ar e report s that , i n culture d feta l ra t hepatocytes, th e ethanol-relate d increase s i n RO S (H2O2, Superoxid e anio n radical ) an d membran e lipid peroxidatio n ar e parallele d b y morphologica l and biochemica l mitochondria l damag e suc h a s reduced comple x I an d I V activitie s an d decrease d synthesis of ATP (Devi et al, 1993, 1994) . Sinc e inhibition o f respirator y chai n component s stimulate s production o f ROS , i t ha s bee n propose d tha t thi s could be the origi n of the increase d RO S levels. More recently, this phenomenon ha s been reporte d i n feta l brain an d i n culture d cerebellar and cortica l neurons exposed to ethanol. Followin g an i n utero binge expo sure to ethanol (G1 7 and G18), mitochondria isolated from feta l ra t brain hav e elevate d level s o f the highl y pro-apoptotic produc t of lipid peroxidation, HNE (Ramachandran e t al., 2001). This i s accompanied by increased mitochondria l permeabilit y transitio n an d release o f pro-apoptotic proteins . Likewise, cerebellar neurons isolate d fro m ethanol-expose d 2-day-ol d ra t

pups exhibit signs of oxidative stress and activate pathways o f apoptosi s alon g wit h altere d mitochondri a morphology and decreased cel l number (d e la Monte and Wands , 2002) . Furthermore , i n vitro exposure of insulin-sensitive immatur e huma n PNET 2 neurona l cells t o ethano l produce s a variet y o f mitotoxi c re sponses, includin g a strikin g reductio n o f comple x IV/Cytochrome C oxidas e (CytOx ) expression . Although n o measures of CytOx activitie s have been reported, inhibitio n of this complex ha s been shown to stimulate formation of ROS (Shigenag a et al., 1994) . Parallel to impaired mitochondrial function is evidence of apoptosis. Similarly , following 24 hr of exposure t o ethanol , ra t postmitoti c cerebella r granular neurons sho w an inhibitio n o f multiple measure s of insulin-stimulated mitochondria l function , sign s o f apoptosis, an d generatio n o f H 2O2 (d e l a Mont e et al, 2001). Thus, disruption o f mitochondrial func tion i s a central even t in th e apoptoti c deat h o f cells, and oxidativ e stress, which i s often a product of mitochondrial damage , i s a prove n initiator of apoptosis. There i s compelling evidenc e tha t th e mitotoxi c effects of ethanol play a role in ethanol-mediated apop totic death o f neurons in the developing brain. Anti-oxidant Interventions Many anti-oxidan t intervention s aimed a t ameliorat ing th e effect s o f ethano l o n th e developin g brai n have use d vitami n E (oc-tocopherol) , a lipid-solubl e anti-oxidant that preferentiall y concentrates i n lipoid compartments o f the cell , wher e i t can bloc k mem brane lipid peroxidation . I t is present i n mitochondr ial membrane s (approximatel y 1 molecule pe r 210 0 molecules o f phospholipid) an d i s a highl y effectiv e peroxyl radica l tra p wit h a one-electro n oxidatio n product tha t ca n underg o several reactions that con sume (bu t ca n als o generate ) radical s (Lieble r an d Burr 1992) . As such, i t is a late-stage defense against the formatio n o f toxi c lipi d peroxidatio n product s such as HNE. This capability, combined wit h its relative low toxicity, makes its use an alluring anti-oxidant intervention strategy. Ethanol-induced deat h o f culture d neonata l ra t hippocampal cell s can be mitigated by treatment wit h vitamin E o r ß-caroten e (Mitchel l e t al. , 1999a , 1999b). Ethano l activate s caspase-3 and increase s at least on e marke r o f apoptosi s (annexi n V binding) . Both vitami n E an d th e anti-oxidan t Pycnogeno l (a bioflavonoi d mixture ) bloc k thes e response s

274 ETHANOL-AFFECTE D DEVELOPMENT (Siler-Marsiglio e t al , 2004) . Likewise , vitami n E enhances th e surviva l o f ethanol-expose d culture d cerebellar granule cells. This surviva l occurs concomitantly wit h increase d amount s o f th e anti-apoptoti c proteins Bcl-2 , Bcl-x l an d activate d Ak t kinas e (Heaton e t al. , 2004) . Th e mechanism s underlyin g the protectiv e effects o f vitamin E remain to be estab lished, bu t i t is tempting t o propose tha t blockad e o f membrane (possibly mitochondrial) lipid oxidation is an importan t factor . The settin g i s complex an d vitamin E treatmen t ca n normaliz e cellula r GS H con tent as well as prevent an ethanol-related reductio n of neurotrophin secretio n (Heato n e t al., 2004). The former manipulatio n can , b y itself , bloc k th e ethanol mediated apoptosi s o f culture d cortica l neuron s (Ramachandran etal., 2003). The ultimat e clinica l valu e o f anti-oxidan t treat ment fo r the neurotoxi c effects o f ethanol i n th e de veloping brai n remain s t o be determined . A possible insight int o th e futur e o f suc h intervention s i s re flected b y a study in whic h behavioral alterations induced b y ethanol wer e at best only partially alleviated by vitami n E treatmen t (Marin o e t al. , 2004 ; Tra n et al. , 2005) . Th e pathogenesi s o f FA S behavioral deficits i s complex an d oxidativ e damage t o neuron s is likely only one underlyin g mechanism. The abov e reports, however , d o suppor t th e concept s tha t ethanol ca n induc e oxidativ e stress i n neuron s fro m the developin g brai n an d tha t thi s i s one mean s b y which apoptotic deat h i s induced.

Although beyon d th e scop e o f this chapter, i t also must be kept in mind tha t ethanol-induced cel l death may also occur by other mechanism s such a s necrosis (Obernier e t al, 2002) . Awareness of the diversit y of death mechanism s i s important whe n conceptualiz ing possibl e interventions . It i s nonetheless unrealis tic t o expec t tha t blockin g th e activit y o f a singl e protein ca n rescu e a cel l fro m death . Indeed , i t ha s been show n tha t blockin g one mechanis m o f deat h may alter the time line and pathway of cell death, but not th e outcom e (Mille r e t al. , 1997 ; Stefani s e t al. , 1999; D'Mell o e t al , 2000 ; Keramari s et al. , 2000 ; Selznick et al, 2000) . Progress i n establishin g a causal link between ox idative stress and cel l death has been made . Throug h documentation o f relevant tim e lines , th e upstrea m effectors o f ethanol-mediate d oxidativ e stress i n th e neuron ca n b e pinpointed an d opportunitie s create d for reversin g or preventing th e relate d apoptosi s with anti-oxidant treatments. The latte r venture is an espe cially excitin g researc h direction , a s i t ma y b e on e path t o an intervention tha t does not have an advers e impact o n norma l brai n development . A s laboratories narro w thei r focu s o n th e underlyin g mecha nisms o f ethanol-mediate d deat h o f neurons , i t i s hoped tha t mor e potentia l therapeuti c intervention s will evolve.

Abbreviations AIF apoptosi s inducin g factor

SUMMARY AND CONCLUSION S

Evidence i s abundant that exposure to ethanol i n vivo or in vitro ca n caus e neurona l deat h an d tha t i t does so vi a multipl e pathways : the caspase-dependen t in trinsic an d extrinsi c pathways, and th e caspase-inde pendent pathway . Th e integration , o r cross-talk , among thes e pathway s i s extensive . Nevertheless , there i s compelling evidenc e tha t eac h o f the thre e pathways i s targete d b y ethanol . Furthe r analyse s of the effect s o f ethanol migh t b e bes t focuse d o n ke y upstream players such as p53 and receptors that medi ate death-relate d signals , a s wel l a s PARP , a playe r presumed t o be commo n t o all three pathways . Th e factors that define the effect s o f ethanol ar e the devel opmental stat e o f the cell s an d th e concentratio n o f ethanol. Thes e issue s ar e addresse d i n th e previou s chapter.

ATP adenosin e triphosphate CNS centra l nervou s system CytOx cytochrome

C oxidase

FADD Fas-associate d death domain FasL Fa s ligand G gestationa l day GSH glutathion e HNE 4-hydroxynonena l MDA malondialdehyd e P postnata l day PACAP pituitar y adenylat e cyclas e activatin g polypeptide PARP poly-adenosin e diphosphat e ribos e poly merase ROS reactiv e oxygen specie s

INTRACELLULAR EVENTS IN ETHANOL-INDUCE D NEURONA L DEATH 27 5 ACKNOWLEDGMENTS Wor k on thi s chapte r wa s supported by the Nationa l Institute o f Alcohol Abuse and Alcoholism an d th e Departmen t o f Veterans Affairs . References Al-Ghoul WM, Mille r M W (1989 ) Transien t expressio n of Alz-50-immunoreactivity i n developin g ra t neo cortex: a marke r fo r naturall y occurrin g neurona l death? Brain Res 481:361-367. Bailey SM, Cunningham C C (2002 ) Contribution o f mitochondria t o oxidativ e stress associated wit h alco holic liver disease. Free Radie Biol Med 32:11-16 . Bauer-Moffett C , Altma n J (1977) Th e effec t o f ethano l chronically administere d t o preweanlin g rat s o n cerebellar development : a morphologica l study . Brain Res 119:249-268. Benn SC , Wool f C ) (2004 ) Adul t neuro n surviva l strategies—slamming on th e brakes . Nat Re v Neurosci 5:686-699. Carloni S , Mazzon i E , Balduin i W (2004 ) Caspase- 3 and calpai n activitie s afte r acut e an d repeate d ethanol administratio n durin g the ra t brain growth spurt. J Neurochem 89:197-203 . Cheema ZF , Wes t JR , Miranda R C (2000 ) Ethanol in duces Fas/Apo [apoptosis]-l mRNAand cell suicide in th e developin g cerebra l cortex . Alcoho l Cli n Exp Res 24:535-543 . Chen J , Schenke r S , Frost o TA , Henderson G I (1998 ) Inhibition o f Cytochrome C oxidas e activit y b y 4hydroxynonenal: rol e o f HN E adduc t formatio n with enzyme subunits. Biochim Biophys Acta 1380: 336-344. Chen J , Schenker S , Henderson G (2002 ) HNE degra dation b y mitochondrial glutathion e S-transferase is compromised b y short-term ethano l consumption . Alcohol Clin Exp Res 26:1252-1258 . Chen, SY , Sulik K K (1996) Fre e radical s an d ethanol induced cytotoxicit y i n neura l cres t cells . Alcoho l Clin Exp Res 20:1071-1076. Climent E , Pascua l M , Renau-Piquera s J , Guerr i C (2002) Ethanol exposur e enhances cel l death i n the developing cerebra l cortex : rol e o f brain-derive d neurotrophic facto r an d it s signalin g pathways . JNeurosci Res 68:213-22 5. de la Monte SM , Neely TR, Canno n J , Wands JR (2001) Ethanol impair s insulin-stimulate d mitochondria l function i n cerebella r granul e neurons . Cel l Mo l Life Se i 58:1950-1960 . de la Monte SM, Wands JR (2001) Mitochondrial DN A damage an d impaire d mitochondrial functio n contribute t o apoptosi s o f insulin-stimulate d ethanol exposed neurona l cells . Alcohol Cli n Ex p Re s 25: 898-906.

(2002) Chroni c gestationa l exposur e t o ethano l impairs insulin-stimulate d surviva l an d mitochon drial function in cerebella r neurons. Cell Mol Lif e Sei 59:882-893 . Devi BG , Henderso n GI , Frost o TA, Schenker S (1993) Effect o f ethanol o n rat fetal hepatocytes: studies on cell replication, lipi d peroxidation an d glutathione . Hepatology 18:648-659 . (1994) Effec t o f acut e ethano l exposur e o n cul tured feta l ra t hepatocytes: relation t o mitochondr ial function. Alcohol Clin Exp Res 18:1436-1442. Devi BG , Schenke r S , Mazlou m B , Henderso n G I (1996) Ethanol-induce d oxidativ e stres s an d enzy matic defense s in cultured feta l rat hepatocytes. Alcohol 16:1—6 . Di Luzi o N R (1963 ) Preventio n o f the acut e ethanol induced fatt y live r b y antioxidants . Physiologis t 6 : 169-173. D'Mello SR , Kua n CY , Flavel l RA , Raki c P (2000 ) Caspase-3 i s required fo r apoptosis-associated DN A fragmentation bu t no t for cell deat h in neurons de prived o f potassium. J Neurosci Re s 59:24-31. Dreosti I E (1987 ) Micronutrients, Superoxide and th e fetus. Neurotoxicology 8:445-450. Dreosti IE , Patric k E J (1987 ) Zinc , ethanol , an d lipi d peroxidation i n adult and feta l rats . Biol Trace Ele ment Res 14:179-191. Garcia-Ruiz C , Morale s A, Colell A, Ballesta A, Rodes J, Kaplowitz N , Fernandez-Chec a J C (1995 ) Feedin g S-adenosyl-L-methionine attenuate s bot h ethanol induced depletio n o f mitochondria l glutathion e and mitochondria l dysfunctio n i n periporta l an d perivenotis rat hepatocytes. Hepatology 21:207-214 . Goodlett CR, Hor n K H (2001 ) Mechanism s o f alcohol induced damag e t o the developin g nervou s system . Alcohol Re s Health 25:175-184 . Heaton MB , Madorsk y I, Paiva M, Maye r J (2002b ) In fluence o f ethano l o n neonata l cerebellu m o f BDNF gene-deleted animals: analyses of effects o n Purkinje cells , apoptosis-relate d proteins , an d en dogenous antioxidants . J Neurobiol 51:160-176 . Heaton MB , Madorsk y I , Paiv a M , Siler-Marsigli o KI . (2004) Vitamin E amelioration o f ethanol neurotoxi city involves modulation o f apoptosis-related protei n levels i n neonata l ra t cerebella r granul e cells . De v Brain Res 150:117-124 . Heaton MB , Moore DB , Paiva M, Gibb s T , Bernar d O (1999) Bcl- 2 overexpressio n protect s th e neonata l cerebellum fro m ethano l neurotoxicity . Brai n Re s 817:13-18. Heaton MB , Moor e DB , Paiv a M, Madorsk y I, Mayer J, Shaw G (2003a ) Th e rol e o f neurotrophic factors , apoptosis-related proteins, and endogenou s antioxi dants i n th e differentia l tempora l vulnerabilit y of

276 ETHANOL-AFFECTE D DEVELOPMEN T neonatal cerebellu m t o ethanol. Alcoho l Cli n Exp Res 27:657-669. Heaton MB , Paiv a M, Madorsk y I, Mayer J, Moore D B (2003b) Effect s o f ethanol o n neurotrophi c factors , apoptosis-related proteins, endogenous antioxidants, and reactiv e oxygen species in neonatal striatum: relationship to periods of vulnerability. Dev Brain Res 140:237-252. Heaton MB , Paiv a M , Madoesk y I , Sha w G (2003e ) Ethanol effect s o n neonatal ra t cortex: comparativ e analysis o f neurotrophi c factors , apoptosis-relate d proteins, an d oxidativ e processes during vulnerable and resistant periods. Dev Brain Res 145:249-262. Heaton MB , Paiv a M , Maye r J , Mille r R (2002a ) Ethanol-mediated generatio n o f reactiv e oxyge n species in developing rat cerebellum. Neurosci Lett 334:83-86. Hirano T , Kaplowit z N , Tsukamot o H , Kamimur a S , Fernandez-Checa J C (1992 ) Hepati c mitochondr ial glutathione depletion an d progressio n o f experimental alcoholi c live r diseas e i n rats . Hepatolog y 16:1423-1427. Inoue M , Nakamur a K , Iwahashi K, Ameno K, Itoh M , Suwaki H (2002 ) Changes o f bcl-2 and ba x mRNA expressions in the ethanol-treate d mouse brain. Nihon Arukoru Yakubutsu Igakkai Zasshi 37:120-129. Jacobs JS, Miller MW (2001 ) Proliferatio n and deat h o f cultured fetal neocortical neurons: effects o f ethanol on th e dynamic s o f cel l growth . J Neurocyto l 30 : 391-401. Jang MH, Shi n MC , Ki m YJ, Chung JH, Yim SV , Kim EH, Ki m Y , Ki m C J (2001 ) Protectiv e effect s o f puerariaeflos agains t ethanol-induced apoptosi s o n human neuroblastom a cel l lin e SK-N-MC . Jp n J Pharmacol 87:338-342. Keramaris E , Stefani s L , MacLauri n J , Harad a N , Takaku K , Ishikawa T, Taketo MM , Robertso n GS , Nicholson DW , Slac k RS, Park D S (2000 ) Involvement o f caspas e 3 i n apoptoti c deat h o f cortica l neurons evoke d b y DNA damage . Mo l Cel l Neu rosci 15:368-379 . Kim YY, Park BJ, Kim DJ, Kim WH, Ki m S, Oh KS , Lim JY, Kim J , Park C , Par k S I (2004 ) Modificatio n o f serine 392 is a critical event in the regulatio n of p53 nuclear export and stability. FEBS Let t 572:92-98. Kornguth SE, Rutledge JJ, Sunderland E, Siegel F, Carlson I, Smollens J, Juhl U , Young B (1979) Impeded cerebellar development and reduce d serum thyroxine levels associated with fetal alcoho l intoxication . Brain Res 177:347-360. Kühn PE , Mille r M W (1998 ) Expressio n o f p5 3 an d ALZ-50 immunoreactivit y i n ra t cortex : effec t o f prenatal exposur e t o ethanol . Ex p Neuro l 154 : 418-429.

Kukielka E , Diche r E , Cederbaum AI (1994) Increase d production o f reactiv e oxyge n specie s b y ra t live r mitochondria after chroni c ethanol treatment . Arch Biochem Biophys 309:377-386. Liebler DC, Bur r JA (1992) Oxidation of vitamin E during iron-catalyze d lipid peroxidation : evidence fo r electron-transfer reaction s o f th e tocoperoxy l radi cal. Biochemistry 31:8278-8284. Light KE , Belcher SM , Pierc e D R (2002 ) Time cours e and manne r o f Purkinje neuro n deat h followin g a single ethano l exposur e o n postnata l da y 4 i n th e developing rat. Neuroscience 114:327-337 . Livy DJ , Maie r SE , Wes t JR (2001 ) Fetal alcoho l expo sure an d tempora l vulnerability : effects o f binge like alcoho l exposur e o n th e ventrolatera l nucleus of the thalamus. Alcohol Clin Ex p Res 25:774-780. Luo J, Miller MW (1999 ) Platelet-derived growth factormediated signa l transductio n underlyin g astrocyt e proliferation: sit e o f ethano l action . J Neurosc i 19:10014-10025. Marino MD , Akseno v MY, Kelly S J (2004 ) Vitami n E protects against alcohol-induced cel l loss and oxidative stres s i n th e neonata l ra t hippocampus . In t J Dev Neurosci 22: 363-377. Miller FD, Poznia k CD, Wals h GS (2000 ) Neuronal lif e and death : an essentia l role for the p5 3 family. Cel l Death Diffe r 7:880-888 . Miller M W (1999 ) A longitudinal study of the effect s o f prenatal ethano l exposur e o n neurona l acquisitio n and deat h i n th e principa l sensor y nucleu s o f th e trigeminal nerve : interaction wit h changes induce d by transection o f the infraorbita l nerve. J Neurocy tol 28:999-1015. (2003) Describin g th e balanc e o f cel l prolifera tion an d death : a mathematical model. J Neurobiol 57:172-182. Miller MW , Al-Ghou l WM , Murtaug h M (1991 ) Ex pression o f ALZ-50-immunoreactivity i n th e devel oping principa l sensor y nucleu s o f th e trigemina l nerve: effec t o f transectin g th e infraorbita l nerve . Brain Res 560:132-138. Miller MW , Küh n P E (1997 ) Neonata l transectio n o f the infraorbita l nerv e increase s th e expressio n of proteins relate d t o neurona l death i n th e principal sensory nucleu s o f the trigemina l nerve. Brai n Res 769:233-244. Miller TM , Moulde r KL , Knudso n CM , Creedo n DJ , Deshmukh M , Korsmeye r SJ , Johnso n E M J r (1997) Ba x deletion furthe r order s th e cel l deat h pathway i n cerebella r granul e cell s an d suggest s a caspase-independent pathwa y to cel l death . J Cell Biol 139:205-217 . Mitchell ES , Snyder-Kelle r A (2003 ) c-fo s an d cleave d caspase-3 expressio n afte r perinata l exposur e t o

INTRACELLULAR EVENTS IN ETHANOL-INDUCED NEURONAL DEAT H 27 7 ethanol, cocaine , or the combination o f both drugs. Dev Brain Res 147:107-117. Mitchell JJ , Paiva M , Heato n M B (1999a ) Th e antioxi dants vitami n E an d beta-caroten e protect agains t ethanol-induced neurotoxicit y i n embryoni c ra t hippocampal cultures. Alcohol 17:163—168 . (1999b) Vitami n E an d beta-caroten e protec t against ethano l combine d wit h ischemi a i n a n embryonic ra t hippocampa l cultur e mode l o f fetal alcoho l syndrome . Neurosc i Let t 263:189 192. Mooney SM , Mille r MW (1999 ) Effect s o f prenatal exposure to ethanol on systems matching: the number of neuron s i n th e ventrobasa l thalami c nucleu s of th e matur e ra t thalamus . De v Brai n Re s 117 : 121-125. (2001) Effect s o f prenatal exposur e to ethanol o n the expressio n of bcl-2, bax and caspas e 3 in the de veloping ra t cerebra l corte x an d thalamus . Brai n Res 911:71-81. Moore DB , Walke r DW , Heato n M B (1999 ) Neonata l ethanol exposur e alter s bcl-2 famil y mRN A levels in th e ra t cerebellar vermis . Alcohol Cli n Ex p Res 23:1251-1261. Morrison RS , Kinoshita Y, Johnson MD , Ghata n S , Ho JT, Garde n G (2002 ) Neurona l surviva l an d cel l death signalin g pathways. Adv Exp Me d Bio l 513 : 41-86. Oberdoerster J , Rabi n R A (1999 ) Enhance d caspas e activity durin g ethanol-induce d apoptosi s i n ra t cerebellar granul e cells . Eu r J Pharmaco l 385 : 273-282. Obernier JA , Bouldi n TW , Crew s F T (2002 ) Bing e ethanol exposur e i n adul t rat s causes necroti c cel l death. Alcohol Clin Exp Res 26:547-557. Olney JW , Tenkov a T , Dikrania n K , Mugli a LJ , Jer makowicz WJ , D'S a C , Rot h K A (2002a) Ethanol induced caspase- 3 activatio n i n th e i n viv o developing mouse brain. Neurobiol Dis 9:205—219. Olney JW , Tenkova T, Dikrania n K, Qin YQ , Labruyere J, Ikonomido u C (2002b ) Ethanol-induce d apop totic neurodegeneration in the developing C57BL/6 mouse brain. Dev Brain Res 133:115-126. Polster BM , Fisku m G (2004 ) Mitochondria l mecha nisms o f neura l cel l apoptosis . J Neuroche m 90 : 1281-1289. Porter A G (1999 ) Protei n translocatio n i n apoptosis . Trends Cell Biol 9:394-401. Rajgopal Y , Chetty CS, Vemur i MC (2003 ) Differentia l modulation o f apoptosis-associate d protein s b y ethanol i n ra t cerebral cortex and cerebellum . Eu r J Pharmaco l 470:117-124. Ramachandran V, Perez A, Chen J, Senthil D, Schenker S, Henderson G I (2001 ) I n uter o ethano l exposure

causes mitochondrial dysfunction which ca n resul t in apoptoti c cel l deat h i n feta l brain : a potentia l role fo r 4-hydroxynonenal . Alcoho l Cli n Ex p Re s 25:862-871. Ramachandran V , Watt s LT , Maff i SK , Che n J , Schenker S , Henderson G (2003 ) Ethanol-induced oxidative stres s precedes mitochondriall y mediate d apoptotic deat h o f cultured feta l cortica l neurons . J Neurosci Res 74:577-588. Resnicoff M , Rubin i M , Baserg a R , Rubi n R (1994 ) Ethanol inhibit s insulin-lik e growt h factor 1—mediated signallin g and proliferatio n of C 6 ra t glioblastoma cells. Lab Invest 71:657-662. Reyes E , Ot t S , Robinso n B (1993 ) Effect s o f i n uter o administration o f alcoho l o n glutathion e level s in brai n an d liver . Alcoho l Cli n Ex p Re s 17 : 877-881. Saito M, Sait o M, Ber g MJ, Guidotti A, Marks N (1999 ) Gangliosides attenuat e ethanol-induce d apoptosi s in ra t cerebellar granul e neurons . Neuroche m Re s 24:1107-1115. Selznick LA , Zheng TS , Flavel l RA , Rakic P, Roth KA (2000) Amyloi d beta-induce d neurona l deat h i s bax-dependent bu t caspase-independent . J Neu ropathol Exp Neurol 59:271-279 . Shaw S, Rubin KP, Lieber CS (1983 ) Depressed hepati c glutathione an d increased diene conjugate s in alco holic live r disease. Evidenc e o f lipid peroxidation . Dig Dis Sei 28:585-589. Shigenaga MK, Hagen TM, Ame s BN (1994) Oxidativ e damage an d mitochondria l deca y i n aging . Pro c Natl Acad Sei USA 91:10771-10778, Siler-Marsiglio KI , Sha w G , Heato n M B (2004 ) Pyc nogenol® an d vitami n E inhibi t ethanol-induce d apoptosis in rat cerebellar granule cells. Neurobiology 59:261-271. Sohma H , Ohkaw a H , Hashimot o E , Saka i R, Sait o T (2002) Ethanol-induce d augmentatio n o f annexi n IV expressio n in ra t C 6 gliom a an d huma n A54 9 adenocarcinoma cells . Alcoho l Cli n Ex p Re s 26-.44S-48S. Stefanis L , Par k DS , Friedma n WJ , Green e L A (1999 ) Caspase-dependent an d -independen t deat h o f camptothecin-treated embryoni c cortica l neurons . J Neurosci 19:6235-6247. Tran TD , Jackso n HD , Hor n KH , Goodlett C R (2005 ) Vitamin E doe s no t protec t agains t neonata l ethanol-induced cerebella r damag e o r deficit s i n eyeblink classical conditioning in rats. Alcohol Clin Exp Res 29:117-129. Valverde F, Lopez-Mascaraque L, de Carlos JA (1990) Distribution and morpholog y of Alz-50-immunoreactive cells in the developing visual cortex of kittens. J Neurocytol 19:662-671 .

278 ETHANOL-AFFECTE D DEVELOPMEN T Vaudry D , Roussell e C, Basili e M , Falluel-More l A, Pa- Youn g C , Klock e BJ , Tenkov a T , Cho i J , Labruyer e J , mantung TF, Fontain e M , Fournie r A , Vaudr y H , Qi n YQ , Holtzma n DM , Rot h KA , Obe y J W Gonzalez B J (2002 ) Pituitar y adenylat e cyclase - (2003 ) Ethanol-induced neurona l apoptosis in vivo activating polypeptid e protect s ra t cerebella r gran - require s BAX in th e developin g mous e brain . Cel l ule neuron s agains t ethanol-induced apoptoti c cel l Deat h Diffe r 10:1148-1155 . death. Proc Nati Acad Sei USA 99:6398-6403.

17

Neural Crest an d Developmental Exposure t o Ethanol Susan M. Smit h Katherine A. Debelak-Kragtor p

The realizatio n that th e developin g neura l cres t i s a target o f ethanol date s t o the origina l description s o f the feta l alcoho l syndrom e (FAS ; Lemoin e e t al. , 1968; Jone s et al. , 1973) . A key observatio n wa s th e finding o f a characteristi c facia l dysmorpholog y tha t often, bu t no t necessarily , accompanies th e neurobe havioral an d neurocognitiv e deficits . Earl y evidenc e that ethanol target s the neura l cres t comes fro m a series o f mous e mode l studie s (Suli k e t al. , 1981).Ye t neural cres t encompasses man y mor e structure s tha n the face , includin g ke y components o f the periphera l nervous system. The presen t chapte r summarize s the literature on neura l cres t and ethanol , an d highlight s issues that can benefi t from furthe r research.

OVERVIEW O F NEURAL CREST DEVELOPMEN T

The neura l crest , sometime s know n a s th e fourt h germ layer , i s a transien t ye t critica l contributo r t o

embryogenesis. I t i s of neuroectodermal origin , yet i t differentiates int o populations a s diverse as peripheral neurons, glia , chondrocytes , melanocytes , an d con nective tissue . It s accessibility an d migrator y properties hav e mad e i t attractiv e fo r stud y fo r ove r 10 0 years, especially in avia n and amphibia n model s an d more recentl y i n mouse . Recen t wor k has greatl y expanded ou r understandin g o f neura l cres t develop ment a t th e cellula r an d molecula r levels . Befor e addressing ho w alcoho l affect s th e developmen t o f this comple x population , it is first helpful to under stand ho w thes e originat e an d t o appreciat e wher e their progeny may be found in the adult . An in-dept h summary o f neural cres t ca n b e foun d i n th e excel lent book by Le Douarin an d Kachie m (1999). Neural cres t arise s at the borde r betwee n th e pre sumptive neura l plat e an d latera l ectoderm . I t ap pears quit e earl y i n development , shortl y afte r th e onset o f neurulation. It s specification an d inductio n involve signal s betwee n ectoder m an d neura l plat e and th e underlyin g mesendoderm. These signal s are

279

280 ETHANOL-AFFECTE D DEVELOPMENT incompletely understoo d an d appea r t o includ e members o f the Wnt, fibroblast growth factor (FGF) , and bone morphogenic protein (BMP ) families (Basch et al., 2004). Their induction occurs in a rostrocaudal manner commensurat e wit h overal l developmen t along tha t axis . Figur e 17- 1 illustrate s these popula tions as defined by the expressio n of the transcriptio n factor slug. The upwar d rollin g o f th e neura l plat e durin g early somitogenesis place s neura l crest populations at the dorsa l lip o f the neura l folds. Shortl y before this, or as the neural folds fuse t o form the neura l tube (depending o n the species), the neura l crest undergoe s a transformation fro m epitheli a to mesenchyme. Subsequently, the cells delaminate an d migrate int o the periphery o f th e embryo . Thei r migrator y route s ar e defined b y extracellula r matri x protein s suc h a s fi bronectin an d specifi c integrin s (notabl y oclß4 ) o n the neura l crest Onc e neura l cres t cell s reac h thei r targets they differentiate int o appropriate cel l types.

Three distinctiv e population s o f neural cres t ar e defined: crania l (o r cephalic) , cardiac , an d truncal . Cranial neura l cres t originate s fro m th e forebrain , midbrain, an d selecte d hindbrai n segment s (rhom bomeres 1/2 , 4, 6, 7/8). Transplantation studie s show that their rostrocaudal identity (for example, mandible vs. hyoid) is dictated b y the leve l of the neuraxi s fro m which they originate, a s defined by their expression of segment polarit y gene s (hox, emx, otx). Recen t evi dence suggest s ther e i s some plasticit y in thi s aspect (Trainor e t al. , 2002) . Crania l neura l cres t cell s have th e mos t divers e fates, and , dependin g o n thei r level o f origi n an d tim e o f emergence , the y differ entiate t o for m th e facia l bone s an d cartilage , melanocytes, toot h papilla , sensor y component s o f certain crania l nerves , an d othe r feature s (Tabl e 17-1). Cell s formin g the connectiv e tissue s emerg e first, followed b y neuronal an d melanocyti c lineages . Thus bot h th e positiona l identit y and differentiatio n fate o f crania l neura l cres t cell s ar e determine d

FIGURE 17- 1 Morphogeneti c sequenc e o f neura l crest inductio n an d migration . Step s describin g chick neural cres t development ar e exemplifie d by changes in th e expressio n o f th e transcriptio n facto r slug. A . Prechordal plat e stage . At this time, neura l cres t cell s are slug negative . Arrow s indicate thei r locatio n (als o see Fig . 17-2A) . Slug expressio n a t thi s stag e i s re stricted t o migratin g cells of the primitiv e streak. B. 3 somite stage . The first pre-migratory neural cres t cell s have appeared at the dorsal margin of the folding neural plate (arrow) . C. 6 somite stage. As the neura l fold s fuse, neura l cres t cells receive their cues to begin mi gration. Younger populations ar e being induced mor e caudally, and these have a low slug expression (arrow). D. 1 0 somite stage. Neural cres t migration occurs i n a

rostral-to-caudal sequence. Cell s tha t are rostral to the otic vesicle have commenced migration. E. 1 5 somite stage. The segmenta i natur e of neural crest migration is depicted. Arrow s indicate th e migrator y waves tha t emerge fro m rhombomere s 2 , 4 , an d 6 . Thes e cell s are fated to form facial bones and cartilage. Within th e hindbrain, neural cres t cells that migrat e slightl y later will attai n neurona l fates . Rostra l neura l cres t cell s have entere d th e mesenchym e tha t form s th e uppe r facial region . F. 1 8 somite stage. Neural cres t populations have reache d thei r targets within the face . With their cessatio n o f migration , the y down-regulat e slug (arrow) and begi n to differentiate. Migration o f neural crest from trun k and cardia c regions is still on-going at this stage.

NEURAL CREST AN D EARL Y DEVELOPMENTAL EXPOSURE TO ETHANO L 28 1 TABLE 17- 1 Neura l crest contributions to the adult Cranial Bones and cartilag e o f the face and skull* Cartilaginous bones: nasal capsule (ect-, mesethmoid, interorbita l septum), basipresphenoid, scleroti c ossicles, ethmoid, pterygoid, Meckel's cartilage, quadratoarticular, hyoid (basihyal, entoglossum, basi- , epi-, ceratobranchial), otic capsule (pars cochlearis [partly] , parotic process ) Membranous bones: nasal, vomer, maxilla, jugal, quadratojugal, palatine, mandibular Membranous bones of skull: frontal (partly) , parietal, squamosal, columell a Cranial nerves * Sensory ganglia: portions of nerves V (posterior portion o f trigeminal ganglion), VII (facial , root ganglion), IX (glossopharyngeal superior ganglion), X (vagal, jugular ganglion) Neurons o f the sympathetic an d parasympathetic ganglia : superior cervical, ciliar y (otic, submandibular, lingual , ethmoid , spheropalatine) Glia and Schwan n cells for all cranial ganglia and nerves Other crania l tissues Melanocytes of skin and internal organs (except the retinal pigmented epithelium ) Dermis of scalp Dermis, smooth muscle, an d adipose tissue of skin in face and ventral neck region Méninges of the forebrain (pachymeninx and leptomeninx) ; not its blood vessels Connective tissu e of cranial glands: lacrimal, pituitary, parathyroid, salivary, thymus, thyroid Connective tissues associated wit h facial muscle s Calcitonin-producing C cells Type I chemoreceptor cell s and type II supporting cells of the caroti d body Portions o f the teeth: odontoblasts, cementoblast s Eye: stromal and endothelia l cells of cornea and orbit* Ciliary muscles Cardiac * Smooth muscl e o f the outflow tract Mesenchymal cell s of the outflo w tract Conotruncal cushion s of the outflo w tract Cardiac gangli a Truncal Sensory neurons and glia l cells of the dorsa l root ganglia1 The entiret y of the autonomie nervou s system: Catecholaminergic an d noncatecholaminergic neuron s and satellite cell s of the sympathetic ganglia and plexuses Cholinergic an d noncholinergic neuron s and satellite cells of the parasympathetic ganglia and plexuses Enteric gangli a Schwann cells of the peripheral nerve s Neuroendocrine (adrena l chromaffin an d small intensely fluorescent) cells of the adrenal medulla t Parasympathetic gangli a of the pancrea s Merkel cells (mechanoreceptor cell s of the mammalian whisker follicle) Melanocytes of skin and interna l organs * Affected b y ethanol, evidence based on in vivo work in animal models or humans. * Affected b y ethanol, evidence based on cell culture work. Source: Adapted from Le Douarin and Kacheim (1999); this list may be incomplete.

predominantly prior to their emigration from the neu- smoot h muscl e an d connectiv e tissu e mesenchym e ral tube. o f the outflo w trac t and aorti c arches. They also par Thé cardia c neural cres t is a relatively small, spe- ticipat e i n th e remodelin g o f thes e earl y structure s cialized subpopulatio n tha t emerge s fro m neura l int o the aorta , pulmonary aorta, and affiliate d vascu tube at the level between the oti c vesicle and somite lature . Deficits i n this neural crest population lead to 4, includin g rhombomere s 6- 8 (Kirb y 1999) . Lik e sever e cardia c defects , suc h a s double-outle t righ t cranial crest , they migrate into the third , fourth, an d ventricle , paten t truncu s arteriosus , an d tetralog y of sixth pharyngeal arches, but instead contribute to the Fallot .

Z82 ETHANOL-AFFECTE D DEVELOPMEN T Neural cres t population s cauda l t o somit e 5 con tribute t o muc h o f th e periphera l nervou s syste m (both sympatheti c an d parasympatheti c neuron s an d their supportin g Schwan n cells) , the enteri c nervou s system, and rnelanocytes . Cells tha t follow ventromedial migratory routes contribute to the peripheral nervous system . Thes e cell s onl y migrat e throug h th e rostal hal f o f eac h sclerotome , an d thi s restrictio n contributes t o th e segmentatio n o f th e sympatheti c and dorsa l roo t ganglia . Cell s fate d t o becom e rnelanocytes follo w a dorsolatera l rout e an d migrat e somewhat later . Trun k neura l cres t cell s appea r t o have a much greater plasticity with respect to fate an d identity and henc e see m t o have a greate r regenera tive capacit y than tha t o f cranial and cardia c populations; whe n on e regio n i s ablated, neighborin g cell s migrate i n an d replac e th e missin g cells . I n general , fairly larg e losse s must occu r befor e anatomica l an d physiological changes are seen. A preponderance o f genetic and experimenta l dat a show that th e basi c rules and signal s governing neural crest development ar e largel y conserved amon g vertebrates. This is best shown in the numerous human neu rocristopathies that are recapitulated b y specific mous e null mutants . Ther e ca n als o b e smal l difference s i n the timin g or identity of regulatory molecules; fo r these finer details investigators are encouraged t o consult the primary literature on their species of interest.

NEURAL CREST POPULATION S AFFECTED B Y ETHANOL

For obvious reasons, the best evidence tha t ethanol exposure disrupt s neural crest development come s fro m animal models. As discussed below, the critical window for huma n neura l cres t development i s between week s three an d eight postconception , an d embryo s tha t young ar e generall y unavailabl e fo r study . A furthe r limitation i s the apparen t lac k of systemic description s of neural crest derivatives and thei r fate i n human s exposed to heavy gestational alcohol exposure . Thus, this section emphasize s anima l models , incorporate s hu man observation s whe n possible , an d highlight s in stances wher e th e huma n evidenc e i s suggestive an d would benefit from clarifying work. The reade r should further not e tha t findings in rodent and chick , the two most popular models for this work, are largely in agree ment. Their concurrence enhance s thei r applicability and relevance for human development.

Ethanol Targets Crania l Skeletal Derivative s The facia l change s observe d i n human s wit h heav y gestational alcohol exposur e are similar to known neu rocristopathies, promptin g th e firs t hypothese s tha t neural crest was targeted by ethanol exposure (Lemoine et al, 1968 ; Jone s et al, 1973) . The facia l change s i n humans includ e midfacia l flattening , shortene d nos e with upturne d nares , micrognathia , an d hypoplasti c maxilla (Clarre n an d Smith , 1978 : Pria s e t al, 1982 ; Johnson e t al., 1996 ; Mattso n e t al, 1997) . Midfacial clefting an d degree s o f holoprosencephaly ar e some times seen . Anthropométri e studie s (Moor e e t al. , 2001, 2002) report that facial dimension s are reduce d overall i n childre n wit h FA S (also know n a s dysmorphic fetal alcohol spectrum disorde r [FASD]) and to a similar, but lesser extent, in those with alcohol-related neurodevelopmental disorde r (ARND; also known as nondysmorphic FASD) . Diagnostic s emphasiz e sof t tissue changes , particularl y the tri o of thin uppe r lip , reduced o r absen t philtrum , an d smal l palpebra l fissures in affected individuals. The grea t valu e of these changes arise s from thei r ease of assessment and thei r relative uniquenes s t o alcoho l (an d toluene ) expo sure. No t surprisingly , the y ar e importan t compo nents of diagnostic screens for ARND and FA S (Astley and Clarre n 1996) . This emphasi s o n sof t tissu e may seem contradictor y because facia l skeleta l muscl e i s not neura l cres t derived . Sof t tissu e shape, however , is dictated b y the underlyin g skeletal element s (proof of thi s i s in th e forensi c scientist' s abilit y t o recreat e a person' s likenes s fro m th e bar e skull) . Thus , th e size and shap e o f the philtrum , lip , and palpebra l fissures ar e a direc t consequenc e o f dysmorpholo gies i n th e underlyin g neural crest-derive d skeleta l elements. Several othe r conclusion s ca n b e draw n from th e human evidence . Th e facia l appearanc e transcend s racial characteristics, reinforcin g the robus t natur e of ethanol-induced changes . Second , i n n o instanc e does ethano l exposur e alte r the segmenta l identit y of the face . Effect s instea d are manifested in dimensional shifts i n otherwis e norma l structures , indicatin g tha t ethanol target s aspect s o f neural cres t cellularit y an d outgrowth, a finding that provide s important clue s to locating the molecular targets of ethanol action . Animal studie s confir m th e hypothese s tha t alco hol targets the developin g neural crest. Studies of rats prenatally expose d t o ethano l hav e th e constellatio n

NEURAL CREST AND EARL Y DEVELOPMENTAL EXPOSUR E T O ETHANO L 28 3

of malformation s i n th e craniofacia l skeleton tha t i s characteristic o f human s wit h FA S (Edward s an d Dow-Edwards, 1991) . Murin e studie s hav e refine d the tempora l windo w o f vulnerabilit y (Suli k e t al. , 1981; Webste r e t al , 1983) . I n mic e o n gestationa l day (G ) 7 , i.e., at the tim e o f gastrulation, ethanol exposure (as two acute dose s 4 hr apart) causes craniofa cial defect s consisten t wit h huma n FAS . Thes e defects includ e midlin e reductions , reduce d midface , and hypoplasti c primordi a o f th e maxill a an d mandible. Ethanol exposur e one da y later (G8, head fold stage ) also affects th e face , but cause s a facial ap pearance more simila r to that o f DiGeorge syndrome (Sulik e t al. , 1986) . Fo r bot h treatmen t windows , these morphologica l change s ar e precede d b y signifi cant cel l deat h withi n thei r respectiv e neura l cres t precursors a s well a s i n adjacen t regions (Kotc h an d Sulik 1992a , 1992b ; Dunt y e t al, 2001) . These two critical windows for mouse correspon d t o weeks 3 and 4, respectively, in human pregnancy. Identical ethano l doses at G9 or G10 d o not appreciably affect facia l appearance (Webste r et al., 1983) , reinforcing the obser vation that ethanol target s early events i n neural crest induction, expansion , and/o r migration , rathe r tha n later events of differentiation . A simila r phenotype occur s i n chic k embryo s exposed t o acute ethanol . A s with mouse, the precis e facial changes depend o n stage of treatment (Cartwrigh t and Smith , 1995b) . Gastrulation-stag e (HH4 ) o r neurulation-stage (HH6 ) exposur e produce s a wider, taller chic k fac e wit h a shorte r maxill a an d nasa l bone, wherea s exposur e a t th e onse t o f migratio n (HH10-12) produces a n overal l hypoplastic fac e (De belak-Kragtorp, Moyers , an d Smith , unpublishe d results). The facia l appearanc e i s preceded b y signifi cant cel l deat h i n region s includin g th e crania l chondrogenic precursors of neural crest, and immunos taining corroborates the los s of these neura l crest populations (Cartwrigh t an d Smit h 1995a ; Cartwrigh t et al. , 1998) . Pretreatmen t o f chic k embryo s wit h a caspase-3 antagonis t prevente d bot h th e ethanol induced cel l deat h an d th e change s i n bea k lengt h (Debelak-Kragtorp, Moyers , an d Smith , unpublishe d results). Thi s finding reinforce s th e concep t tha t cel l death contribute s t o th e facia l dysmorphology . Sus ceptibility to ethanol-induced apoptosi s i s greatest for premigratory neura l crest ; migrator y an d postmigra tory cell s ar e mor e resistan t (Cartwrigh t an d Smith , 1995b). Thi s timin g suggest s tha t uniqu e develop mental event s prio r t o neura l cres t migratio n ma y

govern thei r sensitivity ; some o f these target s ar e dis cussed below . These animal data predict that human s a t weeks 3 and 4 of gestation ar e th e mos t sensitiv e to the facia l teratogenic effects . A stud y i n pregnan t macaque s reinforces thi s prediction . Onc e weekl y dose s o f alcohol (mea n bloo d ethano l concentratio n [BEG ] -220 mg/dl) are associated with facial dysmorpholog y in the offspring onl y when th e exposure coincide wit h G19 o r G20 (Astle y et al, 1999) . Thes e findings suggest that a sufficiently hig h BE G during a critical window i s require d t o induc e th e characteristi c facia l changes o f FAS. The implicatio n i s that i f a pregnan t woman ha s restricte d o r n o acces s t o alcoho l durin g the critica l window, her chil d ma y lack the facia l dysmorphology, despit e havin g profound nervous system deficits fro m late r exposure . The diagnosti c implica tions of this are obviou s for professionals and affecte d families. Ethanol Targets Neuronal Derivatives of Neural Crest The literatur e on this point i s sadly scant with respec t to humans. I n mice , ethano l cause s significan t apoptosis i n th e oti c regio n an d i n th e neura l cres t an d placodal component s o f th e crania l sensor y gangli a (Dunty e t al. , 2001) . Ethanol-expose d chic k embryo s display reductions in cranial nerve cellularity and aberrant migratio n o f their neurona l branche s (Cartwrigh t and Smith , unpublishe d results) . The functiona l con sequences o f suc h losse s hav e no t bee n studied . There are hints, however , that thes e population s als o are targete d i n humans . Fo r example, a n infan t wit h FAS often ha s a reduced rootin g reflex when nursing , a behavio r partl y dependen t o n th e abilit y o f facia l cranial nerve s to sense an d proces s a tactile stimulus. This tactil e los s ca n b e s o significan t tha t som e affected individual s requir e sof t o r liqui d diet s be cause the y hav e troubl e sensin g foo d textur e an d when i t i s appropriate t o swallow . There ca n als o b e significant hearin g losse s and persisten t ear cana l in fections tha t ar e partl y attribute d t o change s i n th e neural crest-derive d bon e an d cartilag e structure s and i n the acoustica l nerves , which ar e supported b y glia an d Schwan n cell s originatin g from neura l cres t (Church e t al , 1996 , 1997) . Furthe r investigatio n into crania l an d periphera l nerves , an d th e conse quences o f any suc h dysfunctions , shoul d b e a hig h priority.

284 ETHANOL-AFFECTE D DEVELOPMEN T The situatio n regardin g neural cres t precursors of the periphera l nervou s system i s even les s clear. Em bryos treated a t G7 and G8 hav e increased cel l deat h within region s populated b y trunk neural cres t (Suli k et al , 1988 ; Kotc h an d Sulik , 1992b) . Th e conse quences o f such losse s hav e ye t to b e detailed . Seg mentation o f the dorsa l roo t gangli a appear s norma l by gros s inspection, althoug h cultur e studie s sugges t that ethano l impair s th e surviva l an d outgrowt h o f DRG neuron s (Do w an d Riopelle , 1990) ; reduce d synthesis of and response s to trophic factor s ar e note d (Dow and Riopelle, 1990 ; Beaton et al, 1992; Bradley et al. , 1995) . Advers e affect s o n th e sympatheti c an d parasympathetic nervou s syste m hav e no t bee n re ported fo r humans an d i n anima l models . Impaire d balance i s observed , bu t follow-up testin g impli cates targets in the centra l nervou s system rather tha n the neura l crest-derive d periphera l nervou s syste m (Church e t al, 1997 ; Roebuc k e t al, 1998) . More over, obstructive bowel problems characteristi c o f impaired enteri c nervou s syste m developmen t ar e no t widely reported. Whereas gli a an d supportin g cells of the brain are appreciated a s a target of ethanol (Chap ter 18) , littl e attentio n ha s bee n pai d t o supportin g cells o f the periphera l nervou s system . I n summary , our bes t answe r t o this questio n i s that these populations ca n b e targete d b y ethanol, bu t th e functiona l consequences ar e essentially unexplored. Ethanol Targets Cardiac Neura l Crest Derivative s Outflow trac t anomalie s ar e mor e frequen t i n chil dren wit h FA S than i n th e genera l populatio n (Abel , 1990), promptin g studie s int o potentia l effect s o f ethanol o n th e cardia c neura l crest . I n mous e an d chick models , ethano l exposur e cause s apoptosi s o f cardiac neura l cres t precursor s (Kotc h an d Sulik , 1992b; Cartwright et al, 1995b) . On th e basis of extirpation studie s (Kirby, 1999) , suc h losse s should resul t in aorticopulmonar y defect s suc h a s persistent trun cus arteriosus , double-outle t righ t ventricle , an d tetralogy o f Fallot . Indeed , ethano l cause s suc h de fects i n bot h mous e (Daf t e t al. , 1986 ) an d chic k (Fang e t al. , 1987 ) wit h a n incidenc e o f 10%-20% . These dysmorphologies are preceded by the expecte d malpositioning an d reduction s o f the cona l cushion s (Daft e t al., 1986) and hypoplasi a of the proxima l bulbar ridge s (Fan g e t al. , 1987 ; Bruyèr e an d Stith , 1993). The critica l window for these effect s i s during

the tim e o f cardiac cres t migration , invasion , and ex pansion int o th e outflo w trac t and pharyngea l arche s (G8.5 fo r mouse, Daf t e t al. , 1986 ; 72-8 0 hr incuba tion in chick, Fang et al., 1987) . But are these change s actually du e t o th e neura l cres t losses ? Ou r vie w is that the heart defects represent defect s in cardiac pop ulations othe r tha n neura l crest . W e expose d chic k embryos to ethanol a t critical times o f cardiac neura l crest an d outflo w trac t development ; despit e signifi cant losse s o f cardia c crest , inspectio n a t late r tim e points foun d normal neura l crest distribution s within the outflo w tract , an d n o aorticopulmonar y o r septa l defects (Caviere s and Smith , 2000) . Bruyère an d colleague s (1994a ) not e inconsisten cies between th e hear t defects commonly see n i n hu man prenata l alcoho l exposur e an d thos e cause d i n the laborator y b y neura l cres t ablation . I n ethanol exposed humans , th e mos t commo n cardia c defect s are no t aorticopulmonary . Rather , the y ar e isolate d ventricular septal defect s (VSDs) and atria l septal de fects (ASDs ; -75 % o f congenital cardia c anomalies) . These tw o septa ar e no t neura l cres t derived . VSD s and ASD s ar e generall y secondar y response s t o pri mary change s i n flow dynamics (Kirby , 1999 ; Firull i and Conway , 2004) . Significant effects i n cardiac output, heart rate , metabolism, an d cardiomyocyte differ entiation ar e documente d i n model s o f prenata l ethanol exposur e (Nyquist-Batti e an d Fréter , 1988 ; Ruckman e t al, 1988 ; Adicke s et al, 1993 ; Bruyèr e and Stith , 1994a ; Bruyèr e e t al , 1994b) . Fan g an d colleagues (1987 ) suggest that ethano l ha s a biphasi c effect o n the developing heart, with lower BECs caus ing VSD s (perhap s targetin g cardiomyocytes ) an d higher concentration s targetin g aorticopulmonar y events (an d cardia c crest) . Lower ethano l exposures , though deletin g som e cardia c crest , ma y allo w th e survivors t o repopulat e an d replac e thei r missin g neighbors. Determinin g th e exten t t o whic h neura l crest losse s contribut e t o cardia c defect s i n FA S will require additiona l research . Carefu l morphologica l studies using , fo r example , th e lacZ-wnf l reporte r mouse (i n whic h neura l cres t ca n b e followe d b y merit o f lac Z expression ) coul d nicel y resolv e thi s question. Ethanol Targets Melanocyte Derivative s Alcohol likel y ha s n o long-ter m consequence s o n melanocyte development , althoug h thi s conclusio n

NEURAL CREST AND EARL Y DEVELOPMENTAL EXPOSUR E TO ETHANO L 28 5 rests o n negativ e data . Melanocyte s ar e th e pig mented cells within the dermis, and to our knowledge there ar e n o report s o f reduce d pigmentatio n o r al tered pigmenta l pattern s in eithe r individual s prenatally exposed t o alcoho l o r expose d mice. Melani n i s still synthesize d i n salamande r neura l cres t culture d with ethanol , an d it s unusual perinuclea r depositio n may b e secondar y to microfilament dysarrangement s (Hassler an d Moran , 1986) . Th e pigmente d chick s we have hatched sho w no sig n of altered feather col oration (Smith , Tessmer, an d Debelak-Kragtorp , un published results) . This lack of effect ma y reflect th e lack of segmental identit y within melanocytes, which facilitates thei r abilit y to expan d an d compensat e fo r losses, Other Neural Cres t Populations Targeted b y Ethanol There ar e hints i n the literatur e that additional neu ral cres t subpopulations may be affecte d b y ethanol . Carones an d colleague s (1992 ) an d Mille r an d Beauchamp (1988) document multiple case s of ocular defect s involving the corne a endothelia , a neural crest-derived populatio n that contributes to the anterior segmen t o f th e eye . Toot h problem s ar e men tioned, althoug h i t wa s no t indicate d whethe r thi s involves th e neura l crest-base d papilla . Similarly , it is no t know n whether problem s o f thymocyte matu ration an d thyroi d hormone productio n are linked to aberrant strom a o f their respectiv e glands. A similar question ca n be raise d with the altere d signal s of the hypothalamic-pituitary-adrenal (HPA ) axis . I n tha t light, i t i s worth notin g tha t a cel l lin e popula r for studies o f ethanol-mediate d neurona l signaling , PC12 cell s (Messing et al, 1986 ; Luo et al, 1999) , is an adrenochromaffi n tumo r originatin g from neura l crest lineage of the adrenal medulla. Again, a systematic stud y o f neura l cres t derivative s i n human s (Table 17-1 ) would provide important clues as to the effects o f ethanol o n nonclassi c neura l cres t populations.

EVENTS OF NEURA L CREST DEVELOPMENT AFFECTED BY ETHANOL

Data fro m anima l models , a s discussed above, poin t to earl y event s of neural cres t induction , determina-

tion, an d migratio n a s th e majo r target s for ethano l teratogenicity (Fig . 17-2) . Evidenc e summarize d be low indicate s tha t n o singl e even t i s affected; rather , multiple target s likely lead t o a cumulativ e effect o n this cell population .

Neural Induction A key, early event of embryogenesis is the anterio r extension o f the prechorda l plat e and it s subsequent in duction o f the neura l plate, includin g neural crest, at the midlin e o f th e overlyin g ectoderm . Prechorda l plate siz e affect s neuronal—an d neura l crest — population size . Ethano l reduce s th e migrator y ca pacity o f thes e anterio r mesoderma l cell s i n bot h chick (Sander s et al. , 1987 ) and zebrafis h embryo s (Blader and Strahle , 1998) . Unfortunately, any consequences fo r neura l cres t population siz e hav e yet t o be tested . Reduce d prechorda l plat e migratio n ma y also contribut e t o th e holoprosencephali c aspect s o f acute prenata l alcoho l exposur e (Suli k e t al. , 1988 ; Blader and Strahle , 1998) , althoug h a major midlin e mediator, sonic hedgehog (shh), does not appea r to be a direct target of ethanol i n this model .

Neural Cres t Migratio n Rovasio an d colleague s hav e detaile d ho w ethano l impairs neura l cres t migration . Co-cultur e o f chic k embryos ( 3 somites ) wit h 0.25 % ethanol cause s significant reduction s i n th e numbe r o f crania l an d truncal neura l cres t cell s exitin g the neura l tube , as visualized i n cross-section vi a immunostainin g wit h the neura l crest-specifi c antibod y NC- 1 (Rovasi o and Battiato , 1995) . Primary cultures o f neural crest cells treate d wit h 15 0 m M ethano l los e thei r mes enchymal appearanc e an d becom e spindl e shaped . They hav e fewe r filopodia , increase d surfac e bleb bing, an d pronounce d rearrangement s o f th e acti n cytoskeleton tha t ar e accompanie d b y dramati c re ductions i n thei r migrator y capacit y (Rovasi o an d Battiato, 2002) . Cranial an d trunca l population s ar e similarly affected . Lowe r BEC s (100-20 0 mg/dl) d o not affec t migration , but the y d o lea d t o thickene d actin and tubulin filaments in cultured primary neural cres t (Hassle r an d Moran , 1986) . Cell-cel l con tacts ar e disrupted . Migrator y pathway s in th e trun k are directed by the somites; the segmenta l anomalie s in alcohol-expose d somite s (Sander s an d Cheung ,

286 ETHANOL-AFFECTE D DEVELOPMEN T

A. Induction events

1990) ma y hav e deleteriou s effect s o n neura l cres t migration and fate .

Reduced prêcherai plate extension would reduce the number of neural crest ceils induced at the neuroepithelial margin.

B. Apoptotic events Apoptosis due to: - oxidative stress; - altered Ça** signals; - loss of L1-mediated cell-cell interactions; - loss of retinoid signals; - loss of trophic signals

C. Migration and differentiation Altered identity „ and/or delayed emergence from neural tube.

Impaired migration and/or cellular expansion into target tissues Delayed or arrested - differentiation within target tissue regions.

FIGURE 17- 2 Candidat e target s o f ethano l durin g neural cres t development . A . Targets durin g the win dow of neural cres t induction . A chick embry o a t th e prechordal plat e stag e (HH5+ ) wa s prepare d t o vis ualize slug transcript s b y i n sit u hybridization . Th e presumptive neura l crest-formin g regio n (shaded ) lies at the boundar y o f the neuroectoder m an d ectoderm . B. Targets tha t coul d caus e neura l cres t apoptosi s are summarized. Show n i s a chic k embry o o f th e 1 7 somite stage . Apoptotic cell s are visualized by the vita l dye acridine orange . Thi s embryo was exposed t o -60 mM ethanol a t gastrulation, as described b y Cartwright and colleague s (1998) . C . Target s durin g th e windo w of neural cres t migratio n and differentiation . Thi s im age depict s a transvers e vie w throug h th e hindbrai n (level o f rhombomer e 6 ) o f a chic k embry o a t th e

Neural Crest Expansion Reduced prechorda l plat e siz e and increase d apopto sis could reduc e th e populatio n size of neural crest. I t should b e noted , however , tha t embryo s hav e robus t repair mechanisms; autoregulator y signals and group effect response s ca n restor e populatio n siz e i n re sponse t o additio n o r loss . Signal s tha t regulat e th e size of the neura l cres t population includ e shh, bmp, fgf, an d wnt. The degre e t o whic h thes e ar e ethano l targets likely depends o n the timin g and dos e of exposure. The homeoti c selecto r gene , msxl, is expressed in premigrator y and migrator y neural crest and i s associated wit h bot h expansio n and apoptosis . On e repor t notes nea r ablatio n o f msx2 expressio n i n ethanol treated mous e embryos and ca l variai osteoblasts (Rifa s et al, 1997) , however , the BECs i n this study are very high. Preparation s expose d t o mor e physiologicall y relevant exposure s d o no t affec t msxl (Cartwrigh t et al , 1998) . Ethano l ma y affec t shh, a mediato r o f directional outgrowth , an d suc h suppressio n migh t contribute t o neura l cres t reduction s (Ahlgre n e t al, 2002). Sh h withi n th e fac e i s transiently reduced b y ethanol exposur e commensurat e wit h reduce d mes enchymal proliferatio n an d elevate d fg f an d ms x expression (Smit h e t al , 2001) . Thes e change s are proportionat e t o late r shift s i n directiona l out growth. Ye t ther e i s n o evidenc e tha t an y suc h changes ar e associate d wit h neura l cres t apoptosi s a t these time s (se e below) . Much mor e wor k is neede d to understan d th e effect s o f ethano l o n thes e mor phoregulatory cues . Apoptosis Cell deat h i s an importan t componen t o f ethanol ter atogenicity. Sando r (1968 ) observe d a prominen t cellular "necrosis " withi n th e neuroepitheliu m o f ethanol-exposed chick embryos, a region that includes neural cres t precursors . Suli k an d colleague s (1981 ) documented cytoplasmi c an d nuclea r extrusion s fro m

36 somite stage . The pharny x lies ventral to the hind brain. Neura l cres t cell s ar e visualize d b y i n sit u hy bridization for slug.

NEURAL CREST AND EARL Y DEVELOPMENTAL EXPOSURE TO ETHANO L 28 7 neural folds of mice treated with ethanol at G8. Similar foci of necrotic and degenerating cells were observed in neuronal population s o f the mous e o n G 9 (Banniga n and Burke , 1982; Banniga n and Cottell , 1984) . Sulik' s work using vital dye s and high-magnificatio n histology had establishe d tha t thi s "necrosis" actually represents pyknosis (Suli k e t al. , 1986 , 1988 ; Kotc h an d Sulik , 1992a, 1992b) . Thi s pyknosi s i s par t o f a n ethanol induced apoptoti c death ; caspas e inhibito r pretreatment prevents it (Cartwright et al, 1998 ) and the DNA is labele d b y termina l transferas e (TUNEL^positive ) (Cartwright et al, 1998; Dunty et al., 2001). Apoptosis i s a well-described , active , energy dependent process that is a mechanism for the removal of unwanted cells (Reed, 2000). It is prominent i n th e developing embryo, for example, i n removing interdigital webbing. It also is used to eliminate cells that have aberrantly activate d thei r proliferativ e machinery , such a s self-recognizin g T cells ; tumor s represen t cells that fai l t o apoptose whe n promitoti c signals become activated . Teratogens frequentl y act by expanding existin g domain s o f endogenou s cel l death . Under thi s model , th e teratoge n recruit s susceptibl e populations by merit of their latent capacit y for apoptosis. This certainl y is the cas e for acute ethanol . Suli k and colleague s (Suli k et al. , 1988 ; Kotc h an d Sulik , 1992b; Dunt y e t al. , 2001 ) hav e comprehensivel y mapped the cell death patterns for normal mouse embryogenesis an d followin g ethano l treatment . Thi s work elegantl y shows that ethanol-induce d apoptosis is selective . Populations ar e no t affecte d uniforml y and th e precis e targe t depends o n th e stag e o f treatment. Region s o f cel l deat h coincide d wit h tissue s and structure s affected b y ethanol treatment , includ ing neural crest of cranial, cardiac, and trunk popula tions. A critical implicatio n o f this work is that ther e are inheren t propertie s withi n certai n cel l popula tions that govern their susceptibility to ethanol. These properties are reviewed in detail below . Our recen t work with chic k embryo s emphasize s the mechanisms underlying ethanol-induced deat h of neural cres t cells . Crania l neura l cres t apoptosi s i n the chic k coincide s wit h th e endogenou s deat h o f neural cres t precursor s i n rhombomere s 3 an d 5 . Thus, we hypothesize that ethanol exposur e might activate thos e endogenou s signals . Thes e signal s ar e well described and involv e the wnf-dependen t activation o f bmp4 synthesi s within rhombomeres 3 and 5 . Bmp4 act s i n a n autocrine-paracrin e manne r t o

stimulate msxl expressio n and the apoptosi s sequenc e (Graham e t al , 1994) . Neithe r bmpi no r msxl, however, ar e elevate d i n th e hindbrain s o f ethanol exposed chic k embryo s (Cartwrigh t e t al. , 1998) . Thus the initia l machinery of ethanol-induced apop tosis mus t lie upstrea m o f the endogenou s deat h signals. We recently ascertained that these signals involve the activatio n by ethanol o f a pertussis toxin-sensitive G protei n an d intracellula r Ca 2+ transient s (Garic Stankovic, et al., 2005; see below).

HOW DOES ETHANOL KILL NEURAL CREST CELLS?

Four majo r mechanism s hav e receive d considerabl e scrutiny: oxidative stress, LI-mediated cell adhesions , induction o f Ca2+ transients, an d retinoid-dependen t signals. It should be stressed that evidence for the first three i s quit e good . Ethano l ha s pleiotropi c effects , and i t is quite likely that ultimately these mechanism s will prove to be interrelated . Oxidative Stress Ethanol exposur e leads to oxidative stress in the neu ral crest . Migrator y an d postmigrator y neura l cres t cells lac k appreciabl e Superoxid e dismutas e (SOD ) activity (Davi s e t al. , 1990) , an d exposur e o f neura l crest t o a free-radica l sourc e (suc h a s xanthin e oxi dase) i s sufficient t o impede their survival (Che n an d Sulik, 1996) . Superoxide , hydroge n peroxide , an d hydroxy radical concentrations all increase after expo sure to 50-100 mM ethanol, an d upo n supplementa tion with SOD thos e concentrations return to normal (Davis e t al. , 1990 ; Che n an d Suli k 1996) . I n whol e embryos treate d wit h ethanol , lipi d peroxidase s an d Superoxide radical s ar e selectivel y enriche d withi n the neura l cres t an d neuroepitheliu m (Kotc h e t al. , 1995). Co-cultur e o f whol e embryo s (Kotc h e t al. , 1995) or postmigratory neural crest (Davi s et al., 1990 ; Chen an d Sulik , 1996 ) wit h free-radica l scavenger s (SOD o r catalase) improve s cell surviva l and reduce s ethanol-induced cytotoxicity . I t shoul d b e noted , however, that thes e treatment s provid e onl y a partial rescue. Iro n chelator s suc h a s deferoxamin e an d phenarthroline als o offe r protectio n agains t ethanolinduced cel l deat h (Che n an d Sulik , 2000) . This is an interestin g finding because ethano l exposur e can elevate iro n concentration s a s i n th e brai n ste m

288 ETHANOL-AFFECTE D DEVELOPMENT (Miller et al., 1995) . In two separate studies, however, vitamin E is less effective a t ameliorating these effects , a surprisin g outcome give n tha t thi s free-radica l scav enger preferentially acts on lipi d an d membran e com partments (Davis et al, 1990; Chen and Sulik, 1996) . There is little question that free radical s contribute to neura l cres t death , jus t a s oxidativ e stres s con tributes t o man y instance s o f neuronal deat h i n th e fetus. Les s clear i s the precis e rol e o f oxidative stress in the sequenc e o f events. Does ethanol itsel f directly cause this stress? Neural crest at these stages does no t express CypZE l (Deltou r e t al , 1996) , bu t i t doe s have a n ADH 4 capable o f metabolizin g ethano l a t high concentration s (Deltou r et al., 1996 ; Haselbec k and Duester , 1998) . Th e resultin g imbalance s i n NADP/NADPH ratio s woul d invok e a metaboli c stress, an effec t wel l documented fo r adult heart an d liver. Alternatively, these increase d free-radica l levels could simpl y reflect tha t the cell s ar e dying, perhaps through changes in the mitochondria l transition por e that ar e par t o f apoptosis itself . Studie s t o dat e hav e emphasized tim e point s o f 1 2 hr an d late r followin g ethanol treatment. Additional studies looking at much earlier times would clarify thi s question. Our curren t view i s that th e pro-oxidan t effects ar e likel y secondary t o th e primar y action o f ethanol, becaus e o f this lack o f earlier time poin t dat a an d becaus e o f the ef fects on Ca2+ signaling detailed below. Cell Adhesion and LI Ethanol also disrupts the development o f neural crest cells and promote s their apoptosis through LI , a cell adhesion molecul e (CAM ) o f th e immunoglobuli n CAM superfamily. Ethano l inhibit s LI-mediated cel l adhesion (Charnes s e t al. , 1994 ; Ramanatha n e t al. , 1996; Wilkemeyer and Charness, 1998) . LI i s strongly expressed b y neural crest (Chen e t al., 2001), and i n this context i t is noteworthy that disruptions of neural crest cell-cel l adhesion , a t leas t a t th e leve l o f gap junctions, result s i n thei r apoptosi s (Bannerma n et al, 2000). LI deficit s caus e craniofacial dysgenesis in human s (CRASH syndrome) that shares aspects of that wit h alcoho l exposur e (Franse n e t al. , 1995) . Ethanol likel y act s o n L I throug h it s abilit y t o si t within hydrophili c pocket s o f membran e proteins , stabilizing protei n structur e an d enhancin g o r re pressing its activity (Mihic et al., 1997). In this model, ethanol woul d bind LI to disrupt crucial trophic cue s that the protein supplies to neural crest.

Low concentrations o f n-octanol block the action s of LI; they block LI-mediate d cel l adhesio n (Wilke meyer e t al. , 2000) . Furthermore , n-octano l amelio rates th e toxi c consequence s o f ethanol o n culture d mouse embryo s (Che n e t al , 2001) . Thei r appear ance i s mor e norma l an d the y exhibi t significantl y less cel l death , includin g neura l crest-populate d re gions. N-octanol ma y act by competitively inhibiting ethanol fro m bindin g t o th e hydrophili c pocket s of LI. Ethanol antagonism o f LI i s prevented by the pep tides NAPVSIPQ (NAP ) and SALLRSIP A (SAL), frag ments o f th e large r glial-derive d activity-dependen t neuroprotective protei n (ADNP ) an d activity dependent neurotrophi c facto r (ADNF) , respectively. These peptide s confe r protectio n agains t numerou s neuronal insults , includin g ethano l (Spon g e t al. , 2001). NA P reduce s severa l of the advers e actions o f ethanol, suc h a s th e antagonis m o f L I adhesio n (Wilkemeyer et al., 2002, 2003), the depletio n of glutathione (Spon g e t al., 2001) , an d th e growt h inhibi tion of embryos, including craniofacial regions (Spong et al, 2001; Wilkemeyer et al, 2003). How NAP does this is unclear . Charnes s and colleague s speculat e that disruptio n of LI adhesion s may trigger an oxidative deat h proces s calle d anoikis (Wilkemeye r e t al. , 2000, 2003). NAP may override these death signal s as part o f its broader neuroprotection . I t may als o have direct effects o n LI action . Further investigation s into LI wil l provide additional, important insights into the molecular mechanisms that underlie the sensitivity of neural crest to ethanol . Ca2+ Signaling The earl y mous e blastocys t respond s t o ethano l (0.10%) wit h immediat e generatio n o f an intracellu lar Ca 2+ transient , th e sourc e o f whic h i s throug h PLC-mediated inosito l 1,4,5-triphosphat e (IP3 ) re lease (Stacheck i e t al, 1994 ; Rou t et al, 1997) . IP3 dependent Ca 2+ transient s occu r spontaneousl y i n neural crest, and thes e ar e essential for the neura l differentiation o f neura l cres t cell s (Care y an d Mat sumoto 1999 , 2000) . Ethanol treatmen t of HH8 chick embryos stimulates an immediate ( < 5 sec) intracellular Ca 2"1" transient. Moreover, this transient is essential to induce neural crest apoptosis, because pretreatmen t with a n intracellula r Ca 2+ chelator (BAPTA-AM ) prevents th e ethanol-induce d bu t no t endogenou s cel l death (Debelak-Kragtor p et al., 2003) . Generation o f

NEURAL CREST AND EARLY DEVELOPMENTAL EXPOSURE TO ETHANO L 28 9 this transien t require s th e activit y o f a pertussi s toxin-sensitive G protein , PLCß , an d ÏP 3 releas e (Garic-Stankovic et al, 2005). Gai2, but not Gail or Gaio, i s expressed i n thes e cells . Extracellula r Ca 2+ contributes abou t 30 % of this Ca 2+ signa l (Debelak Kragtorp e t al. , 2003) . We recentl y detecte d a Ca 2+ oscillation i n ethanol-treate d neura l crest , wit h a repeat ever y 1 5 t o 2 0 sec ove r 1 t o 2 min (Garic Stankovic, Hernandez , an d Smith , unpublishe d results). A potentia l contributio n o f thi s oscillatio n is unde r investigation . Thes e event s ar e sensitiv e to inhibitio n b y decano l (Garic-Stankovi c e t al. , 2006). The sourc e o f thi s Ca 2+ transien t involve s th e Gi/oßy stimulatio n o f a phosphoinositidyl-PLCß . Ethanol-mediated amplificatio n of endogenous Giß y and phosphoinositidyl-PLC ß signal s i s implicated i n the dopaminergi c rewardin g properties o f ethanol i n neurons (Ya o et al. , 2002 , 2003) . Ethano l enhance s the activatio n of Gas, protei n kinase A, and cAM P response elemen t bindin g protei n signals , a s wel l a s Gißy, and synergis m throug h thes e pathways permit s intracellular signalin g a t ligan d level s tha t normall y would no t transduc e signal . Th e identit y o f th e G-protein couple d recepto r an d othe r participant s in this transient is actively being investigated . It i s of in terest that tw o different cellula r target s and outcome s of ethanol, addiction/rewar d an d apoptosis , us e over lapping signaling pathways. Retinoic Acid Signaling One mechanis m propose d fo r ethanol-induced cran iofacial dysmorpholog y involve s th e perturbatio n o f retinoic aci d (RA ) signaling . Some isoform s o f alco hol dehydrogenas e (ADH) , notabl y ADH4, wil l me tabolize bot h ethano l an d retinol , a precursor o f RA (Chou e t al. , 2002) . Severa l group s hav e postulate d that ethano l compete s wit h retino l metabolism , an d thus R A production , i n th e developin g embry o (Duester, 1991 ; Grumme r et al., 1993 ; Deltour et al, 1996). R A signaling through th e nuclea r retinoi c aci d receptors (RARs ) directl y regulate s th e expressio n o f genes critical for the development of numerous embryonic structure s including neura l crest . These includ e the ho x genes, whic h defin e rostral-caudal patternin g of the embry o (Längsten and Gudas , 1994) ; the los s of retinoid signalin g leads t o ablatio n o f posterior hind brain regions , including thei r neura l cres t derivatives (Maden e t al , 1996) . R A also sustain s th e identit y

(Plant e t al. , 2000 ) an d outgrowt h (Schneide r e t al. , 2001) o f the frontonasa l primordial , i n par t throug h its support o f fgß an d sh h pro-proliferativ e cues . R A deficiency a t thes e stage s lead s t o failur e o f fron tonasal an d forebrai n outgrowth a s well a s apoptosis in craniofacial precursors (Zile, 2001). Until recently , thi s hypothesi s wa s challenging t o investigate, becaus e o f the lo w circulating concentra tions o f R A (K d o f R A fo r RAR s i s i n th e 0. 5 n M range) an d th e hig h retino l store s of standard roden t models. To circumvent these difficulties , Dueste r an d colleagues generate d null-mutan t mic e t o identif y ADH1 as a major ethanol dehydrogenase an d ADH4 as a majo r retino l dehydrogenas e (Deltou r e t al. , 1999). Clinicall y relevant ethanol concentration s can compete fo r retino l oxidatio n fo r bot h enzymes ; Molotkov an d Dueste r (2002 ) cit e K { value s o f 0.040-3.8 mM fo r ADH1 an d 6-1 2 m M fo r ADH4. They furthe r sho w tha t bot h ethano l an d adhl nul l mutation prolong s retino l half-lif e throug h suppres sion o f RA synthesis rather tha n R A catabolism. On e hundred m M bu t no t 1 0 mM ethano l inhibit s R A synthesis i n G7. 5 bu t no t G8. 5 mous e embryos , a s measured b y a lacZ-RAR E reporte r construc t (Del tour e t al. , 1996) . I n ra t whole-embry o cultures , retinol and ethano l ha d synergisti c effects o n dysmorphology, bu t n o effec t o n tissu e R A and retina l con tent (Che n e t al , 1996a) . Usin g liqui d diet s (36 % calories as ethanol; BE G ~260 mg/dl) and i n utero exposure, however, Zachman and colleagues report that tissues fro m 12-day-ol d mous e fetuse s ha d two - t o three-fold highe r retino l an d lowe r R A expression, a s well a s altered RA R and cellula r retino l bindin g pro tein transcrip t level s (Zachma n an d Grummer , 1998). In the developin g quail, 1 0 nM R A rescued the cardiac dysmorpholog y cause d b y exposur e t o 1.0 % ethanol (Twal and Zile, 1997) . Evidence o f a specific interaction betwee n ethanol and endogenou s retinoi d signalin g i n neura l cres t is lacking. ADH ma y be expresse d in premigratory neural cres t cells , althoug h thi s particula r expressio n is not alway s observed an d doe s no t appea r t o correlat e with RA production (An g et al., 1996 ; Haselbeck an d Duester, 1998) . Mor e certai n i s th e expressio n o f ADH4 (an d the subsequen t productio n o f RA) in migratory neura l cres t an d th e developin g craniofacial mesenchyme (Ang et al, 1996) . I t is likely, therefore, that any effects o f ethanol on retinoid signaling would act o n th e migrator y and postmigrator y neural crest , rather tha n affectin g premigrator y populations wit h

290 ETHANOL-AFFECTE D DEVELOPMEN T respect t o thei r survival . Indeed , ethanol-exposed , RARE-lacZ reporter mice have reduced expressio n of this RA-dependent construct, an outcome mos t likely explained b y a reductio n o f RA-mediate d signalin g (Deltouretal., 1996) . A major cavea t is that endogenous R A specifically acts o n frontonasa l development . Onc e patterned , there is no apparent retinoid requiremen t for maxilla, mandible, an d hyoi d outgrowt h (e.g. , Wedden , 1987 ; Dickman et al., 1997 ; Schneide r e t al, 2001). At this time, the retinoid hypothesi s doe s no t adequately explain the sensitivity of neural crest to ethanol-induced apoptosis, nor doe s i t wholly account for the change s in maxillar y an d lowe r ja w development . Nonethe less, this remains an attractive hypothesis and i t merits additional, careful investigation . GENETIC FACTORS

Genetic factor s modulat e neura l cres t sensitivit y t o ethanol, jus t a s they modulat e th e ris k o f other con genital anomalie s an d neurobehaviora l outcomes . We screene d 1 1 leghor n chicke n strain s and foun d that they differed widel y in their sensitivity to ethanolinduced apoptosi s withi n neura l cres t an d th e subse quent craniofacia l outcom e (Debela k an d Smith , 2000; S u e t al , 2001) . Culture d neura l cres t cell s from inbre d C57B1/6 J mic e ar e mor e sensitiv e t o ethanol-induced toxicit y tha n thos e fro m outbre d ICR mice (Che n e t al., 2000). Their sensitivit y correlates positivel y with indicator s of membran e fluidit y and negativel y with GM1 gangliosid e content . Moreover, GM 1 applicatio n attenuate s ethanol-induce d damage t o C57B1/6 J neura l cres t (Che n e t al. , 1996b). Chick strain s also differ wit h respect to aorticopulmonary defects associated with the cardia c neural cres t (Bruyèr e an d Stith , 1993) . I n al l thre e models, ethano l metabolism i s not appreciabl y differ ent, whic h suggest s tha t factor s intrinsi c t o th e em bryo and neural crest are responsible . Maternal an d feta l capacit y fo r ethano l metabo lism affect s ris k fo r ethanol-induce d malformation s of th e fetus . Th e mor e efficien t alcoho l dehydroge nase allele IB * 3 confers increase d protection against teratogenic effect s o f ethano l i n a huma n cohor t (McCarver e t al. , 1997) , an d contribution s o f Cy tochrome P45 0 2E 1 ma y b e protectiv e (Rashee d et al., 1997) . These findings extend to facial dysmor phology and reinforc e the notio n tha t alcoho l rathe r

than acetaldehyd e i s th e proximat e teratoge n (Da s et al., 2004) .

FUTURE DIRECTIONS

It i s now clea r that the effect s o f ethanol on facia l ap pearance represen t a respons e a t a relatively narro w developmental window . Facial appearance remain s a highly useful diagnosti c tool, and th e stud y of its neural crest precursors continues to provide important insights int o th e molecula r mechanism s tha t underli e neurotoxic effect s o f ethanol. On e suc h avenu e i s to investigate whethe r ethano l exposur e ultimatel y affects common signaling pathways within otherwise diverse cell types. It i s disconcertin g tha t th e consequence s o f ethanol exposur e t o neura l crest-derive d structure s other tha n th e fac e remai n largel y unstudied , because thes e population s contribut e t o critica l activities suc h a s hearing , vision , textur e sensation , chewing, th e autonomi e nervou s system , an d hor monal action s tha t includ e th e adrena l medulla , pi tuitary, parathyroid , an d thyroi d glands . Studie s o f these less-appreciate d neurochristopathie s an d thei r consequences fo r healt h i s an importan t gap i n FA S research. Th e addressin g of this questio n shoul d be come a high priority in the assessmen t of affected in dividuals an d i n detaile d investigatio n usin g anima l models.

ABBREVIATIONS ADH alcoho l dehydrogenas e ADNF activity-dependen t neurotrophi c facto r ADNP activity-dependen t neuroprotective protein ARND alcohol-relate d neurodevelopmenta l disorder ASDs atria l septal defects BEG bloo d ethano l concentratio n BMP bon e morphogeni c protein CAM cel l adhesion molecul e FAS feta l alcohol syndrom e FASD feta l alcoho l spectru m disorder FGF fibroblas t growt h factor G gestationa l day

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294 ETHANOL-AFFECTE D DEVELOPMENT Roebuck TM , Simmon s RW , Richardso n C , Mattso n SN, Rile y E P (1998 ) Neuromuscula r response s t o disturbance o f balanc e i n childre n wit h prenata l exposure t o alcohol . Alcoho l Cli n Ex p Re s 22 : 1992-1997. Rout UK , Krawet z SA , Arman t D R (1997 ) Ethanol induced intracellula r calcium mobilizatio n rapidl y alters gene expressio n i n the mous e blastocyst . Cell Calcium 22:463-474 . Rovasio RA , Battiato NL (1995 ) Role o f early migratory neural cres t cell s i n developmenta l anomalie s in duced b y ethanol. Int J Dev Bio l 39:421-422 . (2002) Ethano l induce s morphologica l an d dy namic change s on i n viv o an d i n vitr o neural cres t cells. Alcohol Clin Ex p Res 26:1286-1298. Ruckman RN , Messersmit h DJ , O'Brie n SA , Getson PR , Boeckx RL, Morse DE (1988 ) Chronic ethanol exposure in the embryoni c chick heart: effect o n myocardial function an d structure. Teratology 37:317-327. Sanders EJ , Cheun g E (1990 ) Ethano l treatmen t in duces a delayed segmentation anomaly in the chick embryo. Teratology 41:289-297. Sanders EJ , Cheun g E , Mahmu d E (1987 ) Ethano l treatment inhibit s mesoderm cel l spreadin g in th e gastrulating chick embryo. Teratology 36:209-216. Sandor S (1968) The influenc e of aethyl alcohol o n th e developing chick embryo. II. Rev Roum d'Embr et de Cyt Série d'Embr 5:167-171. Schneider RA , Hu D , Rubenstei n JL, Maden M , Helm s JA (2001) Local retinoi d signalin g coordinates fore brain an d facia l morphogenesi s b y maintainin g FGF8 and SHH. Developmen t 128:2755-2767 . Smith SM , S u B, Tessmer LA, Debelak KA, Flentke GR , Hahn S H (2001 ) Prenata l alcoho l exposur e redi rects soni c hedgeho g signalin g during craniofacia l morphogenesis. Alcohol Clin Ex p Res 25:35A. Spong CY , Abebe DT , Goze s I , Brennema n DE , Hil l JM (2001 ) Prevention o f feta l demis e an d growt h restriction i n a mous e mode l o f feta l alcoho l syn drome. J Pharmacol Exp Ther 297:774-779. Stachecki JJ, Yelian FD , Schult e JF , Leach RE , Armant DR (1994 ) Blastocys t cavitation i s accelerate d b y ethanol- o r ionophore-induce d elevatio n o f intra cellular calcium. Biol Reprod 50:1—9 . Su B, Debelak KA , Tessmer LL , Cartwright MM, Smit h SM (2001 ) Genetic influence s on craniofacia l outcome i n a n avia n model o f prenatal alcoho l expo sure. Alcohol Clin Ex p Res 25:60-69. Sulik KK, Cook CS , Webste r WS (1988 ) Teratogens and craniofacial malformations : relationship s t o cel l death. Development 103(Suppl):213-232 . Sulik KK , Johnston MC , Daf t PA , Russell WE, Dehar t DB (1986 ) Feta l alcoho l syndrom e an d DiGeorg e anomaly: critica l ethano l exposur e period s fo r

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18 Glial Targets o f Developmental Exposure t o Ethanol Consuelo Guerri Gemma Ruber t Maria Pascual

The wor d glia i s derived fro m th e Gree k wor d gliok, meaning glue , whic h Vircho w (Letterer , 1958 ) ap plied i n the sens e of nerve glue. The traditiona l point of view of glia a s a passive component of the centra l nervous system (CNS) has changed drasticall y during the pas t decade . Gli a ar e no w recognize d a s activ e partners wit h neuron s a s participant s i n neurotrans mission and they play essential roles in axonal conduction, synapti c plasticity , an d informatio n processin g (Nagler et al, 2001; Ullian e t al., 2001, 2004 ; Fields Graham, 2002) . Further , gli a ca n respon d t o insults and ar e capabl e o f proliferatin g throughou t lif e (Chen and Swanson, 2003). In th e adul t huma n brain , gli a outnumbe r neu rons by one order of magnitude. There are two classes of glia : microgli a (whic h mediat e inflammator y re sponses i n th e CNS ) an d macroglia . The latte r cell s are oligodendrocyte s (whic h for m th e insulatin g niyelin sheaths) and astrocytes, the most paradigmatic glia (whic h are abundan t an d ubiquitou s withi n th e CNS). The presen t chapte r focuse s on astrocytes.

ROLE OF GLI A IN THE DEVELOPIN G CENTRAL NERVOUS SYSTE M

Glia ar e presen t an d integra l throughou t CN S de velopment. Glial-neurona l interaction s play critical roles in multiple developmenta l events . For example, radial glia (RG ) provide physical an d chemica l guid ance fo r th e migratio n o f young neuron s fro m th e embryonic proliferativ e zone s int o th e developin g cortex (Rakic , 1972 ; Hatte n an d Mason , 1990 ) (se e Chapter 3) . When th e migratio n is completed, mos t RG transform into astrocytes. RG als o serve as a rnultipotential precurso r cell , a s the y ar e abl e t o self renew an d generat e neuron s (Malatest a e t al. , 2000 ; Noctor e t al, 2001 , 2002 ; Göt z e t al. , 2002 ; Sana i étal., 2004). Astroglia an d glial-derive d factors ar e key to synaptogenesis, since they promote th e formation of mature functional synapse s (Nagler et al., 2001; Ullian e t al., 2001, 2004). Disturbances o f glia or of neuronal-glial communication durin g th e well-establishe d critica l

295

296 ETHANOL-AFFECTE D DEVELOPMEN T periods o f brain developmen t ca n caus e irreversibl e deficits i n CN S functio n (Hatten , 1999 ; Lammens , 2000; Ros s an d Walsh , 2001 ; Crespe l e t al, 2002). Such findings strongly support the crucia l role of glia in development an d the notion tha t their dysfunction underlies anomalies in brain development . An increasin g number o f studies shows (a) that a prime target of ethanol withi n the developing brain is astrocytes (Guerr i and Renau-Piqueras , 1997 ; Guerr i et al, 2001) and (b) that ethanol impairs astrogliogenesis. The presen t chapte r reviews clinical, experimen tal, and i n vitro evidence about the actions of ethanol on astrogli a and thei r functions . Specia l attentio n i s paid t o the effect s tha t prenatal ethano l exposur e has on neura l ste m cell s (e.g. , RG ) an d t o the action s of ethanol o n astroglia l proliferatio n an d cel l survival . Since astrogli a regulat e synaptogenesi s and synapti c transmission, the potentia l effect s o f ethanol o n thes e processes are also discussed.

GLIAL ABNORMALITIES CAUSE D BY ETHANO L

Since th e first neuropathological studies on childre n with feta l alcoho l syndrom e (FAS ) (Clarre n et al , 1978), abnormalitie s in glial development hav e been suspected a s contributin g t o th e advers e effect s o f ethanol o n th e developin g brain . Ethanol-expose d brains exhibit abnormal glial placement primaril y associated wit h th e méninge s (meningea l neuroglia l heterotopias) (Clarre n e t al., 1978 ; Peiffe r e t al, 1979 ; Clarren, 1986) , gyra l malformations, cerebral dysgenesis, and, in some cases, reactive gliosis (Peiffer e t al., 1979; Wisniewski et al, 1983) . White matter, a repository o f glia , appear s t o b e affecte d b y prenata l ethanol exposur e (PEE) . Severa l studie s usin g mag netic resonanc e imagin g and singl e photo n emissio n computed tomograph y have shown that children with FAS have hypoplasia of the corpu s callosum an d an terior commissure (the anläge of which are formed by glia) (Rile y et al., 1995 ; Johnson e t al., 1996 ; Swayz e et al. , 1997) . PE E ca n lea d t o a reductio n i n whit e matter an d delaye d myelinatio n (Riikone n e t al. , 1999;SowelletaL, 2001b). Animals expose d t o ethano l durin g brai n devel opment exhibi t alteration s i n glia-relate d develop ment, includin g aberrant neuronal migration , delayed astrogliogenesis, an d a reductio n o f cortica l astro cytes (Miller, 1986, 1988 ; Mille r and Potempa , 1990 ;

Gressens e t al , 1992 ; Mille r an d Robertson , 1993 ; Vallès e t al. , 1996) . Moreover , studie s o f a primat e model o f FA S implicate glial involvement . Ethanol exposed monkey s hav e glia l heterotopia s (Clarre n and Bowden , 1984 ) an d dysgeni c corpor a callos a (Miller e t al., 1999) . Thes e alterations ar e consisten t with th e concep t tha t on e o f th e developmenta l stages most vulnerabl e to ethanol i s the brain-growt h spurt (Bonthius and West , 1991) . This period i s characterized b y majo r developmen t o f gli a an d myeli n structures.

ETHANOL AFFECT S ASTROGLIAL ONTOGENY

Radial Glia Normal Ontogeny of Radial Glia Among th e firs t cell s t o differentiat e fro m th e neu roepithelium are RG (Misson et al, 1991) , which appear befor e th e onse t o f neuronogenesi s (Cavines s et al., 1995). RG have a bipolar morphology: each RG has a soma i n th e ventricula r or subventricular zon e and bear s a lon g apica l proces s tha t extend s toward the pia l surfac e an d a shorter basa l proces s tha t con tacts the ventricular wall. The ter m radial glia was introduced b y Rakic (1972) in hi s classic description of neural migration in the feta l primat e neocortex. Most R G transform into mature, stellate-appearin g astrocytes afte r th e neurona l migratio n i s complete d (Schmechel an d Rakic , 1979 ; Pixle y an d DeVellis , 1984; Voigt , 1989; Camero n an d Rakic , 1991; Misson et al, 1991 ; Mille r and Robertson, 1993) . Changes i n the morpholog y of RG are linked to changes in the expression of specific proteins. Vimentin an d nesti n (in cluding its immunorecognized antigenic determinant s RC1 and RC2 ) are prenatally present in RG of rodent CNS, wherea s glia l fibrillar y acidi c protei n (GFAP ) appears in neonates (e.g. , Hockfield and McKay, 1985 ; Millerand Robertson, 1993; Sancho-Tello etal, 1995; Chanas-Sacreetal.,2000). Effects of Ethanol on Radial Glia Development Cerebral Cortex . Ethano l exposure during embryogenesis induce s irreversibl e alteration s i n th e CN S (Guerri, 1998 , 2002). The first suggestion that ethano l

GLIAL TARGETS OF DEVELOPMENTA L EXPOSURE TO ETHANO L 29 7 impairs migratio n originate s fro m neuropathologica l examination a t autops y o f brains fro m childre n wit h FAS (Clarre n e t al, 1978 ; Wisniewsk i e t al, 1983) . Most notable amon g the findings were neuroglial heterotopias located nea r th e pia l surfac e o f the cerebra l cortex. Alteration s observe d i n human s hav e bee n reproduced i n studie s o f animals expose d t o ethano l in utero . Prenata l exposur e t o moderat e amount s o f ethanol (100-20 0 mg/dl) cause s leptomeningea l het erotopias wit h breache s i n th e gli a limitan s an d re duces th e numbe r an d alter s the morpholog y o f RG (Kotkoskie an d Norton , 1988 ; Gressen s e t al. , 1992 ; Komatsu et al, 2001; Moone y e t al, 2004). At least in the rat , heterotopia s persis t into adulthoo d (Komats u et al., 2001). Cultured R G harveste d fro m 12-day-ol d fetuses are affected b y pre - an d postconceptio n exposur e t o ethanol (Vallè s e t al, 1996) . Thes e R G exhibi t shor t glial processes, reduce d number s o f cells, and delays in their transformation int o GFAP-positive astrocyte s (Vallés et al., 1996 ) (Figs . 18- 1 an d 18-2) . A developmen tal dela y in GFA P expressio n ha s als o been describe d in rat whole brains in vivo. During norma l rat brain development, GFA P mRN A appear s o n gestationa l da y (G) 14 , then bot h GFA P transcript and protein expression increas e durin g lat e feta l an d earl y postnata l development. Prenata l ethano l exposur e delays the appearance o f GFA P mRN A unti l G1 9 an d decrease s GFAP expression (Vallès et al., 1996 , 1997) . A compa rable delay is evident among astrocyte s proximal to th e dorsal and medial raphe (Tajuddi n e t al, 2003). An in vivo study focusing on the R G and astrocytes in th e cortica l plate-derive d lamina e o f cortex , i.e. , layers II—Via , show s that GFA P expressio n i s ubiquitously delayed (Mille r an d Robertson , 1993) . I n con trol rats , nestin-positiv e R G ar e presen t throug h th e period o f neuronal generatio n (pas t postnatal da y [P] 8). GFAP-positiv e astrocytes , thoug h common i n th e intermediate zon e (th e anläg e o f the whit e matter) , only appear i n deep layer Via on P3. With time, they progressively appear i n mor e superficia l positions . I n contrast, i n ethanol-treate d rat s R G begi n t o regres s by P 5 an d GFA P immunoreactivit y appear s i n corti cal gray matter o n PI . From th e effect s o f ethanol o n the paire d change s i n nesti n an d GFA P expression , the compellin g conclusio n i s that ethano l cause s th e premature transformatio n o f R G int o astrocyte s i n cortex—this underlies the migratio n of late-generated neurons t o ectopi c site s (Miller , 1986 , 1988 , 1997) . The differen t timin g o f GFA P expressio n i n whol e

control

PEE

FIGURE 18- 1 Effec t of ethanol on nestin and glia l fibrillary acidi c protei n (GFAP ) expression . Corona l sections o f 21-day-ol d contro l (A ) an d prenata l ethanol-exposed (PEE ) (D ) fetuses , staine d wit h hematoxilin-eosin. B, C, E, F . Higher magnificatio n of boxed regions to show nestin-positive glia (RG) and GFAP-positive astrocytes . Brain sections from PE E fe tuses show a reduction i n the corpu s callosu m (CC) , alternations i n th e morpholog y o f RG fibers, and re duction i n the generation of astrocytes.

brain vs . cortex an d th e effec t o f ethanol on tha t tim ing (cf . Mille r an d Robertson , 1993 ; Vallè s e t al , 1996) likel y reflect the effec t o f ethanol on astrocyte s in th e intermediat e zone , th e primar y (sole ) sit e o f GFAP expressio n i n the fetus . Cerebellar Cortex . Th e cerebellu m i s anothe r brain regio n tha t i s sensitive to ethano l toxicit y dur ing developmen t (Guerri , 1998) . Th e cel l bodie s o f Bergmann glia , th e cerebella r subpopulatio n o f R G (Sievers e t al, 1994 ; Yuasa , 1996) , initiall y assum e a position periventricula r t o th e Purkinj e cel l layer . Postnatally, thei r cel l bodie s translocat e peripherall y so that the y ca n guid e th e inwar d migratio n o f granule neurons.

298 ETHANOL-AFFECTE D DEVELOPMEN T

control

PEE

vermis an d th e disruptio n o f th e cerebella r cortical structure.

FIGURE 18- 2 Radia l glia a t 2 an d 7 days of culture. Radial gli a wer e isolate d fro m th e brain s o f 12-day old contro l o r prenata l ethanol-expose d (PEE ) fe tuses. A, B. During the initia l days of culture, control cells expresse d mainl y nesti n (R G marker ) an d formed aggregate s resemblin g neurosphere s (A ) composed o f newly born neuron s (Tujl + ) an d radia l glia (nestin +). C-F. Th e neurosphere-lik e aggregate s increased i n number and siz e with culture time (C) , and radia l gli a wer e transforme d into mor e matur e neurons (MAP2+ ; D , arrowheads ) an d int o GFAP positive astrocyte s (E , F) . Culture d radia l glial cell s isolated fro m PE E fetuse s displaye d morphological alterations an d impaire d neurogeni c potential , a s demonstrated by the decreas e i n numbe r o f neurons and astrocyte s generated . Nucle i wer e staine d wit h Hoechst ( g Rubert, r Miïlana, and c Guerri, unpub lished results) .

Factors Regulatin g Radia l Glia l Transformation . Our curren t understandin g o f mechanism(s) and factors that regulate the transformatio n of RG into astro cytes o r neuron s developmen t i s limited . Extrinsi c cues suc h a s ciliar y neurotrophi c factors , epiderma l growth factors , neureguli n l-erbB 2 signalin g an d members o f the bon e morphogeneti c protei n famil y may induc e cortica l progenitor s t o differentiat e int o astrocytes (Hughe s e t al , 1988 ; Gros s e t al , 1996 ; Johe e t al. , 1996 ; Küh n an d Miller , 1996 ; Burrow s étal., 1997 ; Schmid et al, 2003). Critical determinant s of GFAP expressio n and as trocyte differentiatio n i n feta l brai n ar e change s i n DNA methylation and chromatin structur e (Takizawa et al, 2001 ; Son g and Ghosh , 2004) . These change s are regulate d b y hormone s an d growt h factors . Th e methylation o f genomic DN A a t Cp G dinucleotide s regulates cell - o r tissue-specifi c gen e expressio n (Razin,1998; Bird and Wolffe, 1999) . During embryogenesis an d differentiation , mos t tissue - an d cell specific genes are almost fully methylated and undergo programmed demethylatio n at the momen t of activation and transcriptio n (Barresi et al., 1999) . Chronic i n uter o ethano l exposur e decrease s th e demethylation o f GFAP gene , affectin g bot h it s transcription and expressio n (Vallès et al., 1997) . Ethano l exposure interfere s wit h th e releas e an d actio n o f growth factor s (Luo an d Miller , 1998) , whic h migh t affect th e developmentally regulated epigeneti c mod ifications, leadin g t o th e change s i n bot h DN A methylation and GFA P expression.

Ethanol, given eithe r acutel y o r chronicall y dur ing fetal and neonatal development , delay s the matu ration o f Bergman n glia , decrease s th e numbe r o f GFAP-positive fibers , an d induce s the growt h o f abnormal glia l processe s (e.g. , shorter, thinner, an d ir regular) (Shett y e t al. , 1994 ; Perez-Torrer o e t al. , 1997). Moreover , gestationa l exposur e t o ethano l causes defect s i n th e cerebella r glia l limitan s (formed b y Bergmann glia l en d fee t o n th e cerebel lar surface ) o f the ra t an d th e appearanc e o f solitary or clustered ectopi c granule cells (Sakata-Haga et al., 2001). Damag e t o th e glia l limitan s ma y underli e the fusio n o f th e foli a V an d V I o f th e cerebella r

Cell Adhesion Proteins . Ethano l affect s glia l elab oration o f cel l adhesio n protein s an d growt h factors (e.g., transforming growth factor [TGF ] ßl ) mediatin g neuronal-glial interactions . Among the cel l adhesio n proteins, neural cell adhesion molecule s (nCA M an d LI), integrins , and astrotacti n mediat e neuronal-glia l attachments an d communicatio n (Edelman,1994 ; Hatten, 2002 , Nadaraja h an d Parnavelas , 2002 ; Schmid an d Anton, 2003). Ethanol affects th e expressio n and functio n of several neural cell adhesion proteins both in vitro (Miller and Luo , 2002; Minana et al, 1998 , 2000 ) and in situ (Siegenthaler an d Miller , 2004) . Ethano l als o dis rupts (a) LI-dependent cell adhesion and homophilic interactions among LI molecule s (Wilkemeye r et al.,

GLIAL TARGETS OF DEVELOPMENTA L EXPOSUR E T O ETHANO L 29 9 1999, 2000 ; Bearer , 2001 ) an d (b ) integri n expres sion i n feta l brai n organotypic cultures (Siegenthaler and Miller , 2004). Likewise , TGFßl, which promote s neuronal migratio n i n corte x an d increase s expres sion o f adhesio n proteins , i s profoundl y affected b y ethanol (Mille r an d Luo , 2002 ; Siegenthale r an d Miller, 2004) . Radial Gli a a s Neuronal Precursors . Th e recentl y described rol e o f RG a s neurona l precursor s (Malatesta etal, 2000; Noctor etal. , 2001, 2002; Götz etal., 2002) dramaticall y change s ou r understandin g o f CNS developmen t unde r norma l an d pathologica l conditions. Takin g int o accoun t th e contributio n o f RG a s a neurona l progenito r cell , i t i s plausibl e t o speculate tha t alteratio n o f R G b y ethano l migh t cause dysgeneratio n o f both astrocyte s and neurons . The numbe r o f neuron s an d astrocyte s generate d from culture d R G isolated fro m ethanol-expose d fe tuses is reduced (Rupert , Minana and Guerri , unpublished results ) (Fig. 18-2) . In vivo studies have shown that prenata l exposur e t o ethano l cause s a reductio n in neuronogenesis and gliogenesis (e.g., Miller, 1986 , 1988; Gressen s e t al , 1992 ) (se e Chapte r 13) . Although i t has been interprete d tha t thi s reduction re sults fro m ethanol-induce d change s i n th e outpu t from th e cortica l proliferativ e zones, a contributio n from R G must be considered . It has been suggeste d tha t R G (a ) comprise a het erogeneous population , (b ) var y i n growt h facto r ex pression accordin g to their location withi n the CNS , and (c ) diffe r i n th e type s o f cell s the y generat e (Kriegstein an d Götz , 2003). In contrast , othe r inves tigators argue that RG i n all parts of the CN S serv e as neuronal progenitors (Anthony et al., 2004). In either case, controlle d studie s o f th e potentia l effec t o f ethanol o n th e ste m cel l rol e o f RG ar e require d t o clarify whether ethanol decrease s the pool of stem cell s or onl y induce s abnormalitie s i n th e RG-astrocyte lineage. Astroglial Proliferation Effects of Ethanol on Cell Cycle The mos t activ e perio d o f glia l proliferatio n i s th e brain growth spurt, a time when the brai n undergoes its mos t rapi d growt h (Dobbin g an d Sands , 1993) . In humans , thi s perio d occur s durin g th e fina l trimester o f gestation an d earl y infancy, wherea s i n

the ra t i t occur s durin g th e firs t 2 postnata l weeks. During th e brai n growt h spurt , neura l developmen t is particularl y sensitiv e t o th e toxi c effect s o f envi ronmental agent s suc h a s methylmercury , lead , and ethano l (Cost a e t al. , 2004) . A n implicatio n i s that gli a ar e a targe t fo r thi s neurotoxicity (Aschner etal., 1999) . Ethanol exposure durin g gestatio n ca n impai r th e proliferation an d differentiatio n o f astroglia. The cor tices o f rat s prenatall y expose d t o ethano l hav e one third fewe r gli a (an d als o o f neurons ) tha n control s (Miller an d Potempa , 1990) . Prenata l exposur e t o ethanol reduces th e expressio n o f the astrocyte marke r GFAP durin g postnata l brai n developmen t (Vallè s et al , 1996 ; Tajuddi n e t al , 2003) . Culture d astro cytes fro m th e pup s o f ethanol-fe d rat s exhibi t re duced [ 3H]thymidine and [ 3 H]leucine incorporatio n and GFAP-positiv e cell s (Guerr i e t al. , 1990 ; Guerr i and Renau-Piqueras , 1997) . These data are consistent with a n ethanol-induce d decreas e i n glia l prolifera tion and differentiation . Direct demonstratio n tha t ethano l affect s th e pro liferation o f astroglia l cell s come s fro m experiment s using primary cultures of astrocytes prepared from th e brains of neonatal rats . Ethano l (110-88 0 mg/dl) im pairs cell proliferation , inhibit s th e increas e i n th e astrocyte number , an d reduce s [ 5H]thymidine incorporation (Kenned y an d Mukerji , 1986 ; Davie s an d Cox, 1991 ; Snyde r e t al. , 1992 ; Aroo r an d Baker , 1997; Guizzett i e t al, 1997 ; Lu o an d Miller , 1998 , 1999; Mille r and Luo , 2002) . Ethanol affect s th e cy cling population i n multipl e ways : it inhibits the cel l cycle i n th e GO/G 1 phas e o f the cel l cycle , impede s the progressio n o f cell s int o S phase , an d decrease s the numbe r o f mitoti c cell s (Guerr i e t al. , 1990 ; Mikami e t al, 1997 ; Lu o an d Miller , 1999) . Impair ment o f cel l proliferatio n throug h ethano l exposur e has als o bee n show n i n glia l tumo r cells , principall y C6 astrocytom a cell s (Wazir i e t al. , 1981 ; Isenber g et al, 1992 ; Lu o an d Miller , 1996 , 1998 ; Guizzett i et al , 1997 ; Mille r an d Luo , 2002) . Thi s effec t i s concentration-dependent. Mechanisms of Ethanol Toxicity among Proliferating Astroglial Populations Astrocytic proliferatio n i s regulate d b y mitogeni c growth factor s and growt h inhibitor y agents (Lu o an d Miller, 1998 ) (se e Chapte r 11) . I n turn , thes e pro teins trigge r signalin g pathway s involvin g tyrosin e

300 ETHANOL-AFFECTE D DEVELOPMENT kinases, protei n kinas e C (PKC) , an d mitogen activate protein kinas e (MAPK). Ethanol inhibit s the proliferative effect s o f severa l growth factors , includ ing basi c fibroblas t growt h factor , platelet-derive d growth facto r (PDGF) , an d insulin-lik e growth facto r 1 (Resnicof f e t al , 1994 ; Lu o an d Miller , 1996 , 1999). Thes e factors affec t bot h MAP K and PKCs . I n addition, ethano l potentiate s th e antiproliferativ e activity of TGFßl (Mille r an d Luo , 2002) . Neural cell s actively stimulated b y growth factors are more suscep tible t o th e antiproliferativ e effect s o f ethanol , al though ethano l doe s not affec t th e actio n of all growth factors equivalently (Luo and Miller , 1998 , 1999) . Ethanol potentl y inhibit s mitogeni c signal s initi ated by binding (by carbachol o r acetylcholine) to astroglial muscarinic receptors, particularly M3 subtype (Catlin e t al , 2000 ; Cost a an d Guizzetti , 2002) . Binding o f M3 receptor s increase s DN A synthesi s by glia (Costa e t al, 2001). Ethanol (46-46 0 mg/dl) pre vents thes e mitogeni c effect s i n th e huma n 1321N 1 astrocytoma cells, fetal huma n astrocytes , and ra t cortical astrocyte s (Guizzett i e t al , 1998 , 2003 ; Cost a et al., 2001). Ethanol affect s a t least two pathways triggered by activation of M3 receptors. First, ethanol targets the activatio n of phospholipase D (PLD) , whic h hydrolyzes phosphatidylcholin e t o cholin e an d PA . Thus ethano l decrease s phosphatidi c aci d (PA ) concentration. P A stimulates downstrea m target s suc h a s protein kinases , includin g PKC , an d i s a mitogeni c signaling cascade i n astrocytes (Schatte r et al., 2003). When ethano l i s present, PL D ca n catalyz e a transphosphatidylation reactio n generatin g phosphatidy l ethanol (PEt ) rathe r tha n P A (Kotte r an d Klein , 1999). Thi s diversio n essentiall y block s th e signa l transduction cascad e becaus e PE t doe s no t activat e the downstrea m target s of PA (Schatter e t al, 2003). The secon d pathwa y affected b y ethanol i s the inhibi tion o f atypica l PKCÇ , Subsequently , thi s down regulates th e activatio n o f both the p70s 6 kinas e an d the nuclea r facto r KB (Costa an d Guizzetti , 2002) . A prime metabolite o f ethanol, acetaldehyde , ca n inhibit cel l growt h (Holowni a e t al. , 1996) . Astro cytes expose d t o ethano l ca n produc e acetaldehyd e (Eysseric e t al. , 1997) . Ethano l ca n b e metabolize d into acetaldehyde via alcohol dehydrogenas e (ADH), catalase, and cytochrom e P45 0 2E 1 (CYP2E1) . Th e effects o f ethano l ma y no t b e transduce d throug h ADH, a s ethanol-induce d cel l growt h inhibitio n is observe d i n gli a treate d wit h th e AD H inhibito r 4-methylpyrazole (Isenber g e t al. , 1992) . O n th e

other hand , catalas e an d CYP2E 1 ar e presen t i n astrocytes (Montoliu e t al, 1995 ; Eysseric et al, 2000). These enzymes may be key players in the toxi c effect s of ethanol o n the developing CNS . Astrocytic Death Cell deat h i s a norma l featur e o f CNS developmen t (e.g., Oppenheim, 1991 ; Raffe t al, 1993) . I t is regulated b y growth factors , cytokines, neurotransmitters , and cell-cel l signaling , includin g glial-neurona l in teractions (Johnson and Deckwerth , 1993 ; Raffetal. , 1993). Althoug h mainl y ascribe d t o neurons , deat h among developin g astrocyte s i s als o commo n (Sori ano et al, 1993). Developmental ethano l exposur e affects mos t nor mal regulator y factors, exacerbatin g the processe s associated wit h naturall y occurring neura l deat h i n the ra t developing cortex (Mooney and Miller, 2001; Climent et al., 2002; see Chapters 1 5 and 16) . This ethanol ex posure increases the numbe r of neurons and astroglial cells tha t di e b y necrosi s an d b y apoptosi s (Climen t et al., 2002). Ethanol ca n also induce necrotic (Holownia et al., 1997) or apoptotic (Pascual et al., 2003) death in cultured astrocytes . These effects ar e concentration dependent. Astrocytes die as a result of ethanol-induced activa tion o f sphingomyelinas e (SMase ) (Pascua l e t al. , 2003), leadin g t o th e hydrolysi s of sphingomyelin t o ceramide (Ohania n an d Ohanian , 2001) . Treat ment wit h lo w o r moderat e ethano l concentration s (46-230 mg/dl) eithe r i n vitr o o r in vivo increases astrocyte susceptibilit y to cel l deat h vi a tumor necrosi s factor (TNF)-o t (D e Vit o e t al, 2000) . Conceivably , ethanol shift s the balance of the sphingolipid metabolism b y decreasing the mitogeni c signal s in favo r o f a pathway (SMase/ceramide ) tha t increase s astrocyt e susceptibility to the cytotoxi c effec t o f TNFot. Sphingomyelins ar e cel l membran e phospho lipids that contains ceramid e an d phosphorylcholine . Ceramide i s an importan t regulato r o f cell prolifera tion, differentiation , an d apoptosis . I t ha s trophi c ef fects a t lo w concentration s an d trigger s apoptosi s a t high concentration s (Hannu m an d Luberto , 2000) . Increased ceramid e productio n participate s i n th e cell deat h occurrin g durin g earl y neural differentia tion (Herge t e t al. , 2000 ) an d i n neurona l apoptosi s induced b y nutrient deprivatio n (Toma n e t al., 2002). A variet y o f stimul i an d Stressor s (e.g. , oxidant s and cytokines ) stimulat e SMas e (Andriu-Abadi e and

GLIAL TARGETS OF DEVELOPMENTA L EXPOSUR E TO ETHANO L 30 1 Levade, 2002) , leadin g t o ceramid e generation . Ethanol induce s a stres s respons e (e.g. , oxidativ e stress) b y astrocytes (Montoli u e t al. , 1995) . Stimula tion o f SMas e activit y an d ceramid e generatio n appears t o be a key event i n th e subsequen t ethanol induced cel l deat h (Pascua l e t al., 2003) . Further in vestigation i s require d t o evaluat e th e quantitativ e participation o f thi s mechanis m i n ethanol-induce d astroglial cell death durin g in vivo brain development (Climent e t al., 2002 ) an d t o clarif y i f other mecha nisms are involved.

GLIA AND NEURONA L SYNAPSIS

Pivotal Rol e of Glia in Synaptogenesis It ha s bee n suggeste d tha t gli a pla y a rol e i n synapse formation becaus e astrocyte s an d synapti c terminal s are intimatel y relate d (Ventur a an d Harris , 1999 ; Schikorski an d Stevens , 1999 ; Grosch e e t al, 2002) and glia l developmen t an d synaptogenesi s ar e spa tiotemporally relate d (Aghajania n an d Bloom , 1967 ; Miller and Peters , 1981 ; Parnavela s et al, 1983) . For example, i n ra t cortex , neuron s for m mos t o f thei r synapses during the thir d postnatal wee k after th e differentiation o f astrocytes is largely complete. Further more, culture d retina l ganglio n cells , hippocampa l neurons, an d spina l moto r neuron s for m several-fold more functional synapses when astrocyte s are presen t (Ullian etal, 2001, 2004; Song et al, 2002). Thus, astrocytes appea r t o promot e synaps e formation . In deed, astrocyte s ar e considere d activ e partner s tha t participate with the pre- and postsynaptic terminals in tripartite synapti c structure s (Araqu e e t al. , 1999 , 2001;Haydon,2001). Various glial-derived factor s mediat e synaptogene sis. One suc h factor i s cholesterol, whic h i s produce d by astrocyte s an d secrete d i n apolipoprotei n E-con taining lipoprotein s (Mauc h e t al. , 2001) . Anothe r glial factor , activity-dependen t neurotrophi c facto r (ADNF), i s released b y astrocyte s i n respons e t o va soactive intestina l polypeptide . ADN F ma y pla y a role in synaptic development an d neurona l differenti ation (Blonde l et al., 2000) and i s also a neuroprotector (Gozes and Brenneman, 2000). Not onl y are glia important for synapse formation, they ar e als o key players in synapti c function . The y enhance postsynapti c responsiveness and help to maintain efficient synapti c connectivity. In the presenc e of

glia, retina l ganglio n cell s hav e large r glutamate induced miniatur e postsynapti c current s (Ullia n e t al., 2001, 2004). Experiments conducte d o n both cul tured an d intact-tissu e preparation s hav e demon strated tha t transmitter s release d fro m neuron s ca n stimulate an d caus e th e gli a t o releas e glutamate , ATP, an d othe r neuroactiv e substance s (Bezz i an d Volterra, 2001 ; Field s an d Stevens-Graham , 2002) . These neuroactive substances can fee d bac k onto th e presynaptic terminals , enhancin g o r depressin g fur ther releas e o f neurotransmitters . Moreover , astro cytes ca n respon d t o electrica l neurona l activity , elevating thei r intracellula r Ca 2+ concentratio n an d hence triggerin g glutamat e releas e (Araqu e e t al. , 1998; Innocent i e t al , 2000 ; Pascua l e t al , 2001 ; Pasti e t al. , 2001 ) throug h a Ca 2+-dependent exocy totic proces s (Araque et al, 2001; Bezzi et al, 2004) . Confirmation tha t this release occurs through suc h a process i s provided b y the abolitio n o f glutamate re lease b y tetanu s toxi n (e.g. , Pascua l e t al. , 2001) . Glutamate releas e fro m nerv e terminal s an d astro cytes ca n b e triggere d b y potassiu m chlorid e (KC1 ) and brain-derive d neurotrophi c facto r (BDNF ) (Fig. 18-3) . Glia ca n indirectl y modulat e synapti c transmis sion b y regulatin g th e ioni c extracellula r environ ment, clearin g neurotransmitter s fro m th e synapti c cleft (Bergle s and Jahr, 1998; Anderson an d Swanson, 2000), an d respondin g t o th e metaboli c demand s o f synaptic transmission (Shulman et al., 2001). Glia ca n also affec t glutamatergi c neurotransmissio n b y releasing co-factors. Glia (astrocyte s in the brain and Mülle r cells i n th e retina ) ar e th e onl y source o f D-serine in the CNS . D-serine , rather than glycine , appears t o be the endogenou s agonis t tha t activate s th e glycine binding site a t the N-methyi-D-aspartat e (NMDA ) re ceptors (Wolosker et al, 1999; Steve n et al, 2003). Effects of Ethanol Ethanol exposur e durin g brai n ontogen y alter s brai n synaptology, decrease s synapti c formation , an d im pairs development an d maturation o f synapses in both the hippocampa l formatio n an d neocorte x (Guerri , 1987; Al-Rabiai and Miller , 1989 ; Tanaka etal., 1991 ; Kuge e t al, 1993 ; Sutherlan d e t al, 1997) . Althoug h specific studie s have not ye t broached the rol e of glia in these ethanol-induced synaptogenesis , various data compel u s to consider glial-mediate d synapti c formation as a target o f ethanol.

302 ETHANOL-AFFECTE D DEVELOPMENT /y..

FIGURE 18- 3 Induce d glutamate release from synaptosome s and cortical astrocytes. A spectrofluorimetric assa y wa s used t o determin e th e Ca 2+-dependent release o f glutamat e evoke d b y potassiu m chlorid e (KC1 ; 30 mM) o r brain derived neurotrophi c facto r (BDNF ; 10 0 ng/ml) fro m nerv e terminal s an d from contro l or prenatal ethanol-exposed (PEE) astrocytes (4-day-old cultures). Data ar e means ± standard deviations (n = 3). An asterisk signifies a significant (p < 0.01) difference betwee n PE E an d contro l astrocyte s treated with KC 1 or BDNF.

Astrocytes from prenatall y ethanol-exposed fetuse s release less glutamate after being stimulated with KC1 (Fig. 18-3) . This finding indicates that PEE compro mises th e KCl-stimulate d elevatio n o f intracellula r Ca2+. Thu s prenata l ethano l exposur e likel y affect s the glia l respons e to neuronal activity and cause s abnormalities i n bot h synapti c functio n an d synapti c stabilization. Severa l studie s hav e show n tha t feta l and/or neonata l ethano l exposur e not onl y alter s ligand bindin g t o the NMD A receptor s but als o affect s NMDA receptor function (Savage et al., 1991 ; Vallès et al., 1995; Spuhler-Phillips e t al, 1997; Gruol et al., 1998). Th e compellin g inferenc e i s tha t ethanol induced alteration s i n gli a affec t D-serin e release , thereby altering the function of the NMDA receptors during brain development.

GLIAL DEVELOPMENT IN TH E CORPUS CALLOSUM

The corpus callosum (CC ) i s a larg e fibe r trac t formed fetall y tha t interconnect s neuron s i n th e right an d lef t cerebra l hemispheres . I n humans , th e CC appear s durin g th e sixt h prenata l wee k an d grows i n a rostral-to-cauda l direction . Midlin e glia l populations a s wel l a s axona l guidanc e molecule s

play critica l role s i n C C developmen t (Richards , 2002). Agenesis , dysgenesis , an d change s i n th e shape o f the C C ar e observe d in 7 % of children prenatally expose d t o alcoho l (Rile y e t al. , 1995 ; Book stein étal., 2001, 2002; Sowell étal, 2001a) which is more tha n 20-fol d tha t fo r th e genera l population . Since th e midlin e gli a ar e involve d in th e develop ment o f the CC , ethano l targeting of these cells may initiate CC dysrnorphology . Glia positione d a t the midlin e produc e a number of different molecule s that regulate axonal pathfinding at th e midlin e an d determin e whic h axon s projec t ipsilaterally an d whic h projec t contralaterall y (Fig. 18-4). Thes e gli a ar e hypothesize d t o participat e i n the adhesio n an d closur e o f the interhemispheri c fissure (Silve r et al, 1993) . Concurrently , the y secret e repellent an d growth-suppressive molecules tha t cause callosal axons to turn awa y from th e midline . Th e im portance o f the midlin e glial populations i n the development of the CC i s further supporte d by experiments showing tha t whe n thes e glia l structure s are excise d and replace d b y cortica l graft s tha t d o no t contai n midline glia, the callosal axons actually fail to turn and cross the midlin e (Sh u and Richards , 2001; Richards, 2002). Hypothetically, ethano l alter s glia l productio n and secretion of chemoattractive and chemorepellent

GLIAL TARGETS OF DEVELOPMENTA L EXPOSURE TO ETHANO L 30 3 and changes i n shape) reported i n children prenatally exposed t o alcohol .

GLIAL-DERIVED FACTOR S AND THE NEURON : TH E POTENTIA L

EFFECTS O F ETHANOL

FIGURE 18- 4 Midlin e glia l populations . Thi s schematic vie w o f a corona l sectio n represent s th e various population s o f gli a a t th e leve l o f th e septa l nuclei. GS , glia l sling ; GW , glia l wedge ; IGG , gli a within indusiu m griseum , MG , midlin e zippe r glia. Source: Adapted from Richards , 2002.

molecules tha t affec t th e guidanc e o f midline callosa l axons tha t lead s t o disrupte d C C formation . Thes e molecules ar e secrete d i n th e glia l wedg e an d indu sium griseu m an d mediat e pre - an d postcrossin g axonal guidance ; thei r depletio n ha s bee n foun d t o cause axon s t o defasciculat e o r t o ente r th e septu m aberrantly (Shu e t al., 2003). We have recently found abnormalities in the glia l wedge an d i n indusiu m griseum glia l cel l populations , alon g wit h hypoplasi a of th e CC , i n th e brai n o f 21-day-ol d fetuse s fro m ethanol-fed mother s (Ruber t et al., 2003). Alterations in th e CC wer e accompanied b y cortical atroph y and microcephaly (Fig. 18-1) . In contras t wit h thes e findings , studie s o f rats an d nonhuman primate s sho w tha t th e numbe r o f axons in the CC i s increased following ethanol exposur e between Gl 1 and G2 1 (Qian g et al., 2002) or 1 day per week throughou t gestatio n (Mille r e t al. , 1999) , re spectively. Result s fro m thes e studie s contras t wit h the agenesi s an d dysgenesi s note d i n childre n wit h FAS. Thi s discrepanc y likel y reflect s the pea k bloo d ethanol concentratio n i n th e childre n (estimate d at >3Ö Ö mg/dl) vs . tha t obtaine d i n th e animal s (100-300 mg/dl). In fact, a dose-response relationship in th e amoun t o f hypertrophy ha s bee n describe d fo r monkeys (Mille r e t al. , 1999) . Difference s i n th e amount o f ethano l exposur e and/o r nutritiona l defi ciencies occurrin g durin g critica l stage s of CC devel opment (e.g. , embryogenesis ) ma y als o contribut e t o the variabilit y of effect s (e.g. , agenesis , dysgenesis .

Astrocytes synthesiz e an d releas e a larg e variet y o f compounds, includin g growt h factor s (Lafon-Caza l et al., 2003). These substances have myriad effects o n neurons an d CN S function . Proliferatin g astrocyte s release mor e growt h factor s tha n d o differentiatin g glia (e.g. , Vallès e t al., 1994) , perhaps becaus e o f th e considerably highe r (two-fol d higher ) numbe r o f genes expresse d b y neonatal tha n b y adult astrocyte s (Nakagawa an d Schwartz , 2004) . A particularly interesting factor released b y glia i s the activity-dependen t neuroprotective protein (ADNP ) (Bassa n et al., 1999). This protein, its active fragment NAP (asn-ala-pro-valser-ile-pro-gln), an d th e activ e fragment s o f ADNF are strongly protective against neural insult s (Brenneman e t al, 1998 ; Goze s et al, 2000 ; Beni-Adani et al , 2001), includin g ethanol-induce d insult s (Spon g et al, 2001 ; Wilkemeyer e t al, 2003) . Given th e actions o f ethanol o n glia and the num ber an d importanc e o f th e trophi c an d signalin g functions o f glia , i t i s reasonabl e t o propos e tha t a t least som e o f the detrimenta l developmenta l effect s of ethanol ar e mediated b y defects i n the productio n of glial factors , suc h a s S100B (Erikse n e t al , 2002 ) and ADN P (Pascua l e t al , 2004) . SIOO B i s a glia l trophic facto r tha t i s essentia l fo r th e developmen t of serotonergic neuron s (Li u an d Lauder , 1992) , an d ADNP ha s poten t growth-promotin g an d neuropro tective action s (Bassa n e t al , 1999 ; Goze s e t al , 2003). Astrocytes raise d i n a mediu m conditione d b y ethanol-treated astrocyte s stun t proces s outgrowt h b y neurons an d reduce th e surviva l of serotoninergic neu rons (Ki m an d Druse , 1996 ; Erikse n an d Druse , 2001). Moreover , astrocyte s treate d wit h ethano l ca n increase or reduce the secretio n o f soluble factors tha t influence dendriti c growt h (Yanni et al, 2002) . Secretion o f growt h factor s i s reduce d i n astro cytes isolate d fro m fetuse s o f ethanol-fe d femal e rats (PEE ) an d culture d i n th e absenc e o f ethanol . Ethanol alter s th e intracellula r vesicula r transpor t in astrocyte s (Guerr i e t al, 2001) , leading to impairment o f both th e expressio n o f neurotrophic factor s

304 ETHANOL-AFFECTE D DEVELOPMEN T

FIGURE 18- 5 Co-cultur e of cortical neurons with control or prenatal ethanol exposed (PEE) astrocytes. Neurons grown on control (A , C) or PEE (B , D) astrocytes were staine d th e neurona l marke r MAP 2 an d wit h the synapti c vesicle protein marke r SNAP2 5 (arrows , A, B) . Neuron s co-culture d wit h PE E astrocyte s (B, D) showed a marked reduction in neuronal differentia tion and synaptic connections compared to co-cultures with contro l astrocyte s (A, C). Scal e bar s = 50jlm (A , B) and 1 0 |im (C, D) .

receptors an d th e productio n an d releas e o f growt h factors suc h a s nerv e growt h facto r (NGF ) (Vallè s et al , 1994 ; Climen t e t al , 2000) . PE E astrocyte s also exhibit a remarkable reduction i n cellular NG F (Vallès et al, 1994 ) an d ADNP (Pascua l et al, 2004) mRNA. Co-cultur e o f PE E astrocyte s wit h cortica l neurons fro m contro l rat s lead s t o a reductio n i n neuronal differentiatio n an d synapti c connection s (Fig. 18-5). Interestingly, these effect s ar e blocked b y NAP (Pascual et al, 2004). ADNP and it s active peptide NA P antagoniz e ethanol-induce d inhibitio n of L I adhesio n an d protec t agains t ethano l embry otoxicity (Spon g e t al , 2001 ; Wilkemeye r e t al , 2003). These finding s may for m th e basi s for future therapeutic approache s t o t o protec t neuron s fro m the toxi c effects o f ethanol b y means o f glial-derived factors.

SUMMARY AND CONCLUSION S

Clinical an d experimenta l studie s provid e com pelling evidenc e tha t feta l and/o r neonata l exposur e to ethano l profoundl y affect s astroglia . Amon g th e critical period s o f brain developmen t i n whic h glia l

cells are particularly susceptible to ethanol i s embryogenesis. Ethano l exposur e durin g embryogenesis , a stage durin g whic h R G ar e generated , no t onl y im pairs neuronal migratio n bu t als o can affec t th e gen eration an d maturatio n of astrocytes. Importantly, the novel rol e o f RG a s neural ste m cell s (Nocto r e t al. , 2001; Göt z e t al, 2002 ; Anthony e t al, 2004 ) raises questions as to whether th e effect s o f ethanol o n R G underlie th e reductio n i n numbers o f neurons an d astrocytes generated . The secon d critica l perio d durin g whic h ethano l can affec t gli a i s the brai n growt h spurt . Ethanol an tagonizes th e proliferativ e effect s o f astroglia l mito gens, interfering with signaling transduction pathways associated wit h th e activatio n o f growt h factor s an d neurotransmitters importan t fo r glia l proliferation . Exposure t o ethano l durin g thi s perio d ca n als o en hance neurona l an d astrocyti c apoptotic death . Th e ability of ethanol t o interfer e wit h astroglial mitogens and th e increas e i n cel l deat h ma y contribut e t o ethanol-induced microencephaly . Exposur e t o hig h concentrations o f ethano l durin g th e brai n growt h spurt can induc e reactiv e astrogliosis (Goodlett et al., 1993), thereb y activatin g gli a t o releas e toxi c com pounds tha t damag e neuron s (Lu o e t al. , 2001 ; Blanco e t al., 2004). Ethanol-induced astroglia l damage ca n affec t man y developmenta l process , such a s guidance an d availabilit y o f trophi c suppor t mole cules; modulatio n o f the formation , maturation, an d maintenance o f synapses; an d regulatio n o f synapti c transmission (Field s an d Stevens-Graham , 2002 ; Nedergaardetal.,2003). We are just starting to understand the rol e of glia in many processe s o f th e adul t an d developin g brai n (Ransom et al., 2003 ). The importanc e of glia is illustrated b y th e severit y of th e mor e commo n for m o f Alexander disease, a rare disorder that results from mu tations i n th e gen e fo r GFA P (Brenne r et al. , 2001) . The geneti c modificatio n of Drosophila melanogaster resulting i n glia l ablatio n cause s dramati c defect s in neurona l proliferatio n and differentiation , axona l growth, and neurona l deat h (Jones et al., 1995 ; Boot h et al. , 2000) . This findin g stresse s th e importanc e o f glia fo r neuronal ontogeny . Mor e studie s ar e neede d to confir m th e roles o f astrocyte s an d t o identif y molecular target s o f ethano l o n astroglia . Recen t advances i n thes e topic s hav e relie d o n studie s performed i n cel l culture , wher e glial networ k organization ma y no t exactl y mirro r tha t i n th e intac t brain.

GLIAL TARGETS OF DEVELOPMENTA L EXPOSUR E T O ETHANO L 30 5 Abbreviations ADNF activity-dependen t neurotrophic facto r ADNP activity-dependen t neuroprotectiv e protei n ADH alcoho l dehydrogenas e BDNF brain-derive d neurotrophic factor CC corpu s callosu m CNS centra l nervou s syste m CYP2E1 cytochrom e P450 2E1 FAS feta l alcohol syndrom e G gestationa l day GFAP glia

l fibrillary acidic protein

KC1 potassiu m chlorid e MAPK mitogen-activate d protein kinas e NAP asn-ala-pro-val-ser-ile-pro-gl n nCAM neura l cel l adhesion molecul e NGF nerv e growt h facto r NMDA N-methyl-D-aspartat e P postnata l day PA phosphatidi c acid PDGF platelet-derive

d growt h facto r

PEE prenata l ethanol exposure PEt phosphatidy l ethano l PKC protei n kinas e C PLD phospholipas e RG radia l glia SMase sphingomyelinas e TGF transformin

g growt h facto r

TNF tumo r necrosis facto r

ACKNOWLEDGMENTS Th e author s than k Dr . Vi cente Rubi o for his valuable hel p i n the critica l readin g of this manuscript . Researc h wa s supported b y Spanis h MCYT (project s BF I 2001-0123-01 ) an d SA F (2003 06217) an d b y Conselleria Sanida d (Direc . Gen . Aten cion a la Dependencia). References Aghajanian GK , Bloo m F E (1967 ) Th e formatio n o f synaptic junctions in developing rat brain: a quantitative electro n microscopi c study . Brai n Re s 6 : 716-727.

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Ill NICOTINE-AFFECTED DEVELOPMENT

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19

Tobacco Use During Pregnancy : Epidemiology and Effect s on Offsprin g Jennifer A. Willford Nancy L. Day Marie D. Corneliu s

Tobacco us e durin g pregnanc y affect s feta l develop ment o n multipl e levels. Nicotine readil y crosses th e placenta, concentratin g i n feta l tissue , an d result s in fetal concentration s o f nicotin e tha t ca n b e 15 % higher than those in the pregnant woma n (Luc k et al., 1985). Th e direct action s o f nicotine o n th e fetu s affect growt h an d neura l developmen t durin g th e in trauterine period an d hav e long-term effect s o n brain function, cognition , and behavior (Abreu-Villaca et al , 2004a, 2004b). Tobacco us e durin g pregnanc y ha s indirec t effects o n feta l development . Prenata l tobacc o us e affects th e nutritiona l status of the mother , leadin g to an increase d ris k o f having an infan t wit h lo w birth weight (LBW ) (Hast e e t al , 1990) , an d cause s in creased vascula r resistance i n the placenta , resultin g in reduce d oxyge n flo w t o th e fetu s (Lamber s an d Clark, 1996) . Intermitten t feta l hypoxia , secondary to exposure to carbon monoxid e an d th e metabolit e carboxyhemoglobin, ma y als o affec t oxyge n flo w t o the fetus .

The presen t chapte r review s the epidemiolog y o f tobacco us e durin g pregnanc y an d the n focuse s o n the effect s o f prenatal tobacc o exposur e (PTE).

SCOPE O F THE PROBLE M

Epidemiology of Tobacco Use During Pregnancy In genera l population s surveys , 10 % t o 20 % o f women repor t tha t the y smoke d durin g pregnanc y (National Institut e o n Dru g Abus e [NIDA] , 1996 ; LeClere and Wilson, 1997 ; Center s for Disease Con trol and Preventio n [CDC] , 2002 , 2003) . Dat a col lected o n th e U.S . Standar d Certificate s o f Liv e Births sho w tha t th e proportio n o f wome n wh o smoked durin g pregnanc y decline d fro m 19.5 % t o 11.4% between 198 9 an d 200 2 (Marti n e t al, 2003). This decrease mirror s a general decreas e i n smokin g among wome n o f reproductiv e age , althoug h on e

315

316 NICOTINE-AFFECTE D DEVELOPMENT group, young women aged 18-20 , increased their use slightly fro m 198 7 t o 1996 (Ebrahim e t al , 2000) . This overall decline i n smoking during pregnancy results fro m lo w rates of smoking initiation i n women , rather tha n change s i n smokin g befor e o r durin g pregnancy (Wisbor g e t al. , 1996 ; Cnattingiu s an d Haglund, 1997 ; Department o f Health an d Huma n Services [DHHS], 2001). Smoking durin g pregnanc y i s mor e prevalen t among Caucasia n wome n tha n amon g African American o r Hispani c wome n (NIDA , 1996 ; CDC, 2002). The rate s vary by age (Cornelius e t al., 1999b ; Kahn e t al. , 2002) , wit h younge r wome n smokin g more cigarettes . Us e als o varies by trimester o f pregnancy. A Maternal Health Practices and Child Development (MHPCD ) stud y of teenage mother s report s that th e rat e o f smoking during pregnanc y i s 47% i n the first trimester and 58 % in the third trimester (Cornelius e t al. , 2004) . I n general , wome n wh o smok e during pregnanc y ar e younge r an d les s likel y t o b e married, have less education an d lower incomes, an d attend fewe r prenata l visit s compare d wit h wome n who do not smok e durin g pregnanc y (Da y et al. , 1992; Corneliu s e t al. , 1994 ; DHHS , 2001 ; CDC , 2003). Wome n wh o smok e durin g pregnanc y als o have highe r rate s o f antisocia l behavior , mate s tha t have mor e antisocia l behaviors , highe r rate s o f de pression, an d lowe r socioeconomi c statu s (SES) (Maughanetal., 2004). Women ar e less likely to decrease their tobacco us e during pregnancy than they are to decrease othe r sub stance use, including alcohol, marijuana, and other illicit drug s (Corneliu s e t al, 1995 ; Day et al, 2000), and ar e more likely to continue t o smoke i n the post partum perio d (Corneliu s e t al. , 1999a ; Leech e t al. , 1999). I n a Nationa l Pregnanc y an d Healt h Stud y (NIDA, 1996), approximately two-thirds of the wome n who smoke prior to their pregnancy continue smoking through th e las t trimester . B y contrast , onl y onequarter o f the wome n wh o us e alcoho l prio r t o conception continu e t o drin k i n th e thir d trimester . Women who continue to smoke during pregnancy are more likely to have had previous pregnancies, to have a younger age at smoking onset, to be heavy smokers, and to have less education (Cnattingius, 2004). Women wh o smok e durin g pregnanc y als o continue to smoke in the postpartum period. This mean s that th e offsprin g o f a woman who use s tobacco during pregnanc y ar e expose d continuousl y t o tobacc o smoke i n th e household . Thi s environmental , o r

passive, exposur e als o affect s chil d development . I n the MHPC D cohor t o f adult mothers , fo r example , the correlatio n betwee n prenata l tobacco us e and tobacco us e 1 0 years later wa s 0.63 (Cornelius e t al. , 2000). I n addition , a s man y as 60 % o f wome n wh o quit smokin g durin g pregnanc y bega n agai n withi n 6 months o f delivery (Mullen e t al., 1997; Hajek et al., 2001). Significan t predictor s o f relaps e afte r preg nancy include living with another smoker, less education, and lower income (Kah n et al., 2002). Women who smoke during pregnancy have higher rates of other substance use as well. Among women in the MHPCD project, 76% of the women ove r the age of 1 8 who smoke d durin g the firs t trimeste r o f pregnancy dran k alcoho l durin g thi s perio d (Da y et al. , 1992). Among pregnant teenagers , 61 % of those wh o smoked i n the first trimester drank alcohol (Corneliu s et al , 1995) . Tobacc o us e i s als o highl y associated with th e us e o f illici t drug s durin g pregnancy . I n the Nationa l Pregnanc y an d Healt h Surve y (NIDA , 1996), wome n wh o smoke d cigarette s durin g preg nancy reporte d usin g illicit drug s (26%) , drinkin g (16%), o r bot h (32%) . Therefore , i t i s importan t t o control fo r the covariate s of PTE an d othe r prenata l substance exposures , as well a s curren t materna l tobacco an d othe r substanc e use . In th e absenc e o f considering thes e othe r ris k factors, it i s not possibl e to identif y accuratel y the uniqu e effect s o f prenatal tobacco exposure. Mortality and Morbidity A meta-analysi s estimate s tha t PT E wa s responsibl e for u p t o 480 0 infan t deaths , 61,00 0 infant s wit h LBW, and 26,00 0 neonata l intensiv e care admissions per year (DiFranza and Lew , 1995) . An earlier report estimates tha t materna l smokin g was responsible fo r approximately 10 % of fetal an d infan t death s (Kleinman e t al. , 1988) . Thi s report , alon g wit h anothe r (Singleton et al., 1986) , find that the impac t of PTE is greater amon g Africa n American s tha n fo r Cau casians an d i s greate r amon g th e offsprin g o f olde r mothers (Cnattingius et al., 1988) . PTE i s a significan t risk facto r fo r sudde n infan t death syndrom e (SIDS ) (Nationa l Cance r Institut e [NCI], 1999) . SID S result s fro m th e PTE-relate d chronic hypoxi a (Bulterys et al., 1990) . Postnatal passive exposure of the infan t als o significantly increases the ris k of SIDS (Klonoff-Cohe n e t al., 1995 ; Dwye r étal, 1999) .

EPIDEMIOLOGY OF MATERNA L TOBACC O US E 31 7 GROWTH AN D MATURATION

Human Studie s PTE ha s lon g bee n identifie d a s a significan t ris k factor fo r intrauterin e growt h restrictio n (DHHS , 1980, 2001 ; Stillman e t al., 1986 ; Floy d e t al., 1993 ; NCI, 1999 ; CDC , 2003) . Birt h weigh t decrease s i n direct proportio n t o the numbe r o f cigarettes smoke d (Yerushalmy, 1971 ; Persso n e t al. , 1978) , an d off spring of smokers are 15 0 to 250 g lighter than th e off spring of non-smokers (DHHS , 1980) . Th e effect s o f PTE ca n b e see n eve n a t lower levels of exposure: in one report , 11.5 % o f infant s bor n t o ligh t smoker s (<six cigarettes/day) have LBW, compare d t o 7.5 % of those born t o non-smokers (Marti n e t al., 2003) . Th e reduction i n infan t weight i s not du e t o earlie r gesta tion, a s infants o f smokers exhibit growt h retardation at all gestational ages (NCI, 1999) . Birth lengt h an d hea d an d ches t circumference s are reduce d i n infant s wh o ar e prenatall y exposed t o tobacco (Klin e e t al , 1987 ; Da y e t al , 1992 ; Cor nelius e t al. , 1995) . I n a cohor t o f 151 3 Caucasia n women, infants o f smokers are shorter and hav e lower pondéral indice s an d smalle r mea n uppe r ar m cir cumferences tha n d o th e offsprin g o f non-smoker s (Haste et al, 1991) . In a study of neonatal bod y com position, PT E i s significantly related t o reduce d fat free mas s (Lindsa y e t al. , 1997) , an d th e author s conclude tha t th e lowe r birt h weigh t o f infant s ex posed t o prenatal smoking is primarily due to reduce d fat-free mas s or lean tissue . In a MHPC D stud y o f pregnant teenager s (Cor nelius e t al , 1995 , 1999h) , PT E i s significantl y related t o reduce d birt h weight , length , an d hea d an d chest circumference. These reductions are more pro nounced tha n the effect s of PTE in a similar cohor t of pregnant adul t wome n an d thei r offsprin g a t birth (Day et al., 1992) . The increase d problems associated with younger materna l ag e and poorer feta l outcome s (Ketterlinus et al, 1990 ; Frase r e t al, 1995) , couple d with the hig h prevalence of smoking among pregnan t teenagers (Corneliu s et al., 1994), magnify the risk s to offspring o f pregnant teenagers who smoke . The duratio n and timin g of tobacco exposur e during pregnanc y ar e importan t i n determinin g growt h outcomes. Smokin g onl y durin g th e firs t trimeste r produces a n averag e 5 5 g reductio n i n birt h weight , whereas continuou s smokin g throughou t pregnanc y leads t o a reductio n i n birt h weigh t o f 18 9 g (Cliver

et al. , 1995) . On e stud y report s n o relatio n betwee n PTE an d LB W amon g th e offsprin g o f women wh o stopped smokin g earl y in pregnanc y (MacArthu r and Knox, 1988) . Fo x and colleague s (1990 ) studie d th e height and weight of 714 three-year-old children. Th e children o f wome n wh o qui t smokin g durin g preg nancy ar e heavie r an d talle r tha n thos e o f wome n who do not quit. Adjustment for postpartum exposur e to tobacco smok e reduce s th e differenc e i n th e chil dren's weight , bu t ha s littl e effec t o n th e difference s in height . Anothe r stud y describe s n o difference s i n head circumferenc e amon g childre n whos e mother s stopped smokin g befor e 3 2 weeks o f gestation com pared t o children whos e mother s di d not smoke (Vik et al, 1996) . The Nationa l Healt h Intervie w Survey describes an increased relativ e risk (1.6 times) of having a LBW infant amon g wome n wit h hig h level s of exposure to passive smok e (Mainou s and Hueston , 1994 ) a s have other researcher s (Haddo w e t al., 1986 ; Rubi n e t al. , 1986; Matha i e t al , 1990) . Marti n an d Bracke n (1986) find that passive exposure correlates with LBW among offsprin g o f nonsmoking women , resultin g i n newborns who are, on average, 24 g lighter. The effect s o f PTE o n growth over the longe r term are les s clear. At 8 years of age, there is an associatio n between PT E an d growt h i n expose d childre n (Jone s et al. , 1999) . Childre n wit h PT E hav e lowe r bon e mass at the lumba r spine and femora l neck, althoug h not in the total body. Naeye (1981) detects a small difference i n height an d head circumferenc e i n expose d 7-year-old offspring , usin g dat a fro m th e Collabora tive Perinata l Project . Rantakalli o (1983 ) show s tha t exposed offsprin g ar e shorter at age 14 , and Fogelma n and Mano r (1988 ) repor t decrease d heigh t a t 7 , 11 , and 2 3 years of age tha t i s mediated b y birth weight . These studies do not control fo r the offsprings ' passiv e exposure to tobacco smok e or for other prenatal expo sures. In a MHPCD study of adult mothers an d thei r off spring (Da y e t al. , 1992 ) whic h control s fo r prenata l alcohol an d othe r dru g exposures and curren t mater nal tobacc o an d othe r substanc e use , ther e i s a sig nificant invers e relatio n betwee n PT E an d weight , length, an d hea d circumferenc e at birth. At 8 months of age , onl y lengt h continue s t o b e associate d wit h PTE. B y 1 8 months o f age, ther e ar e n o association s between PT E an d siz e o f the offsprin g afte r control ling fo r the appropriat e covariate s (Da y e t al. , 1992) . Other studie s also have no t foun d growt h retardation

318 NICOTINE-AFFECTE D DEVELOPMENT over the lon g ter m (Hard y and Mellitus , 1972 ; Frie d and O'Connell , 1987 ; Vi k e t al , 1996) . Mor e re cently, i t ha s bee n argue d tha t th e associatio n be tween PTE an d reduced heigh t a t 6.5 and 1 3 month s postpartum i s attributable to prenata l exposure to alcohol (Jacobson et al., 1994) , highlighting th e impor tance o f controlling for the effect s o f other drugs. Recent reports demonstrate that PTE leads to an increase in weight at older ages. Power and Jefferi s (2002) note that althoug h PT E i s associated wit h lower birth weight, thi s relatio n reverse d by adolescence , an d b y age 33 , exposed offsprin g hav e 1. 5 greater odd s of be ing obese . I n a MHPC D cohor t o f teenage mother s and thei r offspring, a positive association is evident between PT E an d increase d skinfol d thickness , an d higher body mass index an indication that the childre n are overweight for their height (Corneliu s e t al, 2002). Similar finding s hav e bee n reporte d b y othe r author s (Vik e t al , 1996 ; Frie d e t al. , 1999 ; Toschk e e t al , 2003). Thus , th e preponderanc e o f evidenc e fro m longitudinal studie s that have controlled fo r passive tobacco exposur e and othe r covariate s of pre- and post natal tobacc o exposur e i s that th e growt h deficit s that are eviden t a t birth ar e no t maintained , an d ove r th e long term there appears to be an increase in weight. The mea n ag e a t menarch e i s approximatel y 6 months earlier among the female offspring expose d to a pac k or more o f cigarettes per da y during gestatio n than that of girls unexposed during pregnancy (Windham e t al, 2004) . Girls with bot h hig h prenata l an d childhood passiv e exposur e to tobacc o hav e onse t o f menarche 4 months earlie r than th e girls who are not exposed a t eithe r time . Earl y onse t o f menarch e i s present afte r adjustin g fo r alcohol, coffee , an d te a exposure, mothers' age at menarche, parity , race, education, and income . The complexit y of relations between prenata l nico tine exposure and growt h are demonstrated i n animal models. Navarr o and colleague s (1989 ) repor t effect s of PTE o n viability, growth, and nervou s system development o f th e offsprin g a t hig h dose s o f nicotin e (>6mg/kg/day). A t lower dose s ( 2 mg/kg/day), whic h are approximatel y equivalent t o
Using osmotic minipumps, nicotine wa s administered to pregnant rats three time s during the pregnancy : on gestational day (G) 8 to G13, G8 to G21, and G 8 to postnatal da y (P) 17 . Nicotine exposur e early in gesta tion doe s not affec t materna l weigh t gain or offsprin g growth outcomes. Continuou s exposur e through much of the pregnanc y an d int o th e earl y postnatal perio d (G4 t o P17 ) cause s significan t growth deficits . Thus, the effect s o f prenata l nicotin e exposur e o n growt h occur later in gestation . The effect s o f prenatal nicotin e exposur e occur in parallel wit h a n increas e i n ornithin e decarboxylas e (ODC) activit y i n th e fetu s (Slotki n e t al , 1986 , 1993). OD C i s a sensitiv e marker of the initia l steps in polyamin e formation , whic h regulate s cellula r development (Heby , 1981 ; Slotki n an d Bartalome , 1986). Thus, the effect s o n growth of prenatal nicotin e parallel th e developmen t o f the nicotini c receptor system an d a t a time o f increased cellula r development , as reflected i n ODC activity . Central Nervous System PTE produce s neonata l centra l nervou s system (CNS ) defects, includin g poore r auditor y orientatio n (Pi cone e t al. , 1982) , autonomi e regulatio n (Picon e e t al., 1982 ; Franc o e t al., 2000) , and increase d tremors and startle s (Frie d an d Makin , 1987) . Usin g dat a from th e Neonata l Intensiv e Car e Uni t Networ k Neurobehavioral Scale , on e stud y demonstrate s dose-response relation s betwee n PT E an d stress/ab stinence sign s and higher excitability scores i n infant s (Law et al., 2003). PTE i s also significantly related t o muscle tone abnormalities after controllin g for prenatal cocain e an d alcoho l exposures , hea d circumfer ence, an d th e qualit y o f prenata l car e (Dempse y et al, 2000). In a sampl e o f predominantly young , unmarried , and lower-SE S mothers , PT E i s associate d wit h a four-point decreas e o n th e Stanford-Bine t Intelli gence Scale at 3 and 4 years of age (Olds et al., 1994). Another stud y shows that cognitive functioning a t age 3 is higher among children whose mothers quit smoking durin g pregnanc y tha n i n childre n whos e moth ers smok e throughou t pregnanc y (Sexto n e t al. , 1990). The Ottaw a Prospective Prenatal Stud y (OPPS ) reports that lower cognitive scores are associated with PTE amon g 2- , 3- and 4-year-ol d childre n (Frie d an d Watkinson, 1988) . This association between PT E an d

EPIDEMIOLOGY OF MATERNA L TOBACCO USE 31 9 IQ i s also evident at age s 9 t o 1 2 years (Fried, 2002 ) and 1 3 to 1 6 years (Frie d e t al, 2003 ) for the OPP S cohort. Anothe r prospectiv e stud y (Streissgut h e t al. , 1989), however , show s n o effec t o f PT E o n I Q i n 4-year-olds. PTE produce s deficits in visual memory and verbal learning o n th e Wid e Rang e Assessmen t o f Memory and Learnin g (Sheslow and Adams, 1990) amon g th e 10-year-old offsprin g o f adult mother s i n a study fro m the MHPCD (Cornelius e t al, 2001). These findings are significan t afte r controllin g for SES, materna l psychological status , hom e environment , othe r prenata l substance exposures , an d curren t materna l substanc e use. I n th e OPP S cohort, whe n th e offsprin g ar e 9 to 12 years old, PTE produce s visua l perceptual deficit s (Fried and Watkinson, 2000). A later report on this cohort a t age s 1 3 t o 1 6 years shows that PT E produce s poorer auditory functioning (Fried e t al., 2003). Poor languag e developmen t i s reporte d i n 2 (Fried and Watkinson , 1988) , 3-, and 4-year-old s with PTE (Frie d an d Watkinson , 1990) . Amon g 9 - to 12 year-old children i n this study, PTE i s negatively associated wit h languag e an d readin g abilitie s (Frie d et al., 1997) . Smokin g during pregnancy is also significantly relate d t o poore r performanc e o n spelling , reading, an d arithmeti c assessment s amon g 5 - to 11 year-olds in another stud y (Batstra et al., 2003). In animals, learning and memor y deficits ar e seen on a numbe r o f comple x cognitiv e behaviora l tasks , including decreased performanc e on a two-way avoidance tas k (Vaglenov a et al. , 2004) , deficit s i n spatia l learning and memor y (Sorenso n e t al., 1991 ; Yanai et al, 1992 ; Levi n e t al, 1993 , 1996) , opéran t learnin g (Martin an d Becker , 1971) , and discriminatio n learning (John s et al., 1982) . These impairment s in cogni tive functio n ar e consisten t wit h th e learnin g an d memory deficits observe d in children with PTE . Behavior Associations between PT E an d error s of omission and commission, measures o f inattention an d impulsivity , respectively, are detectable in 4-year-olds (Streissgut h et al , 1984) . Ther e i s a significan t relation betwee n PTE an d impulsivit y among 6-year-old s in the OPP S cohort (Frie d e t al, 1992) . In a MHPCD study of off spring o f adult mothers , PT E significantl y predicte d increased error s of commission , o r inattention , o n a continuous performanc e tes t i n 6-year-old s (Leec h

et al, 1999) . I n the MHPCD, however , the mother' s current tobacc o us e i s so highly correlate d wit h th e prenatal exposur e tha t th e effect s o f the prenata l an d current exposure s coul d no t b e separated . Kristjans son an d colleague s (1989 ) sho w tha t PT E produce s impulsivity and increase s overal l activit y among 4 - to 7-year-olds. A prospectiv e stud y o f 900 0 childre n i n Finland (Kotima a e t al , 2003 ) found a 30 % higher rate in hyperactivity among 8-year-old s who had PTE than i n thos e wh o di d not , afte r controllin g fo r gen der, family structure, SES, maternal age, and prenatal alcohol exposure . I n th e OPP S cohor t o f 9 - t o 12 year-olds, there was a dose-dependent associatio n be tween PT E an d impuls e contro l (Fried , 2002) . Eskenazi an d Trupi n (1995) , however , repor t tha t there i s no associatio n betwee n PT E an d activit y levels at 5 years of age in their study. In a case-control stud y of children with and with out attentio n defici t hyperactivit y disorde r (ADHD) , Mick an d colleague s (2002 ) repor t tha t 6 - to 7-year old children with ADHD are 2.1 times more likel y to have been expose d t o tobacco during gestation, eve n after controllin g fo r prenatal alcoho l an d dru g expo sures. There i s a positiv e associatio n betwee n mater nal smoking during pregnancy and th e ris k of ADHD in stud y childre n betwee n th e age s o f 6 an d 1 7 (Milberger e t al , 1996) . Materna l smokin g durin g pregnancy i s significantl y associate d wit h ADH D symptoms i n twins , controlling fo r the heritabilit y of ADHD (Thapar et al, 2003) . The effect s o f prenatal nicotin e exposur e o n mo tor activity in laboratory animals are equivocal. Som e studies sho w increase d activit y (John s e t al , 1982 ; Fung an d Lau , 1988 ; Richardso n an d Tizabi , 1994 ; Tizabi e t al , 1997 ; Ajare m an d Ahmad , 1998 ; Thomas e t al, 2000) , wherea s other s sho w n o effec t (Martin an d Becker , 1970) . O n th e othe r hand , pre natal nicotin e exposur e i s linked to abnorma l devel opment o f spontaneous alternation , a behavior that is associated with cholinergic neurotransmitter function and hyperactivit y (Fung, 1989 ; Shack a e t al , 1997 ; Tizabi e t al , 2000) . Studie s als o sho w tha t prenata l nicotine exposur e change s cellula r communicatio n (Maggi e t al, 2003) , electrophysiology (Ehlers e t al , 1997; Slaweck i e t al , 2000) , an d neurotransmitte r function (Tizab i e t al , 2000 ) i n th e hippocampus , changes that could affec t activit y levels. There is consistent evidence that children expose d to tobacco durin g pregnanc y ar e at a n increase d ris k

320 NICOTINE-AFFECTE D DEVELOPMEN T for behaviora l problems, specificall y externalizin g behaviors. I n a n MHPC D cohor t o f adul t mothers , 3-year-old offspring expose d prenatally to tobacco hav e significantly mor e oppositiona l an d aggressiv e behaviors and are more immatur e (Day et al., 2000). These effects persis t after controllin g for SES , factor s in the current hom e environment , materna l psychologica l status, and other prenatal and current drug exposures. In anothe r stud y o f 3-year-old s (Orlebek e e t al. , 1997), there is a significant effec t o f PTE o n external izing behaviors, primaril y higher aggression , i n bot h genders. In a prospective study of 5-year-olds, Williams and colleague s (1998 ) describ e a relativ e risk o f 2. 6 for externalizin g behavior in children wit h PTE com pared t o unexpose d children . Anothe r stud y report s a significan t positiv e associatio n betwee n PT E an d externalizing behavior s i n 5 - to 11-year-old s (Batstr a et al. , 2003) . B y contrast, Weitzma n an d colleague s (1992) d o not fin d significan t effect s o f PTE o n chil dren wh o ar e expose d onl y durin g gestation . The y show that childre n whos e mother s smoke d bot h during an d afte r pregnanc y hav e mor e behavio r prob lems. I n th e latte r group , th e rat e o f behavio r problems i s increased by 1.1 7 times amon g thos e ex posed t o less than on e pac k of cigarettes per da y and 2.04 times among thos e expose d t o one pack or more per day. These behaviora l effect s persis t int o th e adoles cent and adult years. Among children whos e mother s smoked a t leas t 1 0 cigarette s pe r da y durin g preg nancy, ther e i s a fourfol d increas e i n th e rat e o f prepubertal onse t o f conduct disorde r amon g boy s (<13 years old), a twofold increas e in the rat e of ADHD in girls (<13 years old), and a fivefold increase i n the ris k of dru g dependenc e i n girl s age s 13-1 7 (Weissma n et al, 1999) . Thi s associatio n is significant afte r con trolling for the othe r covariate s o f maternal smokin g however, the study does not control for other prenatal substance exposure s o r postnata l environmenta l to bacco exposures . Fergusso n an d colleague s (1993 ) report tha t PT E i s relate d t o childhoo d behavio r problems at 1 2 years of age, whereas current maternal smoking i s not . PT E i s predictive o f higher rate s o f conduct disorder among 16 - to 18-year-old s in this cohort (Fergusso n e t al. , 1998) . Wakschla g an d col leagues (1997 ) als o sho w a significan t relationshi p between PT E an d conduc t disorde r a t age s 13-1 8 years. Materna l smokin g durin g pregnanc y predict s persistent crimina l outcome s i n adul t mal e offsprin g in a Danish prospectiv e study (Brennan et al., 1999) ,

and i n a prospective study in Finland, maternal smoking durin g pregnancy i s significantly associate d with an increas e i n violent offense s amon g adul t mal e off spring (Rasanen et al., 1999) . A significan t relatio n betwee n materna l smokin g during pregnanc y an d conduc t disorde r i s found i n boys 1 2 to 1 7 years ol d (Silber g e t al, 2003) . When maternal conduc t symptom s ar e considered , how ever, that associatio n i s no longer significant . Simila r conclusions are reached b y Maughan an d colleague s (2004), who show that the association s between PT E and childhoo d conduc t problem s ar e confounded b y other risk s for the developmen t o f behavior problems, including parenta l antisocia l behavior , materna l de pression, SES , an d geneti c influences . Th e highe r rates of antisocial behavior in childre n could als o result fro m th e geneti c transmissio n o f anti-social trait s and/or th e effect s o f parenta l trait s o n th e pre - an d postnatal environmen t (Wakschlag et al., 2003) . In a recent twi n study, although nearl y half of the associa tion betwee n PT E an d conduc t problem s i s ex plained by genetic heritability, PTE ha s a direct effec t on conduct problem s eve n after th e genetic contribu tion t o antisocia l behavio r i s controlle d (Maugha n et al, 2004).

TOBACCO US E

Direct Exposur e In a n MHPCD cohor t o f adult women , th e 10-year old offsprin g expose d t o half a pac k or mor e pe r da y during gestation hav e a 5.5-fol d increase d ris k of early tobacco experimentation, controllin g fo r other prena tal substanc e exposures , mothers ' curren t smoking , and othe r covariate s (Corneliu s e t al., 2000). A retrospective stud y report s a comparabl e four-fol d in creased ris k o f tobacco us e amon g girl s 9 to 1 7 years old prenatall y expose d t o tobacc o (Kande l e t al. , 1994). I n follow-u p to th e latte r study , Griesle r an d colleagues (1998 ) confirm th e link between PT E an d offspring behavio r problem s an d demonstrat e tha t these behavior problems mediate the increase d rate of smoking amon g th e adolescen t girl s with PTE . I n a study o f youn g adults, thos e wit h PT E ar e signifi cantly mor e likel y to mee t DSM-I V criteri a fo r life time tobacco dependence , althoug h this analysis does not contro l fo r other prenata l substanc e exposur e o r environmental exposures (Buka et al., 2003).

EPIDEMIOLOGY OF MATERNA L TOBACCO US E 32 1 Abnormalities i n cel l developmen t an d respon sivity associate d wit h prenatal nicotin e exposur e ma y lead t o permanent functiona l deficits whe n brai n re gions and the relate d circuitr y mature i n adolescenc e and adulthoo d (Erns t et al. , 2001) . An up-regulatio n of nicotinic acetylcholine receptor s i s evident amon g adult rat s expose d t o nicotin e i n th e immediat e neonatal period , a tim e comparabl e t o th e thir d trimester i n human s (Slotki n e t al. , 1991) . A recen t study (Klei n e t al. , 2003 ) show s tha t peri-adolescen t male mic e expose d t o gestationa l nicotin e hav e in creased nicotin e preference . Prenata l nicotin e expo sure lead s t o cholinergi c hypoactivity , decrease d cel l numbers i n th e midbrain , an d increase d cel l siz e i n the hippocampu s i n adolescen t animal s (Abreu Villaca e t al , 2004a , 2004b) . Kan e an d colleague s (2004) sho w decrease d responsivit y t o dopamin e i n the nucleu s accumbens , a brai n regio n associate d with rewar d an d th e reinforcin g effect s o f drugs , i n adolescent rats . Thus, there ar e direct biological associations tha t parallel the smokin g behavio r outcome s in human studies . Psychological Status There are few reports of effects of PTE on psychologi cal symptoms . Mother s wh o smoke d durin g preg nancy ar e significantl y mor e likel y t o hav e toddler s who display negativity than mother s who only smoked after deliver y (Brook et al , 2000) . Among 16 - to 18year-olds in another cohort , PT E predict s higher rates of depression (Fergusso n etal., 1998) . Recent anima l studie s identif y link s between pre natal nicotin e exposur e an d anxiety . The open-fiel d and elevate d plus-maz e test s have been use d to evaluate activity, anxiety, and emotionalit y in animals. Pre natal nicotin e i s associated wit h poo r adaptatio n an d anxiety i n a nove l environmen t (Vaglenov a e t al. , 2004). Femal e offsprin g wit h prenata l nicotin e expo sure d o no t sho w a norma l increas e i n activit y in a n open-field tes t across multiple days of testing (Romero and Chen , 2004) . Thi s suggest s an inabilit y to adap t to a novel environment , lear n contextua l cues , o r retrieve learne d informatio n o n th e familiarit y o f th e testing environment. I n an open-fiel d test, male mic e exposed i n uter o t o nicotine sho w increased locomo tor hyperactivity, whereas females have a n attenuate d behavioral respons e (Paul y e t al, 2004) . Thes e ani mal studies demonstrate a link between prenata l nicotine exposur e and anxiety , or the inabilit y to adapt to

new environments, and the y may provide an explana tion fo r some o f the mechanism s underlyin g the be havioral outcomes i n humans . Exposure t o Environmental Tobacco Smoke Pregnant wome n wh o liv e wit h o r spen d tim e wit h smokers expos e thei r childre n t o environmenta l to bacco smok e (ETS) . The level s of newborn exposur e correlate significantl y wit h th e level s of nicotine an d cotinine i n th e mother s (Eliopoulo s e t al. , 1994 ; Os treaetal, 1994) . As with PTE , ther e ar e growth, cognitive , and be havioral effect s fro m exposur e t o ET S durin g preg nancy. ET S exposur e i n earl y pregnanc y (20-2 4 weeks) lead s t o a decreas e i n biparieta l diameter , a measure usefu l i n predictin g feta l weigh t (Hank e et al., 2004) . Kharraz i an d colleague s (2004 ) demon strate a dose-respons e relatio n betwee n ET S expo sure an d birt h weight ; increasin g level s o f materna l cotinine ar e associate d wit h declinin g birt h weight . Other studies have also found that birth weight is negatively associated wit h ET S exposur e (Marti n an d Bracken, 1986 ; Rebagliat o e t al, 1995) . The relativ e risk for LB W i s 2.17-fold highe r amon g th e offsprin g of nonsmoking mothers wh o are exposed to ETS, an d the expose d infants , o n average , are 2 4 g lighter tha n nonexposed infant s (Marti n and Bracken , 1986) . Another study , however , describe s effect s o f ET S o n birth weigh t an d preter m deliver y onl y i n infant s o f older mothers (Ahluwali a et al., 1997) . Makin an d colleague s (1991 ) examine d th e long term effect s o f ETS durin g gestatio n o n 6 - to 9-year olds. They show that on test s of speech an d languag e skills, intelligence , visual/spatia l abilities and o n th e mother's rating s o f offsprin g behavior , th e perfor mance o f offspring o f passive smokers is intermediate to tha t o f th e childre n o f activ e smoker s an d non smokers. Exposure t o ETS i s prevalent i n the postnata l pe riod an d ca n negativel y affec t chil d outcomes . I n one study , 71 % o f inner-cit y childre n o f lo w SE S lived i n household s wit h smoker s (Corneliu s e t al. , 2003). Postnata l exposur e t o ET S increase s th e ris k of SIDS, asthma, respirator y problems, an d otiti s media (DHHS, 1999) . Postnata l ET S i s associated with a reduction i n I Q scores i n 3-year-old s (Johnson e t al. , 1999) an d developmenta l languag e impairment s i n kindergartners (Tomblin et al., 1997) . Note, however,

322 NICOTINE-AFFECTE D DEVELOPMEN T that neither of these analyses controlled fo r the effect s of PTE. ET S i s also associated with decreased perfor mance o n measure s o f receptive vocabulary and rea soning abilit y in a stud y o f 10-year-old s (Bauma n e t al., 1991) . Among 6 - to 11-year-ol d childre n o f non smoking mothers , postnata l exposur e t o ET S result s in deficit s o n centra l auditor y processing task s (Mc Cartney et al., 1994) . Two studie s evaluate the relation s betwee n expo sure to ETS an d chil d outcomes controllin g fo r PTE . In th e MHPC D cohor t o f adolescen t mother s an d their 6-year-old children , the children' s current urin e cotinine level s inversel y correlat e wit h score s o n a measure o f receptiv e languag e controllin g fo r PT E (Cornelius et al., 2000). Five-year-olds wit h postnata l ETS hav e significantly lower scores on the Raven Test and Peabod y Pictur e Vocabular y Test , measure s o f cognitive development , an d ar e rate d a s more activ e by thei r mothers , compare d t o childre n wh o ar e no t exposed to ETS (Eskenaz i and Trupin, 1995) . SUMMARY AND CONCLUSION S

There ar e significan t effect s o f PT E o n th e growth , cognitive development, an d behavio r of exposed chil dren. Children wit h PTE ar e smaller at birth, and recent researc h reports suggest that these childre n hav e a greate r ris k o f obesit y a s the y reac h adolescence . Cognitive deficit s i n reasonin g an d memor y hav e been describe d an d th e expose d children d o less well on test s o f language , reading , an d vocabular y tha n nonexposed children . PTE predict s higher rates of activity, inattention, an d impulsivity . Increases i n oppo sitional an d aggressiv e behavio r an d highe r rate s o f delinquency an d criminalit y i n adolescenc e an d adulthood have been foun d i n a number o f studies. In general, thes e outcome s ar e consisten t whethe r th e mother i s a smoke r o r th e exposur e t o th e mothe r i s through ETS. I n addition, there appear to be differen tial effect s tha t depend on duratio n an d patter n o f exposure and o n whether the offsprin g ar e exposed only during gestation , durin g gestatio n an d afte r birth , o r only afte r birth . Fo r mos t o f the identifie d outcome s of PTE, ther e are parallel data from laborator y studies on animals that lend credenc e t o the human data . Women wh o us e tobacc o durin g pregnanc y ar e often differen t fro m wome n wh o d o no t i n man y ways, includin g i n thei r SES , race/ethnicity , us e o f other substances , an d psychiatri c disorders . I n addi tion, even women wh o do not smoke tobacco ca n b e

exposed t o ETS , resultin g i n exposur e t o th e fetus . Many o f the studie s currently in th e literatur e fai l t o control fo r these significan t covariate s of tobacco us e or for ETS, makin g definitiv e conclusions abou t th e effects o f exposur e mor e difficult . Ther e ar e severa l studies, however , tha t ca n asses s th e competin g effects o f other substanc e us e during pregnancy a s well as the rol e o f the othe r covariate s and ETS . Th e gen eral conclusion s ar e validate d b y the result s o f thes e studies. Characteristics o f th e rearin g environmen t ar e also significantly associate d wit h both PT E an d ETS , as well as with the outcome s i n the offspring . Wome n who smok e durin g pregnanc y ar e highl y likel y t o smoke afte r delivery . Thi s mean s tha t childre n wh o are prenatally exposed to tobacco ar e at higher risk for continued exposur e du e t o ET S exposur e fro m th e mother and/o r othe r househol d smokers . Heredit y is another importan t factor to consider in the interpreta tion o f these findings . A s the mothe r an d chil d shar e genetic components , the y shar e factor s tha t migh t predict thei r respons e t o exposur e o r coul d increas e vulnerability for specific outcomes . Studie s tha t hav e controlled for role of heredity, nonetheless , find independent effect s o f PTE . For al l o f the abov e reasons , i t i s difficult t o enu merate definitivel y th e effect s o f PT E whethe r i t b e from a smokin g mothe r o r fro m exposur e t o ETS . Nevertheless, i t is imperative that the short - and long term effect s o f both prenata l and postnatal exposur e to tobacco o n th e growt h an d neurobehaviora l develop ment o f children b e identified . Although man y of the effects o f PTE ar e subtle , the y hav e importan t effect s on the long-term functionin g of the expose d offspring . Future studie s mus t evaluat e th e separat e an d combined effect s o f exposur e t o tobacc o an d othe r substance exposure s durin g gestation. I t is also critica l to measure the othe r factor s tha t coexist with tobacc o use, a s well a s to evaluat e th e co-occurrenc e o f PT E and ET S bot h durin g pregnancy an d i n the postpar tuni period . Studie s tha t measur e al l o f these factor s across time allows a more specifi c definitio n of the ef fects o f prenata l an d postnata l tobacc o exposur e o n the offspring . Abbreviations ADHD Attentio n Defici t Hyperactivit y Disorde r CNS centra l nervou s system ETS environmenta l tobacco smok e

EPIDEMIOLOGY OF MATERNA L TOBACCO USE 32 3 G gestationa l da y LBW lo w birth weigh t MHPCD Materna l Healt h Practice s an d Chil d Development ODC ornithin e decarboxylase OPPS Ottawa

Prospectiv e Prenata l Stud y

P postnata l da y PTE prenata l tobacco exposur e SES socioeconomi c statu s SIDS sudde n infan t death syndrom e

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20

Prenatal Nicotin e Exposure and Animal Behavio r Brenda M. Elliott Neil E. Grunber g

Cigarette smokin g affect s prenata l brai n develop ment an d subsequen t behavior . Mother s wh o smoke cigarettes durin g pregnanc y ar e mor e likel y tha n nonsmoking mother s t o bea r childre n wh o ar e hy peractive, impulsive, and hav e impairments in learning, memory, and attentio n (Naeye , 1992 ; DiFranz a and Lew , 1995 ; Eskenaz i an d Trupin , 1995 ; Erns t et al, 2001 ; Thapar e t al, 2003) . Children expose d to tobacc o smok e durin g pregnanc y ar e als o mor e likely to smok e cigarette s late r i n lif e (Kande l e t al. , 1994; Abreu-Villac a e t al , 2004a ; 2004b) . Thes e effects ar e i n additio n t o th e retarde d feta l growth , premature births , stillbirths , and increase d neona tal mortalit y associated wit h cigarett e smokin g during pregnancy (Butler and Goldstein , 1973 ; Denso n et al. , 1975 ; Kristjansso n e t al. , 1989 ; Buk a e t al. , 2003). Although cigarette s contai n roughl y 40 0 chemi cals an d cigarett e smok e contain s >400 0 chemicals ,

nicotine is the mos t widely studied chemical . Various properties o f nicotine mak e i t a neurotoxin. Nicotine crosses th e blood-brai n barrier , bind s t o specifi c receptors i n th e brain , an d affect s man y behaviors , including addiction, appetite, stress responses, and attention (Grunberg , 1982 , 1985 ; Luck and Nau , 1985 ; Levin, 1992 ; Scheufel e e t al , 2000 ; Traut h e t al , 2000; Slaweck i an d Ehlers , 2002 ; Slotkin , 2002 ; Slawecki e t al , 2003 ; Farada y e t al , 2003) . Anima l models o f nicotin e an d tobacc o exposur e provid e valuable informatio n on the behaviora l effects of prenatal smoking. The presen t chapte r discusse s knowledge gaine d from studie s of the effect s o f prenatal nicotin e and to bacco exposur e on th e behavio r and developmen t o f the offspring . I t focuse s o n rodent s becaus e mos t o f the researc h on th e effect s o f prenatal nicotine expo sure has been don e i n these animal s and th e findings parallel research o n humans.

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330 NICOTINE-AFFECTE D DEVELOPMEN T VALUE O F ANIMAL MODELS TO ASSESS THE EFFECT S O F PRENATA L SMOKIN G

Animals hav e bee n studie d fo r centurie s t o lear n about anatom y an d physiology . I n ancien t Greece , Galen dissecte d goats , pigs , an d monkey s t o explor e the structur e o f the bod y and to test specifi c hypothe ses about ho w the bod y worked (Nutton , 2002) . Dur ing the eighteent h an d nineteent h centuries , anima l experimentation contributed to our knowledge of physiology, pharmacology , bacteriology , and immunolog y (Nutton, 2002). Darwin's landmar k Origin of th e Species (1859 ) in cluded several concepts that further expande d the study of animals: (1) the continuit y of species, (2 ) natural selection, an d (3 ) spontaneous mutation . Darwi n argued that the senses and intuitions, variou s emotions, an d aspects o f cognition suc h a s memory, attention , curiosity , and reason (attribute s once thought to be unique to humans) migh t b e foun d i n a well-developed for m i n lower animals . Darwin' s compellin g argument s le d t o the stud y of animal biology and behavio r to learn mor e about humans . Animal s were use d t o stud y attention, learning, emotions, and social interaction. Darwin's argumen t tha t ther e i s a continuit y of specie s i n th e anima l kingdo m provide s th e ra tionale fo r studyin g infrahuma n specie s t o lear n about huma n behavior . Ethologists , includin g Lorenz an d Tinbergen, observed th e behavio r o f animals i n thei r natura l habitat s an d provide d com pelling evidenc e tha t supporte d Darwin' s idea s (Dewsbury, 2003) . Pavlov, Thorndike, an d Skinne r separately examine d anima l behavio r i n controlle d laboratory setting s i n a n attemp t t o understan d hu man learnin g an d provide d empirica l evidenc e tha t the stud y of animal behavior i s relevant to the huma n condition. Pavlov (1927 ) develope d th e concep t o f classica l conditioning o n th e basi s o f hi s stud y o f gastri c re flexes i n dog s (Kimbl e an d Schlesinger , 1985) . H e observed tha t dog s respon d t o cue s othe r tha n food , cues tha t ha d bee n previousl y associated wit h food . He labele d thes e ne w response s conditioned (o r conditional) responses , to distinguis h them fro m th e naturally occurrin g responses , tha t occu r withou t prior training . Classica l conditionin g i s a pilla r o f learning principle s an d applie s t o human s a s well as to animals. Around th e sam e tim e tha t Pavlo v wa s experi menting wit h dogs , Thorndik e (1911 ) wa s studying

cats to explore how animal s learn tricks. As part of his investigation, Thorndike constructe d th e puzzl e box , a bo x containin g severa l lever s an d handle s fro m which a ca t mus t lear n ho w t o escape . Fro m carefu l observations o f th e cats ' behaviors , Thorndik e pro posed th e La w o f Effect . Thi s behaviora l la w state s that an anima l wil l repeat a n actio n tha t bring s desirable consequences , bu t i t wil l no t repea t a n actio n that bring s undesirabl e consequences . Thi s la w als o holds for humans. Skinner studie d th e behavio r of rat s an d pigeon s and expande d Thorndike' s finding s (Skinner , 1935) . Building on the concept tha t animals operate on their environment t o obtai n desirabl e consequences o r t o avoid negativ e consequences , Skinne r propose d th e concept o f opéran t conditioning . Specifically , Skin ner foun d tha t th e frequenc y o f a give n behavio r could b e increased b y following the occurrence o f the behavior wit h positiv e consequence s (reinforcement ) or decrease d b y following th e behavio r with negativ e consequences (punishment) . Together, th e studie s by Pavlov, Thorndike, an d Skinne r laid th e groundwork for modern-da y theories of learning and provid e con vincing evidenc e tha t anima l model s ar e a usefu l representation o f human behaviora l an d psychologi cal processes. Animal studie s ar e valuabl e fo r assessin g dru g effects, includin g th e effect s o f nicotin e o n devel opment an d behavior . Anima l model s allo w inves tigators t o evaluat e an d manipulat e separatel y th e components o f tobacco smokin g (e.g. , nicotine ) tha t might affec t prenata l development . Investigator s can control aspect s o f drug deliver y (e.g., dosage , timin g of exposure , duratio n o f exposure , rout e o f administration, and environmenta l context). In addition, the y can isolat e variable s t o focu s o n nicotin e an d avoid variables, suc h a s prenata l care , socioeconomi c sta tus, nutrition , o r us e o f othe r substances , tha t con found huma n studies . Anima l model s offe r uniqu e advantages fo r studying drug effects , includin g thos e of nicotine, on behavior.

ANIMAL MODELS OF NICOTINE AND TOBACCO ADMINISTRATION It i s well establishe d tha t tobacc o smokin g ha s dele terious effect s o n health . A single cigarette , a n objec t smaller tha n a standar d pencil , deliver s mor e tha n 4000 chemical s tha t separatel y o r togethe r ma y

PRENATAL NICOTIN E EXPOSUR E AND ANIMA L BEHAVIOR 33 1

contribute t o thes e effect s (Dub e an d Green , 1982 ; Department o f Healt h an d Huma n Service s 'DHHS], 1989) . Nicotin e ha s been identifie d a s the addictive componen t i n tobacc o smokin g and i s th e centra] dru g contributin g t o th e maintenanc e o f tobacco us e (DHHS , 1988 ; Fieldin g e t al. , 1998 ; Center fo r Diseas e Contro l an d Prevention , 2002) , Nicotine act s i n th e brai n an d result s i n man y be havioral an d psychologica l action s includin g en hancement o f reward , decrease d appetite , focuse d attention, an d change s i n activity . Further , nicotine and it s metabolites readil y cros s th e placenta l mem brane, penetrat e th e preimplantatio n blastocyst , an d cross th e blood-brai n barrie r (Fabr o an d Sieber , 1969; Shiverickand Salafia , 1999) . In rodents , tobacc o ca n b e administere d i n man} ' different ways : through tobacco-extrac t administration, tobacco-smoke exposure , an d topica l applicatio n o f tobacco tars . Tobacco-extract administration refer s t o daily subcutaneou s injection s (SC ) o f nicotine-free tobacco. Som e studie s usin g thi s metho d hav e re ported significan t degenerativ e change s i n variou s organs, includin g th e thyroid , pituitar y gland , an d pancreas (Suematsu, 1931) , or gangrene i n the toe s of male rat s (Friedlande r e t al , 1936 ; Harkavy , 1937) . The tobacco-smoke exposure model involve s exposing rats to cigarett e smoke fo r days o r years and ha s bee n used t o simulat e th e huma n smokin g experience . Kulche and colleague s ( 1952) report that rats exposed to tobacc o smok e exhibi t increase d tea r secretion , salivation, an d copiou s bowe l movements . Othe r studies sho w tha t rodent s expose d t o tobacc o smok e either los e weigh t o r gro w les s tha n contro l animal s (Pechstein and Reynolds , 1937; Passey, 1957) . Tobacc o tar, whe n applie d topicall y t o rats , retard s growth , slows weigh t gain , an d cause s gastri c lesion s (Mc Nallv, 1932 ; Roffo, 1942) . Although tobacco-smok e exposur e i s mean t t o simulate th e huma n smokin g experience, nicotin e i s the actual substanc e i n tobacco smok e that exert s the addictive an d psychobiologica l effect s an d maintain s self-administration (DHHS , 1988 ; Stolerma n an d Jarvis, 1995) . Therefore, nicotin e administratio n paradigms have been th e focu s of animal studies in recen t decades. Nicotin e ca n b e administere d b y oral nico tine administratio n (e.g., nicotine i n drinkin g water), single o r repeate d nicotin e injection s (S C o r intra peritoneal [IP]) , intravenou s self-administration , or sustained nicotin e administratio n (e.g. , a n osmoti c pump). Th e effect s o f nicotin e var y wit h dosage ,

timing, duration of exposure, route o f administration, and chemica l for m o f nicotin e (Levi n e t al. , 1996 ; Slotkin, 1998 ; Phillip s et al, 2004). Oral Nicotine Administration Nicotine i s ingested b y animals via drinking fluids or forced ora l administration . Som e studie s repor t tha t consumption o f nicotin e retard s th e growt h o f mic e and rat s (Nice, 1913 ; Wilson an d D e Eds , 1936 ) o r reduces litte r siz e o f pregnan t mic e (Wilson , 1942) , but othe r studie s report n o suc h effect s (Nice , 1912). The utilit y o f oral-administratio n metho d i s limite d by th e difficult} 7 i n gettin g rat s an d mic e t o drin k nicotine-containing solution s (Murrin et al., 1987 ; Le Houezec e t al. , 1989) . Anothe r disadvantage of using this metho d i s tha t nicotin e i s poorl y absorbe d through th e gastrointestinal tract , leading to lower con centrations of nicotine in th e blood . Therefore , mor e nicotine ma y be neede d t o obtain desirabl e amount s of nicotin e t o serv e a s a mode l o f huma n consump tion. S C injection s and deliver y via a subcutaneously implanted pum p ar e more commonly use d tha n oraladministration paradigm s becaus e th e amoun t o f nicotine administere d ca n b e bette r controlle d an d the absorptio n o f th e dru g i s no t limite d b y passag e through the gastrointestinal tract. Repeated Nicotine Injection s Most studie s examinin g the effect s o f chroni c expo sure t o nicotin e o n behavio r hav e use d on e o f tw o methods: (1 ) repeate d dail y injection s o r (2 ) sus tained nicotin e release . Repeate d dail y injections in volve parentera l injection s —SC o r I P a fe w times a day o r once a da y for several days (Pietila and A h tee, 2000). Thi s approac h ha s bee n widel y use d t o ex amine th e effec t o f nicotine o n behavio r (e.g. , loco motion, acousti c startl e respons e (ASR) , prepuls e inhibition [PPI ] o f the ASR , socia l interaction , ele vated plus maze, an d nociception ) and generate s reli able an d reproducibl e result s (Levi n e t al. , 1997 ; Hahn an d Stolerman , 2002 ; Farada y e t al , 2003 ; Elliott e t al. , 2004 ; Olausso n e t al. , 2004) . Whe n nicotine i s injecte d repeatedl y int o pregnan t dams , similar behavioral disruptions in offsprin g ar e observed (Slotkin, 2004) . I t i s possibl e tha t thes e behaviora l changes reflec t th e consequence s o f uteroplacenta l vasoconstriction an d feta l hypoxi a associate d wit h th e high pea k plasm a concentration s o f nicotin e tha t

332 NICOTINE-AFFECTE D DEVELOPMEN T occur wit h repeate d injection s (Jonsso n an d Hall man, 1980 ; Seidle r an d Slotkin , 1990 ; Carlo s e t al. , 1991; Slotkin, 2004) . That is, nicotine injection s may result in high peak plasma concentration s o f nicotine accompanied b y brie f hypoxi c episode s associate d with eac h dos e (McFarlan d e t al , 1991 ; Slotkin , 1992). These episodi c hypoxi c events ca n alte r brai n development (Jonsso n an d Hallman , 1980 ; Slotkin , 1986; Seidle r an d Slotkin , 1990) . Consequently , an y observed effect s canno t b e full y attribute d t o nicotin e per se . The osmoti c pum p provide s an alternat e an d preferable way of administering nicotin e prenatally . Osmotic Pump Osmotic pump s ar e miniature , subcutaneousl y im plantable pump s use d t o continuousl y delive r drug s or other agents a t controlled rates up to 4 weeks. Th e pump i s a smal l object , resemblin g a pil l capsul e i n shape but large r in size. The pum p ha s two compartments: a n inne r capsul e an d an oute r chamber . Th e inner capsul e i s filled with th e dru g or control liqui d to be delivered. The oute r chamber, whic h surround s the capsule , contain s a sal t an d i s referred to a s th e salt sleeve. When placed underneath th e ski n or in an aqueous medium , a n osmoti c pressur e differenc e de velops between thi s outer salt filled chamber an d th e tissue environment, pullin g water from th e tissu e into the pum p throug h a semipermeable membran e tha t forms th e surfac e o f the pump . Th e pressur e o f th e water i n thi s outsid e chambe r put s pressur e o n the impermeabl e capsule , displacin g th e dru g fro m the inne r cor e int o th e bod y a t a controlle d an d steady rat e muc h lik e th e slo w squeezing o f a tooth paste tube . This method produce s a constant admin istration of nicotine an d a steady-state level o f plasma nicotine (Car r et al, 1989 ; Paul y et al, 1992 ; Slotkin , 1998), therefore , avoiding th e stres s associate d wit h and th e hypoxi a induced b y repeated nicotin e injec tions (Levin etal, 1996 ; Slotkin , 1998) . Grunberg (1982 ) wa s the firs t t o us e a n osmoti c minipump to subcutaneously administer nicotine con tinuously to rats for a week and t o examine their bod y weight and food consumption . Th e findings fro m thi s experiment paralle l result s fro m a stud y o f cigarett e smoking i n humans . Suc h parallel s betwee n dat a generated fro m osmoti c pump-delivere d nicotin e models an d finding s fro m researc h o n huma n ciga rette smokin g hav e bee n describe d i n variou s othe r studies a s well (Bowe n e t al. , 1986 ; Grunber g e t al. ,

1986; Murri n e t al. , 1987 ; Richardso n an d Tizabi , 1994; Levi n e t al. , 1996 ; Farada y et al, 2001 ; Irvine et al., 2001; Malin, 2001) . The osmoti c pum p i s now widely use d t o examin e long-ter m effect s o f nicotin e in rodents. Prenatal Nicotin e Models Studies examinin g prenata l effect s o f nicotin e hav e used mos t o f the method s describe d above. Th e tw o most commo n approache s t o evaluatin g prenata l smoking effect s ar e repeated injection s of nicotine int o the pregnan t da m an d S C administratio n of nicotin e in the pregnant dam via an osmotic pump. Administration o f nicotine t o pregnan t dam s b y repeate d injec tions ha s provide d consisten t evidenc e tha t prenata l nicotine exposur e cause s cellula r damage i n th e feta l brain that may explain subsequen t behaviora l an d cog nitive deficit s (Slotki n et al., 1986) . Repeate d nicotin e injections, however , als o caus e pea k plasm a concen trations o f drug tha t ar e associate d wit h ischemia , a n effect tha t has the potentia l to disrupt fetal brai n development (McFarlan d e t al, 1991 ; Slotkin , 1992) . The osmoti c pum p ha s becom e th e standar d choice for examining prenatal effects o f nicotine (Murrin e t al, 1987 ; Ulric h et al, 1997) . I t delivers drugs or other agents at controlled rate s for up to 4 weeks, avoids peak plasm a concentration s associate d wit h repeate d injections, allow s for better contro l over nicotine delivery, an d eliminate s th e ris k o f hypoxic episodes. Th e continuous infusio n o f nicotine via osmotic minipum p is analogou s t o nicotin e patc h deliver} ' bu t differ s markedly fro m nicotin e self-administratio n throug h smoking tobacco . Overview o f Behavioral Assessmen t in Animals The stud y of animal behavio r ha s become commo n practice an d behaviora l model s ar e frequentl y in cluded i n studie s o f psychology , pharmacology , an d behavioral an d cognitiv e neuroscience . Anima l mod els provid e informatio n abou t norma l behavio r an d responses t o drugs, disease, or adverse environmenta l conditions. Th e behaviora l pattern s observe d rang e from studie s o f basic sensory (e.g., olfactory, taste, au ditory, and tactile) , motor (e.g. , balance), an d neuro logical (e.g. , eye blink, righting reflex, and ea r twitch) functions t o unconditioned behaviors (e.g. , food con sumption, locomotor activity, and sexua l behavior), to

PRENATAL NICOTINE EXPOSUR E AND ANIMAL BEHAVIOR 33 3 more comple x assessments of attention, learning , and memory (e.g., Morris water maze, T-maze , radial arm maze, an d PPI) . The followin g section discusse s key findings from prenata l nicotine studies in which some of thes e behaviora l paradigm s wer e use d an d offer s suggestions for future studies.

EFFECTS O F PRENATA L EXPOSURE TO NICOTINE IN ANIMAL MODELS Outcome measure s o f prenatal effect s includ e birt h weight and feta l liability , locomotor activity, and cog nition. Th e effec t o f nicotin e o n thes e variable s is a function o f th e amoun t o f nicotin e administered , route of administration, timing of administration, and animal specie s (Erns t e t al., 2001) . Prenatal nicotin e exposure compromises th e health of the newbor n an d can hav e long-lasting deleteriou s effect s o n offsprin g behavior. Studies o f prenata l nicotin e effect s dat e bac k t o the earl y 1900 s (Nakasawa , 1931 ; Sodano , 1934 ; Grumbecht an d Loeser , 1941) . Mos t o f thes e earl y studies wer e conducte d i n rabbit s an d dogs . Gener ally, thes e studie s focuse d o n effect s o f nicotin e o n pregnancy duratio n an d th e siz e an d viabilit y o f th e young. Earl y studie s i n th e rabbi t repeatedl y sho w that dail y injection s o f tobacco-smok e solutions , chronic nicotine infusions , or nicotine administration caused spontaneou s abortions or premature deliveries (Guillain and Gy, 1907 ; Benigni , 1911 ; Perazzi , 1912). Tissue s fro m fetuse s o f rabbit s tha t receive d daily SC injection s of nicotine fo r up t o 1 2 weeks are damaged (Var a an d Kinnunen , 1951) . Hofstatte r (1923) note s that pregnancie s occurring durin g nicotine administration frequently abort. Body Weight Animals expose d t o nicotin e prenatall y consistentl y weigh les s than animal s not expose d t o nicotin e pre natally (Bass i e t al. , 1984 ; Paulso n e t al , 1993 ; Ajarem and Ahman , 1998 ; Vaglenov a et al , 2004) . Animals receivin g 4. 0 mg/kg/day o f nicotin e prena tally (ora l administration ) weig h les s tha n controls , and thes e effect s persis t unti l postnata l da y (P ) 2 9 (Paulson e t al, 1993) . Ajarem an d Ahmed (1998 ) administered nicotin e (0.5 0 mg/kg) or saline to pregnan t mice via SC injection s for 9-10 day s and assesse d offspring development . Th e nicotine-expose d animal s

gained weigh t a t a slowe r rate tha n tha t o f controls . Such findings are interesting because they parallel reports from human s an d sugges t that prenatal smokin g affects prenata l an d postnata l development . Specifi cally, wome n wh o smok e durin g pregnanc y hav e a greater numbe r o f spontaneous abortions , premature births, an d infant s with low-birt h weight s (Eskenaz i etal, 1995; Hofhuis etal, 2003). In addition, nicotineinduced reductio n i n postnata l weigh t gai n als o has been reporte d i n huma n offsprin g (Marti n e t al. , 1977). Hyperactivity Several studies have reported hyperactivit) ' in th e offspring o f nicotine-treate d animal s (Marti n e t al. , 1976; Lichtensteige r e t al, 1988; Ajarem and Ahmad, 1998; Fun g and Lau, 1998 ; Newma n e t al, 1999 ; Ti zabi and Perry , 2000). In rats , hyperactivity is indexed by measurin g open-field activit y o r activit y i n othe r novel environments such as the elevate d plus maze. Open-field activity refers to the behavio r of an animal whe n i t i s place d i n a nove l environment . Ini tially, activity levels are high whe n an animal explores its ne w environment . A s th e environmen t become s familiar, ambient activity decreases. Persistent heightened activit y is interpreted a s evidence o f hyperactivity, a behavior frequentl y observed i n th e offsprin g o f mothers who smoked during pregnancy. Rats exposed to nicotine (1. 5 mg/kg/day via SC in jections) throughout gestatio n hav e increased sponta neous locomoto r activit y compare d t o tha t o f saline-exposed control s (Fun g and Lau , 1988) . Simi larly, Tizabi an d colleague s (1997), who administered nicotine (9. 0 mg/kg/day) vi a osmoti c minipump s t o pregnant dam s throughout gestation, show that 20-24day-old offsprin g prenatall y expose d t o nicotin e ex hibit mor e locomoto r activit y tha n tha t o f salin e controls. The elevate d plu s maze i s used t o test anxiety and to measur e activit y and impulsivity . Accordingly , a n animal is placed i n the center of an elevate d four-arm maze tha t include s tw o ope n an d tw o close d arms . The tota l numbe r of times an animal enters any of the four arm s provides an inde x of activity and impulsiv ity. Ajarem an d Ahma d (1998 ) administered nicotine (0.50 mg/kg) or saline to pregnant mic e for 9-10 days and measure d activit y on th e elevate d plu s maz e of weaned offspring . Nicotine-expose d offsprin g mad e more entrie s in the arm s of the maz e tha n di d saline

334 NICOTINE-AFFECTE D DEVELOPMENT controls. This finding suggests that prenata l exposure to nicotin e promote s hyperactivit y an d impulsivity . Sobrian an d colleague s (2003 ) repor t simila r effect s of prenata l nicotin e exposur e o n activit y i n a n ele vated plu s maze . The y administere d nicotin e (2. 5 or 5.0 mg/kg) or saline via an osmotic pump t o pregnant dams throughout gestation . Adult rats exposed to high doses o f nicotine prenatall y ar e les s timi d an d mor e impulsive than controls. These findings are consistent with reports from huma n studies , that prenatal smoking t o nicotin e result s i n hyperactivit y i n offsprin g (Milberger e t al , 1996 ; Mic k e t al , 2002 ; Thapa r et al, 2003). Attention Prenatal smokin g disrupt s attentio n i n expose d offspring (Kristjansson e t al., 1998) . In rats, attention ca n be measure d usin g the PP I of the ASR . PPI refer s t o the reductio n o f a startle respons e whe n th e startlin g stimulus is preceded b y a nonstartling stimulus. Con trol animal s exhibi t a reduce d startl e respons e t o a sudden, loud nois e if the nois e i s preceded b y a softe r sound. Disorders that disrupt attention, suc h as schizophrenia an d attentio n defici t hyperactivit y disorder (ADHD), disrup t PP I (Swerdlo w et al, 2000 ; Geye r et al., 2001). Reports o f prenata l nicotin e o n attentio n i n rat s are limited , but dat a tha t ar e available show that rat s respond to prenatal nicotine exposure . Prenatal nicotine exposur e (6mg/kg/da y vi a osmoti c minipump ) throughout gestatio n reduce s th e PP I of the ASR to a 98 dB stimulu s i n femal e offsprin g (Popk e e t al. , 1997). Thes e finding s ar e consisten t wit h th e ob served attentiona l deficit s reporte d i n human s ex posed t o nicotin e prenatally . Futur e studie s usin g other attentional paradigms (e.g., five-choice serial reaction tim e test ) will furthe r hel p t o characteriz e the attentional domain s affecte d b y prenata l nicotin e exposure. Learning an d Memor y Prenatal nicotin e exposur e alter s learnin g an d mem ory (Cornelius et al., 2000). In rats , the effect s o f prenatal exposur e to nicotin e o n learnin g and memor y have been evaluate d primaril y by use o f a radial arm maze, whic h present s a challeng e tha t assesse s th e ability o f an anima l t o learn an d remembe r multipl e

spatial locations . Th e anima l i s placed i n th e cente r of a maz e containin g eigh t o r mor e radiatin g arms. Food i s placed a t th e en d o f four arms . Th e anima l must ente r a n arm to retrieve the food . Th e number s of repeat entries by a rat into the arm s already entered are recorde d as working memory errors (Olton, 1987 ; Hodges, 1996) . Th e number s o f reentries int o arm s never havin g containe d foo d ar e recorde d a s refer ence memor y errors. Several studie s repor t impairment s o n th e radia l arm maz e followin g prenatal nicotin e exposure . Fol lowing administration o f nicotine (6. 0 mg/kg) to pregnant dam s i n thei r drinkin g wate r throughou t gestation, offsprin g exhibi t impaired performanc e i n the radia l arm maz e (Sorenso n e t al., 1991) . Severa l other studie s describ e simila r finding s (Yana i e t al. , 1992; Levin et al., 1996) . Levin and colleague s used a radial arm maz e t o assess the effect s o f prenatal nico tine exposur e (2.0mg/kg/da y via osmotic minipump ) on learnin g an d memory . The y repor t tha t nicotine exposed animals do not diffe r fro m control s in the ac quisition o f the task , but thes e rat s make mor e errors when spatia l cue s ar e changed , henc e addin g t o th e demands o f the task. Prenatal effect s o f nicotin e hav e bee n evaluate d using the two-wa y active avoidance (shuttle box) task. In thi s task , classica l conditionin g procedure s ar e used t o asses s learnin g an d long-ter m memory . Th e apparatus consist s of two equall y divide d compart ments connecte d b y a cente r opening . Durin g th e training th e anima l i s presente d wit h a combine d light an d ton e (conditione d stimuli) , followed by the presentation o f shock o n a grid floo r (unconditione d stimulus [UCS]) . Followin g presentatio n o f the con ditioned stimulus , th e anima l ca n avoi d th e UC S by passing fro m on e compartmen t t o another . Thi s avoidance behavio r i s evidence o f learning, wherea s failure t o avoi d th e UC S i s evidence o f no learning . Memory i s assessed b y re-exposing th e anima l t o th e apparatus severa l days later an d measurin g avoidanc e activity. Greate r avoidanc e behavio r is evidenc e of long-term memory . Two-month-old mal e offsprin g o f dam s injecte d with nicotin e (0.50mg/kg/day ) durin g pregnanc y exhibit deficit s i n learnin g th e avoidanc e respons e (Genedani e t al. , 1983) . Mor e recently , Vaglenova and colleague s (2004 ) administere d nicotin e (6.0 mg/kg/day) vi a osmoti c minipum p t o pregnan t dams an d measure d activ e avoidanc e behavio r o f

PRENATAL NICOTINE EXPOSUR E AN D ANIMA L BEHAVIO R 33 5 offspring o n P45 . Animals of both sexes prenatally exposed t o nicotin e hav e a significantl y lowe r numbe r of avoidance behavior s durin g all days of training an d during the retentio n test . These data are indicative of impaired learnin g an d memory . Th e result s of thes e animal studie s parallel report s of memory an d learn ing problems i n human s an d provid e further suppor t for th e valu e o f animal s fo r investigatin g prenata l smoking effects o n memor y an d learnin g in offsprin g (Naeye an d Peters , 1983 ; Rantakalli o an d Koiranen , 1987; DiFranz a an d Lew , 1995 ; Slotkin, 1998) . Addiction Liability Prenatal tobacco smokin g increase s the likelihoo d of smoking amon g expose d offsprin g (Kande l e t al. , 1994; Niaur a et al, 2001; Chassin et al, 2002; AbreuVillaca et al, 2004a, 2004b). In humans, prenata l exposure to nicotine is an important predictor of smoking in adolescents . (Corneliu s e t al., 2000 ; Niaur a et al. , 2001). Interestingly , thi s vulnerabilit y exists , regard less of whether parents continue t o smoke. Recent anima l studie s examinin g th e effect s o f prenatal nicotin e o n late r biologica l response s t o nicotine paralle l th e finding s o f huma n studie s an d provide underlying mechanisms fo r these observed effects (Abreu-Villac a et al, 2004a, 2004b) . I t has been suggested tha t th e increase d likelihoo d o f smokin g among exposed offsprin g reflect s long-lasting changes in neural cell number, neural cell size, and cell surfac e area. These changes are evident in the neonate an d become exaggerate d whe n nicotin e i s re-administere d during adolescence . Anothe r propose d mechanis m for th e relationshi p between prenata l nicotin e expo sure and adolescen t smokin g is that prenatal nicotin e exposure disrupt s th e programmin g o f cholinergi c function (Abreu-Villac a et al., 2004b). After prenata l nicotin e exposure , ther e i s impairment o f nicotin e acetylcholin e recepto r (nAChR ) up-regulation an d recepto r desensitizatio n whe n nicotine i s administere d durin g adolescence . Thes e factors ma y contribute to increased cigarett e smokin g by adolescent s (Abreu-Villac a e t al, 2004b) . A rela tionship betwee n gestationa l nicotin e exposur e an d nicotine preferenc e i n peri-adolescen t offsprin g ha s also been examine d i n mice (Klei n et al., 2003). Pregnant female s wer e administere d saccharin-flavore d water containin g 5 0 [Ig/ml nicotin e o r saccharin flavored water without nicotine fro m th e nint h da y of

gestation throug h P21 . Th e nicotin e preferenc e o f adolescent mal e offsprin g expose d t o nicotin e prena tally i s increase d compare d t o tha t i n age-matche d controls. The mechanis m fo r this effec t ha s not bee n established.

CONCLUSIONS AND FUTURE IMPLICATIONS

Animal models of nicotine administratio n and animal studies o f behavior provide investigators wit h valuabl e ways t o examin e th e relationshi p betwee n prenata l smoking (especiall y prenatal nicotine ) an d cognitiv e and behaviora l outcome s i n affecte d offspring . Th e fact that findings from prenata l nicotin e studies in animals parallel and predic t findings from human s sup port th e validit y o f thes e studies . These model s ar e well establishe d an d hav e provide d reliabl e result s that hel p expan d ou r understandin g o f the effect s o f tobacco. Despit e thi s knowledge , man y question s re main unanswered . Fo r example , althoug h w e no w have a better understandin g o f specific brai n region s affected b y prenata l nicotin e exposure , th e mecha nisms b y which thes e effect s occu r ar e poorl y under stood. Littl e researc h ha s bee n conducte d o n higher-animal species ; mos t studie s examinin g th e effects o f prenata l exposur e t o nicotin e hav e bee n conducted i n rodents . Studie s conducte d i n anima l species that more closel y parallel human s (i.e. , mon keys) ma y provide additiona l informatio n about rele vant mechanisms . Ou r understandin g ma y offe r opportunities fo r th e developmen t o f intervention s that reverse damaging effects o f nicotine o n the fetus . Future studie s shoul d includ e othe r behaviora l measures. I n humans , prenata l nicotin e exposur e results i n attentio n deficit s i n offspring , however , mod eling anima l studie s tha t examin e attentio n ar e limited. Comple x measure s of attention d o exis t and should b e include d i n futur e studie s of the effect s o f prenatal exposur e to nicotine. Examination o f sensor y an d moto r function s would b e instructive , bu t fe w animal studie s hav e in cluded suc h behaviora l measure s (Ajare m an d Ah mand, 1998) . Huma n offsprin g prenatall y exposed t o nicotine exhibi t impairment s i n sensor y processin g and basi c moto r function s tha t persis t eve n a yea r postdelivery. Saxto n (1978) , fo r example , examine d babies born to mothers who smoked with the Brazelton

336 NICOTINE-AFFECTE D DEVELOPMEN T Neonatal Behavioral Assessmen t Scale . These infant s have reduce d auditor y acuit y compare d t o tha t of infants of nonsmokin g mothers . Further , Gusell a and Fried (1984) report a decrease i n motor scores, a s mea sured b y the Bayle y Mental and Moto r Scales, among 13-month-old offspring whose mother s smoked in dur ing pregnancy. Thes e findings are importan t because they sugges t tha t prenata l smokin g affect s no t onl y complex behaviors suc h a s learning an d memory , bu t also fundamenta l sensor y an d moto r processes . Fur ther studie s examinin g thes e relationship s ar e impor tant, a s disruption s i n sensor y an d moto r processin g can explai n learnin g problems , behaviora l problem s such a s hyperactivity, an d perhap s eve n sensitivit y t o drug exposur e i n adolescence or adulthood. Abbreviations ADHD attentio n deficit hyperactivity disorde r ASR acousti c startl e respons e IP intraperitonea

l

nAChR nicotin e acetylcholine receptor P postnata l da y PPI prepuls e inhibition SC subcutaneou

s

UCS unconditione d stimulu s

ACKNOWLEDGMENTS Thi s wor k wa s supporte d b y the Rober t Wood Johnso n Foundatio n an d the U.S . De partment of Defense. References Abreu-Villaca Y , Seidle r FJ , Slotki n T A (2004a ) Doe s prenatal nicotin e exposur e sensitiz e th e brai n t o nicotine-induced neurotoxicit y in adolescence? Neu ropsychopharmacology, 29:1440-1450. Abreu-Villaca Y , Seidle r FJ , Tät e CA , Cousin s MM , Slotkin T A (2004b ) Prenata l nicotin e exposur e alters th e respons e t o nicotin e administratio n i n adolescence: effect s o n cholinergi c system s durin g exposure an d withdrawal . Neuropsychopharmacology, 29:879-890. Ajarem JS , Ahmad M (1998 ) Prenatal nicotine exposur e modifies behavio r o f mic e throug h earl y develop ment. Pharmaco l Biochem Beha v 59:313-318. Bassi JA, Rosso P, Moessinger AC, Blan c WA, James LS (1984) Feta l growt h retardatio n du e t o materna l

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21

Neuronal Receptors for Nicotine: Functional Diversity and Developmental Changes Huibert D. Mansvelder Lorna W. Role

The nicotini c acetyleholin e recepto r (nAChR ) i s the principal target-mediato r o f nicotin e fro m smokin g and chewin g of tobacco (Hog g et al., 2003; Picciotto, 2003; Gott i an d dementi , 2004 ; Wonnacot t e t al, 2005). Th e effect s o f nicotin e o n offsprin g o f preg nant smoker s ar e widespread , rangin g fro m altere d neural developmen t t o increase d susceptibilit y t o addiction i n adolescenc e (Kande l e t al. , 1992 ; Broide and Leslie , 1999 ; Everitt e t al , 2001 ; Frank e t al , 2001; Walsh e t al, 2001; Slotkin , 2002, 2004; AbreuVillaca e t al , 2004a , 2004b ; Cohe n e t al , 2005 ; Slotkin e t al. , 2005) . Thes e facts , couple d wit h epidemiological finding s tha t >20 % o f pregnant wome n smoke, underscor e th e importanc e o f understanding nAChR function s i n th e prenata l mammalia n brai n (Chassin e t al , 1996 ; Ebrahim e t al, 2000 ; Wals h et al, 2001; Cohen et al, 2005). Intrinsic cholinergi c signalin g participates i n be haviors a s fundamenta l a s memory , motivation , an d movement (Picciott o an d Corrigall , 2002 ; Picciotto , 2003; Gott i an d dementi , 2004 ; Laviolett e an d van 341

der Kooy , 2004 ; Wonnacot t e t al, 2005) . Alterations in nACh R number s an d profile s ar e associate d wit h numerous disorders , includin g Alzheime r demen tia, schizophrenia , and , not surprisingly , addictio n (Cordero-Erausquin e t al. , 2000 ; Marubi o an d Changeux, 2000 ; Changeu x an d Edelstein , 2001; Sabbagh e t al, 2002 ; Freedma n e t al, 2003 ; Hog g et al , 2003 ; Gott i an d Clementi , 2004 ; Newhous e et al, 2004b ; Sacc o e t al, 2004). I n view of the central role of nAChRs in such divers e conditions, i t may seem surprisin g that mor e i s not know n o f th e precise composition , localization , an d overal l rol e o f nAChRs i n synapti c signalin g in th e centra l nervou s system (CNS) . A majo r obstacl e t o advancin g th e fiel d i s tha t CNS cholinergi c synapses , i n contras t t o thei r cousins i n th e periphera l nervou s syste m (PNS) , have largel y elude d direc t electrophysiologica l scrutiny. I n defens e o f efforts t o date , several difficul ties shoul d b e noted . First , centra l cholinergi c neurons ar e fe w i n numbe r an d thei r projection s ar e

342 NICOTINE-AFFECTE D DEVELOPMEN T diffuse (Woolf , 1991) . Second , ther e ar e multipl e subunit-combination variant s of nAChRs. This structural medle y i s reflected i n a comparabl e functiona l diversity that include s differences i n nACh R sensitiv ity t o both endogenou s agonist s and exogenousl y ad ministered nicotin e (L e Novere e t al. , 2002a , 2002b ; Hogg e t al, 2003 ; Gott i an d Clementi , 2004) . Th e cellular targetin g o f nAChR s i s als o diverse . Som e nAChRs ar e shippe d t o presynapti c o r pretermina l sites, other s t o postsynapti c an d perisynapti c locales , and al l nAChR subtypes are expressed at strikingly low amounts compare d wit h ionotropi c receptor s fo r glutamate o r y-aminobutyri c aci d (GABA ) (Colquhou n and Patrick, 1997; Tembumi et al, 2000; Le Novere et al., 2002a, 2002b; Hogg et al, 2003). Perhaps most discouraging i s how quickl y nAChRs transi t int o close d (desensitized) state s i n respons e t o eve n minut e con centrations o f agonist . Thi s trait , too , varie s amon g neurons and amon g nAChR subtype s (Lu et al., 1999 ; Quick an d Lester , 2002 ; Wooltorto n e t al , 2003 ; Alkondon and Albuquerque, 2004, 2005). Although suc h feature s presen t seriou s obstacle s to investigators , thes e sam e characteristic s underli e the importan t contributio n of nAChRs t o the compu tational flexibilit y o f circuit s wher e signalin g is sub ject t o modulation, rathe r than jus t "on/off " control . Recent molecula r biological , biochemical, an d phar macological studie s have generated a powerful armamentarium o f ne w probe s an d approache s fo r analyzing nAChR subuni t compositions an d subtypes (Gotti an d Clementi , 2004 ; Laviolett e an d va n de r Kooy, 2004 ; Nick e e t al , 2004 ; Wonnacot t e t al , 2005). Th e challeng e no w i s to advanc e th e curren t understanding o f ho w cholinergi c synapses , i n gen eral an d pre - and postsynapti c nAChR s i n particular , contribute t o th e function s an d dysfunction s of th e developing nervous system. The las t decad e ha s witnesse d considerabl e ad vances i n basi c knowledg e o f th e function , develop ment, an d plasticit y o f nAChRs . Severa l recen t reviews hav e presente d nove l perspective s on nACh R signaling mechanism s an d o n emergen t view s o n nAChR involvemen t i n neuropsychiatri e disorder s (Jones e t al, 1999 ; Picciott o et al, 2001 ; Hogg e t al, 2003; Picciotto, 2003 ; Dajas-Bailador an d Wonnacott , 2004; Fucile , 2004 ; Gotti and Clementi , 2004 ; Laviolette and van der Kooy, 2004; Lester et al, 2004; Newhouse e t al , 2004a , 2004b ; Sacc o e t al , 2004 ; Wonnacott e t al. , 2005) . The presen t chapte r focuse s on fundamenta l aspect s o f th e acetylcholin e (ACh )

receptors with which nicotine interacts in the context of potential effects o n neura l development and plasticity.

STRUCTURAL DIVERSIT Y OF NEURONA L NÏCOTINIC ACETYLCHOLINE RECEPTORS Twelve separat e gene s encodin g distinc t neurona l nicotinic recepto r subuni t proteins have bee n identi fied an d iterativel y labeled a s oc2 , ot3, cc4, oc5 , ot6 , Ot7 , cx8, cc9 , an d oclO , o r ß2 , ß3 , an d ß 4 (L e Nover e an d Changeux, 1995 ; L e Nover e e t al , 2002b ; Hog g et al., 2003 ; Gott i an d Clementi , 2004) . Each o f the five subunits that compris e the assemble d oligomeri c complex i s a four-transmembran e domai n protei n with considerabl e homolog y amon g th e membran e spanning regions (Fig. 21-1 A, B). All nicotinic recep tor subunit s are characterize d b y a tw o C-loop con taining N-termina l domain , th e signatur e o f thei r membership i n th e broade r famil y o f ligand-gate d ion channels , alon g wit h GABA A, glycine, an d mus cle nicotini c recepto r subunit s (L e Nover e an d Changeux, 1995 ; Le Novere et al, 2002a; Hogg et al., 2003; Gotti an d Clementi , 2004 ; Leste r e t al, 2004). Similar t o th e muscl e nicotini c Ot l recepto r subunit , neuronal a subunit s are identified by the presence of vicinal cystein e residue s withi n th e N-termina l do main, a uniqu e ta g o f th e ligand-bindin g regio n (Brejc e t al , 2001 ; Karlin , 2002 ; L e Nover e e t al , 2002a, 2002b) . The assemble d recepto r protei n i s a pentamer wit h a hydrophili c cor e line d primaril y by the secon d transmembran e domai n o f eac h subuni t (Fig. 21-1B , C) . Th e contributio n o f specifi c sub units t o assemble d receptor s an d ke y feature s of th e ligand-binding domains an d io n channel por e ar e being scrutinize d wit h "knock-down, " single - an d double-iiAChR subuni t gen e knockou t mic e (L e Novere e t al., 2002b ; Champtiau x e t al, 2003 ; Hog g et al, 2003 ; Leste r e t al. , 2003) . The ligand-bindin g domain i n neurona l nAChRs , lik e muscle-typ e nAChRs, i s formed b y th e N-termina l domain s o f o e subunits an d a t th e interfac e of adjacent subunit s i n the assemble d pentamer . Th e identificatio n o f se quences involve d i n th e ioni c "gate " o f nAChRs ha s emerged fro m studie s usin g ingeniou s combination s of mutagensi s t o incorporat e cystein e residue s at key sites, an d subsequen t biochemica l an d biophysica l probing wit h specia l cysteine-reactiv e reagent s (Karlin, 2002; Jensen et al, 2005).

DEVELOPMENT OF NICOTIN E RECEPTOR S 34 3

FIGURE 21- 1 Curren t model s o f neuronal nicotini c receptor (nAChR ) subuni t complexes. Panel s A and B illustrate singl e a - o r ß-type nicotinic recepto r sub unit protein in the membran e bilayer . A. Each o f the neurona l nicotini c recep tor subuni t gene s encode s a protei n tha t traverse s the membran e fou r time s (labeled 1-4) . Th e N-termina l domain is on the external face of the membrane , the first and secon d transmembran e domain s (TM ) ar e linke d by short amin o acid sequence s o n th e interna l face , an d th e secon d an d thir d T M ar e con nected b y a short external linker . The cytoplasmi c loop betwee n T M 3 and 4 is the mos t variabl e portion o f the subuni t sequences . I t has bee n implicate d i n both th e modulatio n an d membran e targetin g o f assemble d nACh R com plexes. Th e C termina e ar e short extracellula r domains . B . Dispositio n o f the TM withi n a single o c subunit relative to each othe r an d t o the Hydrophobi e surrounds. Th e linke r betwee n TM 1 an d TM 2 an d th e intracellula r sid e TM 2 amino acids comprise th e io n channe l gat e (se e tex t and Karlin , 2002) . Panels C an d D sho w model s o f the arrangemen t o f five nAChR subunit s (e.g. , oc 7 alone o r a an d ß subunits) assembled as neuronal nACh R complexes . C . This simplified structura l mode l o f a neuronal-typ e nACh R pentame r i s largely inferred fro m studie s of the muscl e an d torped o electropla x nAChR. Ke y features include th e "stave s o f a barrel " arrangemen t o f eac h subuni t aroun d th e hy drophilic pore , an d th e contributio n o f the TM 2 t o the linin g of the channel . D. Example s o f propose d subuni t composition s o f neurona l nACHR s an d experimentally derive d difference s i n Ca 2+:Na permeabilit y o f th e illustrate d subtypes.

The multiplicit y of nAChR-subunit types beg s the question, "wh y so many ?" . Thi s issu e received a con siderable researc h effor t ove r the las t decad e resultin g in th e conclusio n tha t differen t subuni t combination s are functionall y an d pharmacologicall y distinc t fro m one anothe r (Fig . 21—ID ; se e Diversit y o f Nicotini c Acetylcholine Receptors, below). The physiologica l justification fo r th e subuni t diversity, however , raise s th e difficult questio n o f how t o identif y th e combination s

and stoichiometrie s o f subunit s tha t compris e native nAChRs (Colquhou n an d Patrick , 1997 ; Champtiau x et al , 2003 ; Na i e t al , 2003 ; Alkondo n an d Albu querque, 2005) . Despit e a concerte d effort , thi s issue remain s largel y unresolved. Nevertheless , sev eral generalization s abou t nACh R compositio n have hel d u p t o scrutiny : ther e i s reasonable agree ment tha t th e oc4 , ß2, and oc 7 subunits ar e the majo r players i n nAChR s expresse d i n th e CNS , wherea s

344 NICOTINE-AFFECTE D DEVELOPMEN T oc3, ß4 , an d a ? subunit s ar e mor e typica l compo nents o f PN S nAChR s (Cordero-Erausqui n e t al. , 2000; Picciotto et al, 2001; Le Novere et al, 2002b; Hogg et al, 2003; Gotti and démenti, 2004). Likewise, i t i s generally agreed tha t (1 ) a2 , oc3 , an d oc 4 require co-assembl y with a ß subuni t t o mak e func tional channels ; (2 ) ß 2 an d ß 4 requir e co-assembl y with a n a subuni t to make functiona l channels; (3) oc5, ot6 , an d ß 3 requir e co-assembl y with bot h an other a an d ß subunit to make functional channels; and (4 ) onl y oc7 , oc8 , oc9 , an d ocl O ca n for m func tional homopentamers (Wang et al., 1996 ; Cordero Erausquin e t al. , 2000 ; Changeu x an d Edelstein , 2001; Ber g an d Conroy , 2002 ; L e Nover e e t al , 2002b; Hogg et al, 2003; Gotti an d Clementi, 2004; Nicke, 2004). This i s what nAChR subunit s can d o i n term s of co-assembly. Wha t happen s i n viv o i s les s clear , al though a cleare r pictur e i s emergin g a s nativ e nAChRs becom e bette r define d throug h combine d molecular, pharmacological , an d biophysica l ap proaches (Sivilott i e t al, 1997 ; Y u and Role , 1998a , 1998b; Cueva s et al, 2000; Champtiaux e t al, 2003; Nai et al, 2003; Fucile, 2004 ; Fischer e t al, 2005). In recognition o f compositiona l uncertainty , a consor tium o f "nicotinologists " hav e adapte d a nomencla ture in which the inclusio n of a subunit in an nACh R complex i s indicate d b y a n asteris k (Luka s e t al , 1999) Fo r example, the designatio n oc4ß2* i s used for nAChR complexe s tha t includ e oc 4 an d ß 2 subunits , regardless o f stoichiometry , i.e. , (oc4) 2 (ß2) 3 o r (oc4) 3 (ß2) 2 , and allows for the possibility that other subunits may participate in th e nativ e complex. S o far, reports on nativ e nACh R compositio n includ e everythin g from th e simples t configuratio n (i.e. , homopen tameric, suc h a s (oc7) 5 t o th e mos t complicate d ver sions (i.e., different a an d ß subunits) of nAChRs (Le Novere e t al., 2002b). Mercifully, studies of the over laps in nAChR-subuni t gene expressio n pattern s sug gest that the subuni t composition o f native nAChRs is not as horrific as the simple mathematical calculatio n with 1 2 subunits in groups of five would predict (>10 5 possible outcomes!) . I n addition , th e expressio n pat terns for several subunits are relatively restricted (e.g., oc5, a6, and ß3 ) (Le Novere et al., 1996 ; Klin k et al, 2001; Le Novere e t al, 2002b ; Zoli e t al, 2002) ; cx8 has no t bee n identifie d i n mammalia n system s an d oc9 an d ocl O appea r t o b e largel y restricte d t o inner vated sensory epithelia (Elgoyhe n et al., 2001; Keiger et al. , 2003) . I n sum , th e subuni t compositio n an d

stoichiometry o f nativ e neurona l nAChR s i s mor e complicated tha n ca n be resolved with any one o f the current analyti c techniques , bu t i s probabl y les s baroque tha n straigh t combinatoria l analyse s migh t suggest.

DIVERSITY OF NEURONA L NICOTINI C ACETYLCHOLINE RECEPTORS

Functional Group s The functiona l distinctions among nAChR s of differ ent compositio n ar e no t trivial . Eac h i s an importan t determinant o f ho w th e gatin g o f a particula r nico tinic receptor coul d contribute to synaptic excitability and/or be affecte d b y exogenously delivered nicotine. In th e beginnin g o f the 1990s , biochemica l an d bio physical analyse s combined t o rescue oc5 , oc6 , and ß 3 from thei r orpha n statu s (L e Nover e e t al. , 1996 ; Ramirez-Latorre e t al , 1996 ; Wan g e t al , 1996 ; Groot-Kornielink et al, 1998; Lena et al, 1999 ; Kury atov et al., 2000) . More recen t wor k has honed thes e techniques t o bette r defin e the functiona l properties that distinguis h on e nACh R subtyp e fro m anothe r (Nai e t al., 2003). It is now clear that the inclusio n of each o f thes e subunit s i n nACh R complexe s ca n change th e kinetics , unitar y conductance , an d cal cium permeabilit y o f th e resultan t channel s (Ramirez-Latorre e t al., 1996 ; Wan g e t al, 1996 ; Yu and Rôle , 1998a , 1998b ; Nai et al, 2003). For example, biophysica l studie s sho w tha t Ot3ß4 * an d a3oc5ß4* nAChRs , whic h ar e presen t a t man y pe ripheral site s an d involve d in th e cardiovascula r effects o f nicotine pass more picoamp s per millisecon d than Ot4ß2 * nAChR s (Gerzanic h e t al, 1998) . Th e activation profile s an d desensitizatio n rate s o f thes e nAChRs are also dissimilar from on e another an d ar e very differen t fro m oc 7 homomeri c nAChR s (Ber g and Conroy , 2002 ; Quick an d Lester , 2002 ; Khiroug et al, 2004). Rectification propertie s o f th e variou s channe l subtypes a s wel l a s agonis t an d antagonis t bindin g properties are distinct, but perhaps the most functionally importan t differenc e amon g nACh R subtype s is in their relative Ca2+ permeability (e.g., Fig. 21-1D) . The subjec t o f Ca 2+ conductanc e an d nAChR s re quires an extra comment on <x7 * nAChRs, th e poster child equivalen t for decades o f "grind an d bind " an d autoradiographic assay s o f CN S oc-bungarotoxi n

DEVELOPMENT OF NICOTIN E RECEPTOR S 34 5 (otBgTx) sites . The dogge d effort s o f severa l investigators brough t th e beguilin g finding that th e arche typal muscl e aBgTx-bindin g sit e wa s peppere d al l over the CNS, to the ultimat e resolutio n o f a unique set of nicotine-gated io n channel s (Wonnacot t e t al., 1982; Clark e e t al, 1985 ; Dajas-Bailador an d Wonnacott, 2004 ; Fucile , 2004 ; Nick e e t al, 2004). Th e calcium permeabilit y o f oc7 * nAChR s i s greate r than tha t o f othe r neurona l nAChR s an d muc h larger tha n tha t o f the muscl e nAChR . I n fact , th e fractional curren t throug h a? * nAChR s tha t i s attributable t o Ca 2+ i s comparabl e t o N-methyl-D aspartate (NMDA)-typ e glutamat e receptors , bu t without the extr a control afforde d b y the voltag e an d Mg-dependent gatin g that constrain s NMDA recep tors (Dajas-Bailado r and Wonnacott , 2004). I n othe r words, a7* nAChR s ar e essentiall y Ca2+ flood gate s just waiting for ligand to happen! Judiciou s targeting of suc h receptor s t o presynapti c domain s ca n (and apparently does ) provid e a loca l trigge r fo r Ca 2+dependent transmitte r release . Mor e subtl e t o a n electrophysiologist, bu t no t t o th e nAChR-bearin g neurons, ar e th e man y downstrea m molecula r targets o f calciu m influ x (Dajas-Bailado r and Wonna cott, 2004) . Thes e sort s o f Ca 2+-dependent tuning , by bot h oc 7 an d non-oc 7 nAChRs , appea r t o b e im portant loc i o f nicotine' s effect s o n th e developin g nervous syste m (se e Nicotinic Acetylcholin e Recep tors, Synapti c Plasticity , an d Brai n Development , below). The essentia l contributio n o f oc2ß4 * nAChR s a t synapses an d circuit s relate d t o nicotine-elicite d re sponses ha s bee n repeatedl y an d compellingl y demonstrated (Picciott o an d Corrigall , 2002 ; Leste r et al , 2003 ; Picciotto , 2003 ; Gott i an d dementi , 2004; Laviolett e an d va n der Kooy , 2004; Wonnacot t et al. , 2005) . Sinc e Changeu x an d colleague s (Picciotto et al., 1995 ) first reported th e altere d avoidance learning i n knockou t mic e o f th e high-affinit y ß2 * nAChRs, th e choru s ha s mounte d o n th e ke y role of these nAChR s i n CN S function , culminatin g wit h the recen t identificatio n of a singl e sit e deeme d es sential t o Ot2ß4*-mediate d addictio n (Tappe r e t al , 2004). The rol e of changes i n the numbe r o f nAChRs (not confined to, but exemplifie d by, oc2ß4* nAChRs) in th e effect s o f long-ter m nicotin e exposur e i s stil l under debat e (Gentr y an d Lukas , 2002 ; Wonnacot t et al., 2005). In additio n to compositional diversit y in nAChRs , other factor s an d condition s that ma y diffe r fro m cel l

to cell an d fro m synaps e to synapse regulate the func tional efficac y o f nAChRs. Thus , marke d difference s in the physiologica l profile o f native nAChRs vs. their recombinant version s expressed i n heterologou s systems underscor e th e potentia l rol e o f loca l o r cell specific regulator y molecule s in determinin g nACh R function i n vivo. Extracellular modulators of nAChRs abound, includin g endotoxins , endocannabionoids , and neuropeptide s (Miw a e t al., 1999 ; Di Angelanto nio e t al , 2003 ; L e Foi l an d Goldberg , 2004 ; O z et al, 2004). A striking example o f native receptors being mor e functionally comple x tha n thei r heterologous equivalents ha s emerge d fro m studie s o f oe7-containin g nAChRs (Y u an d Role , 1998b ; Ber g an d Conroy , 2002; Khirou g e t al. , 2002 ; Drisde l e t al , 2004) . Comparison o f many i n vitro expression studies with neuronal studie s has led t o the conclusio n tha t mos t native oc7 * nAChR s ar e homopentameri c complexe s with ver y rapi d desensitization , lo w agonis t affinity , and long-lastin g otBgT x binding . Othe r wor k indi cates that a subset of native a7* nAChRs may include other subunits , o r oc 7 subunit s tha t hav e bee n subjected t o post-translationa l modifications , yieldin g nAChRs wit h ver y differen t agonist-induce d activa tion an d desensitizatio n fro m (oc7) 5 (Y u an d Role , 1998a; Cueva s et al, 2000; Khiroug et al, 2002; Drisdel étal, 2004). Diversity in Targeting o f Nicotinic Acetylcholine Receptors i n the Central Nervous System The developmen t o f better pharmacologica l tool s and new molecula r probe s t o nAChR s hav e unveile d impressive intricacie s in the cellula r traffickin g of nAChRs (William s e t al. , 1998 ; Temburni e t al , 2000, 2004; Ber g and Conroy, 2002; Brumwell et al, 2002; Blank et al., 2004; Liu et al., 2005). Dependin g on subuni t compositio n (an d in th e cas e o f oc3 , o n identified subuni t sequences ) a n nACh R comple x can b e selectively targeted t o somatic-dendritic vs. axonal domains , a s well a s t o synaptic , pre-, or perisy naptic subdomains within a neuron . As ACh an d nicotin e ca n influenc e synaptic transmission vi a pre- and postsynapti c nAChRs , and sinc e "extrasynaptic" nAChR s ma y exert importan t regula tory effect s o n neurona l activity , the traffickin g t o al l loci i s likely important (Fig. 21-2) (Hogg et al., 2003; Dajas-Bailador an d Wonnacott , 2004 ; Gott i an d

346 NICOTINE-AFFECTE D DEVELOPMENT A. POSTSYNAPTIC NiCOTINIC ÄChRs

B. PRE-SYNÄPTIC NICOTINiC AChRs

FIGURE 21- 2 Propose d dispositio n and physiological effects o f pre-, post - and perisynapti c nicotini c recep tors. A . Thi s imag e depict s neuron-neuro n connec tions where postsynaptic nAChRs participate i n direc t transmission. Acetylcholin e (ACh ) act s a s a primar y synaptic transmitte r i n periphera l autonomi e gangli a and a t som e CN S sites . ACh i s released fro m closel y apposed presynaptic elements, leadin g to the direc t activation o f postsynapti c nAChRs . Not e tha t despit e their perisynaptic location o n ciliary ganglion neurons , oc7* nAChR s participat e i n direc t synapti c responses . Administration of nicotine could activate and/or desensitize receptor s a t both peri - an d directl y postsynapti c locales. B . Presynapti c (and/o r preterminal ) nAChR s contribute t o the modulatio n o f synaptic transmission. ACh ma y be released from nearby cholinergic projec tions, leading to the activatio n of nAChRs localize d a t or near presynaptic terminals. Depending o n the posi tion an d subtyp e of nAChR, as well as the ambien t vs. stimulated concentratio n o f ACh , th e spontaneou s and/or evoked release of transmitter is facilitated o r depressed b y adde d nicotine . Th e ambiguit y o f thes e statements, and thos e in the text, as well as the fancifu l nature of the diagram reflect the very real difficulties i n assessing the actions of nicotine in vivo.

Clementi, 2004) . Man y group s hav e advance d th e proposal that , despit e th e relativel y lo w concentra tions of nAChRs i n the CNS, nAChR s do play an important role in fine-tuning CNS synapti c transmission because o f their strategi c localizatio n t o presynapti c sites (Dani, 2001; Fucile, 2004 ; She r et al, 2004).

Early demonstration s o f presynapti c effect s o f nicotine recepto r activatio n cam e fro m studie s o f synaptosome preparation s b y Wonnacott , Collins , and colleague s (Rapie r e t al , 1988 ; Wonnacot t et al, 1989 ; Grad y e t al, 1992 ) an d fro m electro physiological studie s o f Changeu x an d colleague s (Vidal an d Changeux , 1993 ; Gioann i e t al, 1999) . The presynapti c modulatio n o f central glutamater gic transmissio n by nAChR s a t severa l sites was initially distinguishe d b y it s ocBgT x sensitivit y and it s ablation followin g knock-dow n o f oc 7 subunit s (McGehee e t al, 1995 ; Gra y et al, 1996) . The hig h Ca2+ permeabilit y o f th e oc7 * nAChR s serve s t o escort Ca 2+ into axon terminals, thus facilitating spon taneous an d stimulus-evoke d transmissio n at numer ous site s o f glutamat e release . Non-ot7-containin g presynaptic nAChR s ha s bee n implicate d i n th e regulation o f glutamat e an d GAB A releas e (Vida l and Changeux , 1993) . Th e cumulativ e literatur e suggests tha t th e recepto r pharmacolog y an d pre sumed compositio n o f presynaptic nAChR s appea r to b e jus t a s elaborat e a s those o f peri- an d postsy naptic nAChR s (Mull e e t al. , 1991 ; Jone s e t al , 2001; Jone s an d Wonnacott , 2004 ; Fische r e t al , 2005). Despite evidenc e tha t nAChR s ca n participat e i n the modulatio n o f transmitte r releas e i n th e CNS , one shoul d no t get too carried away. There is increasing evidenc e fo r direc t synapti c transmissio n (i.e. , mediated b y AC h releas e an d postsynapti c nACh R activation) i n severa l CN S regions , includin g th e hippocampus, nigrostriata l system , an d i n sensor y cortex (Jone s an d Yakel , 1997 ; Alkondo n an d Albu querque, 2004 ; Matsubayash i e t al, 2004) . The vari ety o f nACh R subtype s tha t hav e bee n accuse d o f mediating postsynapti c transmissio n i n th e CN S i s also ver y broad , includin g bot h Ot7 * an d non-a7 * nAChRs. Last , bu t no t least , regardles s o f whethe r the nACh R populatio n densit y i s sufficien t t o sup port a synaptic current, activation of pre- and/or post synaptic nAChRs ma y be sufficient t o elicit importan t downstream signalin g and metaboli c response s (Ber g and Conroy , 2002 ; Dajas-Bailado r an d Wonnacott , 2004). The mechanism s underlyin g th e release , an d th e ambient vs . stimulated concentrations , o f ACh i n th e CNS remai n a bi t o f a mystery . Both publishe d an d unpublished report s hint at less than classica l mecha nisms o f ACh releas e (d e Rove r et al. , 2002 ; Lester , Jo, an d Role , persona l communication) . Electro n

DEVELOPMENT OF NICOTIN E RECEPTOR S 34 7 microscopy studie s examinin g th e localizatio n o f cholinergic synapti c terminal s hav e reveale d consid erable distanc e fro m cholinoceptiv e terminals , consistent wit h a volume mod e o f cholinergic trans mission (Descarrie s e t al. , 1997 ; Zol i e t al. , 1999 ; Jones and Wonnacott, 2004) . Suc h vagarie s in the local concentration s o f AC h a t pre - o r postsynapti c cholinocepetive site s i n th e CN S mus t b e addresse d before th e contributio n o f nAChR activatio n (and inactivation) t o th e developin g nervou s syste m ca n b e understood.

DEVELOPMENTAL EXPRESSION OF NICOTINI C ACETYLCHOLINE RECEPTOR S

Prenatal Expressio n o f Neuronal Nicotinic Acetylcholine Receptor s Constituents of the Cholinergic System Acetylcholine an d nAChR s pla y critica l role s i n virtually al l phase s o f brai n maturation , durin g embryogenesis a s wel l a s postnata l development . Choline acetyltransferas e (ChAT), the syntheti c en zyme fo r ACh, appear s very earl y during embryoge nesis (Smit h e t al , 1979) , an d AC h influence s development a s early as gastrulation (Fig. 21-3). Initially, AC h promote s cel l divisio n a t a stag e when ACh synthesi s occur s i n preneura l undifferentiate d cells. Durin g gastrulatio n an d postgastrulation , ACh regulate s cel l movement s tha t ar e blocke d b y ACh antagonist s i n se a urchi n embryo s (Falugi , 1993; Laude r an d Schambra , 1999) , and se a urchin oocytes an d earl y embryo s contai n nAChR s (Ivon net an d Chambers , 1997 ; Buzniko v an d Rakich , 2000). Thi s findin g suggest s tha t i n additio n t o ChAT, other protein s involve d in cholinergic signaling ar e expresse d b y embryoni c cell s durin g thes e stages. Indeed, acetylcholinesteras e (AChE), the en zyme tha t break s dow n ACh , i s present durin g se a urchin gastrulatio n (Falugi , 1993) . Messenge r RN A coding for nAChR subunit s i s detected in cultures of premigratory neura l cres t cell s (Howar d e t al. , 1995), which i n th e fetu s will give rise to most neu rons in th e PNS . Thus, eve n befor e neuronal differ entiation, cholinergi c signalin g throug h nAChR s occurs an d ma y eve n contribut e t o developmen t o f non-neuronal cells .

In prenata l neura l tissue , bot h muscarini c acetyl choline receptor s (mAChRs ) an d nAChR s ar e de tected, bu t nAChR s ar e expresse d earlie r tha n mAChRs (Laude r an d Schambra , 1999) . nACh R ex pression as well as ChAT immunoreactivity and ACh E activity show a caudorostra l gradien t o f appearance i n the fetal brain (Zoli et al., 1995; Lauder and Schambra , 1999). Functiona l nACh R expressio n ha s bee n de tected i n ste m an d progenito r cell s o f the ra t cerebral cortex at gestational day (G) 1 0 (Atluri et al., 2001) and in mesencephalon of developing brai n at Gil (Zol i et al., 1995) . Earl y expression o f subunit mRNA s seem s characteristic o f nAChR, sinc e othe r ligand-gate d io n channels becom e expresse d somewha t later . GAB A and NMD A receptor mRNA s ar e no t detecte d i n ra t brain unti l G14-G15 (Lauri e e t al, 1992 ; Monye r e t al., 1994) . Durin g thi s stage, ACh promote s the switch from cel l divisio n to neurona l differentiatio n (Slotkin , 1998), an d guidanc e o f nerve growth cones appear s to be on e o f th e role s o f cholinergi c signalin g throug h nAChRs (Hum e e t al. , 1983 ; Zheng e t al, 1994) . At the onse t o f neurona l nAChR-subuni t expression , innervation-independent factor s ar e responsibl e fo r neuronal differentiatio n before cholinergic projection s arrive (Zol i et al., 1995) . Indeed, althoug h cholinergi c neurons are among the first neurons to differentiate i n the CNS , i n ra t fetuses , cholinergi c neuron s ar e no t generated befor e Gi l an d ar e generate d firs t i n th e spinal cord , brai n stem , an d basa l ganglia . I n mos t other brai n regions , cholinergi c neuron s ar e no t de tected until G12 to G16 (Lauder and Schambra, 1999) . ChAT-immunoreactive nerv e terminal s ar e de tectable during the first postnatal weeks , especially i n the forebrain . Th e majo r cholinergi c innervatio n originating fro m th e nucleu s basali s o f Meynert en ters th e cerebra l corte x a t abou t postnata l da y (P ) 0 (Hohmann an d Berger-Sweeney, 1998) , bu t i s ini tially confine d t o th e subplate , a transient , synapse rich laye r o f neuron s locate d directl y unde r th e cortical plate . Thus , th e initia l profil e o f nACh R ex pression may be induced by local endogenous differen tiation signals rather tha n b y cholinergic input s per se. This contrast s wit h finding s o n cholinergi c synapse s in th e developin g autonomi e nervou s syste m (Deva y et al. , 1999) . Here , nAChR-subuni t expressio n i n neuronal cholinergi c synapse s is regulated b y signals from th e autonomi e targe t tissu e tha t i s innervated . Depending o n th e innervate d tissu e type , differen t types of nAChRs ar e expresse d tha t hav e differen t ki netic properties (Devay et al., 1999) .

348 NICOTINE-AFFECTE D DEVELOPMEN T

FIGURE 21- 3 Developmenta l expressio n of nicotinic acetylcholine receptors (nAChRs) i n ra t forebrai n and cortex . nACh R subunit s ar e expresse d i n ra t forebrain an d corte x a t differen t site s an d a t differen t time s durin g develop ment. Interestingly , the expressio n of nAChRs precedes th e birt h o f cholinergic cell s wit h a neurona l phenotype , suggestin g tha t cholinergi c signalin g through nAChR s ma y be involve d i n th e differentiatio n o f neurons. Durin g early postnatal development, when cholinergi c projections arrive in the cortex, different nACh R subtype s show differen t expressio n dynamics , indicatin g that cholinergic signalin g through nAChR s i s involved in synapti c refinement of the neuronal circuitry.

Spatiotemporal Patterns Expression of nAChRs i s not constant durin g fetal development; i t varie s amon g region s an d subunit . At G10, when the mouse corte x consists of dividing stem and progenito r cells, thes e cortica l cell s expres s oc3, oc4, an d oc 7 recepto r subunit s that giv e ris e to ACh evoked inward currents (Atlur i et al., 2001). Although these nAChR subunits can be detected fro m G1 0 un til birth, the incidenc e o f these subunits declines with increasing gestational age . I n feta l ra t brain , oc3 , oc4 , ß2, an d ß 4 nACh R subuni t mRNA expression is detectable in the hindbrain and midbrain , but no t fore brain, at Gl 1 (Zoli et al, 1995) . In forebrain, mRNA for oc3 , oc4 , an d ß 2 subunit s become s detectabl e a t G12. ß 2 mRN A seem s t o b e th e mos t widel y expressed an d a t relatively stable level s throughout th e remaining prenatal development. oc4-subuni t mRNA is at lower levels than ß 2 mRNA throughout gestation,

but show s a similarl y wid e distribution . In contrast , Ct3 and ß 4 mRNA are less abundantly distributed and expression can be transient in some brain regions. For instance, oc 3 mRNA is present in th e ra t forebrain o n G12, G13 , an d G15 , bu t i s not detecte d ther e any more a t G17 , G19 , an d P O (Zoli e t al , 1995) . ß 4 mRNA i s absent fro m forebrai n durin g embryogenesis, but show s transien t expressio n in othe r brai n areas. It is detected i n midbrain on G12 and G15 , but is not detecte d i n midbrain nucle i a t G19 and P O (Zoli et al. , 1995) . I n developin g chic k brain , simila r regional specificit y an d transien t expressio n occu r throughout gestationa l stage s (Torra o e t al. , 2000) . oc2, oc3 , oc5 , an d ß 2 mRNA s are expresse d very early on i n gangli a a t embryoni c da y 4, and ar e expresse d throughout th e chic k brai n a t G18 . oc 7 mRN A was detected i n Purkinj e neuron s o f chick cerebellum a t G12, bu t expressio n decrease d afte r G1 8 (Kanek o et al., 1998) . Taken together, the earl y onset, regional

DEVELOPMENT OF NICOTIN E RECEPTOR S 34 9 specificity, an d transien t nature o f nAChR expression suggest that they may play important role s during embryonic developmen t o f rodent an d chic k brain. This theory i s underscored b y finding s i n transgeni c mic e with inducibl e expression o f oc2 subunits. Restoratio n of ß2 subunit expression durin g development i n thalamocortical projection s of ß2 knockout mice normal izes passive-avoidanc e learnin g i n thes e animals , suggesting that ß2 expression in these projections during developmen t i s critical fo r norma l developmen t of this behavior (King et al, 2003). In fetal huma n brain , nAChR gen e transcripts and proteins ar e als o detecte d a t ver y earl y gestationa l stages. nACh R protei n an d gen e transcript s are read ily detecte d b y 4 t o 5 weeks o f gestation (Hellstrom Lindahl et al, 1998) . Messenger RNA s of a3, a4? a5, oc7, ß2 , ß3 , an d ß 4 ar e foun d widel y distribute d over the feta l brai n betwee n 4 an d 1 2 weeks o f gestation . In mos t brai n areas , radio-ligand bindin g of nAChR s by [ 5H] epibatidine and [ 3 H] cysteine increase d fro m weeks 4 t o 12 . The highes t specifi c bindin g of thes e ligands wa s detecte d i n spina l cord , pons , an d medulla oblongata , wherea s bindin g b y th e oc 7 subunit-specific prob e [ 125I]aBgTx was found t o b e strongest i n pons , medull a oblongata , an d midbrai n (Hellstrom-Lindahl and Court , 2000) . Periods of transient hig h nACh R densit y were found i n fronta l cortex, hippocampus , cerebellum , an d brainste m o f humans durin g mid-gestatio n an d neonata l period s (Kinney et al, 1993; Court et al, 1997) . A significant positive correlatio n betwee n gestationa l ag e an d th e expression o f oe7 mRNA was observed i n al l brai n re gions excep t corte x (Fal k e t al., 2002). From th e lat e fetal stage , human brai n nAChR expressio n has bee n shown t o fal l wit h increasin g ag e (Hellstrom-Lindah l etal, 1998; Hellstrom-Lindahl an d Court, 2000; Falk et al, 2003) . Thus , nAChR s als o appea r t o pla y a n important role in human development , neurogenesis , and synaptogenesis .

to exogenou s nicotin e exposure . A substantia l percentage o f pregnant woman continu e smokin g during pregnancy. I n th e Unite d States , nearl y hal f o f al l women smoker s continue t o smoke during their pregnancies (Ebrahi m e t al, 2000) , o r abou t 12 % of all women wh o giv e birt h (Mathews , 2001) . Mor e tha n 500,000 infant s eac h yea r ar e expose d t o cigarett e smoke i n utero . Fetal exposur e t o nicotin e damage s th e develop ing brain, interferin g wit h cel l replicatio n an d differ entiation. I t als o evoke s apoptosis , culminatin g i n reduced cel l numbe r an d aberran t cel l proportion s (Slotkin, 2002; Abreu-Villaca e t al, 2004a). I n ra t fetus, durin g neurulatio n i n th e neura l tub e stag e a t G9.5, nicotin e exposur e cause s cytotoxicit y i n al l brain area s an d incidence s o f apoptosi s sharpl y in crease (Ro y et al. , 1998) . Prenata l exposur e t o nico tine increase s cortica l mRN A expressio n o f oc4 , ß2 , and <x 7 nACh R subunit s i n rat s (Shack a an d Robin son, 1998a ; Frank e t al., 2001) . Also, when pregnan t primates are exposed to nicotine a t concentrations experienced b y smokers, feta l brai n damag e i s induced (Slotkin e t al., 2005). Nicotine exposur e durin g 3 0 to 160 days of gestation induce d a4ß 2 an d oc 7 nACh R upregulation i n severa l brai n areas , includin g cortex . In addition , cel l loss , reductio n i n cel l siz e an d re duced neuriti c outgrowt h wa s observed. I n humans , these prenata l nicotin e effect s lea d t o behaviora l deficits durin g late r lif e (Slotkin , 2004) . Nicotin e treatment o f primar y neurona l culture s o f huma n prenatal brai n increase s expressio n o f Ot 3 an d oc 7 nAChR subunit s (Hellstrom-Lindah l e t al., 2001) . In longitudinal studie s o f prenatally expose d infants , fetal tobacco exposur e affects attentio n an d impulsivit y behavior (Leec h e t al., 1999) , and ca n lea d t o lowe r intelligence quotien t an d conduc t disorde r i n late r life (Erns t et al, 2001; Wakschlag et al, 2002). Postnatal Expressio n o f Neuronal Nicotinic Acetylcholine Receptor s

Response to Prenatal Nicotine Exposure Depending o n th e developmenta l phase , AC h promotes o r prevent s neurona l apoptosis : whe n cell s are poorl y differentiated , th e effec t i s primaril y proapoptotic, wherea s i n differentiate d cells , i t i s anti apoptotic (Hohman n an d Berger-Sweeney , 1998) . The earl y expression of nAChRs during fetal develop ment give s rise to a vulnerability of the huma n fetu s

Expression Patterns and Integration with Other Neurotransmitter Systems In human s a s well as in rodent s an d othe r mammals , brain developmen t i s far from complet e a t birth, an d many neuronal systems need t o mature i n response to ongoing interactio n wit h th e e x uter o environment . Indeed, neuroproliferation , apoptosis , an d synapti c

350 NICOTINE-AFFECTE D DEVELOPMEN T rearrangement persis t int o adolescence . Cholinergi c signaling continues to play an importan t role in postnatal brai n development , whic h i s emphasize d b y brain region-specific an d transient expression profiles of nAChR subunits (Fuchs, 1989 ; Broide et al, 1995 , 1996; Zhan g e t al., 1998) . Disruptio n o f cholinergi c innervation durin g earl y postnata l developmen t re sults i n delaye d cortica l neurona l developmen t an d permanent change s i n cortica l cytoarchitectur e an d cognitve behavior s (Hohmann an d Berger-Sweeney , 1998). oc3 , a.7, an d ß 4 subuni t mRN A expressio n is low durin g mid-gestatio n an d increase s durin g lat e gestation and earl y postnatal life (Winzer-Serha n an d Leslie, 1997) . Durin g th e firs t fe w postnata l weeks , various subunit s ar e transientl y expresse d i n selec t sites i n th e brain : a 2 i n th e hippocampu s an d i n brain stem ; cx 3 i n th e striatum , cerebellum, an d cor tex; cc 4 i n hippocampus , striatum , an d cerebellum ; oc7 i n the cerebellu m an d cortex ; and ß 2 i n the striatum (Broid e et al, 1995 , 1996 ; Zhang e t al, 1998) . In rat cortex , oc 4 mRNA is maximal b y P14 after whic h its expressio n remain s high. ß 2 mRN A is low o n P 7 but peak s a t P14 . ot 7 mRN A expressio n reache s a maximum o n P 7 (Shack a an d Robinson , 1998b) . These expressio n pattern s sugges t tha t nACh R sub units pla y specifi c role s durin g postnata l develop ment. Geneti c silencin g o f oc4 , ß2 , an d ß 3 nACh R subunits shows that each subunit is involved in particular neurona l an d behaviora l phenotypes, hintin g a t their uniqu e role s i n brai n developmen t (Cordero Erausquin e t al. , 2000) . Only th e oc3-subuni t knockout mice have a high fractio n of mortality during th e first postnatal wee k (Xu et al, 1999) , mos t likel y because thi s subuni t i s a n essentia l componen t o f nAChRs that mediate fas t synaptic transmission i n the autonomie nervou s system, and, a s such, i s essential for perinata l survival. T o illustrat e the contribution s of cholinergi c signalin g t o postnata l brai n develop ment, developmenta l change s i n nACh R expression and functio n in primary sensory cortex are describe d below. Cholinergic transmissio n is presen t i n th e earl y developing ra t parieta l corte x a t birt h (Descarrie s et al., 1997 ; Mechawa r and Descarries , 2001). In th e first 2 week s afte r birth , th e cholinergi c innervatio n increases greatl y in numbe r o f varicosities and num ber o f branche s pe r axon . Th e associatio n o f thes e new cholinergic terminals with postsynaptic densities is lo w (<15%) , b y contrast , an d remain s constan t

throughout adulthoo d (Mechawar et al., 2002). Ultrastructural analyse s o f som e monoaminergi c nerv e terminals i n th e corte x (Aude t e t al , 1988 , 1989 ) and hippocampu s (Viz i an d Kiss , 1998 ) sho w tha t most o f these nerv e terminal s do no t mak e contac t with structurall y identifiable postsynaptic elements . These terminals are equippe d fo r vesicle release despite no t makin g direc t synapti c contact s (Seguel a et al, 1989 , 1990 ; Descarrie s and Mechawar, 2000) . This phenomeno n suggest s tha t the y communicat e with postsynapti c target s throug h volum e transmis sion (Zol i et al., 1999) . By comparison, th e percent age o f GABAergi c an d glutamatergi c axon s tha t terminate o n a postsynapti c sit e approache s 100 % (Seguela e t al, 1990 ; Umbriac o e t al, 1994 , 1995) . Cholinergic innervatio n i s almos t exclusivel y ex trasynaptic i n severa l areas of the ra t brain , includ ing th e parieta l corte x (Umbriac o e t al. , 1994 ; Mechawar e t al., 2002) , hippocampus (Umbriac o et al, 1995) , neostriatu m (Contan t e t al, 1996) , an d visual, sensory , an d parieta l cortice s (Avendan o et al, 1996 ; Turrin i e t al, 2001) . Cholinergic com munication throug h volum e transmissio n has wideranging implications fo r nAChR impac t on neurona l function, especiall y whe n on e take s difference s i n activation an d desensitizatio n propertie s o f nAChR s into accoun t (Mansvelde r e t al, 2002 ; Quic k an d Lester, 2002). Functionality of the Cholinergic Receptors During postnata l developmen t o f sensor y cortice s there i s a dramatic , transient increas e i n th e expres sion of AChE (Robertso n etal., 1991) . Concurrently, nAChR oc 7 subuni t gen e expressio n als o transientl y increases i n sensor y cortices. Binding of [ 125I]ocBgTx in ra t sensor y cortex start s a t PO , peaks a t P10 , an d then decline s to adult concentratio n by P20 (Fuchs, 1989). Expression of oc7 subunit mRNA follows a similar time course (Broide et al., 1995) . During fetal de velopment, oc 7 mRNA is expressed i n low amounts o n G13 in the ventricula r zone o f the neocortex , and o n G15 i n the thalami c neuroepithelium . A marked in crease i n ot 7 mRN A concentrations i s observed dur ing th e lat e prenata l perio d i n bot h sensor y an d nonsensory regions of the corte x and thalamus . Mod erate to high levels of messenger RNA are maintained into th e firs t postnata l week , followe d b y a declin e into adulthoo d (Broid e et al., 1995) . The increas e i n

DEVELOPMENT OF NICOTIN E RECEPTOR S 35 1 cortical oc 7 mRN A follow s th e arriva l o f AChE labeled thalamocortical afférents . Preventin g these afférents fro m reachin g th e corte x strongly reduces th e a? subuni t mRN A and [ 125I]ocBgTx binding i n layers I V and V I (Broid e et al, 1996) . Thes e dat a sug gest tha t th e expressio n o f oc 7 subunit-containin g nAChRs i s regulate d b y thalami c inputs . Thes e oc 7 subunit-containing nAChR s coul d b e located postsynaptically as well as presynaptically on thalami c affér ents. I t ha s bee n hypothesize d tha t presynapti c oc 7 nAChRs i n primar y auditory corte x ar e involve d i n the maturatio n o f glutamat e synapse s by facilitatin g the conversio n o f "silent synapses " (define d a s thos e containing onl y NMD A receptors ) int o matur e ot amino-3-hydroxy-5-methyl-4-isoxazole-propionic aci d (AMPA) an d NMD A receptor-containin g synapse s (Metherate an d Hsieh , 2003 ; Metherate, 2004) . Th e authors o f thi s hypothesi s sugges t tha t throug h thi s mechanism o f nAChR-induce d maturatio n o f glutamate synapses , the expressio n o f oc 7 subunit s i n th e auditory corte x coul d defin e a critica l perio d o f sensory corte x developmen t i n whic h synapti c refine ment of cortical circuitry and tuning to sensory inputs takes place. nACh R activation has been show n to alter synapti c strength o f glutamatergic synapti c transmission i n othe r brai n area s a s well , a s describe d below. In developin g hippocampus , nAChR s containin g <x7 subunits can als o activate silent synapses that show a lo w probability of being activ e an d tur n the m int o high-probability synapses (Maggi et al., 2003) . Schaf fer collatera l to CA1 synapses that have a high proba bility o f bein g activ e durin g developmen t ca n b e down-regulated b y a 7 an d ß 2 subunit-containin g nAChRs (Magg i et al, 2004). These findings suggest that nicotini c receptor s als o ca n pla y a rol e durin g postnatal developmen t o f excitator y glutamatergi c connections an d can contribute t o shaping of the hip pocampal neuronal circuitry. To understand th e physiologica l effect o f the acti vation of nAChRs by neuronal activit y during postna tal development , i t i s importan t t o conside r thei r cellular an d subcellula r distribution . A s mentione d above, nAChR s modulat e presynapti c glutamat e re lease (McGehe e e t al , 1995 ; Gra y e t al , 1996 ; McGehee an d Role , 1996 ; MacDermot t e t al. , 1999). I n addition , nAChR s ca n modulat e GABAer gic transmissio n in multipl e brai n areas , suc h a s th e ventral tegmenta l are a (VTA) , thalamus , neocortex ,

and hippocampus (Len a e t al., 1993 ; Alkondon e t al., 1997, 2000 ; Lena an d Changeux, 1997 ; Fishe r e t al, 1998; Radcliff e e t al, 1999 ; Mansvelde r and McGe hee, 2002) . Modulatio n o f GABAergi c neuron s b y nAChRs ha s bee n mos t extensivel y studie d i n th e hippocampus, wher e GABAergi c loca l circui t neu rons (LCNs ; als o know n a s interneurons ) expres s multiple nACh R subtype s (Alkondo n e t al. , 1997 , 1999; Jone s an d Yakel , 1997 ; Frazie r e t al , 1998 ; McQuiston an d Madison , 1999 ; J i and Dani , 2000 ; Alkondon an d Albuquerque , 2004) . Ther e i s evi dence fo r nAChR expressio n at two sites: (1) at presynaptic terminal s (wher e the y directl y modulat e GABA release , independen t o f actio n potentia l fir ing) (Fishe r e t al, 1998 ; L u e t al, 1999 ; Radcliff e et al. , 1999 ) an d (2 ) awa y fro m synapti c terminals (where modulation o f GABA release is dependent o n action potentia l firing) (Alkondon et al, 1997 , 1999 ; Frazier e t al. , 1998) . Th e expressio n of nAChRs de pends o n th e typ e o f LC N (McQuisto n an d Madi son, 1999 ; Alkondo n an d Albuquerque , 2004 ; se e below). Activation of nAChRs o n cortical and hippocam pal LCN s result s i n eithe r inhibitio n o r disinhibi tion o f projectio n neuron s (PNs ) (Alkondo n e t al. , 2000; J i and Dani , 2000 ; Ji et al, 2001) . Inhibitio n is likel y t o b e induce d vi a nAChR-mediate d in crease i n th e GABAergi c transmissio n directl y ont o PNs. Disinhibitio n o f PNs result s fro m a n increas e of inhibitor y GABAergic transmissio n t o GABAer gic LCN s b y activatio n o f nAChRs . Consequently , PNs ma y receiv e les s GABAergi c inpu t an d ar e dis inhibited. Effect on Neuronal Subpopulations There are few data o n nicotini c modulatio n o f differ ent type s of neurons i n th e prefronta l corte x (PFC) . As in other part s of the neocortex , th e PF C ha s a layered structure i n which most of the neuron s ar e PNs . PNs an d LCN s ca n b e distinguishe d o n th e basi s of distinctive physiological , morphologically , an d im munocytochemical criteri a (Peter s an d Jones , 1984 ; Kawaguchi, 1993 , 1995 ; Gabbot t e t al, 1997 ; Con nors an d Telfeian , 2000 ; Kawaguch i an d Kondo , 2002; Gabbott e t al, 2003). In rat , among th e target s of thalamocortica l afférent s ar e PN s wit h somat a i n layers III, IV, or V of the PFC . Thes e inputs are mod ulated b y a4ß2-containin g nAChR s (Vida l an d

352 NICOTINE-AFFECTE D DEVELOPMEN T Changeux, 1993 ; Gil etal, 1997; Gioanni etal., 1999; Lambe e t al., 2003) . Nieotinic modulatio n o f thalamocortical projection s by oc4ß2-containing nAChRs ap pears t o b e a genera l phenomeno n throughou t th e cortex (Metherate , 2004) . An elegant stud y in the ra t motor cortex identified distinct LC N subtype s tha t bot h expres s nACh R mRNA for oc4, a5, and ß 2 subunits and sho w somatic nicotinic currents (Porter et al., 1999) . PNs, as well as LCNs expressing either parvalbumin or somatostatin , showed n o effec t o f agonist applicatio n i n thi s study. LCNs expressin g vasoactive intestinal peptide (VIP) and cholecystokini n (CCK) , b y contrast , di d sho w nicotinic currents , and pharmacologica l analysis implicated non-oc 7 nAChRs. I n human cerebra l cortica l slices, bipola r an d multipola r LCNs exhibite d eithe r a? o r a4ß 2 nAChR-rnediate d current s (Alkondo n etal., 2000). An interestin g bu t seldo m addresse d aspec t o f cholinergic signalin g in the cortex is the role of cholinergic LCNs i n cortical microcircuit function. Cholinergic LCN s i n th e nucleu s accumben s (NAc ) can affect GABAergi c transmissio n within th e NA c itsel f via nACh R activatio n (de Rover et al, 2002) . In addition, these cholinergic LCN s could be important i n the productio n o f lasting changes i n microcircuitries that affec t anima l behavior, since their intrinsic firing properties ar e altere d durin g behavioral sensitization to amphetamin e (d e Rove r e t al. , 2004) . Further more, immunohistochemica l dat a sho w tha t a smal l fraction o f bipolar LCNs i n laye r II/II I o f the roden t sensory and moto r cortice s are cholinergic (House r et al., 1985) . A mor e recen t stud y usin g single-cel l reverse-transcriptase polymeras e chai n reactio n found a subgrou p o f cortica l LCN s tha t ar e im munopositive for VIP an d calretini n and als o express ChAT transcript s (Caul i e t al. , 1997) . Thes e sam e VIP-positive cells are the mai n LCN subtyp e expressing nicotini c current s an d nACh R mRN A (Porte r et al. , 1999) . Th e Nationa l Institut e o f Neurological Disorders an d Strok e (NINDS ) GENSA T BA G Transgenics Projec t show s tha t ChAT-positlv e stain ing i s eviden t i n almos t ever y par t o f th e cortex , including th e PF C (http://www.gensat.org/makecon nection.jsp). Th e putativ e presenc e o f cholinergi c LCNs in the corte x introduces the possibilit y of cortical nAChR activatio n independent o f cholinergic terminals tha t originat e fro m lowe r brai n areas . Sinc e the PF C i s implicated in behavioral sensitization (see

below), intrinsi c properties o f cholinergic LCN s an d ACh releas e i n the PF C coul d als o be modifie d during behaviora l sensitizatio n followin g th e intak e o f drugs o f abuse , a s describe d i n th e NA c (d e Rove r et al, 2004) .

NICOTINIC ACETYLCHOLINE RECEPTORS, SYNAPTI C PLASTICITY, AND BRAI N DEVELOPMEN T

Activation o f oc 7 nAChR s i n primar y sensory cortex during postnata l developmen t ma y alte r synapti c strength of glutamatergic synapses (Metherate, 2004) . In recent years, several studies i n differen t brai n area s have reveale d lastin g effect s o f nicotin e o r AC h o n synaptic connection s (Mansvelde r an d McGehee , 2000; Dan i e t al. , 2001 ; Girod an d Role , 2001 ; J i e t al., 2001; Mansvelder et al., 2002). Long-term modu lation o f glutamatergi c an d GABAergi c synapti c connections by nAChRs ca n alter functioning of neuronal circuit s for many days and possibl y induce lasting change s tha t coul d b e importan t fo r postnata l development o f these brai n areas. These properties of cholinergic signalin g throug h nAChR s ar e discusse d in th e contex t o f findings on nicotini c modulatio n o f the mesolimbic DAergi c system. In rat , the D A rewar d system show s considerabl e plasticity during postnatal lif e an d doe s no t complet e maturation unti l lat e adolescence or early adulthood (Benes et al., 2000) . Since this system exhibits a similar make-u p i n th e huma n brain , adolescence migh t well represen t a time windo w prone t o aberrations by exposure to drugs of abuse. The mammalia n brai n is particularly sensitive t o developing addiction s durin g adolescence. Th e uniqu e behaviora l feature s of adolescence ar e drive n largel y by maturationa l change s in th e CNS , a process prone t o environmenta l influ ences suc h as drug exposure. Epidemiological studie s show that exposur e t o addictiv e drug s durin g adoles cence increase s th e ris k o f becomin g a dru g addic t later i n lif e (Kande l et al, 1992 ; Grant an d Dawson , 1997). Adolescen t smokin g i s clearly correlated wit h smoking i n late r life . Moreover , adolescen t smokin g is a strong predictor o f the occurrenc e of adult depression (Chassi n et al., 1996) . In th e VTA , nAChRs ar e expresse d o n DAergi c neurons, GABAergi c neurons , an d glutamatergi c terminals (Fig . 21-4 ) (Pidoplichk o e t al. , 1997 ;

DEVELOPMENT OF NICOTIN E RECEPTOR S 35 3 Charpentier e t al., 1998 ; Mansvelde r an d McGehee , 2000; Klin k e t al , 2001 ; Champtiau x e t al , 2002 ; Mansvelder e t al. , 2002 ; Pidoplichk o e t al , 2004) . VTA DAergi c neuron s expres s thre e pharmacologi cally identifiable nAChRs—one that i s likely a homomeric oc 7 nAChR an d tw o that d o not contai n th e oc 7 subunit. Mos t DAergi c neuron s expres s nAChR tha t can b e blocke d b y mecamylamine, whereas less tha n half of the DAergi c neuron s expres s a7 nAChRs (Pi doplichko et al., 1997 ; Klin k et al, 2001; Wooltorto n et al , 2003 ; Pidoplichk o e t al. , 2004) . GABAergi c neurons i n th e VT A expres s a simila r variet y o f

FIGURE 21- 4 Nicotini c modulatio n o f synaptic com munication i n ra t ventra l tegmenta l are a (VTA) . I n the VTA , nAChR s ar e presen t o n dopamin e (DA)er gic neurons , GABAergi c neurons , and glutamatergi c terminals. Arrival of low concentrations o f nicotine, as experienced b y smokers, rapidl y activates and desen sitizes non-cc 7 nAChRs , wherea s al nAChR s o n glutamatergic terminal s ar e activate d bu t suffe r muc h less fro m desensitization . (Source: Modifie d fro m Mansvelder and McGehee, 2002)

nAChR subunits . A s wit h D A neurons , onl y a mi nority o f GABAergi c neuron s expres s oc 7 nAChR s (Wooltorton e t al, 2003) . Th e majorit y o f the VTA GABAergic neuron s expres s nAChRs tha t mos t likel y contain a 4 an d ß 2 subunits , whic h ar e blocke d b y dihydro-ß-erythroidine (DHßE ) (Mansvelde r e t al. , 2002). Glutamatergic transmissio n ont o DAergi c neuron s is enhance d b y activatio n o f presynapti c nAChR s (Mansvelder an d McGehee , 2000 ; Jone s an d Wonna cott, 2004). Interestingly, cholinergic synaptic terminals are no t foun d i n clos e vicinit y to glutamatergi c termi nals expressing Ot7-containing nAChR, consisten t wit h a "volume " mode of cholinergic signalin g (Descarrie s et al. , 1997 ; Zol i e t al. , 1999 ; Jone s an d Wonnacott , 2004). Whe n nicotine arrives in the VTA, i t stimulates glutamatergic terminal s as well a s dopamine neurons , thereby favorin g condition s o f pre - an d postsynapti c paired activation and a Hebbian typ e of synaptic plasticity7. Nicotine-induce d pairin g resulted i n long-ter m po tentiation (LTP ) o f glutamatergi c input s (Mansvelde r and McGehee , 2000) . Nicotin e als o induce d LT P i n vivo, measured a s an increase i n AMPA/NMDA recep tor rati o (Saal et al., 2003) . Taken together , thes e find ings suggest tha t synapti c plasticit y in the VT A may be induced afte r smoking a single cigarette and mos t likely underlies th e persistent effect s o f the dru g o n D A re lease in the NAc and PFC . The oc7-containin g nAChR s involve d i n thi s mechanism ar e no t desensitized b y low nicotine con centrations associate d wit h tobacc o smokin g (Mans velder et al, 2002; Wooltorton e t al, 2003). However, the non-ot 7 nAChRs on GABAergi c neurons underg o rapid desensitizatio n withi n minute s afte r th e star t of nicotine exposur e and, a s a consequence, reduc e th e inhibitory inpu t t o th e D A neuron s (Mansvelde r et al, 2002 ; Wooltorton e t al, 2003). Desensitizatio n of the nAChR s o n GABAergi c neuron s no t onl y pre vents further activatio n by nicotine bu t als o precludes the contributio n o f thes e receptor s t o endogenou s cholinergic transmissio n (Mansvelde r e t al. , 2002) . VTA dopamine neuron s ar e thu s disinhibite d by desensitization o f non-oc7-typ e nAChR s (Mansvelde r et al., 2002). Despite rapi d desensitization properties , genetically engineere d mice lacking ß2 subunits (Pic ciotto e t al. , 1998 ) o r expressin g oc 4 subunit s hyper sensitive t o nicotin e (Tappe r e t al. , 2004 ) hav e bee n used t o sho w tha t thes e subunit s ar e ke y in nicotin e addiction.

354 NICOTINE-AFFECTE D DEVELOPMENT NICOTINIC ACETYLCHOLINE RECEPTORS AND SYNAPTI C PLASTICIT Y IN OTHE R BRAIN AREAS

Currently, i t is unknown whether long-term modula tion o f synapti c connection s b y nicotin e occur s within th e PFC . Dat a o n long-ter m modulatio n o f synaptic contact s b y nicotine anywher e i n th e neo cortex are lacking. On th e othe r hand , nicotin e doe s modulate synapti c plasticity in brain areas other than the VTA . I n ra t spinal cord , the tx 7 subunit contain ing nACh R affect s th e inductio n o f synaptic plasticity (Genzen an d McGehee , 2003). In the absenc e of nicotine, pairin g of pre- an d postsynapti c activit y in dorsal hor n neuron s induce s LT P i n som e o f th e neurons. Wit h nicotine , th e prevalenc e o f LT P in duction i s enhanced . I n th e hippocampus , i n a minority o f th e glutamatergi c synapse s nicotin e in duces LT P b y itself , bu t i n mos t o f glutamatergi c synapses nicotin e modulate s th e inductio n o f synaptic plasticit y (Fujii e t al, 1999 ; J i et al., 2001; Man n and Greenfield , 2003) . Activatio n o f postsynapti c nAChRs o n CA 1 PNs ca n boos t short-term plasticity into LT P i n Schaffe r collatera l synapse s (J i e t al. , 2001). Activation of nAChRs o n LCN s tha t synaps e on PN s ca n preven t LT P i n glutamatergi c synapses (Ji e t al. , 2001) . Thus , timin g an d localizatio n o f nAChR activit y in th e hippocampu s ca n determin e whether LT P will occur o r not . These type s of nicotinic mechanism s o n LT P inductio n ma y contribut e to the well-know n effects o f nicotine o n learning an d memory.

axon guidanc e t o fine-tunin g neurona l circuits , ac tivating silen t synapses , an d modulatin g synapti c strength. Th e sam e hold s tru e fo r nicotine exposur e during development: dependin g o n the developmen tal stage , th e magnitud e o f damage induce d an d th e specific target s affected b y smoking during pregnancy will vary. The finding s reviewe d i n thi s chapte r describ e part of what is surely a much bigge r picture of the rol e of nAChR-signalin g i n development ; man y gap s i n this picture remain . Molecular and electrophysiological techniques, despit e their great sensitivity, still lack the flexibility to precisely define whic h combination s of nACh R subunit s combin e t o determin e th e ful l spectrum o f responses to ACh. Cholinergi c signalin g during developmen t i s th e culminatio n o f th e inte grated actio n o f different nAChRs , actin g i n somato dendritic vs . axona l domains , transducin g eithe r electrical informatio n o r calciu m signaling . Futur e studies wil l nee d t o addres s thes e question s befor e sketches of cholinergic signaling in development ca n emerge i n more vibrant detail. Abbreviations ACh acetylcholin e AChE acetylcholinesteras e OcBgTx oc-bungarotoxi n AMPA a-amino-3-hydroxy-5-methyl-4-isoxazole propionic aci d CCK cholecystokini n ChAT cholin e acetyltransferas e

SUMMARY AND CONCLUSION S

Over the past 30 years, research o n cholinergic signal ing in the developin g nervous system has shown tha t nAChR pla y a n importan t rol e i n shapin g neurona l circuits. The subuni t compositio n of nAChR s expressed i n different brai n regions changes during preand postnata l development ; th e subuni t mRN A profiles are highl y dynami c durin g thi s time . Give n th e variety of biophysical and pharmacologica l properties conferred b y differences in nACh R composition , th e types o f signals mediated b y nAChR s ar e mos t likely subject t o important change s throughou t normal de velopment. I n thi s regard , cholinergic signalin g during gestatio n an d postnata l developmen t change s from facilitatin g cell division , cell differentiation , an d

CNS centra l nervou s system DA dopamin e DHßE dihidro-ß-erythroidin e G gestationa l day GABA y-aminobutyri c acid LCN loca l circuit neuron LTP long-ter m potentiatio n mAChR muscarini c acetylcholine recepto r NAc nucleu s accumbens nAChR nicotini c acetylcholine recepto r NMDA N-methyl-D-aspartat e P postnata l day PFC prefronta l cortex

DEVELOPMENT OF NICOTIN E RECEPTORS 35 5 PN projectio n neuron PNS periphera l nervou s syste m VIP vasoactiv e intestina l peptide VTA ventra l tegmenta l are a

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22

Neural Precursors a s Preferential Target s for Drug Abuse: Long-Term Consequences and Latent Susceptibility to Central Nervous System Disorders Kurt F. Hauser Nazira El-Hage Shreya Buc h Gregory N. Barnes Henrietta S . Bada James R. Pauly The productio n o f ne w neuron s an d gli a occur s throughout lif e an d i s exceedingly sensitive to disruption b y drug abuse. Ne w neural cells originate within specialized region s o f th e centra l nervou s syste m (CNS) tha t maintain an environmental milie u neces sary for cell proliferation, and they appear to be essen tial fo r th e maintenanc e o f ste m cell s (Fig . 22-1) . The germina l zone s als o provid e spatial cues tha t establish cel l polarit y an d organization . Th e appear ance o f germinal zone s i s highly ordered an d follow s precise spatiotempora l guideline s tha t aris e i n th e ventricular zon e (VZ ) an d ar e sustaine d i n th e sub ventricular zone (SZ) , cerebellar external granular (or germinal) laye r (EGL), an d intrahila r zone (IHZ ) of the dentat e gyru s (Ramo n y Cajal , 1960 ; Boulde r Committee, 1970 ; Goldman , 1998 ; Alvarez-Buyll a and Garcia-Verdugo, 2002; Taupin an d Gage, 2002). Considerable evidenc e indicate s tha t drug s wit h abuse liabilit y disrupt developmen t b y affectin g th e production o f new neuron s an d glia . Moreover , th e effects o f dru g abus e ar e no t limite d t o a particula r

cell type , germinal zone, or stage of development, bu t rather the y uniquel y affec t individua l neuronal an d glial precurso r type s withi n variou s germina l zones . Germinal zone s ar e targete d t o varyin g degree s b y most substance s wit h abus e liabilit y (Laude r e t al. , 1998; Lu o and Miller , 1998 ; Hildebrand t et al., 1999 ; Teuchert-Noodt e t al, 2000 ; Buzniko v e t al, 2001 ; Duman e t al, 2001; Radle y and Jacobs, 2002 ; Crew s and Nixon , 2003 ; Lin an d Rosenthal , 2003 ; Gordo n and Hen , 2004 ; se e Chapter s 1 1 an d 12) . Like thei r progeny, neura l precursor s expres s a n inexplicabl y rich variet y o f recepto r an d neurotransmitte r types , which seemingl y contribute s t o thei r pronounce d sensitivity to multipl e neurotransmitter s and drug s of abuse that mimic the endogenou s neurochemica l systems. Drug s o f abus e tha t effec t th e genesi s o f ne w neural cell s likel y includ e cocaine , methampheta mine, opiates , nicotine , alcohol , an d perhap s ecstas y (via serotonergi c actions ) (Hause r an d Mangoura , 1998; Laude r e t al, 1998 ; Hildebrand t e t al, 1999 ; Teuchert-Noodt e t al, 2000 ; Buzniko v et al, 2001 ;

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364 NICOTINE-AFFECTE D DEVELOPMEN T

FIGURE 22- 1 Schemati c diagra m showin g site s o f neural precurso r productio n throughou t roden t on togeny. Neura l cells are initially produced i n the ven tricular zon e (VZ ) an d the n i n th e subventricula r zone (SZ) . Th e cerebella r externa l granula r (o r germinal) laye r (EGL ) i s a secondar y proliferativ e zon e that arise s from th e brai n stem an d exclusivel y generates neurons (Hauser et al., 2003). The S Z becomes a major source of supragranular neurons an d macrogli a relatively earl y durin g maturatio n (approximatel y a t birth i n rodents an d durin g the thir d trimeste r i n hu mans), wherea s th e intrahilala r zone (IHZ ) o f th e dentate gyru s is a majo r sit e of adult neuronogenesis. There i s compelling evidenc e tha t opiate s an d nico tine affect germina l cell s i n all four o f the zones .

Duman et al., 2001; Radley and Jacobs , 2002; Hauser et al. , 2003 ; Li n an d Rosenthal , 2003 ; Gordo n an d Hen, 2004) . Although w e will focu s o n th e effect s o f opiates an d nicotin e o n th e genesi s o f neura l cells , most othe r drugs with abuse potential also seem t o affect neura l developmen t t o varyin g degrees throug h direct actions o n the CNS . As noted earlier , it i s assumed tha t drug s of abuse disrupt th e genesi s o f neurons an d gli a b y disruptin g the endogenou s neurochemica l system s tha t nor mally regulat e maturatio n (Hause r et al. , 2003) . I t is also importan t to conside r that , beside s mimicry , the unique pharmacologica l action s o f drug s o f abus e may activate novel signaling events and genes , resulting i n qualitativel y differen t response s fro m thos e seen with normal neurotransmitte r systems.

OPIATES Opiates, whic h w e wil l defin e as being derive d fro m the opiu m popp y (e.g., heroin an d morphine) , act by mimicking endogenous "opioid " peptide s an d prefer entially activatin g (i-opioid receptor s (MOR) . Opiat e

exposure can modulat e th e production o f neural cells in th e V Z an d S Z (Dodg e Mille r e t al , 1982 ; Reznikov et al, 1999 ; Stiene-Marti n et al, 2001) and the EG L (Zago n an d McLaughlin , 1983 , 1987 ) dur ing maturation i n experimental anima l models. Mor e recently, opiates have been additionally implicated i n affecting neurogenesi s i n the adult IH Z (Eisc h e t al, 2000; Mandyam et al, 2004), which suggest s that th e ability o f opiates t o disrup t the genesi s o f neural pre cursors i s lifelong . Th e exten t t o whic h cellula r changes in the germinativ e zone s translate s int o neu rocognitive defect s i n opiate-expose d childre n i s un certain. Exposur e t o opiate s i n utero , however , i s highly correlate d wit h lo w infan t birt h weigh t an d length, reduce d hea d circumference , a s well as alterations i n cognitive , motor , and/o r behaviora l mile stones during the first 3 years (Messinger et al., 2004 ; Shankaranetal., 2004). Although providin g essentia l evidenc e tha t opi ates effec t CN S developmen t irrespectiv e of a variety o f psychosocia l factors , whole-anima l studie s present som e limitation s i n approachin g certai n mechanistic questions . Fo r example, th e endogenou s opioid syste m —i.e., endogenou s peptide s an d MOR , 0- (DOR ) an d K-opioi d receptors (KOR)—regulate s a variety of physiological functions that ma y secondarily effect developmen t (Hause r an d Mangoura , 1998) . Besides direc t effect s o n opioid-recepto r expressin g neural progenitors , opioi d exposur e ca n induc e changes in , fo r example , respiration , nutrition , an d endocrine functio n (Martin , 1983 ; Aki l e t al, 1984 ; Simon, 1991 ; Simo n an d Hiller , 1994 ; Roger s an d Peterson, 2003) , whic h ma y i n tur n affec t neura l development. Dissociatin g th e direc t fro m indirec t effects o f opiate s i n vivo ca n b e quit e challengin g (Hammer, 1993) . Fo r thi s reason , tissu e cultur e ha s been prove n a n invaluabl e too l i n isolatin g differen t neural cel l type s an d studyin g the rol e o f opioids i n the regulatio n o f the proliferation , differentiation, o r death o f neura l cell s (Hause r an d Stiene-Martin , 1993; Hauser an d Mangoura , 1998) . Endogenous opioid s and opiate drug s have marke d effects o n cel l neurona l differentiatio n (Zago n an d McLaughlin, 1986a , 1986b ; Vernadaki s e t al, 1990 ; Mangoura an d Dawson , 1993) . I n particular , Ham mer ha s show n tha t morphine , th e majo r bioactiv e form o f heroi n i n th e brain , inhibit s pyramida l cel l differentiation i n th e cerebra l cortica l pyramida l neurons (Hamme r e t al. , 1989 ; Ricald e an d Ham mer, 1991) . Althoug h th e effect s o n differentiatio n

NICOTINE, OPIATES , AND THE GENESI S O F NEURA L CELLS 36 5 during developmen t ar e significant , man y aspects of the effect s o f opioid on CNS maturatio n ha s been extensively reviewe d i n th e pas t (Zagon , 1987 ; Ham mer an d Hauser , 1992 ; Hammer , 1993 ; Hause r an d Stiene-Martin, 1993 ; Hause r e t al, 2003 ; Eisch an d Mandyam, 2004 ; Slotkin , 2004). For this reason , the present chapter focuses on the effect s o f opiates as related to the genesi s of neurons an d gli a i n the CNS. Neuronogenesis Alterations i n th e endogenou s opioi d system , eithe r by manipulatin g the endogenou s opioid s themselve s or throug h opiat e dru g treatment , caus e profoun d changes i n th e developin g nervou s system . Opioid s affect multipl e regions , includin g th e cerebellu m (Zagon an d McLaughlin, 1986a , 1987 ; Hause r étal, 1989; Lorbe r et al, 1990 ; Hause r and Stiene-Martin , 1991) an d forebrai n region s such a s the cerebra l cortex an d hippocampu s (Zago n an d McLaughlin , 1986b; Hause r et al., 1989; Hause r and Stiene-Martin , 1991). In excess, opiates reportedly inhibit the prolifer ation o f neuroblasts and/o r glia (Steel e an d Johannes son, 1975 ; Vertes et al., 1982 ; Zagon and McLaughlin , 1986a, 1986b , 1987 ; Kornblu m etal., 1987b ; Hamme r et al, 1989 ; Schmah l e t al., 1989 ; Lorbe r e t al, 1990; Seatriz an d Hammer , 1993) , althoug h thes e initia l studies relie d o n labelin g indice s wit h thymidin e analogs t o asses s cel l proliferation , which ma y onl y yield a partia l understandin g o f cel l cycl e change s (Mandyam e t al. , 2004) . B y contrast , continuou s treatment with opioid receptor antagonist s during development generall y ha s th e opposit e effect s o f con tinuous agonis t treatment . Continuou s opioi d receptor blockad e increase s (1 ) the rat e o f thymidine incorporation int o DNA , (2 ) the are a an d volum e o f cortical cel l layers , (3 ) neurona l an d glia l numbers / unit area, (4) dendritic size and the numbe r of spines/ unit length , an d (5 ) increase d differentiatio n an d synaptogenesis (Zago n an d McLaughlin , 1986a , 1986b, 1987 ; Hauser etal., 1987, 1989) . Many o f th e abov e developmenta l studie s suggested tha t opiate drugs could modulat e the genesis of neurons an d gli a i n th e V Z o r SZ . Initia l studie s showed that opiate exposure (morphine or methadone) markedly reduced cel l numbers i n the forebrai n whe n the genesi s o f th e cell s originate s fro m th e VZ/S Z (Steele and Johannesson, 1975 ; Zagon and McLaugh lin, 1977b , 1982b) . Initially, it was uncertain whethe r neural progenitor s expresse d opioi d receptor s o r

whether th e effect s o f opioids were direct. Later studies used radioligan d binding (Kornblum et al., 1987a ; Leslie an d Loughlin , 1993) , i n sit u hybridizatio n (Leslie et al, 1998 ; Zh u et al, 1998) , and immunocy tochemistry (Rezniko v e t al. , 1999 ; Stiene-Marti n et al , 2001 ) t o identif y MOR , DOR, and KO R o n germinal cell s i n th e VZ/SZ . Conversely , i t was later shown tha t chroni c opioi d recepto r blockad e in creased thymidin e incorporatio n an d cel l number s (Zagon an d McLaughlin , 1983 , 1986b, 1987) , whic h was generally the opposit e effec t o f opiate drug treatment Importantly , the findings from opioi d antagonist studies additionally provided circumstantial evidenc e that th e endogenou s opioi d syste m i s tonically active and inhibit s development. Opioid peptide s an d receptor s ar e highl y ex pressed b y immatur e EG L cells , whic h consis t o f neuroblasts an d immatur e post-mitoti c neurons , bu t are no t expresse d b y mor e matur e granul e neuron s derived fro m thi s germinativ e laye r (Zago n e t al. , 1985;KinneyandWhite, 1991; Osborne étal, 1993). This finding suggests that the expressio n of the opioi d system b y EG L cell s i s unrelate d t o th e onse t o f expression of adult neurochemical systems and there fore likel y t o b e involve d i n maturationa l processe s (Hauser e t al. , 2003) . I n th e cerebellum , manipula tion o f th e opioi d syste m alter s th e proliferatio n o f cerebellar EG L neuroblast s and/or their granule neuron progen y (Zago n an d McLaughlin , 1983 , 1986a, 1987; Hause r e t al, 1987 , 1989; Lorbe r e t al, 1990) , as well as their differentiation (Zago n an d McLaugh lin, 1986a ; Hause r e t al. , 1989 , 2000) an d surviva l (Hauser et al., 2000). Similarly, exposure to preferential MOR-actin g opiate s suc h a s morphin e o r methadone cause s decreased thymidin e incorporation and/or reduction s i n cel l number s (Zago n an d McLaughlin, 1977a , 1977c , 1982a) . I n cell s isolate d from th e murin e EGL , morphine exposur e signifi cantly reduce s neuroblas t number s an d DN A synthe sis an d th e effect s o f morphin e ca n b e prevente d b y the selectiv e MOR antagonis t ß-funaltrexamine , find ings suggesting that MOR regulate s cell proliferation. By contrast , DO R agonist s selectively reduce neurit e outgrowth fro m granul e neuron s a s they differentiat e from EG L precursors , while morphin e ha s n o effec t on this measure (Häuse r et al., 2000). This action suggests tha t individua l opioi d recepto r type s ma y regu late different aspect s of development i n the sam e cell. The deat h o f neuronal precursor s i s an occasion ally reporte d consequenc e o f exposur e t o opiate s

366 NICOTINE-AFFECTE D DEVELOPMEN T alone (Merine y e t al. , 1985) . Opioid s ca n modulat e the effect s o f existing apoptotic signal s (especiall y in immune cell s [Singha l e t al. , 2001 , 2002]) , an d opiates may be co-factor s i n regulating neural cell deat h (Gurwell e t al, 2001 ; Khurdaya n et al, 2004) . Cel l death i s evident in culture d Purkinj e cell s with mor e chronic exposur e (Hauser et al., 1994) . Purkinje cell losses hav e bee n reporte d i n chroni c heroi n abuser s who are HIV seronegative (Oehmichen e t al., 1996) . Although the mechanisms underlying opiate-induce d cell deat h ar e incompletely understood an d likel y differ amon g cell types , opiate-induce d modulatio n o f cell deat h ha s been proposed t o involve phosphoinos itol 3-phosphokinas e an d extracellula r signa l regu lated kinase (ERK) (Polakiewicz etal, 1998). Singhal and coworker s report tha t p3 8 mitogen-activate d pro tein kinase (MAPK), p53, and caspase s 3 and 8 medi ate morphine-induced deat h i n macrophages (Singha l et al, 2002), whereas morphine-induced T cell losses involve c-jun-N-terminal kinase activation and reduc tions i n ER K phosphorylation (Singha l e t al. , 2001) . By contrast, cell death i s not seen with concentration s of morphine exceedin g 1 jim in cultured mous e EG L cells (Hause r e t al. , 2000 ) o r i n astrocyte s (Gurwel l and Hauser , 1993) . Thus , i n som e instances , opiate s may affec t cel l surviva l i n additio n to thei r effect s o n proliferation, although thi s is not a consistent observa tion. Gliogenesis There are considerable indication s that glial-restricte d precursors (GRPs) , a s wel l a s thei r astroglia l an d oligodendroglial progeny, are a direct target of opiates during maturation . Mos t glia l type s i n th e forebrai n arise from th e S Z and gliogenesis typically follows th e production o f neuron s i n a particula r brai n regio n (Fig. 22-2). Many hav e propose d tha t astrogli a ar e target s for drugs wit h abus e liabilit y (Eriksso n e t al. , 1991 ; Stiene-Martin an d Hauser , 1991 ; Beitner-Johnso n e t al, 1993 ; Hauser and Mangoura, 1998 ; Fattor e et al, 2002; Belchev a e t al, 2003) . Astroglial growt h i s inhibited b y opioid s i n cell cultur e an d i n vivo (Schmahl e t al , 1989 ; Stiene-Marti n an d Hauser , 1990, 1991 ; Hause r and Stiene-Martin , 1991 ; Stiene Martin e t al, 1991 , 2001 ; Zago n an d McLaughlin , 1991; Hause r e t al. , 1996 ; Hause r an d Mangoura , 1998). Astrogli a ca n expres s MOR , DOR , and/o r

FIGURE 22- 2 Presenc e o f opioid and nicotini c acetylcholine receptor s (nAChRs ) o n neurona l an d glia l precursors, fi- , Ô- , an d K-opioi d receptor s (MOR , DOR, an d KOR , respectively ) ar e variably expresse d (±) o n neurona l an d astroglia l precursors , an d nAChRs ar e variabl y expressed b y neurona l precur sors (±). IHZ, intrahilar zone; O2A , oligodendrocyte type 2 astrocyte progenitor; SZ, subventricular zone.

KOR (Eriksso n et al, 1990 , 1991 ; Stiene-Marti n an d Hauser, 1990 , 1991 ; Ruzick a e t al., 1995 ; Gurwel l e t al, 1996 ; Hause r e t al. , 1996 ; Stiene-Marti n e t al , 1998, 2001 ; Thorlin et al., 1998) , and opioid recepto r expression is developmentally regulated and/o r differ s among brai n region s (Hause r an d Stiene-Martin , 1991; Eriksso n e t al , 1992 ; Ruzick a e t al , 1994 , 1995; Gurwel l e t al, 1996; Stiene-Marti n e t al, 1998 ; Thorlin e t al, 1999) . Activatio n o f MOR , KOR , o r DOR cause s reduction s i n cel l proliferatio n an d in creased cellula r hypertroph y (Stiene-Marti n an d Hauser, 1991 ; Gurwel l e t al , 1996 ; Hause r e t al , 1996). Th e inhibitio n o f cel l proliferatio n an d as troglial hypertroph y are mediate d b y opioid-induce d increases i n intracellula r concentratio n o f Ca 2+ ([Ca2+]i) (Gurwel l e t al, 1996 ; Hause r e t al, 1996) . The "reactive " cellula r hypertroph y i s accompanie d by increased glia l fibrillary acidic protei n (GFAP ) immunoreactivity an d aspect s ma y mimi c reactiv e gliosis in vivo (Hauser et al, 1996 , 1998) . Unlike in neurons an d astroglia , where MOR acti vation inhibits cell replication, i n immature oligoden droglia, MO R activatio n i s mitogeni c (Knap p e t al. , 1998). By contrast to MOR, KO R is expressed in more mature, nondividin g oligodendroglia . KO R blockad e increases th e deat h o f culture d oligodendrocytes ,

NICOTINE, OPIATES , AND THE GENESI S OF NEURA L CELLS 36 7 inferring tha t KO R couple s t o enhance d surviva l i n this cell typ e (Knap p et al., 2001). Similar to granul e neurons, differen t aspects o f oligodendroglia l devel opment appea r t o b e independentl y regulate d b y MOR an d KOR . A s mentioned, unlik e granul e neu rons and thei r EG L precursors , which d o not express KOR in vitro (Hauser et al, 2000), MOR activatio n is mitogenic i n oligodendrogli a (Knap p e t al. , 1998) . Collectively, th e findings in neuron s an d gli a suggest that opioi d effect s ar e highl y complex—affecting th e maturation of each cel l type differently . Adult hippocampa l progenitor s (AHPs ) fro m th e rat IH Z expres s ß-endorphi n ( a post-translationa l product o f th e proopiomelanocorti n gene ) (Persso n et al., 2003a) . ß-endorphi n stimulate s the maturatio n and perhap s cel l fat e decision s o f IHZ cell s throug h autocrine/paracrine mechanisms o f action (Persso n et al, 20 0 3a). Interestingly , AHP s expres s MO R an d DOR, an d blockad e o f thes e receptor s b y naloxon e increases the AHPs, resultin g in increase s in neurob last replicatio n wit h apparen t compensator y reduc tions i n th e genesi s o f astrogli a an d oligodendrogli a (Persson et al., 2003a , 2003b) . These findings suggest that, i n additio n t o modulatin g th e proliferatio n and death o f neurona l an d glia l precursors , opioid s ma y influence cel l fat e decision s o f bi - o r multipotentia l neural cell precursors. Importantly, opioids can modulate basi c fibroblast growth factor (bFGF ) and/or epiderma l growt h facto r (EGF) activatio n of ERK in C6 gliom a cells or in primary astrocyte s (Boh n e t al. , 2000 ; Belchev a e t al. , 2002, 2003) . The proliferativ e effect s o f ß-endorphi n in adul t hippocampa l progenitor s wa s mediate d through a signalin g pathwa y involvin g PI3-kinase , [Ca2+]j, and MAP K activation (Persso n et al, 2003a), which overlap s wit h event s associate d wit h MOR mediated cel l deat h i n Chines e hamste r ovar y cells (Polakiewiczetal, 1998) .

NICOTINE In adults, acetylcholine is a significant neurotransmitter i n severa l neura l pathway s (Rand , 1989 ; Luetj e et al, 1990 ; Heineman n e t al. , 1991a ; Albuquerqu e et al., 1995) . During development, acetylcholin e (and perhaps relate d substances ) additionall y provide s trophic suppor t t o guide th e maturatio n an d surviva l of incipien t cholinergi c neuron s (Zahalk a e t al. ,

1992; Rol e and Berg , 1996) . Nicotin e i s known t o alter adul t neura l function . I n th e maturin g nervou s system, ther e i s considerabl e evidenc e tha t nicotin e exposure (as a component of cigarette smoke ) disrupts the norma l interaction s betwee n acetylcholin e an d the cellula r targets that expres s subtypes of nicotini c acetylcholine receptors (nAChRs) . Cigarette smokin g durin g pregnanc y ca n hav e multiple direc t an d indirec t effect s o n feta l develop ment an d i s strongly associate d wit h developmenta l and behaviora l abnormalitie s i n newborn s (Econo mides an d Braithwaite , 1994 ; Mclntos h e t al, 1995 ; Shu et al., 1995; Lambers and Clark, 1996) . Epidemi ological studie s sugges t tha t lo w infan t birt h weight , delayed feta l development , an d prematur e birth s ar e closely correlate d wit h smokin g (Opanashu k e t al. , 2001). Neurobehaviora l developmen t i s also altered . In addition , childre n bor n t o smokin g mother s hav e an increase d incidenc e o f attention defici t hyperactiv ity disorde r (Mclntos h e t al. , 1995) . Smok e exposur e may als o increas e th e incidenc e o f sudde n infan t death syndrom e (Slotki n et al., 1995) . Although ciga rette smoke contains over 4000 chemical compounds , many studie s have show n tha t nicotin e i s the princi pal insulting agent in development . In anima l models , prenata l exposur e to tobacco o r nicotine disrupt s somati c (Paulso n e t al. , 1991 ) an d CNS developmen t an d cause s defect s in morphologi cal, neurochemical, an d behaviora l indices (Slotki n et al., 1987 , 1993 ; Fuxe étal, 1989; Paulso n étal., 1994a, 1994b; Penningto n e t al, 1994 ; Witsch i e t al, 1994 ; Court e t al, 1995 , 1997 ; Ro y et al., 1998 ; Paul y et al, 2004). Interestingly , nicotine ca n affec t brai n develop ment i n the absenc e o f overt changes i n body weight, which suggest s that effec t o f nicotine o n th e brai n is direct (Zahalka et al, 1992) . Additional evidence sug gests tha t th e effect s o f nicotin e ar e gender-specifi c (Pauly e t al, 2004) . Althoug h assessin g the effec t o f in uter o exposur e t o cigarett e smok e i s potentiall y confounded b y reduced maternal-placental-feta l cir culation (Birnbau m e t al. , 1994 ) o r amin o aci d up take (Sastry , 1991) , studie s in chic k embryo s sugges t that nicotin e retard s neurobehaviora l developmen t (Pennington e t al. , 1994) ; studie s i n vitr o similarl y suggest tha t nicotin e ca n directl y affec t developin g neural cell s (Opanashuk e t al., 2001). Perinatal expo sure t o nicotin e cause s lastin g change s eviden t i n adult brains (Nordberg et al, 1991 ; Mia o e t al, 1998 ; Eriksson et al, 2000).

368 NICOTINE-AFFECTE D DEVELOPMENT Epidemiological and/o r experimenta l studie s o f Parkinson an d Alzheime r disease s hav e show n tha t nicotine alon e (o r fro m cigarett e smoking , chewin g gum, and/o r derma l patches) ma y have neuroprotec tive properties (Baron, 1986; Sershe n e t al., 1987 ; van Duijn and Hofman, 1991 ; Jones et al., 1992 ; Ishikawa and Miyatake , 1993) . Som e interes t originate s fro m studies showin g that ther e i s a selectiv e reductio n i n neuronal nAChRs , i n compariso n t o age-matche d controls, i n severa l neurodegenerative disease s (Perry et al. , 2002) . Acute exposur e t o nACh R agonist s en hances som e measure s o f cognitiv e function , an d chronic exposur e t o nicotin e agonist s paradoxically increases th e numbe r o f neuronal nAChR s (Jone s e t al, 1992 ; Wilso n e t al. , 1995) . Nicotin e ca n protec t against ß-amyloi d toxicit y i n vitr o (Kihar a e t al. , 1997), prompting a n interes t i n attemptin g t o under stand interaction s betwee n nAChR s an d th e patho physiology of these neurodegenerative diseases. In th e aging nervous system, nicotine appear s t o be trophi c in neuron s that express nAChRs an d perhap s second arily in other non-nAChR-expressin g cells . Although the trophi c effect s o f nicotine i n aging may be neuro protective, th e developmenta l trophi c action s o f nicotine may be detrimental to the fetus , an d i t is important to note that an inappropriat e blockade o f normal programme d cel l deat h o r a n abnormall y hig h rate o f cell proliferatio n is also likely to b e detrimen tal (Opanashuk et al, 2001). Nicotine ha s lon g bee n propose d t o modulat e neural developmen t b y influencing the proliferation and/or surviva l o f neura l precursor s (Slotki n e t al. , 1987, 1993 ; Slotkin , 2004). Recen t findings that perinatal exposur e t o nicotin e ha s long-lastin g conse quences o n behavio r i n rats , combined wit h clinica l observations o f low infant birth weight , th e potentia l for psychiatric problems, and predilection towar d substance abus e (Lamber s and Clark , 1996 ; Erns t e t al., 2001), underscor e th e importanc e o f examinin g effects o f nicotine i n development . Targete d deletio n of specifi c nACh R subuni t genes , includin g oc3 , ß2 , and ß 4 subunits , can b e letha l earl y during development, wherea s deletio n o f man y othe r nACh R subunits significantl y modifie s adul t behavior s (Cordero-Erausquin e t al. , 2000) . Finally , nicotin e decreases neurogenesi s in th e IH Z o f adult male rats (Abrous e t al. , 2002) , a findin g suggesting tha t nico tine's abilit y to influence the productio n o f new neu ral cells is lifelong.

Neuronogenesis The appearanc e o f cholinergi c syntheti c enzyme s and multipl e nACh R subtype s in the germina l zone s of th e developin g CN S suggest s tha t acetylcholin e might potentiall y influenc e CN S maturatio n i n th e VZ/SZ an d EG L (Goul d an d Butcher , 1987 ; Clo s et al , 1989 ; Perr y e t al , 1993 ; Cour t e t al , 1995 ; Atluri e t al, 2001 ; Opanashu k e t al, 2001 ; Hause r et al, 2003; Gai et al, 2004). For example, thé matu ration o f cholinergi c system s i n th e cerebellu m (Brooksbank e t al, 1989 ; Perr y et al, 1993 ; Cour t et al., 1995 ; Jaarsma et al., 1997 ) coincide s with critical periods o f neurogenesi s i n thi s brai n regio n i n ro dents (Mial e an d Sidman , 1961 ; Altman , 1972a , 1972b). Th e mechanism s b y which nicotin e modu lates maturatio n ar e likel y to b e comple x an d diffe r among cel l types . nAChR s ar e member s o f th e ligand-gated io n channel recepto r superfamily . Binding t o nAChR s induce s a conformationa l chang e i n an intrinsi c ion channel, whic h increase s Na + influ x (Heinemann e t al. , 1991b ; Dan i an d Heinemann , 1996). Althoug h nicotin e ma y acutely activate cells , desensitization and/o r possibl y permanen t inactiva tion follow s th e activatio n o f th e recepto r (Collin s and Marks , 1996) . I t has been show n tha t individual neurons ca n expres s multipl e nACh R subtype s (i.e. , differing subuni t composition) , wit h eac h servin g unique function s (Conro y an d Berg , 1995) . I t ha s long bee n know n tha t nicotin e induce s inwar d Ca2+ currents, especiall y throug h oc 7 nACh R subunit containing complexes, as well as through som e othe r nAChR subtype s (Dajas-Bailado r an d Wonnacott , 2004). [Ca^J i ha s well-documente d effect s o n cel l proliferation, differentiation , and survival . Interest ingly, th e expressio n o f som e nicotini c recepto r types ma y b e linke d t o ke y regulator s wit h neura l cell development . So x 10, an importan t transcription regulator o f neura l maturatio n an d cel l fat e deci sions, activate s ß 4 an d oc 3 subunit s i n a cel l type-specific manner , and So x 10 directly affects reg ulatory regio n o f th e ß 4 nACh R subuni t promote r (Liu et al, 1999) . Nicotine increase s the rat e of proliferation o f neuroblasts in the EGL (Opanashu k e t al, 2001), which are predecessor s o f granule neurons . Whe n isolate d EGL precursor s ar e continuousl y expose d fo r 7 days to selectiv e oc3/oc 4 (epibatidine ) o r ct 4 (cytisine ) nAChR agonist s or to partial agonists, epibatidine, but

NICOTINE, OPIATES, AND THE GENESI S O F NEURA L CELL S 36 9 not eytisine , concentration-dependen t increase s i n DNA synthesis and content are produced (Opanashu k et al , 2001) . Thi s resul t indicate s tha t Ot 3 iiACh R stimulation is mitogenic to cerebellar granule neuroblasts. Moreover , significan t effect s wer e significantl y attenuated b y concurren t administratio n o f th e iiAChR antagonis t dihydro-ß-erythroidin e (DHßE) , suggesting th e involvemen t o f specifi c nAChRs . I n summary, thes e dat a provid e nove l evidenc e tha t nAChRs directl y affec t th e developmen t o f granul e cell precursor s an d furthe r sugges t tha t th e effect s are mediate d throug h specifi c oc 3 nACh R subtype s (Opanashuk e t al , 2001) . Importantly , oc 7 nACh R subunits ar e expresse d b y differentiatin g granul e neurons, thu s othe r nACh R subtype s ma y regulat e alternative aspect s o f development suc h a s differen tiation, includin g synaptogenesi s and/o r surviva l (Didier e t al. , 1995 ; Fucil e e t al , 2004) . Th e rol e of nicotin e i n th e EG L o f th e developin g cerebel lum ha s bee n reviewe d i n detai l elsewher e (Hause r et al., 2003). Importantly, beside s regulatin g cel l proliferation , nicotine can modulat e programme d cel l deat h (Ren shaw et al, 1993 ; Ro y et al, 1998 ; Opanashu k e t al, 2001; Prendergas t e t al, 2001 ; Abrou s e t al, 2002) . Chronic nicotin e exposur e i s neuroprotectiv e i n organotypic culture s o f th e ra t hippocampu s b y up regulating calbindi n expression , whic h buffer s toxi c increases i n intracellula r Ca 2+ (Prendergas t e t al. , 2001) an d modulate s nAChR-specifi c membran e currents i n neura l ste m cell s (Ca i e t al., 2004) . Similarly, i n culture d cerebella r EG L neuroblast s (Opanashuk e t al. , 2001 ) o r thei r progen y (Fucil e e t al., 2004), chroni c activatio n o f oc3 nAChRs prevent s cell deat h an d th e activatio n o f oc 7 nACh R subunit s protects spina l cor d neuron s agains t arachidoni c acid-induced deat h (Garrid o e t al. , 2003) . B y contrast, th e activatio n o f <x 7 nACh R subtype s permit s Ca2+ influ x an d cause s cel l deat h i n dissociate d hip pocampal neuron s (Delbon o e t al. , 1997 ; Berge r et al, 1998 ; Ferchmi n e t al. , 2003) , feta l ra t neura l cells (Ro y e t al , 1998) , an d vascula r cell s (Vill ablanca, 1998). Taken together, thes e findings suggest that nicotin e ca n modulat e th e proliferatio n an d death o f neuron s an d thei r precursors , an d thes e ef fects ma y diffe r markedl y amon g differen t cel l type s and subtypes . Like opioids , nicotine ca n elici t highly individualized an d divers e intracellula r responses . The varie d responses are likely determined b y varying

nAChR subuni t composition s an d b y nACh R cou pling to particular intracellula r effectors . Gliogenesis There is emerging evidenc e tha t the maturation o f astroglia, oligodendroglia , an d potentiall y GRP s i s directly affected b y nicotine. As noted earlier , most glia arise fro m th e SZ . Importantly , glia l precursor s iso lated largel y from th e S Z express nicotini c receptors , and suggestin g immatur e gli a ar e importan t target s for nicotin e i n neura l developmen t (Layer , 1991) . Despite recen t finding s tha t astrogli a ca n expres s nicotinic receptor s (Hosl i et al., 2000; Mesulam e t al., 2002; Lim an d Kim , 2003; Gahring e t al., 2004), few studies have assessed the rol e o f nicotine i n astroglial function an d especiall y durin g development . As troglia expres s a4 and ß4 nAChRs, an d likely express other nACh R subuni t type s (Hosl i e t al. , 2000) . Iso lated astrocyte s treated wit h nicotin e i n cultur e sho w disruptions i n glutamat e uptak e tha t wa s influenced by th e duratio n o f nicotine exposure , an d precondi tioned astrocyte s displaye d sensitizatio n t o nicotine . Glutamine synthetas e activit y wa s als o disrupte d in astroglia , a s wa s sodium/potassium-dependen t ATPase activity , which tende d to respond inversel y to glutamine synthetas e activit y (Li m an d Kim , 2003). Butyrylcholinesterase, which ma y pay play an impor tant ancillar y rol e t o acetylcholinesterase i n nicotin e degradation, i s predominantly expresse d b y astrogli a (Mesulam e t al. , 2002) . Collectively , thes e result s suggest that astrocytes, in addition to neurons, may be direct target s fo r the action s o f nicotine. Thi s target ing ma y hav e importan t consequence s durin g CN S development. Oligodendrocyte precursor s (O2 A progenitors ) can expres s oc3 , a4, oc5 , oe7 , ß2 , an d ß 4 nicotini c re ceptors subunit s (Roger s e t al. , 2001) . Activatio n o f nAChRs cause s long-lastin g oscillator y increase s i n intercellular calcium , althoug h i t i s not entirel y cer tain whic h subunit(s ) mediate d thi s effect . A s note d elsewhere, Ot 7 nAChRs permi t sustaine d inwar d Ca2+ currents followin g exposur e to nicotine . Man y o f the Ca2+ current s i n thes e cell s coul d b e reverse d b y DHßE an d ar e no t mimicke d b y th e activatio n o f L-type calciu m channel s or carbachol . Interestingly , acetylcholine receptor-inducin g protei n (ARIA) , a member o f the neureguli n famil y (Falls , 2003; Corfas et al. , 2004) , i s widely expressed b y oligodendroglial

370 NICOTINE-AFFECTE D DEVELOPMEN T progenitors in the S Z and triggers nAChR expression in skeleta l muscl e an d neura l cell s (Vartania n et al. , 1994). ARIA exposure causes O2A progenitors t o develop along an oligodendroglial fat e i n vitro, althoug h the maturationa l change s resul t fro m neureguli n sig naling and i t is unclear whether ARIA drives nAChR expression i n oligodendroglia . Taken together , thes e findings sugges t tha t nicotin e ma y hav e a profound influence o n th e maturatio n o f GRP s an d direc t them t o a n oligodendroglia l fate . Importantly , un timely exposur e t o nicotin e ma y disrup t gliogenesi s and, in particular, the production of oligodendroglia.

DRUG INTERACTION S WITH DISEASE OR TRAUMA

Considerable evidenc e her e an d elsewher e suggest s that drug abuse can influenc e neurogenesis and gliogenesis an d affec t long-ter m organizationa l change s in the brain. On th e basi s of this insight, we question whether th e maladaptive , organizationa l change s might predispose the CN S t o failure whe n confronte d with non-drug-relate d insults . A remarkable aspec t of the CN S i s that despite significan t insults during critical developmenta l periods , opposin g neuroadaptiv e processes ar e capabl e o f considerabl e recovery . Th e neuroplastic respons e t o perinatal dru g exposure may itself be maladaptive when the CNS i s challenged wit h other insult s unrelate d t o dru g abuse . Fo r example , does exposure to nicotine or opiates before disease onset or concurrently increase the risk and/or severity of diseases not typically attributed to deficits i n the gene sis o f ne w cells , suc h a s Alzheime r o r Parkinso n disease, neurological acquired immunodeficienc y syndrome (AIDS) , o r neurotrauma ? As with dru g abuse , disruptions i n adul t neurogenesi s hav e bee n impli cated i n Alzheime r diseas e (Haughe y e t al. , 2002) , traumatic brai n injur y (Brau n e t al. , 2002 ; Chiru mamilla e t al. , 2002) , neuropsychiatrie disorders (Jacobs, 2002 ; Kempermann , 2002 ; McEwen , 2002) , and us e of psychotropic drugs (Duma n e t al., 2001) , as well a s in schizophreni a (Corfa s et al. , 2004 ; Ste fansson e t al., 2004), depressio n (Jacob s et al., 2000) , and seizur e disorder s (Parent and Lowenstein , 2002 ; Ribak an d Dashtipour , 2002) . Moreover , althoug h neural progenitor s may be vulnerabl e t o a singl e in sult, whe n multipl e insult s ar e combined , suc h a s drug abuse and disease, this combination ma y exacerbate losses in neural progenitors .

Assuming dru g abus e modulate s CN S organiza tion and plasticity , might the developmenta l effect s of opiates and/o r nicotin e exposur e o n CN S organiza tion b e reveale d o r exaggerate d b y aging, disease , o r environmental Stressors ? Although thi s i s an impor tant issue , i t i s difficul t t o addres s becaus e o f chal lenges i n modelin g dru g abuse an d CN S disease s in the laboratory. HIV-Drug Interaction s To addres s whether dru g exposur e migh t exacerbat e CNS disorders , we have bee n explorin g opiate-HIV interactions i n several murine and huma n model s of HIV. There ar e several key reasons for choosing HI V as a diseas e t o explor e dru g abuse-diseas e interac tions. First , dru g abus e an d HI V are interlinke d epi demics. I n th e Unite d States , AID S i s no w largel y spread throug h injectio n dru g use or through th e exchange o f sex for drugs (Nath et al, 2000, 2002). Second, opiat e drug s exacerbat e th e pathogenesi s an d neurological complication s o f HIV (Bel l et al., 1998 ; Nath e t al., 2002) . Opiates likel y exacerbate th e neu ropathogenesis o f HIV indirectl y b y modulatin g im mune functio n (Peterso n e t al. , 1998 ; Roger s an d Peterson, 2003 ; Donahoe, 2004) and directly by modulating th e intrinsi c respons e o f neuron s an d gli a to HI V and/o r vira l product s (Gurwel l e t al. , 2001 ; Khurdayan et al, 2004; Ei-Hage e t al, 2005). The HI V gene regulatory protein Tat has been implicated a s a mediato r o f HIV-induced neurotoxicity . Tat i s a transactivating , nonstructura l vira l nuclea r regulatory protei n compose d o f 10 1 amino acid s en coded b y tw o exon s (Atwoo d e t al. , 1993) . Ta t i s released by infected lymphoid cells (Ensoli et al, 1993) and glia (Tardieu et al, 1992). Tat is intrinsically cytotoxic t o neurons (Sabatie r e t al, 1991 ; Magnuson e t al, 1995 ; Week s e t al. , 1995 ; Marago s e t al, 2002 ; Singh e t al., 2004). Extracellular Tat is neurotoxic, i n part throug h interaction s wit h excitator y amino aci d receptors (Magnuso n e t al, 1995 ; Nat h e t al., 1996 ; Haughey an d Mattson , 2002) , whic h ar e accompa nied b y increase s i n [Ca 2+]j an d reactiv e oxyge n species (Nat h e t al , 1996 ; Kruma n e t al , 1998 ; Bonavia et al, 2001). Neuronogenesis Although HIV- 1 infectio n result s i n neurodegenera tion, the neurons themselve s ar e not directly infected .

NICOTINE, OPIATES , AND THE GENESI S O F NEURA L CELL S 37 1 Instead, HIV-1 affects microgli a and astroglia , subsequently contributin g t o neurodegeneratio n throug h inflammatory signalin g and th e releas e o f toxic viral products b y gli a (Brack-Werner , 1999 ; Nath , 1999 ; Kaul e t al, 2001; Garden , 2002) . A novel findin g is that multipotentia l neura l progenitor s ca n becom e infected an d serv e as a reservoir for HIV-1 (Lawrence et al., 2004). Interestingly , if the infecte d neura l pro genitors ar e allowe d t o differentiat e towar d a n as troglial phenotype , ther e ar e marke d increase s i n viral production . Thi s resul t suggest s tha t neurona l progenitors ca n becom e infected , bu t commitmen t to a neurona l fat e i s incompatible wit h vira l expres sion. Beside s bein g a sit e fo r vira l infection , neura l precursors expres s severa l ke y chemokin e receptor s that ar e co-factor s involve d i n HI V binding , fusion , and infectio n (Lazarin i e t al. , 2000 ; Ka o an d Price , 2004; Pen g e t al. , 2004) , includin g CXCR 4 an d CCR5 (Pen g e t al , 2004) . I n addition , immatur e neurons expres s majo r histocompatibilit y comple x (MHC) clas s I and othe r key immune signalin g molecules tha t blu r distinctions between neuroplasticit y and neuropatholog y (Boulange r an d Shatz , 2004) . Interestingly, bot h monotropi c an d T-tropi c strain s of HIV, respectively , inhibit the proliferatio n of neural progenitors through CCR5 - or CXCR4-mediate d activation o f ER K (Krathwoh l an d Kaiser , 2004) . Although ther e i s evidence tha t neuroblast s are a target for HIV, no studies have examined the combine d effects o f opiates an d HI V o n neurogenesis . Gliogenesis Unlike neurona l precursors , preliminar y i n vitro evi dence suggest s tha t opiate s ca n exacerbat e th e cyto toxic effect s o f HI V i n glia l precursor s (Khurdaya n et al., 2004). O n th e basi s of findings that neura l pro genitors ar e target s o f HIV , w e hav e bee n assessin g whether dru g abuse migh t furthe r disrup t the action s of HIV i n neura l progenitors . Interestingly , glia l pro genitors appea r t o b e especiall y vulnerabl e t o th e combined effec t o f opiates an d HIV , resultin g i n in creases i n activate d caspase- 3 an d increase d cel l death a s assessed by a failure o f cells to exclude ethid ium monoazide (Khurdayan et al., 2004). Most of the dying cell s ar e glia l precursors having characteristic s of O2 A glia l progenitor s (Raf f e t al. , 1983 ; Fulto n et al., 1992) , wherea s there i s a tendency fo r some im mature oligodendrocyte s an d immatur e astrocyte s t o be preferentiall y lost . A vast majority o f cultured O2 A

progenitors woul d hav e develope d int o oligoden droglia wit h furthe r maturation , thus , i t appear s tha t cells committe d t o a n oligodendroglia l fat e ma y b e preferentially vulnerabl e t o combine d opiate-HIV- 1 toxicity (Khurdaya n et al. , 2004) . Interestingly , ther e tended t o b e greate r number s o f dyin g oligodendro cytes wit h combine d morphine-Ta t exposure , al though thi s tren d wa s no t statisticall y significan t a t 96 hr. Since morphine an d Tat in combination prefer entially kil l O2 A cells , significan t oligodendrocyt e losses migh t becom e apparen t wit h exposur e tha t i s more prolonged. I t can be predicted tha t gliogenesis is affected an d tha t oligodendroglia l progenitor s ar e especially vulnerabl e i n HIV-infecte d individual s wh o abuse opiates. The consequence s o f losin g glia l precursor s ar e uncertain. Fate d glia l precursor s ma y hav e lifespan s lasting years, so that deficits i n gliogenesis would only become eviden t with time. A tentative notion i s that a sustained destruction o f glial precursors wit h chroni c opiate abus e i n HIV-infecte d individual s and subse quent los s in tota l glia l numbers migh t contribut e t o accelerated neurocognitiv e defects . Thus , chroni c opiate abuse may have debilitating effects o n the long term stabilit y of glial population s an d CN S functio n in HIV-infected individuals.

SUMMARY AND CONCLUSION S

Collectively, th e abov e findings suggest that glia l pre cursors are novel targets for HIV and opiate abuse, an d disruptions in th e genesi s and fat e o f new neura l cell s may contribut e t o th e pathogenesi s o f neuro-AIDS . The exten t t o which finding s i n opiat e an d HI V Tat treated gli a can b e generalize d to other drugs of abuse or disease s i s uncertain, however , considerin g th e ap parent susceptibilit y o f neura l progenitor s t o sub stances with abuse liability and pathologi c insults , it is likely that precursors will be affected . Furthermore , as suming the los s of precursors inevitabl y contributes t o the pathology of the disease , the prognosis for slow, progressive cognitive decline may be assured. Abbreviations AHP adul t hippocampal progenito r AIDS acquire d immunodeficienc y syndrome ARIA acetylcholin e receptor-inducing protei n

372 NICOTINE-AFFECTE D DEVELOPMEN T bFGF basi c fibroblast growth facto r 2+

2+

[Ca ]A intracellula r concentration o f Ca CNS centra l nervou s syste m DHßE dihydro-ß-erythroidin e EGF epiderma l growth facto r DOR 0-opioi d receptor s

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23

Nicotinic Receptor Regulation of Developing Catecholamine System s Frances M . Leslie Layla Azam Kathy Gallardo Kathryn O'Leary Ryan Frank e Shahrdad Lotfipou r

Central catecholamin e systems , consisting of dopaminergic, noradrenergic , and adrenergi c cell groups , serve numerous integrativ e neura l function s an d criticall y regulate action , emotion , motivation , an d cognitio n (Berridge an d Robinson , 1998 ; Berridg e and Water house, 2003 ; Nieoullon an d Coquerel , 2003) . These neural pathway s ar e functiona l a t a n earl y stag e o f brain developmen t an d hav e importan t role s i n neu ronal maturatio n an d behavior s tha t ar e critica l fo r neonatal surviva l (Levit t et al., 1997 ; Sullivan , 2003). Dysregulation of these systems has been implicate d in numerous diseas e states , particularl y those i n whic h there ar e cognitive , emotional , and/o r moto r deficit s (Aston-Jones e t al. , 2000 ; Nieoullo n an d Coquerel , 2003). Numerous clinical studies have shown that smoking during pregnancy can lea d to long-standing neurobehavioral deficit s in th e offsprin g tha t ma y resul t from catecholaminergi c dysfunction , includin g at tention defici t hyperactivit y disorde r (ADHD) , con duct disorder, cognitive deficits, an d substanc e abuse

(Ernst et al. , 2001 ; Fried an d Watkinson , 2001 ; Buka et al, 2003 ; Kah n e t al., 2003 ; Thapar e t al, 2003). Whereas tobacc o smok e contain s ove r 400 0 con stituents, many of which may be harmful to the developing fetus , anima l studie s hav e focuse d o n an d confirmed a neuroteratogeni c rol e fo r nicotin e (Slotkin, 1998) . Gestationa l nicotin e exposur e in ro dents result s i n cognitiv e impairment s (Levi n e t al. , 1996), enhance d spontaneou s locomoto r activit y (Fung, 1988 ; Newman et al., 1999 ; Paul y et al, 2004), and increase d nicotine-induce d locomotio n (Shack a et al. , 1997) . Neurochemica l studie s provide furthe r evidence o f a n advers e effec t o f prenata l nicotin e exposure o n centra l catecholamin e developmen t (Ribary and Lichtensteiger , 1989 ; Slotkin , 1998 ; Mu neoka e t al, 1999) , wit h norepinephrin e (NE ) mor e highly affected tha n dopamine (DA). Route of administration is a critical variable in such studies , however , with continuou s administratio n producing result s opposite to those of intermittent injection (Navarro et al., 1988). Suc h findings have led Slotki n and colleague s

381

382 NICOTINE-AFFECTE D DEVELOPMEN T to propos e tha t nicotine-induce d hypoxi a ma y pro duce man y o f the observe d deficit s i n catecholamin e development (Slotkin, 1998) . Nicotine bind s to specfic cholinergi c receptors , A nicotinic acetylcholin e receptor (nAChR ) i s a ligand gated catio n channel . Eac h recepto r consist s o f five subunit proteins surroundin g a channel por e tha t me diate man y o f th e biologica l effect s o f acetylcholin e (ACh) (Leonar d an d Bertrand , 2001 ; Picciott o e t al., 2001). Radioligan d bindin g an d mRN A expressio n studies have show n substantia l expression o f nAChRs in feta l huma n an d roden t brai n (Naef f e t al, 1992 ; Zoli e t al , 1995 ; Ospin a e t al. , 1998 ; Hellstrom Lindahl an d Court , 2000) . Therefore, thes e receptor s may pla y a critica l physiologica l rol e i n regulatin g brain developmen t (Rol e and Berg , 1996 ; Broid e and Leslie, 1999 ; Metherat e an d Hsieh , 2004 ) an d ma y also serv e a s a targe t fo r th e neurotoxi c action s o f nicotine on developing brain . As outlined i n the pres ent chapter , a combination o f biochemical, anatomi cal, an d behaviora l approache s hav e bee n use d t o evaluate th e hypothesi s tha t functiona l nAChR s ar e expressed o n catecholamin e neuron s durin g critica l phases of brain development .

DOPAMINE NEURONS

Adult

The forebrai n dopaminergi c syste m arise s fro m tw o main cel l group s locate d i n th e midbrain , th e sub stantial nigr a (SN ) and ventra l tegmental area (VTA). The nigrostriata l system , arisin g fro m th e SN , con trols sensorimoto r integratio n an d moto r output . Mesolimbic an d mesocortica l projections , arisin g from th e VTA , regulat e rewar d an d motivate d behav ior. A wid e variet y o f nACh R subunits , oc3-ot 7 an d ß2-ß4, are expressed on DAergic neuron s bot h in the SN an d VT A o f adul t rodent s (Klin k e t al , 2001 ; Azam et al., 2002), giving rise to a rich pharmacologi cal complexity . At least two classe s of nAChR are expressed o n DAergi c terminal s withi n th e striatum , which ar e discriminate d o n th e basi s of their affinit y for th e antagonis t a-CtxMI I (Kula k e t al , 1997 ; Kaiser e t al, 1998 ; Gu i e t al, 2003 ; Marubi o e t al, 2003). oc-CtxMII-sensitive nAChRs consis t of Ot6ß2ß3 and oc4oc6ß2ß 3 subunits . Thos e no t blocke d b y oc CtxMII contai n oc4ß 2 an d oc4oc5ß 2 subunit s (Salmi nen e t al. , 2004) . A separat e populatio n o f a ?

FIGURE 23- 1 Expressio n o f nicotini c recepto r sub units i n dopaminergi c (DAergic ) neurons . Th e pho tomicrographs sho w expressio n o f oc 3 (A ) and oc 4 (B ) nAChR subuni t mRN A within substantial nigr a (SN) and ventral tegmental are a (VTA ) at Gl 5. mRNAs for nAChR subunit s (silve r grains ) are expresse d i n bot h DAergic cell s expressin g tyrosin e hydroxylas e (TH ) mRNA (dar k cells ) an d non-DAergi c neurons . Not e the differentia l distributio n o f cc 4 mRN A withi n S N and VT A at this age.

NICOTINIC RECEPTO R REGULATIO N OF CATECHOLAMIN E 38 3

nAChRs ha s als o bee n identifie d o n DAergi c cel l bodies (Pidoplichko et al, 1997 ; Klink et al, 2001). Perinatal Period DA-containing neurons of the S N and VT A are born between gestationa l da y (G ) 1 2 an d G1 4 (Voorn et al. , 1988) , wit h nACh R bindin g appearing i n th e anläge shortly thereafter (Naeffet al. , 1992 ; Zoli et al, 1995). B y G15, severa l nACh R subuni t mRNA s ar e expressed b y DAergic neuron s includin g a3, a4, oc5 , oc7, ß2, and ß4 (Fig . 23-1, Table 23-1) . At this age, both high-affinit y [ 3H]nicotine bindin g an d oc 4 mRNA expressio n are highe r i n th e S N tha n i n th e VTA. A t late r stage s o f feta l an d postnata l develop ment, however, no differences ar e detectable betwee n nAChR expressio n i n DAergi c neuron s o f these tw o nuclei (Aza m e t al., 2002) . The postnata l declin e i n [ 3 H] nicotine binding to lower adult concentrations in the S N an d VT A parallels a decreas e i n th e expres sion o f mRNAs for oc3 , oc4 , an d ß 2 subunits . I n con trast, ot6 and ß 3 mRNAs are expressed at low amounts in feta l DAergi c neurons , wit h transcrip t level s in creasing durin g th e postnata l period . Suc h finding s suggest tha t th e type s o f nAChR s expresse d o n D A ergic neuron s chang e wit h age . Functiona l studie s support this view (Azam et al., 2005). By examinin g i n vitr o neurotransmitte r releas e from striata l slices , th e presenc e o f functiona l nAChRs o n ra t striatal DA terminals a s early as G17 has bee n show n (Aza m e t al. , 2005 ) (Tabl e 23-2) .

Maximal nicotine-stimulate d [ 3 H]DA releas e de clines b y 50 % at birth , then graduall y increases during th e postnata l perio d t o reac h adul t level s b y postnatal day (P) 21. There is a concomitant increas e in nicotine potency, as indicated by the concentratio n inducing a half maximum response (EC 50 ) at P21 being almost an order of magnitude lowe r than tha t observed i n feta l brain . Thus, during the first postnatal month ther e i s a switc h i n th e typ e o f nAChR(s) expressed on striata l DAergic terminals from thos e with low affinity fo r nicotine to those wit h high affinity . Data on the functional influence of a4-containing nAChRs on developing DAergi c neuron s are consis tent wit h thos e fro m studie s o n a mutan t mous e model i n whic h th e a 4 subuni t wa s modified to b e hypersensitive t o nicotin e an d endogenou s ligand s (Labarca e t al., 2001). Whereas fetuses of these transgenie mice hav e the same number o f midbrain DAergic neurons as wild-type controls on G14, they exhibit a dramati c declin e i n D A cel l numbe r b y G1 6 an d G18. Thus, fetal DAergi c neurons in these transgenic lines degenerate followin g hyperactivation of the mu tant <x4* nAChRs by endogenous ACh o r choline. Adolescence Adolescence, define d a s P28 t o P4 2 i n rodents , i s a time a t whic h youth s star t t o execut e higher-leve l cognitive function s an d establis h independenc e (Spear, 2000) . Novelt y seekin g an d impulsivit y are hallmark s o f thi s developmenta l period , a s i s

TABLE 23- 1 Nicotini c acetylcholin e recepto r subuni t mRNAs and bindin g sites in th e developing substantial nigra and ventral tegmental area Prenatal

oc3 oc4 06 oc6 al ß2 ß3 [*H]NIC [125I]a-BgTK

++++ -H-+/I 1 1 1 * -H-/+ + +++ +

++ÂH-H-* -

Early Postnatal

Late Postnatal Adolescence

+++

-H+ + + ++

+/++ +++ +

Adult +

++ +

++ + ++ +/+ ++ +/+

++ + ++ +

++ ++ + ++

+ +

+ + + + + + + +

a-BgTX, a-bungarotoxin; NIC, nicotine ; 4-H-, concentrations a t least threefold over those i n adults; -H-, concentrations about twofol d ove r adult; +, concentrations similar to adult; -/+, concentrations below adult; -, concentrations at background. *Higher in substantia nigra than in ventral tegmental area on Gl 5.

384 NICOTINE-AFFECTE D DEVELOPMEN T TABLE 23- 2 Nicotine-induce d [ 5 H] dopamin e re lease fro m striata l slice s durin g th e firs t 3 postnata l weeks Age

G17-G18

PI P4 P7 P14 P21

EC50 fmM ) 92*** (5.7-15) 4.0 (0.4-42) 3.09 (1.5-6.6) 5.78** (2.0-17) 2.48 (1.3-4.8) 1.65 (0.8-3.3)

Maximum Release (%)

4.8410.55 2.50 ±0.58**

<£ Qo

F*

2.59 ±0.30** 3.15±0.56** 3.76 ±0.48* 6.19 + 0.86

EC50 values (95% confidence intervals) and maximum release (mean± SEM) ar e fro m thre e t o fou r independen t experiments . G , gesta tional day; P, postnatal day. */> < 0.05, **p < 0.01, ***p < 0.001 values significantly different o n P21.

initiation o f addictive behaviors. There i s increasing evidence fro m bot h roden t an d primat e studie s tha t DAergic systems continue t o mature throughou t thi s developmental period . DAergi c innervatio n o f pre frontal corte x increase s unti l youn g adulthoo d (Benes e t al, 2000 ; Lamb e e t al, 2000) , with som e evidence o f a n "overshoot " a t pubert y an d subse quent prunin g o f nonspecifi c innervatio n (Lewis , 1997). Bot h D l an d D 2 recepto r level s in prefrontal cortex reac h pea k concentration s i n youn g mal e adult rats , the n declin e substantiall y (Anderse n e t al., 2000 ; Taraz i an d Baldessarini , 2000). Overpro duction o f Dl an d D 2 receptor s als o occur s i n th e caudate nucleu s an d putamen , wit h pea k conten t reached a t puberty followed by substantial declines . A gende r differenc e ha s bee n note d i n thi s effect , with striata l recepto r prunin g occurrin g onl y i n males an d no t i n female s (Anderse n an d Teicher , 2000). Whereas th e majorit y o f nACh R mRNA s i n th e SN an d VT A reach matur e expressio n prior t o ado lescence, transcrip t amount s fo r oc5 , Ot6 , an d ß 4 subunits continu e t o decreas e towar d lowe r adul t concentrations (Tabl e 23-1) . Nicotine-stimulate d DA releas e i n th e ventra l striatum , whic h include s the nucleu s accumbens , i s also highe r durin g early adolescence (P30 ) tha n durin g lat e adolescenc e (P40) o r adulthoo d (Fig . 23-2) . Thi s transien t

FIGURE 23- 2 Releas e of dopamine (DA ) from striatal slices. Thes e dose-respons e curve s depic t nicotine stimulated [ 3 H]DA release fro m dorsa l (A ) and ventral (B ) striata l slice s fro m adolescen t mal e rat s o n P30 an d P4 0 an d fro m adults . Maximal release i s significantly highe r fro m ventra l striatum at P30 than a t P40 or in adults . *p < 0.05, Dunnett's post-hoc analysis with adult as control.

increase i n nicotin e efficac y i s no t observe d i n th e caudoputamen. Suc h finding s suggest a n enhance d sensitivity o f motivationa l circuitr y to th e activatin g effects o f nicotin e durin g earl y adolescence . Al though a recen t microdialysi s study suggest s tha t a similar enhance d effec t o f nicotine o n th e releas e of DA fro m th e nucleu s accumben s i s no t detectabl e in viv o (Badanic h an d Kirstein , 2004), othe r neuro chemical finding s ar e consisten t wit h evidenc e tha t

NICOTINIC RECEPTO R REGULATIO N OF CATECHOLAMIN E 38 5

early adolescenc e i s a perio d o f uniqu e sensitivit y to the rewardin g effects o f nicotine (Vastol a et al., 2002; Laviola e t al, 2003 ; Belluzzi et al, 2004, 2005 ; Rezvani and Levin , 2004).

TABLE 23. 3 Nicotin e EC 50 value s fo r stimulatio n o f [ 3 H] norephinephrine release from brai n slices in th e neonate and adul t Nicotine EC 50 (|lM) Brain region

NOREPINEPHRINE NEURON S Adult

The noradrenergi c system projects extensively throughout the central nervous system (CNS) and modulates a variety of cognitive and viscera l functions. NE projec tions stem fro m nucle i in the pons an d medulla , eac h with a rol e i n regulatin g response s t o sensor y inpu t and t o environmental an d viscera l Stressors. Nicotinic receptors ar e widely expressed throughou t thes e cate cholamine nuclei . Locus Coeruleus A dorsa l pontin e tegmenta l nucleus , th e locu s coeruleus (LC) , i s the CN S structur e wit h th e mos t widespread projection s (Loughli n e t al. , 1986) . I t gives ris e to a networ k o f fiber s that exten d through out the neuraxis . The L C provide s th e exclusiv e N E input t o th e majorit y o f brai n structures , includin g the neocortex , hippocampus , an d cerebellum , an d plays a critical role in attention an d arousal. A wealth o f nAChR subunit s ar e expresse d i n th e adult L C (Len a e t al, 1999) . I n sit u hybridization studies sho w hig h expressio n o f several nACh R sub unit mRNAs , includin g a6, ß2 , an d ß3 , wit h lowe r levels o f expressio n o f oc4 , oc5 , an d oc 7 (Gallard o et al. , 2005) . Consistent wit h th e lo w expressio n o f oc4 and ot 7 subunits , ver y low amount s o f [ 3 H]nicotine an d [ 125I]oc-bungarotoxin binding , respectively , are present in the adul t LC. Bot h oc 3 and ß 4 mRNA s are topographicall y distributed , wit h muc h highe r expression i n dorsa l tha n i n ventra l cells . Thes e findings ar e consisten t wit h a single-cell reverse transcriptase-polymerase chai n reactio n stud y i n which ot 3 and ß 4 mRNAs were found to be expressed in les s tha n hal f o f al l L C cell s teste d (Len a e t al. , 1999). LC efferen t projection s are organize d topographi cally, with dorsal cells projecting to more rostral brain regions (Loughli n e t al, 1986) . Th e topographi c dis tribution o f nAChR mRNA s in LC cell s suggests that different termina l field s expres s nAChR s wit h differ -

Cortex Cerebellum Hippoeampal formation Hypothalamus

neonate

adult

1.4 9.1

14.1 10.3 39 75

26 20

ing pharmacologica l properties . Thi s issu e ha s no t been examine d systematically . A preliminary comparison o f nicotine potenc y t o stimulate [ 3 H]NE release in slic e preparation s fro m fou r brai n region s suggests that ther e ma y b e pharmacologica l difference s i n nAChR regulatio n (Tabl e 23-3) . Immunoreactivit y for oc 6 an d ß 3 subunit s ha s bee n show n i n th e hip pocampal formation , leadin g t o th e suggestio n tha t the nACh R o n L C terminal s i n thi s structure consis t of a n oc6ß2ß 3 subuni t combinatio n (Len a e t al. , 1999). O n th e othe r hand , nAChR s regulat e hip pocampal [ 3 H]NE release b y both direct an d indirec t mechanisms, wit h nicotine-stimulate d GAB A releas e leading t o a n anomalou s excitatio n o f LC terminal s (Leslie e t al. , 2002) . I n organotypi c slic e culture s of parieta l cortex , i n contrast , nicotine-stimulate d [ 3 H]NE releas e i s mediate d entirel y b y nAChR s lo cated o n LC terminals (dat a not shown) . Medullary Nuclei Medullary catecholamin e neuron s cell s ar e foun d within th e nucleu s tractu s solitariu s (NTS) an d th e ventrolateral medulla (VLM ) an d consis t of both NE and epinephrine-containin g cells . Th e NT S i s th e initial site of integration fo r central an d peripheral au tonomie systems , with afférent s projectin g from mul tiple visceral modalities (Andrese n and Kunze, 1994) . The NT S send s reciproca l projection s to numerou s brain ste m nucle i tha t mediat e periphera l autonomi e outflow an d t o th e hypothalamus , wher e i t serve s a critical rol e i n regulatin g plasm a glucos e vi a activation o f the hypothalamic-pituitary-adrena l (HPA ) axis and food intake (Ritter et al, 2001, 2003). The cauda l and rostra l VLM generall y hav e opposin g effect s o n autonomie output , suc h a s centra l flui d an d cardio vascular homeostasis (Heslop et al, 2004). The NEer gic neurons of the rostra l VLM als o provide the major

386 NICOTINE-AFFECTE D DEVELOPMENT inhibitory input t o the LC, thus providing coordinate regulation o f cognitiv e an d autonomi e response s t o external stimul i (Aston-Jones et al., 1992) . Data fro m i n situ hybridization studies provide evidence fo r expression of nAChRs o n adul t medullar y catecholamine neurons (Gallard o e t al., 2005). Unlik e

the LC , wher e mos t neuron s exhibi t a rich complex ity of subunit mRNAs, fewe r nACh R subunits ar e expressed withi n thes e neuron s (dat a no t shown) . I n contrast to the LC , ther e i s no expression of oc6 and ß 3 subunits i n th e NTS . Th e predominan t subunit s expressed i n catecholaminergi c neuron s o f this nucleu s

FIGURE 23- 3 Quantitativ e analysi s of nicotine bindin g site and nACh R sub unit mRN A expressio n i n the locu s coemleus a s a function o f age. A . Spe cific [ ?H]nicotine bindin g (fmol/m g tissue) . B . oc 4 (triangles ) an d ß 2 (squares) mRNA expression. C. a5 mRNA expression . D. oc 6 (triangles) and ß3 (squares) mRNA expression. E. Ot 3 mRNA expression, dorsal (d; triangles) and ventra l (v; squares) subdivisions. F. ß4 in dorsal (d; triangles) and ventral (v; squares) subdivisions. The da y of birth wa s G22.

NICOTINIC RECEPTO R REGULATIO N O F CATECHOLAMIN E 38 7 are Ot3 , oc5, and ß2/4 . I n th e VLM , ther e i s expression of ß 3 mRN A i n scattere d cells , wit h a mor e wide spread expressio n o f oc 6 mRN A i n -50 % o f cate cholamine neurons. I n contrast to both LC and NTS , almost al l cell s o f the VL M expres s both a? an d ß 2 subunit mRNAs , wit h 30%-50 % als o expressin g Ot3-a5. Suc h finding s ar e consisten t wit h functional studies i n whic h nicotin e microinjection s int o brain stem nuclei have been reported t o increase NE releas e in th e hypothalamu s (F u e t al., 1997 ) an d t o regulate both HP A axi s (F u e t al. , 1997 ) an d cardiovascula r functions (Ferreir a et al., 2000). Ontogeny Locus Coeruleus Neurons o f the L C ar e bor n betwee n G1 0 an d G1 3 in rat , and first exhibit the biosyntheti c enzym e tyrosine hydroxylas e (TH ) o n G1 2 (e.g. , Lande r an d Bloom, 1974 ; Spech t et al, 1981 ; Koni g et al, 1988) . These neuron s rapidl y extend axon s int o immatur e brain structures , wher e the y ar e believe d t o serv e a n

important morphogeneti c rol e (Rui z e t al. , 1997 ; Naqui e t al, 1999) . L C cell s are also highly sensitive to earl y sensory inpu t (Nakamur a e t al. , 1987 ; Mar shall e t al. , 1991 ) an d regulat e function s critica l t o the surviva l o f the neonat e suc h a s olfactory imprinting (Sullivan , 2003). A numbe r o f studies sho w tha t the hyper-reactivit y of the L C t o innocuou s sensor y stimuli decrease s afte r birth , an d tha t thes e change s are accompanie d b y alteration s i n th e propertie s o f adrenoceptors expresse d b y LC neuron s (Kimur a an d Nakamura, 1987 ; William s an d Marshall , 1987 ; Nakamura e t al. , 1988) . Interestingly , littl e attentio n has been give n t o date t o possible change s i n th e ex pression of other recepto r types . Anatomical an d functiona l studie s demonstrat e major change s i n th e expressio n o f nAChR subunit s in th e L C ove r the cours e o f development (Gallard o et al. , 2005) . Feta l L C neuron s exhibi t high-affinit y [ 3 H]nicotine bindin g tha t decrease s t o lo w adul t amounts durin g th e earl y postnata l perio d (Figs . 23-3, 23-4 , an d 23-5) . Consisten t wit h earlie r findings tha t high-affinit y [ 3 H]nicotine binding site s ar e comprised o f a4 and ß 2 subunit s (Zoli e t al. , 1998) ,

Age FIGURE 23- 4 Stimulatio n o f norepinephrin e (NE ) i n developin g forebrain. Developmental change s i n nicotine-stimulate d [ 3 H]NE fro m slice s o f hip pocampus (A) , cerebellum (B) , parieta l corte x (C) , an d hypothalamu s (D) . Significant difference s ( p < 0.01, Dunnett's test) relativ e to the adul t are signi fied by asterisks.

NICOTINE-AFFECTED DEVELOPMEN T

FIGURE 23- 5 Propertie s o f nAChR s tha t stimulat e [ 3 H]norepinephrine (NE ) releas e fro m locu s coeruleus terminals i n developin g parieta l cortex . A. Nicotine concentration-respons e curve s on P I (triangles), P 4 (diamonds) , an d P6 0 (adult ; circles). Nico tine potenc y an d maxima l releas e value s diffe r wit h age. B . DHß E inhibitio n o f nicotine-induce d [ 5 H]NE release from cortica l slice s on P 4 (diamonds) and P6 0 (adult; squares). Antagonist inhibition o n P 4 is biphasic, indicatin g that nicotine act s a t two differ ent type s of nAChR. I n contrast , th e antagonis t inhi bition curv e is monophasic i n adults. high amount s o f expression of ot4 and ß 2 mRNA s occurs i n th e L C durin g th e perinata l period , alon g with ot3 , a5 , an d ß 4 mRNA s (Fig . 23-3 ) (Gallard o et al , 2005) . Expressio n o f a4 an d a 5 mRNA s de clines during the first postnatal week , in parallel with the los s of high-affinity [ 3H]nicotine binding. Wherea s perinatal expressio n o f oc 3 an d ß 4 subuni t mRNA s is

homogeneously distribute d throughout th e entire nu cleus, there i s an emergenc e o f a topographic expres sion durin g th e earl y postnata l period , wit h highe r levels of expression in the dorsa l LC. At the sam e time, the phenotyp e o f th e matur e L C nACh R emerges , with increasin g postnata l expressio n o f ot 6 an d ß 3 mRNAs. Fetal L C cell s dissecte d fro m dorsa l rhomben cephalon o n G14 and culture d fo r 4 days have bee n shown t o tak e u p an d releas e [ 3 H]NE i n respons e to appropriat e physiologica l stimul i (Raymo n an d Leslie, 1994) . Nicotin e stimulate s [ 3 H]NE releas e from feta l culture d L C cell s vi a activatio n o f a re ceptor wit h hig h affinit y fo r nicotine an d propertie s consistent wit h tha t o f an a4ß2 * nACh R (Gallard o and Leslie , 1998) . The EC 50 value for nicotine stim ulation o f thi s nACh R wa s foun d t o b e < 1 jilM, which i s withi n th e rang e o f bloo d nicotin e level s found i n moderat e t o heav y smoker s (Benowitz , 1990). In orde r to evaluat e possibl e postnata l change s i n the propertie s o f nAChR s tha t regulat e N E release , similar studie s wit h slice s take n fro m L C termina l fields were conducted (Fig . 23-4) (Lesli e et al, 2002). Using thi s approach , a remarkabl e regiona l hetero geneity i n nACh R regulatio n o f transmitte r releas e from L C terminal s durin g th e postnata l perio d wa s shown. I n th e hippocampus , a late-maturin g struc ture, ther e i s n o majo r developmenta l switc h i n nAChR regulatio n o f NE releas e (Lesli e et al, 2002). There is a monotonie increas e in nicotine-stimulate d NE releas e fro m hippocampa l slice s durin g th e first postnatal wee k to reac h adul t concentration s b y P10. There i s als o n o chang e i n th e pharmacologica l profile o f hippocampa l nAChRs , wit h majo r contri butions o f both direc t an d indirec t pathway s at eac h postnatal stag e (Lesli e e t al, 2002) . I n contrast , an other structur e tha t mature s largel y durin g th e post natal period i n rat, the cerebellum, exhibit s substantial postnatal change s i n nAChR regulation of NE releas e (O'Leary and Leslie, 2003) . During th e first postnatal week, nicotin e i s highly efficacious , releasin g almos t 40% o f cerebella r N E store s a t a maxima l effectiv e concentration. Ther e i s a gradual decline i n nACh R efficacy durin g the nex t month to reach lo w adult levels. Pharmacologica l analysi s has show n tha t neona tal an d adul t nAChR s o n cerebella r L C terminal s have differen t subuni t compositions , wit h decreasin g involvement o f ß 2 subunit s i n th e adul t recepto r (O'Leary and Leslie , 2003).

NICOTINIC RECEPTO R REGULATIO N O F CATECHOLAMIN E 3 8 In parieta l cortex , ther e i s increase d maxima l nicotine-induced releas e in the neonatal perio d with a subsequent declin e t o lowe r adul t concentration s b y P7 (Fig. 23-4C) (Gallardo etal., 2005). More detaile d pharmacological analysi s ha s show n tha t ther e i s a switch in the properties of nAChRs on cortical LC ter minals durin g th e firs t postnata l wee k (Fig . 23-5 ) (Gallardo e t al , 2005) . Wherea s a t birt h nicotin e stimulates cortical NE releas e with high affinity , ther e is a n orde r o f magnitude decreas e i n potenc y b y P4 . At this age, nAChRs o n cortica l LC terminal s are in a period o f transitio n betwee n th e neonata l recepto r that ha s hig h affinit y fo r th e Ot4ß2-selectiv e antago nist, DHßE, and the adult low-affinity form . Hypothalamus wa s examined because , unlik e th e other brai n region s studied , i t ha s substantia l inpu t from th e medullar y catecholamin e nucle i a s well a s the LC . Thi s regio n als o shows enhanced efficac y o f nicotine t o releas e [ 3 H]NE during th e first postnatal week (Fig . 3-4D) . Subsequently , ther e ar e comple x changes i n nicotine-induce d transmitte r release , with adult profile s no t reache d unti l adolescenc e (P30) . Pharmacological comparison s o f nACh R propertie s at P 7 and adul t hav e show n that , simila r t o the cere bellum an d neocortex, the neonata l recepto r i n hypothalamus ha s distinc t propertie s fro m tha t o f adult . The neonata l for m o f th e recepto r i n al l thre e ter minal field s appear s t o hav e a predominanc e o f ß 2 subunits compare d t o adul t amounts , however , th e neonatal recepto r i s no t identica l acros s regions . I n the hypothalamu s and cerebellum , unlik e neocortex , the neonata l recepto r doe s no t hav e hig h affinit y fo r nicotine (Tabl e 23-3) . Althoug h hypothalamu s re ceives mixed inputs from NEergi c neurons o f both th e pons and medulla , lesio n studies in adult show that hypothalamic nAChR s selectivel y regulat e N E releas e from L C terminal s (Sperlag h e t al. , 1998) . Similarly , nAChRs are exclusively localized on hypothalamic LC terminals i n th e neonate , sinc e nicotine-stimulate d pH]NE release is abolished by the LC-selective neurotoxin DSP-4 (O'Leary and Leslie , 2005). These finding s ar e consisten t wit h a n importan t and changin g rol e of nAChRs i n regulating release of NE fro m L C terminal s throughou t development . Unique pattern s o f nicotinic regulatio n o f individual LC termina l field s indicat e tha t th e specifi c rol e o f nAChRs may vary by brain region. Despite thes e individual differences , ther e i s a consistent tren d i n mos t brain region s fo r enhance d nACh R efficac y durin g the perinata l perio d whe n L C i s hyperreactiv e t o

external stimuli. Thus cholinergic input s may provide an importan t mechanis m fo r regulatin g L C respon siveness during this critical neonatal period . Medullary Nuclei The VLM , whic h provides the majo r inhibitor y input to the L C (Aston-Jone s e t al, 1992) , exhibit s coordi nated developmenta l regulatio n o f nACh R subuni t expression. Ther e i s high perinata l expressio n o f oc 4 and oc 5 mRNAs , with a slow decline t o adult concentrations after birth . There is also a parallel increase i n oc6 and ß 3 mRNA expression during the postnata l pe riod, indicatin g tha t som e cell s o f th e VL M ma y switch their nAChR phenotype . In contrast to the L C and VLM , th e NT S exhibit s less dynami c alteration s in nACh R subuni t expressio n throughou t develop ment. Other than a brief up-regulation o f oc4 mRN A in feta l NTS , n o transien t nACh R expressio n ha s been observed. Rather, there i s a slow maturation dur ing the postnata l perio d to adult concentrations . The absenc e o f nAChRs o n medullar y hypothalamic terminal s precludes th e typ e o f functional analysis o f developmenta l change s i n th e propertie s o f nAChRs describe d for the L C (Gallard o e t al, 2005). More comple x i n vivo studies are required to evaluate this issue. Whereas nicotin e administratio n in vivo activates the HP A axis via stimulation o f nAChRs withi n the medull a (F u e t al. , 1997) , n o suc h stimulatio n is evident durin g earl y adolescenc e (dat a no t shown) . Such finding s sugges t tha t ther e i s a lat e postnata l maturation o f nACh R regulatio n o f HP A function , consistent wit h the lat e developmenta l maturatio n of nAChR expression withi n th e NTS .

DEVELOPMENTAL EXPOSURE

Gestation Although ther e ar e functionally active nAChRs o n fetal D A terminals , the y hav e relativel y lo w affinit y fo r nicotine (~10|iM ) (Aza m e t al. , 2005) . Thus , give n the lo w concentratio n o f nicotin e i n bloo d (-500 nM), one would expect there to be minimal occupancy o f these sites . I n contrast , nAChR s o n feta l LC neuron s an d neonata l cortica l terminal s expres s nAChRs wit h high affinit y fo r nicotine tha t woul d b e activated b y blood concentration s o f nicotine i n pregnant femal e smokers . Suc h finding s predic t tha t

390 NICOTINE-AFFECTE D DEVELOPMEN T prenatal exposure to nicotine has a more harmfu l effect o n developin g NEergi c system s than o n DAer gic systems . Althoug h a numbe r o f studie s sugges t that gestationa l nicotin e treatmen t doe s affec t th e development o f the striata l DAergic pathwa y (Olif f and Gallardo , 1999) , chroni c prenata l infusio n o f nicotine i n moderat e dose s mor e profoundl y affect s postnatal ontogen y o f NEergi c system s (Slotkin , 1998). Gestational treatmen t wit h nicotin e infusio n (3 mg/kg/day) induces a long-term chang e i n the effi cacy of nAChRs on cortica l LC terminal s (Fig. 23-6 ) (Gallardo e t al. , 2005) . Maximal nicotine-stimulate d [ 3 H]NE releas e i s greatl y increase d durin g th e firs t postnatal month and , although reduced , i s still signif icant in the adult. This does not result from a n overall increase i n th e excitabilit y o f L C terminals , sinc e

potassium-stimulated transmitte r releas e i s un changed b y gestationa l nicotine . No r doe s i t resul t from a delayed transition in the propertie s of terminal nAChRs; bot h anatomica l an d functiona l studie s have demonstrate d tha t ther e i s a norma l postnata l switch i n nACh R phenotype o n L C cell s an d corti cal terminal s i n gestationall y treate d animals . On e change i n nACh R properties , however , i s a muc h greater acut e nicotine-induce d recepto r desensitiza tion, tha t i s most eviden t a t lo w drug concentration s (data no t shown) . This finding may explain an appar ent discrepancy between i n vitro data (Gallardo et al., 2005) an d thos e o f a n earlie r i n viv o stud y (Seidle r et al, 1992 ) o f N E turnover , whic h foun d prenata l nicotine treatmen t t o induc e hypoactivit y i n th e re sponse o f N E system s t o acut e postnata l nicotin e treatment.

FIGURE 23- 6 Effec t o f gestationa l treatmen t wit h o f nicotine - (triangles ) o r saline - (squares ) treate d nicotine (3. 0 mg/kg/day) o n subsequen t nicotine - dam s o n P 4 (A) , P1 4 (B) , P3 0 (C) , o r P6 0 (adult ) induced [ 3 H]norepinephrine (NE ) releas e fro m (D) . Significan t difference s relativ e t o th e saline locus coeruleu s terminal s i n parieta l cortex . Nico - treate d grou p ar e identifie d b y **(£<0.01 ) an d tine concentration-respons e curve s ar e fro m pup s * * * ( / > < 0.001).

NICOTINIC RECEPTO R REGULATIO N O F CATECHOLAMIN E 39 1 Adolescence

Physiological Implications

It is well established that catecholamine systems, particularly DA , ar e no t full y mature d b y adolescenc e (Spear, 2000 ; Chamber s e t al , 2003) . Functiona l studies sho w tha t nACh R regulatio n o f mesolimbi c DAergic an d medullar y NEergic system s has no t yet fully mature d i n adolescen t rat s (Aza m e t al. , 2005). Such finding s sugges t tha t brai n catecholamin e systems ar e vulnerable to nicotin e exposur e durin g adolescence. A growin g bod y o f evidenc e support s thi s view. Exposur e t o dru g fo r relativel y shor t period s during adolescenc e i n roden t induce s permanen t changes i n behavior s mediate d b y forebrai n D A systems, includin g locomotio n an d rewar d (Slotkin , 2002; Adrian i e t al. , 2003 ; Farada y et al, 2003 ; Bel luzzi e t al. , 2004) . Simila r change s ar e no t observe d following equivalen t exposure in adult . Neurochemical studie s confir m tha t chroni c nicotin e treatmen t during adolescenc e produce s permanen t alteration s in D A an d N E system s i n ra t brai n (Traut h e t al. , 2001). Thus , catecholamin e system s withi n adoles cent brai n may be particularly vulnerable to the neu roteratogenic effects o f nicotine.

nAChRs are expressed on immature DA and NE neu rons and functio n to regulate transmitte r release in fetal brain . Suc h finding s sugges t a critica l interaction of cholinergic an d catecholamin e brai n system s during the earlies t phase o f brain development . Ther e is a lat e postnata l maturatio n o f mos t cholinergi c sys tems (Hohman n and Ebner , 1985 ; Dinopoulo s e t al., 1989; Kis s and Patel , 1992) , however, a brief perinatal phase o f cholinergi c neurona l expressio n ha s bee n described i n severa l brain regions, includin g neocor tex and cerebellu m (Goul d an d Butcher , 1987 ; Dor i and Parnavelas, 1989) . Thus, transient cholinergic innervation ma y provid e critical regulatio n o f developing catecholamine system s via activation of nAChRs. Although ther e ar e regiona l difference s i n th e properties o f nAChR s tha t regulat e termina l cate cholamine releas e durin g th e perinata l period , ther e are som e commonalities . Mos t feta l catecholaminer gic cells , bot h DAergi c an d NEergic , expres s hig h amounts o f [3H]nicotine binding and a4/ß2 mRNAs. Many cell s als o expres s th e oc 5 structura l subunit , which combine s wit h othe r subunit s t o alte r agonis t affinity, calciu m permeability , an d agonis t desensiti zation (Wan g et al, 1996 ; Gerzanic h et al, 1998) . Although quit e rar e in adul t brain , nAChR s comprise d of oe4oc5ß 2 subunit s ar e highl y expresse d durin g th e perinatal perio d an d ma y serv e uniqu e functiona l roles (Conro y an d Berg , 1998) . Afte r birth , ther e i s a declin e i n oc 4 an d oe 5 subuni t expressio n an d o f [3H]nicotine binding. During thi s period , ther e i s increased expressio n of oe6 and ß 3 subunits . These subunits do not appear to contribute to fetal nAChR s bu t are importan t constituent s o f nAChRs i n adul t cate cholamine termina l field s (Len a e t al. , 1999) . Concomitant change s i n th e pharmacologica l propertie s of nAChR s tha t regulat e termina l catecholamin e re lease ar e observed . Thus , i n bot h N E an d D A neu rons ther e i s a postnata l switc h i n th e propertie s o f terminal nAChR s t o th e matur e phenotype . Thes e findings suggest that nAChR s serv e critical regulatory roles fo r catecholamin e pathway s at al l stage s of on togeny bu t tha t th e natur e o f the functiona l interac tion changes with age.

PHYSIOLOGICAL AND CLINICAL IMPLICATIONS

Methodological Issue s Whereas earl y i n viv o studie s hav e show n lastin g changes induce d b y gestationa l nicotin e treatmen t (Slotkin, 1998 ; Erns t e t al , 2001) , th e underlyin g mechanisms ar e unclear . A systematic i n vitr o analysis wa s performe d t o clarif y mechanism s underlyin g nAChR regulatio n o f developin g catecholaminergi c neurons. Accordingly, it was demonstrated tha t nAChR are ke y regulator s o f developin g catecholaminergi c neurons. Interpretation o f these data mus t be qualifie d because nACh R pharmacolog y i s extremely complex, and subuni t composition o f native receptors is difficult to asses s (Cordero-Erausquin e t al. , 2000 ; Whiteaker et al. , 2000) . Furthermore , severa l type s o f nACh R may b e expresse d withi n a singl e cell , wit h differen tial localizatio n o f receptor subtype s in termina l an d somatodendritic region s (Champtiau x e t al , 2003) . Given thes e caveats, it is difficult t o draw conclusions about the natur e of nAChR regulatio n of somatodendritic area s tha t hav e bee n show n t o hav e critica l functional roles .

Clinical Implication s Fetal L C neuron s an d thei r cortica l terminal s hav e nAChRs wit h sufficiently hig h affinit y fo r nicotine t o

392 NICOTINE-AFFECTE D DEVELOPMEN T be activate d b y materna l smoking . Chroni c gesta tional exposure to nicotine, at concentrations approximating bloo d concentration s i n smokers , induce s long-term change s i n th e propertie s o f nAChR s o n cortical L C terminal s suc h tha t thei r efficac y i s in creased, but the y are more readil y desensitized by agonist exposure . Given th e critica l role o f central N E in modulatin g cognitiv e an d viscera l function s an d the apparen t functiona l importanc e o f nAChR s i n regulating NEergic system s throughout development , such findings have substantial implications for understanding the etiolog y of many of the advers e effect s o f maternal smoking . In particular , altered sensitivit y of central NEergic systems to nAChR stimulation and to perinatal hypoxia has been implicate d i n the etiolog y of sudde n infan t deat h syndrom e (Slotki n e t al. , 1995). Give n th e critica l role o f the L C i n cognitio n and attentio n a t al l stage s o f developmen t (Aston Jones et al, 1999 ; Moricea u an d Sullivan , 2004), i t is likely that disrupte d developmen t o f forebrain NEer gic system s may also contribut e t o the cognitiv e dys function an d ADHD that are associated with maternal smoking. DA system s ar e les s severel y disrupted b y gesta tional nicotin e exposure , however , ther e i s evidenc e for long-ter m alteration s i n forebrai n DAergi c func tion (Slotkin , 1998 ; Muneok a e t al, 1999) . Striata l DAergic terminal s expres s a lower-affinit y nACh R during the feta l period , which would no t be activated by blood-born e nicotin e i n th e pregnan t femal e (Azam e t al., 2005) . Som e type s of nAChR, however , are desensitize d b y nicotin e a t fa r lowe r concentra tions than thos e require d to activate the recepto r (L u et al , 1999 ; Wooltorto n e t al, 2003) . Furthermore , somatodendritic nAChRs , whic h ar e criticall y implicated i n the effect s o f systemic nicotine on D A release (Champtiaux e t al., 2003) , have no t bee n evaluated . Thus, gestationa l nicotin e exposur e ma y directl y in fluence th e ontogen y of DAergic system s via modula tion of nAChR function. Adolescence i s also a critica l tim e fo r maturatio n of forebrai n limbi c system s an d thei r afferen t cate cholamine input s (Spear , 2000 ; Chamber s e t al. , 2003). DAergi c systems , i n particular , are stil l quit e immature an d termina l nAChR s i n ventra l striatu m are more highly activated by nicotine a t this age tha n in the adult . Biochemical and behavioral studies indicate tha t bot h acut e an d chroni c nicotin e hav e unique effect s o n catecholamin e systems during adolescence tha t ar e no t observe d i n adul t (Slotkin ,

2002). Thus, early smoking i n adolescence may rein force subsequen t addiction , a s has been suggeste d b y epidemiological studie s (Kande l an d Logan , 1984 ; Kandel e t al. , 1992) , b y modifyin g forebrai n cate cholamine development . Tobacco smok e contain s thousand s o f chemicals , many o f which disrup t development. Thi s has le d t o the recommendatio n tha t nicotin e replacemen t ther apy (NRT ) b e use d extensivel y a s a n alternativ e t o smoking by pregnant women (Dempse y and Benowitz, 2001). Some studies , however, have questioned th e efficacy of NRT a s a smoking cessation ai d for pregnant women an d adolescent s (Hur t e t al, 2000 ; Wisbor g et al , 2000) . Furthermore , a growin g bod y o f evi dence show s (a ) tha t nAChR s pla y a critica l rol e i n modulating neura l developmen t an d (b ) tha t expo sure o f immatur e brai n t o nicotin e ca n disrup t nor mal cholinergi c regulatio n o f ontogenetic processes . Thus, the safet y of NRT as a therapeutic regimen dur ing pregnancy and adolescence need s to be evaluated more thoroughly . Abbreviations ACh acetylcholin e ADHD attentio n defici t hyperactivit y disorder CNS centra l nervous syste m DA dopamin e EC 5o concentratio n effectin g a half maximal response G gestationa l day HPA hypothalamic-adrenal-pituitar y LC locu s coeruleus nAChR nicotini c acetylcholine recepto r NE norepinephrin e NRT nicotin e replacemen t therap y NTS nucleu s tractu s solitarius P postnata l day SN substantia l nigra TH tyrosin e hydroxylase VLM ventrolatera l medull a VTA ventra l tegmental are a

ACKNOWLEDGMENTS Thi s wor k wa s supported by a grant from th e Nationa l Institute of Drug Abuse and b y

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Index

apoptosis protease-activating facto r (APAF) , 74, 78, 93-98, 268 neural crest, 286-290 tumor necrosi s factor (TNF) , 95, 97, 98, 167 , 169 , 171 , 268, 269 , 272 , 300 Arginine vasopressin (AVP), 156 , 161, 162 , 171 Astrocyte. See Glia Astrotactin, 31 Attention, 129 , 130 , 134 , 319 , 320 Attention deficit hyperactivit y disorder (ADHD), 129 , 130 , 134,319,367,381,392 Autophagy, 74, 92, 246 Axon bundling, fasciculation , 51 , 55 effect o f ethanol, 233, 23 4 fate, specification , 46, 48, 23 4 growth, 47, 49, 50 , 58, 111, 233, 235, 238 growth con e chemotaxis, directed growth, 50, 54 filopodia, 28, 45,48-50,58, 236, 237 lamellipodia, 28, 45, 48, 49, 236, 237 motility, 48, 49 structure, 48, 347 guidance, 50-56,233,235 , 354 pruning, 60, 233, 384

Academic performance, 13 3 mathematics, 133 , 134 Acetylcholine (ACh), 155 , 300, 346, 347 , 352 , 367, 36 8 acetylcholinesterase, 347, 351 , 369 cholinergic neurotransmission , 341-354 receptors. Se e Receptor s Acquired immunodeficienc y syndrom e (AIDS ) 370-371 . See also Human immunodeficienc y virus (HIV) Actin. See Cytoskeleton Activity-dependent neurotrophic protei n (ADNP) , 303 Activity-dependent neurotrophic facto r (ADNF), 301,303 Adolescence, 126 , 131 , 134, 135 , 316, 341 , 350, 352 Adrenalectomy (ADX), 159-162 , 169 Adrenal gland, 153-155 , 159 , 160,281,28 5 Adrenocorticotrophic hormone (ACTH) , 156 , 158 , 160-165, 168 , 169 , 17 1 Alcohol dehydrogenase (ADH) , 106, 288-290, 300 Alfred E . Newman, 4- 6 Anxiety. See Mood disorders Apoptosis (programmed cel l death), 73 , 73, 76-79, 81, 85, 92-99, 163 , 171 , 206, 207, 248, 253-255 , 267-274, 349, 366 , 368 , 369 . See also DNA , fragmentation apoptosis inducing factor (AIF) , 95-98, 268-271

399

400 INDE X Basal forebrain, 17 Basal ganglia (including caudate nucleus, putamen, and striatum), 9, 17-21, 145 , 148-150, 210, 346, 347 , 350, 382-384 Basement membrane, 32 bel, 74, 79, 84, 93,96-98 bax isoform, 74, 94-97, 255 bcl-2 isoform, 74, 85, 93, 97, 98, 249, 253, 255, 257 bcl-2 homology (BH), 74, 93, 94 Behavior cognition. See Cognitio n conduct disorder, 134-136, 320 aggression, 320 externalizing, 320 oppositional, 320 delinquency, 13 5 executive function, 130-132 , 134 impulsivity, 319 motor, 128 , 319, 321 sensory, 128 , 129 social, 134 emotional functioning, 134 Bergmann glia. See Cerebellu m Brain-derived neurotrophic factor. See Neurotrophins Bromodeoxyuridine (BrdU), 15 , 19 , 78, 206-211 Cadherin. Se e Cell adhesion molecul e Caenorhabditis elegans, 74, 92-94 Cajal-Retzius neuron, 50 , 57, 60, 91, 222 Calcium, 208 , 209, 222, 238, 239, 288, 289 , 301 , 302, 344-346, 353, 366-368 Cancer cell line, 300 B104 neuroblastoma cell, 188-190, 192 C6 astrocytom a cell, 188 , 189 , 192 , 270, 299, 367 PC 12 pheochromocytoma cell, 252 Caspase, 74, 84, 92-99, 208, 248-250, 255, 257, 268-271, 273, 283, 289, 366, 371 Catecholamine, 59 , 15 6 dopamine (DA), 231, 352, 353, 381-384, 389-392 epinephrine, 155 , 171 norepinephrine (NE), 155 , 162, 163 , 171 , 381, 385-392 Catenin, 62 , 95 Caudate nucleus . See Basal ganglia CD133. SeePromini n ced, 74, 93, 94, Cell adhesion molecule (CAM) , 49, 55 , 114, 219, 224 cadherin, 55 , 58-60,62 integrin, 31-33, 35, 55, 59, 109, 220-222, 224, 236,299 LI, 55,219,234,288,29 9 neural cell adhesion molecule (nCAM) , 31, 219, 220,299 Cell cycle, 77 GO phase, exit, 10, 13, 15-17, 187 , 188 , 202, 208, 218,299 Gl phase , 10 , 11, 14 , 15, 19, 20, 187 , 188 , 202-207, 211,299 kinetics, 10 , 14-18, 187-192, 201, 205, 299 M phase, mitosis, 14, 15, 83, 84, 187 , 202-206, 210, 211,299

S phase, 10 , 11, 14, 15, 19,20,84,187, 188 , 202-207, 299 Cell fate, 15 , 74, 75, 79, 113 , 114 , 200, 201, 347, 366, 371 Cerebellum, 114 , 127 , 144-147, 150 , 208, 217, 269, 271-273, 297, 298, 348-350 , 365-369, 385, 387, 390 Bergmann glia, 298 external granular layer (EGL), 187 , 207 , 363-366, 368, 36 9 granule neuron, 114 , 187 , 248, 207, 231, 234, 254-257, 271,272,297,367-369 neuronogenesis, 186—18 7 Purkinje neuron , 78, 114 , 144 , 147 , 246, 247 , 252, 253, 259,269,271,272,297,348,366 Cerebral cortex , 9, 11 , 13 , 17, 18, 20, 76, 78, 81, 83-85, 91, 146 , 205 , 206, 210, 231, 233, 269, 271-273, 296, 297, 301 , 346-352, 385, 387, 389, 391 . For developmental references, se e also Cerebral wall death, 246 , 249-250, 252 neuronogenesis, 187 , 249 Cerebral wall cortical plate (CP) , 10 , 11, 28, 29, 32, 24, 75, 76, 80, 84, 297, 348 intermediate zone (IZ) , 10 , 11, 28, 30, 75, 76, 80, 84, 218,220,297 marginal zone (MZ) , 10 , 11, 13 , 20, 28-30, 32, 34, 35, 51,75,76,91,220-222,224 preplate, 29, 32 subplate, 29, 32, 51,76,91 subventricular zone (SZ) normal, 10 , 11, 13, 17, 19-21, 28-30, 33, 35, 36, 75, 76 , 84 ethanol-affected, 184-193 , 200, 205, 206, 208, 210, 211,216,297 nicotine-affected, 363-366 , 368, 369 ventricular zone (VZ) normal, 9, 10 , 13, 19-21, 28, 29, 33, 75, 76, 80, 84 ethanol-affected, 184-193 , 200, 205, 216, 297 nicotine-affected, 350 , 363-365, 368 Chemotaxis Cholinergic receptor. See Receptor, acetylcholine Cobblestone complex, 3 4 Cocaine, 363 Cognition, 107 , 108 , 143 , 148 , 150 , 315, 381, 383, 364 , 385, 386, 392 intelligence quotien t (IQ), 128 , 129 , 132 , 143 , 148 , 318,349 learning, 129 , 143,319,32 2 memory, 130-132, 319, 322, 341 intentional, 13 2 metamemory, 131 , 13 2 working, 132 , 13 4 test California Verbal Learning Test, 131 , 132 Continuous Performance Test, 31 9 McCarthy Scales , 131 Peabody Picture Vocabulary Test, 322 Ravens Test, 32 2 Stanford Bine t Intelligence Scale , 31 8 Tower of London, 13 3

INDEX 40 1 Wisconsin Car d Sor t Task, 13 3 Wide Range Assessment of Memory and Learning , 131,319 visuospatial, 129 , 13 4 Collapsin respons e mediato r protein (CMRP) , 47 Conduct disorder . See Behavior Congenital muscula r dystrophy, 34 Corpus callosu m fiber bundle, 127 , 144, 145 , 147 , 148 , 150 , 233, 234, 297, 302, 303 neuronogenesis, 18 4 neurons of origin, 18 4 Cortical plate. See Cerebral wall Cortisol, 156-160 , 162 , 171 Corticosterone (CORT), 156 , 158-162, 169 , 170 , 171 Corticotropin releasing hormone (CRH) , 155 , 156, 158, 160-162, 164 , 165 , 168,170 , 171 Cotinine 321 , 322 Craniofacial malformation . See Facial dysmorphia Critical period, 60, 115 , 252, 282-284, 286, 351 , 368, 37 0 Cyclic nucleotide (cAM P and cGMP), 20, 50, 54, 208, 256 Cyclin. 13 , 14, 188, 191 , 201-205, 207, 208, 21 1 Cyclin-dependent kinase (cdk), 13 , 14, 17, 32, 33 , 188 , 201,202,204-208,211 Cyclin-dependent kinas e inhibitor "p!7,pl8,p57, 202, 204, 206 P21, 14,17 , 96, 191 202, 204-208, 211 P27, 13 , 14, 17, 20, 113 , 188 , 191 , 202 , 204, 205 , 207,211 Cytochrome C, 94-98, 268-271, 273 Cytochrome P450, 290, 300 Cytokine, 156 , 168-171 Cytoskeleton, 285 "actin, 33, 47-50, 54, 58, 60, 202, 222, 224, 236-238 microtubule, 34, 47-50, 202, 224, 236, 238 regulation, 236-238 Death ethanol-induced in vitro, 232, 250, 252-259, 267 , 268, 270, 271-274, 286-290 in vivo, 188, 245-250, 252-259, 267-274 , extracellular regulation, 288 intracellular regulation, 255-258, 287-289 naturally occurring, 92, 246, 249, 250, 253, 259, 269, 270,287 nicotine-induced, 368 , 369 opiate-induced, 364-36 7 pathway intrinsic, 96-99, 267-269, 274, 287 extrinsic, 96-99, 267, 268 , 270 , 274 caspase-independent, 97-99 Dendrites effect o f ethanol, 230-23 3 fate, specification, 46 growth, 58,235,239 remodelling, 231, 236 Depression. Se e Mood disorder s Disabled, 32 , 33 Distalless, dix, 4, 18 , 30

DNA aneuploidy, 81, 82 endjoining, 79-8 1 fragmentation, 73 , 77, 79, 80, 81-92, 94-96, 98, 206, 207,248,250,255,268 Dopamine. See Catecholamine s Dose-response, 124 , 126 Doublecortex syndrome, 33, 34 Doublecortin (dcx), 32-34 , 36 Endocrine, 153 , 364 Ephrin, 52 , 53, 58,61 Epidermal growth factor. Se e Growth factor s Epigenetic/epigenomic, 172 , 173 Epilepsy, 2 7 Epinephrine. Se e Catecholamine s Ethanol metabolism, 10 6 threshold o f consumption, 123 , 12 4 genetic influences , 290 Evolution, 104-106 , 109-112, 115 , 116 duplicate and vary , 110—111 redundancy. 11 1 Excitotoxicity, 115 , 254 Extracellular matrix (ECM), 55 , 58-60, 171 , 202, 219, 221,222,224 chondroitinsulfate proteoglycan , 36 , 58, 60 dystroglycan, 3 5 filamin A , 3 3 fukutin, 3 5 laminin, 32 , 35 mannosyl glycans, 35 polysialic acid, 3 1 reelin (r/n) , 4, 32, 33, 36, 114,224 Extracellular signal-regulated kinase (ERK). See Mitogen activated-protein kinase External granular layer. See Cerebellu m Facial dysmorphia , 4, 94, 107 , 109 , 123-126 , 143 , 148 , 246, 282-29 0 Fasligand,97,98,268,270,271 Fetal alcoho l spectrum disorder s (FASD) 126 , 129 , 131 , 134, 135 , 143-150,153, 156 , 158 , 159, 166 , 167 , 199,211,282 alcohol-related neurobehaviora l disorder (ARND), 124 , 134,135 fetal alcoho l syndrome (FAS), 4-6, 107 , 123 , 124 , 126,130,132-135, 143-146, 148-150, 217, 223, 233,234,274,296 Fetal programming , 153 , 154 , 157 , 162 , 172 , 173 Filopodia. Se e Axon, growth con e 4-hydroxynonenal, 96, 271-273 Fukuyama congenital muscula r dystrophy, 34 Gl phase . See Cell cycle y-aminobutyric acid (GABA) , 9, 13 , 17, 18, 20, 21, 31, 59-61, 155 , 162, 163 , 342 , 346, 350-35 4 Ganglionic eminenc e (GE), 12 , 13, 17-19, 28, 30, 36 Ganglioside, 290 , 300 Gap junction , 1 1

402 INDE X Glia, 113,252,281,283,28 4 astrocyte, 17 , 29, 31, 78, 184 , 188-190 , 200, 210 , 223, 236, 295-304, 366 , 367 , 369, 371 ependyma, 200 glial-derived factor, 60 oligodendrocyte, 369—37 1 radial glia, 191,200,209,21 0 Bergmann cell . See Cerebellum guides for migration, 29, 31 , 51, 55, 219, 220, 224, 295, 296, 29 8 Müller cell. See Retina neural stem cell. See Stem cel l transformation t o astrocytes, 29, 220-224, 295, 297, 298 Gliogenesis effects o f ethanol, 29 6 effects o f nicotine, 369, 37 0 effects o f opiates, 366 , 367 , 37 1 Glucocorticoid, 154,155 , 158 , 167-173 Glutamate, 60 , 164 , 301 , 302, 342, 346, 350 , 351, 353, 354 a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) 20 N-methyl-D-aspartate (NMDA) 20, 31, 221, 231, 254-256 receptors. See Receptor Gonadotropin releasin g hormone (GnRH) , 165 , 171 Growth cone . See Axon Growth factors bone morphogeni c protei n (BMP) , 222, 223, 280, 286,287 epidermal growth factor (EGF), 30 , 200, 298, 367 fibroblast growth factor (FGF), 83, 109, 188-190 , 192 , 200,203,280, 286, 300, 367 hepatocyte growt h factor (HGF), 30 , 31 insulin-like growth factor (IGF) , 109 , 188 , 190 , 192 , 203,251,253-254,300 platelet-derived growth factor (PDGF) , 188 , 189, 192, 193,203,300 transforming growth factor (TGF ) a 30 TGF ß 18, 31,188-193, 203, 222-224, 234, 298, 300 Growth obesity, 318, 322 retardation, low birth weight, 123, 126 , 134 , 315 , 317 Hippocampal formation, 246, 248-249, 301 , 364 death, 24 9 dentate gyrus , 60, 186 , 249, 364 normal, 47, 60, 12 , 114 ethanol-affected, 148 , 156 , 162-164, 172 , 185, 186 , 200,231,233,236,237,239,249 nicotine-affected, 319 , 321, 346, 349-351, 365, 385,387 neuronogenesis, 182 , 185 , 186 hippocampus, 24 9 intrahilar zone (IHZ) , 12 , 182, 185 , 192, 206, 249 , 363, 364 , 36 7 Homeostasis, 155 , 156, 158, 168, 238, 385 Human immunodeficienc y virus (HIV), 370 , 371 neuronogenesis, 370 , 371 gliogenesis, 37 1

Hypothalamus, 153, 154, 156, 158, 160, 162-164 , 171 , 172,385,387, 389 hypothalamic-pituitary adrenal (HPA) axis, 153-165 , 168, 169 , 171-173 , 285, 385, 387 , 389 hypothalamic-pituitary gonadal (HPG) axis, 161, 164 , 165 paraventricular nucleus (PVN) , 156 , 161 , 163 , 164 , 17 2 Imaging, 12 7 confocal, 238 , 239, 272 diffusion tenso r imagin g (DTI), 12 7 computed tomography , 149 , 150 magnetic resonanc e imaging (MRI), 34, 127, 145 , 147-149,233,296 positron emissio n tomograph y (PET) , 149 , 29 6 ultrasonography, 149 voxel-based morphometry , 145 , 146 Immune cells B cells, 165-16 9 T cells, 76, 165-167, 169-171 Immune response , 154 , 163 , 165 , 168 , 17 1 Immunity, adaptive, 166 cell-mediate (CMI), 166-168, 170 humoral, 166 , 168 , 17 0 In situ end labeling (ISEL) . See DNA fragmentation Insulin-like growth factor. See Growth facto r Integrin. Se e Cell adhesion molecul e Interleukin (IL), 94, 164 , 169-17 1 Intelligence quotient . Se e Cognition Intermediate zone. See Cerebral wall Ki-67,187,210,211 LI. See Cell adhesion molecule s Lamellipodia. See Axon, growth cone Leading process, 28, 34 Lipopolysaccaharide (LPS) , 161 , 164 , 168 , 16 9 Lisl, 32, 34 Lissencephaly, Se e Migration, defect s Local circuit neuron (LCN) , 17 , 18, 30, 31, 35, 76, 245,351 Locus coeruleus, 155 , 156, 165, 168, 385-39 2 Luteinizing hormone (LH), 165 Luteinizing hormone releasin g hormone (LHRH) , 27, 30 Lysophosphatidic aci d (LPA) , 83-85 Major histocompatibility complex (MHC) , 165 , 169 , 171,371 Marginal zone . Se e Cerebral wal l Mental retardation , 27, 128 , 13 4 Microtubules. Se e Cytoskeleton Microtubule-associated protein (MAP), 34, 47, 232 Migration, 124,238,24 6 defect ectopia/heterotopia, 33 , 34, 114, 144 , 216 , 217 , 220-224, 296, 297 lissencephaly, 216 , 217 neural crest, 27, 166 radial, 29, 114 , 115 rate, 29, 218, 219 tangential, 3 0

INDEX 40 3 Mitogen-activated protein kinas e (MAPK), 300 , 36 6 extracellular signal regulated kinase , 189 , 190 , 192 , 256-257, 366 jun kinase, 366 Monoamines. See Catecholamines Mood disorder s anxiety, 32 1 depression, 32 1 Motor activity , 128 M phase. See Cell cycle Müller cell. See Retina Muscle-eye-brain disease, 34 Necrosis 73, 92, 99, 254, 274, 28 6 Neostriatum. See Basal ganglia Nerve growth factor. See Neurotrophi n Neural cell adhesion molecule . Se e Cell adhesio n molecules Neural cres t cardiac development , 281, 284 derivatives, 166 , 170,280-28 5 effects o f ethanol, 222 , 271, 279-290, also see facial dysmorphia homeobox genes, 280 , 282 , 286, 287 , 289 neural induction, 285 origin, 280-282 peripheral nervou s system (PNS), 280-284 Neuroblastic layer. See Retin a Neuroendocrine, 153 , 154 Neurogenesis, 17 , 18, 112, 113, 205, 206, 209 , 21 0 Neuromuscular junction. 5 9 Neuronogenesis, 365 , 366, 368 , 36 9 effects o f ethanol, 218 , 249, 29 9 effects o f nicotine, 368 , 36 9 effects o f opiates, 364-36 6 Neuropilin, 36 , 52 Neurotrophin, 236 , 251-253 , 258 , 274, 28 8 nerve growth factor (NGF) , 74, 76, 251-253 brain-derived neurotrophic factor (BDNF) , 30 , 50,54, 60, 74, 76, 223, 224, 251-253, 255, 301,302 neurotrophin 3 (NT-3), 74 , 83, 251, 252 neurotrophin 4 (NT-4), 30 , 74, 251 Neurotrophin receptor . See Receptor Nicotinamide-adenine dinucleotid e (NAD), 95 Nicotine, 4, 108 , 341-354 , 367-370 Nicotinic receptor . Se e Receptor, acetylcholin e Nitric oxide (NO), 162-164 , 232 N-methyl-D-aspartate (NMDA). Se e Glutamat e Norepinephrine. Se e Catecholamine Nucleus tractus solitarius (NTS), 385, 386-389 Olfactory bulb , 35 , 36, 200 Opioids, 15 6 ß-endorphin, 156 , 160 , 162 , 169 , 36 7 effects o n neurona l developmen t death (apoptosis) , 365-367, 371 differentiation, 36 5 proliferation, 211 , 365-367 Optic nerve, 111 , 148 , 14 9

Ornithine decarboxylase , 318, 321 Oxidative stress , 95, 96, 108 , 115 , 271-274, 287, 28 8 P 53,

80, 95-98, 202-204, 207, 208, 268 , 270 , 271 , 274, 36 6 Phosphotidylinositol 3 /-phosphokinase (PI3K), 237, 255, 257,258,367 Pial basement membrane , 32 , 34, 35, 221, 222, 224 Pituitary adenylyl cylcase activating polypeptide (PACAP) , 251,255-259,270 Placenta, 153 , 156, 158, 159 enzymes, 15 8 feto-placental unit , 159 , 170 Platelet-activating factor acetylhydrolas e isoform Ib . See Lis l Polarity, 45-48, 234, 235 Poly-adenosine diphosphate ribos e polymerase (PARP) , 95, 97, 98, 268, 27 4 Poly-drug use, 124,316,32 2 Preplate. See Cerebral wall Principal sensory nucleus of the trigemina l nerve, 186 , 188,246,247,249 Process outgrowth , 230-24 0 axon, 45, 48 dendrite, 45, 48 Proliferation. See Cell cycle ; Ste m cell s neuronal site s of origin. See Cerebral wall; Cerebellum ; Hippocampus Programmed cell death. See Apoptosi s Projection neuron , 17 , 18, 60, 76, 231-234, 245, 351, 354 Proliferation, 182-193 , 246, 250 , 363-371, See also Cell cycle; Cerebellum; Cerebral wall ; Hippocampal formation, Neuronal site s of origin; Ste m cell s Pro-opiomelanocortin (POMC), 155 , 156, 160-162 , 169, 17 0 Protein kinas e A (PKA), 256-258 Protein kinas e C (PKC) , 189 , 237 Protein-O-mannosyl transferase (POMT), 34 Pseudostratified ventricula r epithelium (PVE) , 9-1 8 Pyknosis, 246 Pyramidal (corticospinal) tract 184 , 23 3 Pyramidal neurons . See Projection neuron s Radial glia, 200, 209 , 21 0 guides for migration. Se e Gli a neural stem cell . See Stem cel l Reactive oxygen species (ROS) , 96-98, 248, 268 , 271 , 273, 287, 28 8 Receptor desensitization, 344 , 345 , 350 , 353, 368, 390 , 39 1 growth factor epidermal growt h factor (EGFr), 3 0 insulin-like growt h factor , 191 , 253 , 254 platelet-derived growt h factor , 189 , 192 , 19 3 transforming growt h factor , 189 , 191 , 19 3 neurotransmitter acetylcholine, 59 , 367 muscarinic (mAChR), 207, 300 , 321 , 347, 382-39 2 nicotinic (nAChR), 341-354 , 368-370 acetylcholine receptor-inducin g protein , 369 , 37 0

404 INDE X Receptor (continued) dopamine (DI and D2), 19-21 , 384 y-aminobutyric acid, 20,21, 347 glutamate a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), 20, 21,58,59, 351, 353 N-methyl-D-aspartate (NMDA), 20, 21, 58-61, 164 , 221, 231, 301, 302 , 345, 347 , 351 , 353 opioid 5-opioid receptors (DOR) , 364-367 K-opioid receptors (KOR), 364-367 H-opioid receptors (MOR) , 364-367 neurotrophin p75, 74, 75, 251-253, 25 9 trk30, 74,75, 223,251-253,259 others corticotropin releasin g hormone typ e l (CRH-R1) , 161,162,171 glucocorticoid (GR) , 161, 162 , 170, 172 mineralocorticoid (MR), 161 , 162 phosphatidylserine, 73, 78, 84 very low density lipoprotein (VLDL) , 3 2 Reeler mouse, 32,91 , 11 4 Retina, 79 , 111,113, 149 ganglion cells, 301 Müller cells , 113,301 neuroblastic layer topographic maps , 50-53 , 79 Retinoblastoma protein (Rb) , 13, 14 , 202, 204, 20 6 Retinoic acid, 289, 290 Robo, 36,51 , 52,5 5 Schwann cells , 280-284 Secondary proliferative populatio n (SPP) , 10-15, 17, 18, 36 4 Serotonin, 155 , 162, 163, 36 3 Slit, 36,51,52 , 55 Socioeconomic status (SES), 107 , 108 , 124 , 129 , 133,134 , 316,318-320,322 Sonic hedgehog , 51,286 , 289 S phase. See Cell cycle Stem cell , 76, 79-83, 85 embryonic stem cell, 199 , 203-205 , 208 , 211 marker nestin, 29 , 185 , 200, 201, 209, 210 , 296, 297 prominin(CD133), 201,209 vimentin, 210,296 neural stem cell (NSC) , 11 , 17 , 18, 29, 185 , 192 , 199-211,295-299,347,363,369 Stress, 154 , 155 , 162, 164 , 168-17 3 concept, 15 4 response, 154 , 160 Subplate. See Cerebral wall

Substantiel ragra , 231 , 346, 38 2 Subventricular zone. See Cerebral wall Sudden infan t deat h syndrom e (SIDS), 316, 321 , 367 , 392 Superior colliculus , 52 Sympathetic adrena l medullary system, 15 5 Synapse chemical, 56-61 , 342 electrotonic, 56 , 61, 62 extra-synaptic communication, 35 0 formation, synaptogenesis , 56-62, 76, 111 , 232, 246, 249, 250 , 259, 295, 296 , 301 , 351 , 354 molecular constituents, 57 , 58 post-synaptic density , 56-5 8 "silent," 59, 351,354 structure, 56 , 61 synaptosome associated protein, 58 vesicular traffic, 57 , 58 Telomerase, 200, 207, 208, 211 Teratogen, 109 , 127,315-32 2 Terminal deoxynucleotidy l transferase mediate d deoxyuridine nick end labeling . See DNA fragmentation Thalidomide, 109 Thalamus, 149 , 349 , 35 0 death, 92, 109,249,250 cortical connections, 51,55 , 249, 350 , 351 Thymidine, 14 , 192, 217 , 218 , 221 , 299, 36 5 Thymus, 154 , 165 , 167 , 170 Tobacco, 108 , 124 , 315-322 , 341 , 349 , 35 3 composition, 329-331 , 367 effect o n growth, 317 , 318, 367 effect o n behavior, 318-32 2 environmental exposure , 316, 317, 321 , 322, 367 Topographic maps , 52 , 53 Transcription factor s activating protein 1 , 207, 20 8 nuclear factor KB , 207, 30 0 sox, 201,368 Transforming growth factor. Se e Growth factor s Transplantation, 114 , 201, 209, 210 Trapezoid body , 60 Tumor necrosi s factor. Se e Apoptosis UDP-N-acetylglucosamine protein-O-mannos e ßl ,2-N-acetylglucosaminyltransferase, 34 Vasoactive intestinal polypeptide , 3 0 Ventral tegmental area , 351-354, 382-384 Ventricular zone. See Cerebral wall Vitamin E , 288 Walker-Warburg syndrome, 34