Development of Dopaminergic Neurons (Neuroscience Intelligence Unit)

NEUROSCIENCE I N T E L L I G E N C E U N I T 4

Umberto di Porzio Roberto Pernas-Alonso Carla Perrone-Capano

Development of Dopaminergic Neurons

R.G. LANDES C O M P A N Y

NEUROSCIENCE INTELLIGENCE UNIT 4

Development of Dopaminergic Neurons Umberto di Porzio, Ph.D., M.D. International Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche Naples, Italy

Roberto Pernas-Alonso, Ph.D. International Institute of Genetics and Biophysics Consiglio Nazionale delle Ricerche Naples, Italy

Carla Perrone-Capano, Ph.D. Faculty of Pharmacy University of Catantaro "Magna Grecie" Roccelletta di Biorgia (cz), Italy R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

NEUROSCIENCE INTELLIGENCE UNIT Development of Dopaminergic Neurons R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright © 1999 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN:1-57059-565-8 While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Development of Dopaminergic Neurons/ [edited by] Umberto di Porzio, Roberto Pernas-Alonso, and Carla Perrone-Capano p. cm. -- (Neuroscience intelligence unit) Includes biographical references and index ISBN 1-57059-565-8(alk. paper) 1. Development neurobiology. 2. Neurons -- Growth. 3. Dopaminergic mechanism. 4. Dopamine -- Physiological effect. I. Di Porzio, Umberto. II. Pernas-Alonso, Roberto. III. Perrone-Capano, Carla. IV. Series. [DNLM: 1. Neurons --physiology. 2. Dopamine --physiology 3. Biogenic Amine Neurotransmitters--physiology. WL 102.5 D4895 1999] QP363.5.D4726 1999 573.8'536--dc21 DNLM/DLC 95-52676 for Library of Congress CIP

CONTENTS 1. Specification and Patterning of the Rostral Neural Tube ....................... 1 Salvador Martinez and Antonio Simeone CNS Axial Patterning and Retinoic Acid ............................................... 2 Early Neural Plate Specification .............................................................. 3 Patterning Mechanisms in the Neural Plate .......................................... 4 Induction of Longitudinal Domains ...................................................... 4 Induction of Transversal Domains ......................................................... 6 Development of Regional Organizers .................................................... 7 Different Histogenetic Competence ....................................................... 7 Local Patterning Centers in Further Defining the Organization of the Forebrain ................................................................................... 8 2. Specification of Dopaminergic Neurons: Inductive Factors and Their Receptors ................................................................................ 15 Mary Hynes and Arnon Rosenthal Induction Along the D-V Axis .............................................................. 16 Induction of a Supernumerary Floor Plate In Vivo Results in the Appearance of Ectopic DA Neurons ...................................... 18 Induction of Midbrain DA Neurons is Mediated by Contact with the Floor Plate ........................................................................... 18 Shh Mediates the Induction of DA Neurons by Floor Plate ............... 18 Shh Receptors and Intracellular Signaling Molecules ......................... 19 Intracellular Mediators of the Shh Signal ............................................. 21 Induction Along the A-P Axis ............................................................... 23 Intersections of Shh and FGF8 Specify the Position and Identity of DA and 5HT Neurons ................................................................... 25 Conclusion ............................................................................................. 29 3. Development of Midbrain Dopaminergic Neurons .............................. 37 Carla Perrone-Capano, Roberto Pernas-Alonso, and Umberto di Porzio The Birth of Mesencephalic DA Neurons ............................................ 39 DA Cell Lineage Specification ............................................................... 40 Specific Gene Expression During Differentiation ................................ 42 Phenotypic Maturation ......................................................................... 45 Conclusion ............................................................................................. 50 4. Growth Factor Actions on Developing Midbrain Dopaminergic Neurons .................................................................................................... 57 J. Engele and N. Bayatti Growth Factors Have Multiple Effects on Cultured Midbrain Dopaminergic Neurons .................................................................... 57 Some Growth Factors Affect Dopaminergic Neurons by an Indirect Glial-Mediated Mechanism ...................................... 59 Growth Factors with Direct Effects on Dopaminergic Neurons ........ 61 Growth Factor Sensitivity Defines Distinct Subpopulations of Dopaminergic Neurons ................................................................ 64

Fitting Growth Factors into the Developmental Schedule of Midbrain Dopaminergic Neurons ............................................... 66 5. The Effects of Sex and Sex Hormones on the Development of Dopaminergic Neurons ...................................................................... 75 Christof Pilgrim, Cordian Beyer, and Ingrid Reisert Sex Differences of Adult Dopamine Systems ....................................... 75 Developmental Mechanisms ................................................................. 76 6. Neural Development of the Striatal Dopamine System ........................ 87 Fu-Chin Liu and Ann M. Graybiel Dopamine as a Potential Regulator of Neuronal Development: Expression of Dopamine Receptors in the Germinal Zones of Striatal Anglage ............................................................................. 88 Developmental Regulationn of the Mesostriatal Dopamine-Containing Neurons by Striatal Target Cells ................ 90 Developmental Regulation of Striatal Neurons by the Mesostriatal Dopamine Neurons .......................................... 90 Modulation of Mosaic Structure of Striatal Compartments by the Mesostriatal Afferents During Development ........................ 92 Dopamine Activity-Dependent Modulation of Compartmental Phenotypes During Striatal Development ....................................... 93 Specification of Topographic Projections of the Mesostriatal and Mesolimbic Afferents During Development ........................... 94 Conclusion ............................................................................................. 95 7. The Involvement of Dopamine in Various Physiological Functions: from Drug Addiction to Cell Proliferation ....................... 101 Daniela Vallone, Roberto Picetti, and Emiliana Borrelli Pharmacological Profiles of Dopamine Receptors ............................ 101 Distribution of Dopamine Receptors ................................................. 102 The Dopamine Receptor Genes .......................................................... 107 Signal Transduction ............................................................................ 108 Dopamine and Locomotion ................................................................ 111 Dopamine and Drugs of Abuse .......................................................... 112 Molecular Responses toTreatments with Drugs of Abuse ................ 113 Proliferative Role of Dopamine .......................................................... 114 Dopamine Acts as an Antiproliferative Factor in Pituitary Cells .................................................................................................. 115 8. Dopamine Neuron Grafts: Development and Molecular Biology ................................................................................................... 123 Lauren C. Costantini and Ole Isacson Establishment of Surviving Dopamine Neuron Grafts ..................... 123 Regulation of Axonal Outgrowth from Dopamine Grafts ................ 129 Reconstructing Synaptic Connections with Dopamine Grafts ................................................................................................ 132

Clinical Relevance ................................................................................ 134 Conclusion ........................................................................................... 137 9. Dopaminergic Neurons in the Olfactory Bulb .................................... 145 S. Denis-Donini Anatomy and Circuitry of the Olfactory System ............................... 145 Evidence and Possible Role for Dopamine in the Olfactory Bulb .................................................................................................. 147 Ontogeny and Differentiation: Neurotransmitter Plasticity ............. 150 Conclusion ........................................................................................... 152 10. Dopamine in Drosophila: Neuronal and Developmental Roles ........ 157 Wendi S. Neckameyer Biosynthetic Pathways and Evolutionary Considerations ................. 157 The Role of Dopamine in Central and Peripheral Nervous Tissues .............................................................................................. 160 Dopamine and Fertility ....................................................................... 169 Dopamine as a Developmental Signal in Other Tissues .................... 171 Conclusion ........................................................................................... 171 11. Genetic Analysis of Dopaminergic Neurons in the Nematode Caenorhabditis elegans ......................................................................... 175 Robyn Lints and Scott W. Emmons C. elegans Dopaminergic Cells: Structure and Function .................. 176 Sex-Specific Development ................................................................... 179 Hierarchical Specification of Neuronal Properties and the Role of bHLH Transcription Factors ................................ 179 Regulatory Genes Specifying Neuronal Differentiation .................... 181 Heterochronic Genes Define Types of Neuronal Subprograms .................................................................................... 182 Genes of Dopamine Biosynthesis, Metabolism and Utilization ................................................................................. 182 A TGF-β Signal Induces Expression of Dopamine by Ray Neurons ............................................................................... 184 The TGF-β Signal Specifies Dopaminergic Cells within an Equivalence Group ..................................................................... 184 A Hox Gene May Define the Dopaminergic Equivalence Group ............................................................................................... 186 Conclusion ........................................................................................... 187 Index ....................................................................................................... 191

EDITORS Umberto di Porzio, Ph.D., M.D. International Institute of Genetics and Biophysics Consiglio Nazionale delle Ricerche Naples, Italy Chapter 3 Roberto Pernas-Alonso, Ph.D. International Institute of Genetics and Biophysics Consiglio Nazionale delle Ricerche Naples, Italy Chapter 3 Carla Perrone-Capano, Ph.D. Faculty of Pharmacy University of Canantaro "Mana Grecie" Roccelletta di Borgia (cz), Italy Chapter 3

CONTRIBUTORS Nadhim Bayatti, M.Sc. Department of Anatomy and Cell Biology University of Ulm Ulm, Germany Chapter 4

Lauren C. Costantini, Ph.D. Neuroregeneration Laboratory Harvard Medical School McLean Hospital Belmont, Massachusetts, USA Chapter 8

Cordian Beyer, Ph.D. Department of Anatomy and Cell Biology University of Ulm Ulm, Germany Chapter 5

Suzanne Denis-Donini, Ph.D. Department of Biology University of Milan CNR Center of Cytopharmacology Milan, Italy Chaper 9

Emiliana Borrelli, Ph.D. Institut de Génétique et de Biologie Moléculaire et Cellulaire Illkirch Cedex, France Chapter 7

Scott W. Emmons, Ph.D. Department of Molecular Genetics Albert Einstein College of Medicine Bronx, New York, USA Chapter 11

Jurgen Engele, Ph.D. Department of Anatomy and Cell Biology University of Ulm Ulm, Germany Chapter 4

Wendi S. Neckameyer, Ph.D. Department of Pharmacological and Physiological Science Saint Louis University Medical Center St. Louis, Missouri, USA Chapter 10

Ann M. Graybiel, Ph.D. Department of Brain and Cognitive Sciences Massachusetts Institute of Technology Cambridge, Massachusetts, USA Chapter 6

Roberto Picetti, Ph.D. Institut de Génétique et de Biologie Moléculaire et Cellulaire Illkirch Cedex, France Chapter 7

Mary Hynes, Ph.D. Genentech Inc. San Francisco, California, USA Chapter 2 Ole Isacson, M.D. Harvard Medical School McLean Hospital Belmont, Massachusetts, USA Chapter 8 Robyn Lints, Ph.D. Department of Molecular Genetics Albert Einstein College of Medicine Bronx, New York, USA Chapter 11 Fu-Chin Liu, Ph.D., M.D. Institute of Neuroscience and Department of Life Science National Yang-Ming University Taipei, Taiwan, Republic of China Chapter 6 Salvador Martinez, Ph.D., M.D. Department of Morphological Sciences Faculty of Medicine University of Murcia Murcia, Spain Chapter 1

Christof Pilgrim, M.D. Department of Anatomy and Cell Biology University of Ulm Ulm, Germany Chapter 5 Ingrid Reisert, M.D. Department of Anatomy and Cell Biology University of Ulm Ulm, Germany Chapter 5 Arnon Rosenthal, Ph.D., M.D. Department of Neuroscience Genentech Inc. San Francisco, California, USA Chapter 2 Antonio Simeone, Ph.D., M.D. International Institute of Genetics and Biophysics Naples, Italy Chapter 1 Daniela Vallone, Ph.D. Institut de Génétique et de Biologie Moléculaire et Cellulaire Illkirch Cedex, France Chapter 7

PREFACE The catecholamine dopamine (DA) plays a key role in the physiology of most vertebrate and invertebrate organisms. The biochemical pathway is highly conserved throughout evolution.1 In addition to its fundamental role as a transmitter in the nervous system, there is evidence for a role of dopamine in vertebrate and invertebrate development, as well as an involvement for dopamine in the physiology of peripheral structures in invertebrates. “The role of dopamine in the modulation of various kind of behavior may arise from an ancient signaling pathway.” This hypothesis is in line with the view that the evolution of the nervous system is not predominantly dependent upon the formation of new or better transmitter substances, receptor proteins, signal transducers and effector proteins, but instead it invents a new and more complex utilization of these elements in creating highly advanced and refined circuitry. As Sir Peter Medawar well described for the endocrine system “…endocrine evolution is not an evolution of hormones but an evolution of the uses to which they are put; an evolution not, to put it crudely, of chemical formulae but of reactivities, reaction patterns and tissue competence.”2 The relatively few DA neurons in the mammalian brain subserve an important regulatory role for many neural functions, including fine motor integration, neuroendocrine hormone release, cognition, emotive behaviors, male sexual behavior, reward, and possibly memory. The role of dopamine in hedonistic pleasure is at the basis of the use of various addictive drugs that enhance dopamine neurotransmission. In the late 1950s the elucidation of dopamine biochemistry and physiology and the development of the Falck-Hillarp fluorescence method allowed visualization of dopaminergic neurons and provided insights on their function.3 These data later led to the discovery that dopamine is the lacking neurotransmitter in the crippling Parkinson’s disease. In the mid 1960s, Cotzias of Brookhaven laboratories found that the oral administration of high doses of L-DOPA, the dopamine precursor that crosses the blood brain barrier, resolved dopamine deficiency and relieved the symptoms of Parkinson’s disease.4 In those years L-DOPA was also used to treat lethargic encephalitis patients, as movingly described in Oliver Sacks’ book Awakenings (1973). Since then, a lot has been learned about the role of midbrain dopaminergic neurons in human physiology. A large deal of information has been accumulated on the complex mechanisms of midbrain patterning, dopamine phenotype induction and maturation, and the role of epigenetic factors involved in specification, development and maintenance of midbrain dopaminergic functions. In this book we will discuss the molecular and morphological events required for the correct induction of a neural plate and for the establishment of regional identities. Here is work presented that pertains to inductive

factors and their receptors, which play a role in the specification of DA neurons in the brain. Emphasis is given to the peculiar asynchronous development of midbrain DA functions and the cellular and molecular events responsible for their differentiation and maturation. In particular, in this book we address the fundamental role of striatal target cells in the midbrain DA neuron development and the most recent findings of striatal neuron development and their anatomical and functional compartmentation. The understanding of the complex integrative functions exerted by dopamine on target cells has been greatly expanded by the molecular characterization of the DA receptor gene family. Great importance and possible clinical relevance attain to the increasing list of “dopaminotrophic” factors and their receptors. In the last several years a great debate has arisen on the possible use of embryonic or engineered DA cells to treat Parkinson’s disease symptoms. Strikingly, the in situ maturation and phenotypic specialization of DA neurons grafted into the adult striatum/ caudate-putamen parallels the normal development of committed fetal dopamine neurons during neurogenesis. The correct matching between the right presynaptic and postsynaptic neurons is also required for grafted DA cells. More has been learned on the role of dopamine in the diencephalon, namely in the hypothalamus and in the olfactory bulbs. The outline of the most recent progress in understanding CNS DA neuronal development in mammals is followed by a brief excursus on the role of dopamine in the two most studied invertebrates, C. elegans and Drosophila. We hope that the chapters collected in this book will provide new and extensive information that will enable students and scientists to further the knowledge of the basic mechanisms underlying DA neuron development and function. We also hope that the content of this book can stimulate more studies in this field, which will be useful for future clinical application to achieve functional restitution to patients with dopamine neuron dysfunction and degeneration. References 1. For a review, see Venter JC, di Porzio U, Robinson DA et al. Evolution of neurotran mitter receptor systems. Prog Neurobiol 1988; 30:105-169. 2. Danielli FJ. On some physical and chemical aspects of evolution. Soc Exp Biol 1953; 7:440-448. 3. Carlsson A, Falck B, Hillarp A. Cellular localisation of brain monoamines. Acta Physiologica Scandinavica (suppl) 1962; 196:1-27. 4. Cotzias GC, Papavasiliou PS, Gellene R. Experimental treatment of parkinsonism with L-DOPA. Neurology 1968; 18:276-277.

CHAPTER 1

Specification and Patterning of the Rostral Neural Tube Salvador Martinez and Antonio Simeone

T

he morphogenesis of the brain and the differentiation of neural structures are highly complex processes that are sequentially established during embryonic development. The first event is represented by the so-called neural induction and gives rise to an early neural plate. This phenomenon is defined as an interaction between an inducing and a responding tissue, the result of which is a change in the differentiative fate of the latter.1 In fact, when induced by an organizer, the responding ectoderm undergoes morphogenetic changes and gives rise to an early neural plate.2,3 The early neural plate is then transformed into a neural tube composed of large domains with distinct fates, corresponding to the prosencephalon, mesencephalon and rhomboencephalon. The establishment of these large territorial identities also coincides with the appearance of transverse neuroepithelial constrictions. 4 The phenomenon of regional differentiation in the induced neural plate is called regionalization.5-10 To the early regionalization follows a complex temporally and spatially regulated series of morphogenetic events (e.g., cell differentiation and migration) giving rise to smaller areas which are phylogenetically, functionally and morphogenetically different. Repeated regions have been interpreted as segment-like structures. This architecture is particularly evident in the rhomboencephalon, where additional transverse constrictions highlight its segmental nature and define smaller transverse neuromeres called rhombomeres (reviewed in Lumsden and Krumlauf).11 In the rostral vesicles, the first overall division is followed by a subsequent differentiation of various neuroepithelial domains, resulting in the identification of prosencephalic neuromeres (prosomeres).10,12-17 Anatomical, as well as histological, studies postulate the existence of genetic fate determinants which subdivide the large neural regions into smaller longitudinal and transverse domains.4,11-13,15,17 The expression of specific gene combinations, spatially and temporally regulated during early and subsequent events leading to the patterning of the central nervous system (CNS), might supply positional and differentiative information to define regional identities and morphogenetic boundaries.11,12,17 Gene candidates for establishing these events are transcription factors or signaling molecules, and most of them are the vertebrate homologs of Drosophila regulatory genes that operate in the fly to subdivide the embryonic body into segments as well as to control the development of head and brain segments.10,17-21 The analysis of mouse models carrying deletions for some of these genes greatly contribute to the molecular knowledge of morphogenetic events predicted on the basis of histological, embryological and anatomical studies. The Development of Dopaminergic Neurons, edited by Umberto di Porzio, Roberto Pernas-Alonso and Carla Perrone-Capano. ©1999 R.G. Landes Company.

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CNS Axial Patterning and Retinoic Acid A large body of data indicates that fate and patterning of tissues depend on the activity of cells able to induce differentiative changes in the responding tissue.1 The first evidence of an inducing region comes from transplantation experiments in amphibians, in which the dorsal lip of an early blastopore induces an ectopic complete secondary axis when transplanted on the ventral side of a host embryo. Because of this ability, the dorsal lip of the blastopore has been called the organizer.2 When induced by a grafted organizer, responding ectodermic tissue undergoes morphogenetic changes and gives rise to a complete, correctly patterned CNS.5-7,9 Transplantation experiments in amphibian and chick embryos suggest that the age of the organizer tissue influences the extension of the neural plate as well as its regional characteristics. An organizer deriving from an early gastrula induces anterior as well as posterior neural tissue, whereas a late organizer induces only posterior tissue.5,9,22-24 Previous studies suggest that the signals required to regionalize the induced neural tissue derive from the mesoderm contained in the organizer.7,9 A large body of evidence suggests the presence of vertical transactivating signals deriving from the prechordal plate and notochord and transmitted to the overlaying neural tissue,7,9,25,26 and of so-called planar signals, acting through the plane of the ectoderm and deriving from the organizer.27-29 Although it is clear that both vertical and planar signals are required for the correct antero-posterior (A-P) and dorso-ventral (D-V) patterning of the neural tissue, it is unclear yet whether these signals coexist at the same stage of development or follow a temporal hierarchy.7,10,30-32 CNS axial patterning is strongly affected by retinoids, naturally occurring derivatives of vitamin A with pleiotropic effects on development and cell differentiation.33-38 Retinoids are present at active concentrations in embryonic structures with a proven role in pattern formation, such as the zone of polarizing activity (ZPA),33-35 the node39,40 and the floor plate of the neural tube.41,42 In mouse embryos, the node is a site of retinoid synthesis already at the 0-1 somite stage and increases its production as it regresses after gastrulation, suggesting a possible mechanism for the establishment of temporal and/or spatial gradients.39 Active retinoids are present along the A-P axis of most of the CNS excluding the head,43,40 although even anterior-most structures have a potential competence to respond to retinoid signals.45 Systemic or local administration of excess all-trans-retinoic acid (RA) to developing embryos has profound effects on axial patterning and specification of regional identities in CNS, as well as in other districts such as the axial skeleton and the limbs.36,37,46 In general, excess RA has a posteriorizing effect, more or less severe depending on the dosage and the time of administration to developing embryos.38,47-52 Retinoids are therefore likely to play a key role in the establishment of regional identities along the A-P axis, although they might not represent the actual morphogens but rather have an instructing role on specific structures (e.g., the ZPA), which would in turn provide morphogenetic signals.53,54 RA-induced alterations in the establishment of A-P identities in the CNS are invariably accompanied by repatterning of homeobox-containing Hox gene expression domains in the hindbrain and spinal cord.45,54 Studies in cell culture models indicate that Hox genes are regulated by retinoids in a time- and dose-dependent fashion,55,56 thus suggesting a specific role for Hox gene products as molecular transducers of the signals triggered by retinoids in the posterior CNS. In the rostral CNS, evidence does exist that RA might play a potential role in the early distinction between rostral CNS territories fated to give fore-midbrain and more posterior neuroectoderm of the hindbrain.57-59 Morphological and molecular analyses of a statistically significant number of embryos generated in a detailed time-course experiment performed to decipher the effect of RA on the early development of the murine CNS indicate that RA is able to induce stage-specific alterations of the rostral CNS with perturbation of different morphogenetic steps during

Specification and Patterning of the Rostral Neural Tube

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the establishment of neural pattern.57-59 Molecular analysis carried out with markers of fore-, mid- and hind-brain regional identities suggests that exogenous RA might affect both the regions supposed to be sources of morphogenetic vertical signals (the head process and rostral-most endoderm) and those supposed to be targets of planar and vertical signals deriving from the organizer (the head folds).57,58 Therefore, these data support the possibility that retinoid signals coming from the node may contribute to the early distinction of head from trunk structures by selecting different sets of regulatory genes, while other signal molecules are required for patterning of the rostral CNS.

Early Neural Plate Specification The distinction between rostral neuroectoderm corresponding to fore-midbrain and more posterior neuroectoderm (hindbrain) is evident at the end of gastrulation. Tissue transplantation and explant recombination experiments indicate that different inductive events contribute to specifying rostral and posterior neuroectodermal territories. In fact, as previously mentioned, an early organizer is able to induce anterior structures while a late organizer specifies only more posterior structures of branchial/trunk regions. Furthermore, these organizing properties are time-dependent, since early organizers contribute to form the anterior axial mesendoderm and induce anterior structures, and late organizers only more posterior structures, indicating that at the end of gastrulation the early node is split into two more restricted derivatives organizing head and trunk structures, respectively. Nevertheless, new increasing data indicate that in mouse the anterior visceral endoderm (AVE), and in Xenopus the leading edge of the involuting endoderm, play a crucial role in head organizer activity.32,33,60-64 In vivo manipulation experiments indicate that the AVE as well as the node-derived axial mesendoderm play important roles in specification and maintenance of signals required for head specification. The sample evidence proving a role for the AVE may be summarized as follows: 1. Transplantation of node-derived axial mesoderm in mouse induces a secondary axis lacking anterior-most neural tissues;64 2. In zebrafish, graft of shield induces a secondary axis lacking the most anterior region of the central nervous system;65,66 3. Removal of a patch of cells expressing the Rpx/Hesx1 gene prevents the subsequent expression of the gene in the rostral headfolds; the result is reduced and abnormally patterned;32 4. Chimeric embryos composed of wild type epiblast and nodal–/– visceral endoderm are heavily impaired in rostral CNS development;63 5. In Xenopus the secreted molecule coded by the cerberus gene is restricted to the leading edge of the involuting endoderm, and microinjection of its mRNA into embryos induces the formation of ectopic head-like structures;31 6. Chimeric embryos containing Otx2–/– epiblast cells (Otx2–/– embryos are headless, see below) and wild type visceral endoderm (VE) rescue an early neural plate but fail to develop a brain, whilst chimeric embryos containing Otx2–/– VE and wild type epiblast display all the features of Otx2–/– embryos.67 On the other hand, considerable evidence has also suggested a role of the axial mesendoderm in the maintenance and/or specification of anterior character. In fact: 1. Explant recombination experiments in mouse embryos show that a positive signal from anterior mesendoderm of headfold stage is able to maintain Otx2 expression in the anterior ectoderm of early streak embryos, and a negative signal from posterior

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The Development of Dopaminergic Neurons

mesendoderm represses Otx2 expression in anterior ectoderm of late streak embryos;59 2. Transplantation of an early organizer in Xenopus induces both anterior and posterior nervous systems;2 3. In chick the prechordal region contributes during early development of the central nervous system to instructing cells to acquire anterior regional identities.68 Interestingly, most of the genes having head organizer activity such as Otx2, Lim1, HNF-3b and cerberus are expressed either in the AVE or in the node and/or node-derived axial mesendoderm (Fig. 1.1), suggesting that AVE and axial mesendoderm might share common regulatory properties taking place at different developmental stages. Mice lacking the homeobox-containing genes Lim1 and Otx2 display a headless phenotype. The headless phenotypes have been interpreted as deriving from a failure of head-organizing properties of prechordal mesendoderm,60-62,69 although the earliest impairment in Otx2–/– mice was observed at the pre-early-streak stage in the VE,60 thus suggesting that contrary to previous evidence an impairment of VE was responsible for the onset of head organizing properties. Based on previous findings, it can be argued that specification and patterning of the early neural plate begins at the early-pre-streak stage and is mediated by head organizing properties of the AVE, where several genes might contribute to the establishment of the signal pathway leading to the first specification of the neural plate. This signal(s) might persist throughout the gastrulation process until the late-streak stage. At late-streak/headfold stage, signals from the node-derived axial mesendoderm might act to maintain the earliest specification and to induce more posterior trunk structures. At this stage, signals throughout the neuroectodermal plane might coexist with vertical signals from the surrounding mesendoderm, to refine and distinguish early neural plate fated to give fore-midbrain from more posterior neuroectoderm. This early regionalization is first evident at the healdfold stage when the Otx2-expressing domain (future fore-midbrain) is adjacent to Gbx2-expressing territory (rostral hindbrain).70

Patterning Mechanisms in the Neural Plate After the neural induction and rostral specification of the neural plate, development progresses with the regionalization of the planar sheet of pseudostratified neuroepithelium. This regionalization generates different histogenetically specified anlages that develop into structural and functionally different areas of the adult CNS. Fate maps of the neural plate show that the arrangement of histogenetic primordia in the neural plate is basically a flattened representation of the topological relationships in the mature brain.10 These prospective maps and gene expression patterns in the neural plate suggest that some aspects of the neural plate patterning can be simplified to a two-dimensional problem: patterning along the longitudinal and transversal dimensions.

Induction of Longitudinal Domains This process is related to the medio-lateral (M-L) patterning of the neural plate. Four longitudinal columns can be described along the CNS: the floor, basal, alar and roof plates (Fig. 1.2). These longitudinal columns are specified in a medio-lateral dimension in the neural plate. During the process of neurulation the edges of the neural plate thicken and move upward to form the neural folds, the movement generating the neural groove. Then the neural folds migrate toward the dorsal midline and fuse, closing the neural tube. Thus medio-lateral (M-L) patterning of the neural plate is topologically equivalent to the ventro-dorsal patterning of the neural tube. It has been established that within the posterior neural plate the M-L regional identities are specified in part by molecules produced by adjacent non-neural tissues.71 The molecular

Specification and Patterning of the Rostral Neural Tube

AVE cer-l Hesx1 Otx2

Lim1 gsc

5

Epiblast

Epi

Visceral Endoderm Mesoderm

VE

Fig.1.1. Schematic representation of the early-mid-gastrula mouse embryo showing major tissue components (epiblast, mesoderm along the primitive streak, and visceral endoderm). Several genes at this stage appear expressed within the AVE or in the node at the anterior tip of the primitive streak or in both tissues. Most of these genes (cerberus in Xenopus, Lim1, Otx2, Hesx1) have proven head organizing properties. Therefore, there is now increasing evidence that as early as early-mid-streak stage in mouse two different organizers might be identified: the AVE (head organizer) and the node (trunk organizer).

mechanisms underlying the inductive process of longitudinal regionalization are being elucidated. Both gain-of-function and loss-of-function experiments demonstrate that medial inductive signaling is regulated by Shh protein. Shh is first expressed in the notochord and then induced in the floor plate of the epichordal neural plate and tube.72-79 Because the notochord does not underlie the anterior forebrain (the anterior limit underlies the posterior diencephalon), it is unclear whether patterning of medial forebrain is regulated by mechanisms distinct from more posterior regions. Several lines of molecular and genetic evidence now suggest that medial and ventral specification of the forebrain is regulated by the prechordal plate using molecular mechanisms (e.g., Shh) that are also employed in more posterior CNS regions. Several genes are expressed along the longitudinal columns. For example, the expression patterns of Nkx2.2, Shh and HNF-3b are distributed all along the entire medial (ventral) neural tube,80,81 while the expression of other genes is localized in a precise segmental pattern inside a columnar domain. For example, the medial region of the anterior neural plate expressing Nkx2.1 is then localized in the anterior ventral prosencephalon17 and is essential for development of the anterior forebrain basal plate.82 Shh is expressed in the endoderm underlying the forebrain anlage, thus suggesting that it could be a signal required for patterning of the ventral forebrain. In fact, Ericson et al79 showed that Shh can induce Nkx2.1 in forebrain neural plate explants. Recently, Chiang et al78 showed that mice lacking a functional Shh gene lack essential functions required in

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The Development of Dopaminergic Neurons

Fig. 1.2. Longitudinal columns in the neural plate. (A) Scheme of a mouse neural plate (stage E8, dorsal view). Four longitudinal columns are descrived along the CNS. From the midline to lateral these columns are called: floor, basal, alar, and roof plates. Transversal lines represent limits between prosencephalic segmental domains. (B) Representation of vertical inductive influences at the neural plate stage. Morphogenetic signals are coming from the notochord (black arrows) and prechordal plate (AVE; gray arrows) to regionalize the neural plate in antero-posterior segments and longitudinal columns. At the top of the notochord the zona limitans (ZL) is specified in the neural plate and at this point bilateral symmetry is transformed in radial symmetry.

ventral patterning of the entire brain. Zebrafish mutant analyses, such as of cyclops, one-eye-pinhead and uncle freddy,83-85 have recently reinforced the idea favoring a common signaling mechanism for medial patterning in the CNS. These studies strongly suggest that a “vertical induction” from the prechordal plate is required for initial patterning of medial prosencephalic neural plate. Shimamura and Rubenstein86 reported that the prechordal plate may function alone in the initial specification of the medial prosencephalon.

Induction of Transversal Domains The A-P patterning leads to the generation of distinct transverse domains at different longitudinal positions in the CNS. There is evidence that A-P patterning begins during early gastrulation. We have described how vertical signals from underlying tissues (mesoderm and endoderm) and planar signals from the organizer may contribute to the initial specification of A-P regional differences in the neural plate.10,87 We now analyze how A-P

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patterning generates transverse zones that act as local organizers and how differential territorial competence is established.

Development of Regional Organizers Three regions having inducing properties have been described within the anlage of CNS: the node, the anterior boundary organizer and the isthmic organizer (Fig. 1.3). The node is required in neural plate specification and initial regionalization of the brain anlage; the anterior boundary organizer (ANB) in zebrafish embryos is an ectodermal region in the prospective head that is required for patterning of the anterior brain;88 and the isthmic organizer represents the organizing center for normal development of the mid-hindbrain area.89 The latter corresponds to the isthmus, which is a neural tranversal domain localized in the constriction between mesencephalic and rhombencephalic vesicles, at the midbrain-hindbrain junction. The region develops to produce cerebellar and isthmic structures.89 Experimental manipulations have demonstrated morphogenetic-inducing properties of this neuroepithelium and suggested a fundamental role in the normal specification and patterning of cerebellar, isthmic and mesencephalic territories. This region influences development of adjacent neuroepithelial zones, either in its normal position, or in ectopical graft in a more rostral position of the neural tube.89,90 The isthmic organizer region retains a characteristic pattern of gene expression (Fig. 1.4). Four different transcription factors, encoded by the homeobox genes En1 and En2 and the paired box genes Pax2 and Pax5, are expressed in the mouse isthmus together with two secreted signaling molecules, Wnt1 and Fgf8.21,91,92 Both the activation and expression pattern of these genes are required for proper development of the mid-hindbrain regions including the organizer.21,91,92 Very recently, new findings have contributed to the knowledge of isthmic organizer development. Early expression of Otx2 and Gbx2 highlights the initial regionalization of the anterior neural plate. In fact, these genes are expressed in complementary adjacent territories of the anterior neural epithelium, and at the late gastrula stage the caudal edge of Otx2 coincides with the rostral limit of Gbx2 expression (Fig. 1.4). The isthmic organizer develops exactly in the region where these two genes contact each other, suggesting that the interaction between their borders of expression may establish an initial signal to specify or stabilize the position of the organizer. Loss-of-function of Gbx293 and low doses of Otx2 gene products94 affect the molecular pattern of the isthmic organizer as well as the mid-hindbrain development. Heterotopic quail/chick grafts showed that ectopic contact of Gbx2 and Otx2 expressing domains induce ectopic expression of the Fgf8, the isthmic organizing molecule (Cobos, Garda, Martínez, in preparation). In fact, beads soaked in FGF8 protein can induce, when ectopically inserted in chicks embryos, morphogenetic effects similar to those observed after ectopic grafts of the isthmus.91

Different Histogenetic Competence There is evidence that AP patterning can generate transverse blocks of neuroepithelium that have distinct fate and competence to respond to the same inductive signal.16,17,75,79,95-97 FGF8 is an example of an inductive signal that generates distinct molecular responses at different axial levels. This signaling molecule either recapitulates many of the inductive properties of the isthmic organizing center, or induces the expression of distinct genes such as BF1 anteriorly and En2 posteriorly.86 The boundary between these two different responses to the same inductive signal may correspond to a single boundary of the zona limitans intrathalamica (ZL; Fig. 1.2).10 The ZL is a transverse boundary located between prosomere 2 and 3 in the diencephalon.17

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The Development of Dopaminergic Neurons

Fig. 1.3. Neural organizers. Schematic representation of a mouse embryo at stage E7.5. Lateral view (at the left) and cephalic pole of hte embryo in a midsagital section (at the right). The three organizers have been identified (large black arrows). The planar morphogenic activities from each organizer are represented by empty arrows. Additional interneuromeric boundaries define transversal segments in the rhombencephalon and anterior brain.10-12

Local Patterning Centers in Further Defining the Organization of the Forebrain Besides the general mechanisms specifying A-P and M-L patterning, there is evidence that the local patterning centers underlie further levels of complexity. Some of these local patterning centers arise in specialized ectoderm tissues such as the olfactory and lens placode, the anterior neural ridge and the Rathke’s pouch.98-103 In summary, regionalization of the anterior neural plate appears to result from the contribution of multiple distinct patterning mechanisms. A-P patterning creates transverse zones with differential competence within the neural plate. Patterning along the M-L axis generates longitudinally aligned domains. The combination of M-L and A-P patterning then generates a grid-like organization of distinct histogenic brain primordia. Additional levels of regional complexity are then generated by local sources (Fig. 1.5).

Acknowledgments We thank D. Acampora and M. Gulisano for helpful discussions and A. Secondulfo for typing the manuscript.

Specification and Patterning of the Rostral Neural Tube

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Fig. 1.4. The ismic organizer. Representation of a mouse embryo at stage E8 (lateral view) A sagital section of the cephalic neural plate is schematized in the drawing on the right. The domains of several gene expressions are mapped by different frames (described in square boxes). AVE: anterior visceral endoderm.

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Fig. 1.5. Prosomeric model and local sources of morphogenetic activity. Drawing of a lateral view of a mouse embryo neural tube at stage E10.5. Identified sources of planar morphogenetic boundaries. AN: anterior neuropore region; D/M: di/mesencephalic boundary; IsO: Isthnic organizer; ZL: zona limitans. 16. Puelles L, Amat JA, Martinez del la Torre M. Segment-related, mosaic neurogenetic pattern in the forebrain and mesencephalon of early chick embryos: I. Topography of AChE-positive neuroblasts up to stage HH18. J Comp Neurol 1987; 266:247-268. 17. Rubenstein JLR, Martinez S, Shimamura K et al. The embryonic vertebrate forebrain: The prosomeric model. Science 1994; 266:578-580. 18. Lewis EB. A gene complex controlling segmentation in Drosophila. Nature 1978; 276:565-570. 19. Finkelstein R, Boncinelli E. From fly head to mammalian forebrain: The story of otd and Otx. Trends Genet 1994; 10:310-315. 20. Krumlauf R. Hox genes in vertebrate development. Cell 1994; 78:191-201.

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21. Joyner AL. Engrailed, Wnt and Pax genes regulate midbrain-hindbrain development. Trends Genet 1996; 12:15-20. 22. Nieuwkoop PD, Albers B. The role of competence in the cranio-caudal segregation of the central nervous system. Dev Growth Differ 1990; 32:23-31. 23. Saxén L. Neural induction. Int J Dev Biol 1989; 33:21-48. 24. Ruiz i Altaba A, Melton DA. Interaction between peptide growth factors and homeobox genes in the establishment of anterior-posterior polarity in frog embryos. Nature 1989; 341:33-38. 25. Ang S-L, Rossant J. Anterior mesendoderm induces mouse Engrailed genes in explant culture. Development 1993; 118:139-149. 26. Dixon J, Kintner CR. Cellular contacts required for neural induction in Xenopus embryos: Evidence for two signals. Development 1989; 106:749-757. 27. Papalopulu N, Kintner CR. Xenopus distal-less related homeobox genes are expressed in the developing forebrain and are induced by planar signals. Development 1993; 117:961-975. 28. Eagelson GW, Harris WA. Mapping of the presumptive brain regions in the neural plate of Xenopus laevis. J Neurobiol 1989; 21:427-440. 29. Jessell TM, Melton AD. Diffusible factors in vertebrate embryonic induction. Cell 1992; 68:257-270. 30. Smith JC. Hedgehog, the floor plate and the zone of polarizing activity. Cell 1994; 76:193-196. 31. Bouwmeester T, Kim SH, Sasai Y et al. Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann’s organizer. Nature 1996; 382:595-601. 32. Thomas P, Beddington R. Anterior primitive endoderm my be responsible for patterning the anterior neural plate in the mouse embryo. Curr Biol 1996; 6:1487-1496. 33. Thaller C, Eichele G. Identification and spatial distribution of retinoids in the developing chick limb bud. Nature 1987; 327:625-628. 34. Thaller C, Eichele G. Isolation of 3,4-didehydroretinoic acid, a novel morphogenetic signal in the chick wing bud. Nature 1990; 345:815-819. 35. Summerbell D, Maden M. Retinoic acid, a developmental signalling molecule. Trends Neurosci 1990; 13:142-147. 36. Tabin CJ. Retinoids, homeoboxes, and growth factors: Toward molecular models for limb development. Cell 1991; 66:199-217. 37. Pijnappel WWM, Hendriks HFJ, Folkers GE et al. The retinoid ligand 4-oxo-retinoic acid is a highly active modulator of positional specification. Nature 1993; 366:340-344. 38. Hogan BLM, Thaller C, Eichele G. Evidence that Hensen’s node is a site of retinoic acid synthesis. Nature 1992; 359:237-241. 39. Chen YP, Huang L, Russo AF et al. Retinoic acid is enriched in Hensen’s node and is developmentally regulated in the early chicken embryo. Proc Natl Acad Sci USA 1992; 89:10056-10059. 40. Wagner M, Thaller C, Jessell T et al. Polarizing activity and retinoid synthesis in the floor plate of the neural tube. Nature 1990; 345:819-822. 41. Wagner M, Han B, Jessell TM. Regional differences in retinoid release from embryonic neural tissue detected by an in vitro reporter assay. Development 1992; 116:55-66. 42. Rossant J, Zirngibl R, Cado D et al. Expression of a retinoic acid response element-hsplacZ transgene defines specific domains of transcriptional activity during mouse embryogenesis. Genes Dev 1991; 5:1333-1344. 43. Balkan W, Colbert M, Bock C et al. Transgenic indicator mice for studying activated retinoic acid receptors during development. Proc Natl Acad Sci USA 1992; 89:3347-3351. 44. Conlon RA, Rossant J. Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 genes in vivo. Development 1992; 116:357-368. 45. McGinnis W, Krumlauf R. Homeobox genes and axial patterning. Cell 1992; 68:283-302. 46. Durston AJ, Timmermans JPM, Hage WJ et al. Retinoic acid causes an anteroposterior transformation of the developing central nervous system. Nature 1989; 340:140-144. 47. Sive HL, Draper BW, Harland RM et al. Identification of a retinoic acid-sensitive period during primary axis formation in Xenopus laevis. Genes Dev 1990; 4:932-342.

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48. Ruiz i Altaba A, Jessell TM. Retinoic acid modifies the pattern of cell differentiation in the central nervous system of nerula stage Xenopus embryos. Development 1991; 112:945-958. 49. Morriss-Kay GM, Murphy P, Hill RE et al. Effects of retinoic acid excess on expression of Hox-2.9 and Krox-20 and on morphological segmentation in the hindbrain of mouse embryos. EMBO J 1991; 10:2985-2995. 50. Papalopulu N, Clarke JDW, Bradley L et al. Retinoic acid causes abnormal development and segmental patterning of the anterior hindbrain in Xenopus embryos. Development 1991; 113:1145-1158. 51. Holder N, Hill J. Retinoic acid modifies development of the midbrain-hindbrain border and affects cranial ganglion formation in zebrafish embryos. Development 1991; 113:1159-1170. 52. Wanek N, Gardiner DM, Muneoka K et al. Conversion by retinoic acid of anterior cells into ZPA cells in the chick wing bud. Nature 1991; 350:81-83. 53. Noji S, Nohno T, Koyama E et al. Retinoic acid induces polarizing activity but is unlikely to be a morphogen in the chick limb bud. Nature 1991; 350:83-86. 54. Marshall H, Nonchev S, Sham MH et al. Retinoic acid alters hindbrain Hox code and induces transformation of rhombomeres 2/3 into 4/5 identity. Nature 1992; 360:737-741. 55. Simeone A, Acampora D, Arcioni L et al. Sequential activation of the HOX2 homeobox genes by retinoic acid in human embryonal carcinoma cells. Nature 1990; 346:763-766. 56. Simeone A, Acampora D, Nigro V et al. Differential regulation by retinoic acid of the homeobox genes of the four Hox loci in human embryonal carcinoma cells. Mech Dev 1991; 33:15-228. 57. Simeone A, Avantaggiato V, Moroni MC et al. Retinoic acid induces stage-specific antero-posterior transformation of rostral central nervous system. Mech Dev 1995; 51:83-98. 58. Avantaggiato V, Acampora D, Tuorto F et al. Retinoic acid induces stage-specific repatterning of the rostral central nervous system. Dev Biol 1996; 175:347-357. 59. Ang S-L, Conlon RA, Jin O et al. Positive and negative signals from mesoderm regulate the expression of mouse Otx2 in ectoderm explants. Development 1994; 120:2979-2989. 60. Acampora D, Mazan S, Lallemand Y et al. Forebrain and midbrain regions are deleted Otx2-/- mutants due to a defective anterior neuroectoderm specification during gastrulation. Development 1995; 121:3279-3290. 61. Matsuo I, Kuratani S, Kimura C et al. Mouse Otx2 functions in the formation and patterning of rostral head. Genes Dev 1995; 9:2646-2658. 62. Ang S-L, Jin O, Rhinn M et al. Targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. Development 1996; 122:243-252. 63. Varlet I, Collignon J, Robertson EJ. nodal expression in the primitive endoderm is required for specification of the anterior axis during mouse gastrulation. Development 1997; 124:1033-1044. 64. Beddington RSP. Induction of a second neural axis by the mouse node. Development 1994; 120:613-620. 65. Shih J, Fraser SE. Distribution of tissue progenitors within the shield region of the zebrafish gastrula. Development 1995; 121:2755-2765. 66. Shih J, Fraser SE. Characterizing the zebrafish organizer: Microsurgical analysis at the early-shield stage. Development 1996; 122:1313-1322. 67. Rhinn M, Dierich A, Shawlot W et al. Sequential roles for Otx2 in visceral endoderm and neuroectoderm for forebrain and midbrain induction and specification. Development 1998; 125:845-856. 68. Foley AC, Storey KG, Stern CD. The prechordal region lacks neural inducing ability, but can confer anterior character to more posterior neuroepithelium. Development 1997; 124:2983-2996. 69. Shawlot W, Behringer RR. Requirement for Lim-1 in head organizer function. Nature 1995; 374:425-430.

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70. Montzka Wassarman K, Lewandoski M, Campbell K et al. Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer is dependent on Gbx2 gene function. Development 1997; 124:2923-2934. 71. Tanabe Y, Jessell TM. Diversity and pattern in the developing spinal cord. Science 1996; 274:1115-1123. 72. Echelard Y, Epstein DJ, St-Jacques B et al. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 1993; 75:1417-1430. 73. Roelink H, Augsburger A, Heemskerk J et al. Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord. Cell 1994; 76:761-775. 74. Roelink H, Porter JA, Chiang C et al. Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis. Cell 1995; 81:445-455. 75. Hynes M, Poulsen K, Tessier-Levigne M et al. Control of neuronal diversity by the floor plate: Contact-mediated induction of midbrain dopaminergic neurons. Cell 1995; 80:95-102. 76. Martì E, Bumcrot DA, Takada R et al. Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cells types in CNS explants. Nature 1995; 375:322-325. 77. Tanabe Y, Roelink H, Jessell TM. Induction of motor neurons by Sonic hedgehog is independent of floor plate differentiation. Curr Biol 1995; 5:561-558. 78. Chiang C, Litingtung Y, Lee E et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 1996; 383:407-413. 79. Ericson J, Morton S, Kawakami A et al. Two critical periods of Sonic hedgehog signaling required for the specification of motor neuron identity. Cell 1996; 87:661-673. 80. Sasaki H, Hogan LM. Differnetial expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development 1993; 118:47-59. 81. Shimamura K, Hartigan DJ, Martinez S et al. Longitudinal organization of the anterior neural plate and neural tube. Development 1995; 121:3923-3933. 82. Kimura S, Hara Y, Pineau T et al. The T/ebp null mouse: Thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain and pituitary. Genes Dev 1996; 10:60-69. 83. Hatta K, Puschel AW, Kimmel CB. Midline signalling in the primordium of the zebrafish anterior central nervous system. Proc Natl Acad Sci USA 1994; 91:2061-2065. 84. Hammerschmidt M, Pelegri F, Mullins MC et al. Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio. Development 1996; 123:143-151. 85. Schier AF, Neuhauss SCF, Harvey M et al. Mutations affecting the development of the embryonic zebrafish brain. Development 1996; 123:165-178. 86. Shimamura K, Rubenstein JLR. Inductive interactions direct early regionalization of the mouse forebrain. Development 1997; 124:2709-2718. 87. Doniach T. Planar and vertical induction of anteroposterior pattern during the development of the amphibian central nervous system. J Neurobiol 1993; 24:1256-1276. 88. Houart C, Westerfield M, Wilson SW. A small population of anterior cells patterns the forebrain during zebrafish gastrulation. Nature 1998; 391: 788-792. 89. Martinez S, Wassef M, Alvarado-Mallart R-M. Induction of a mesencephalic phenotype in the 2 day-old chick prosencephalon is preceded by the early expression of the homeobox gene en. Neuron 1991; 6:971-981. 90. Marin F, Puelles L. Patterning of the embryonic avian midbrain after experimental inversions: A polarizing activity from the isthmus. Dev Biol 1994; 163:19-37. 91. Crossley PH, Martinez S, Martin GR. Midbrain development induced by FGF8 in the chick embryo. Nature 1996; 380:66-68. 92. Bally-Cuif L, Wassef M. Determination events in the nervous system of the vertebrate embryo. Curr Opin Genet Dev 1995; 5:450-458. 93. Montzka Wassarman K, Lewandoski M, Campbell K et al. Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer is dependent on Gbx2 gene function. Development 1997; 124:2923-2934.

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94. Acampora D, Avantaggiato V, Tuorto F, Simeone A. Genetic control of brain morphogenesis through Otx gene dosage requirement. Development 1997; 124:3639-3650. 95. Yamada T, Placzek M, Tanaka H et al. Control of cell pattern in the developing nervous system: Polarizing activity of the floor plate and notochord. Cell 1991; 64:635-647. 96. Simeone A, Acampora D, Gulisano M et al. Nested expression domains of four homeobox genes in developing rostral brain. Nature 1992; 358:687-690. 97. Simon H, Hornbruch A, Lumsden A. Independent assignment of antero-posterior and dorso-ventral positional values in the developing chick hindbrain. Curr Biol 1995; 5:205-214. 98. Jacobson AG. The determination and positioning of the nose, lens and ear. I. Interactions within ectoderm, and between the ectoderm and underlying tissues. J Exp Zool 1963; 154:273-284. 99. Daikoku S, Chikamori M, Adachi T et al. Ontogenesis of hypothalamic immunoreactive ACTH cells in vivo and in vitro: Role of Rathke’s pouch. Dev Biol 1983; 97:81-88. 100. Graziadei PPC, Monti-Graziadei AG. The influence of the olfactory placode on the development of the telencephalon in Xenopus laevis. J Neuroscience 1992; 46:617-629. 101. Saha MS, Servetnick M, Grainger RM. Vertebrate eye development. Curr Opin Genet Dev 1992; 2:582-588. 102. Byrd CA, Burd GD. The quantitative relationship between olfactory axons and mitral/tufted cells in developing Xenopus with partially deafferented olfactory bulbs. J Neurobiol 1993; 24:1229-1242. 103. Webb JF, Noden DM. Ectodermal placodes: Contributions to the development of the vertebrate head. Am Zool 1993; 33:434-447.

CHAPTER 2

Specification of Dopaminergic Neurons: Inductive Factors and Their Receptors Mary Hynes and Arnon Rosenthal

T

he vertebrate nervous system is composed of multiple cell types which develop in stereotypic positions along the dorso-ventral (D-V) and anterior-posterior (A-P) axes of the neural tube. While the mechanisms controlling this process are not fully understood (reviewed in refs. 1, 2), it has been proposed that signaling centers which operate along the two main axes of this system establish an epigenetic grid of Cartesian coordinates, and that neural progenitors assume distinct cell fates according to their location on this grid (see, e.g., refs. 3, 4). Consistent with the idea that neuronal cell fate is specified by epigenetic factors, grafting experiments have demonstrated that neural progenitors can acquire new identities if moved to ectopic locations.5-9 In addition, transplantation and explant culture studies have confirmed the existence of signaling centers which can change the fate of juxtaposed neural progenitors. For example, the dorsal ectodermal epidermis, roof plate, floor plate and notochord have been shown to instruct cell fates along the D-V axis (reviewed in ref. 2),while the prechordal plate, paraxial mesoderm, mid-hindbrain boundary (isthmus) and the anterior neural ridge (ANR) were shown to change cell fate along the A-P axis of the neural tube.1,7,8,10-14 In addition, a number of secreted proteins and chemicals have been identified which can modify cell fate in a characteristic fashion. Thus, Shh and BMP proteins influence cell fate along the D-V axis (reviewed in ref. 2), and FGF2, FGF8, retinoic acid and Wnt1 can change cell fate along the A-P axis (reviewed in refs. 1, 13, 15). The idea that neuronal cell fate is specified by signaling centers and secreted molecules was used as a framework to determine how dopaminergic (DA) neurons in the anterior part of the brain (midbrain and forebrain) are specified during embryogenesis. Midbrain DA neurons innervate the striatum, limbic system and neocortex, and reside in the ventral midbrain together with several other classes of neurons including motor neurons (Fig. 2.1A). The loss of midbrain DA neurons results in the motor disorders of Parkinson’s disease,16 and their abnormal function has been associated with schizophrenia and drug addiction.17-19 Forebrain DA neurons reside in the hypothalamus,20 and a population of these neurons sends axons to the pituitary to regulate hormonal release into the circulation. Despite the clinical importance of dopaminergic neurons, the mechanisms that direct their development were unknown until relatively recently. This chapter summarizes work that pertains to inductive factors and their receptors, which play a role in the specification of DA neurons in the anterior brain. The Development of Dopaminergic Neurons, edited by Umberto di Porzio, Roberto Pernas-Alonso and Carla Perrone-Capano. ©1999 R.G. Landes Company.

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Induction Along the D-V Axis Ontogenetic studies in the rat showed that midbrain dopaminergic (DA) neurons are born between embryonic days ~12-15 (E12-15), near the midbrain-hindbrain junction (rhombic isthmus) (see refs. 21-25; Fig.2.1A). These neurons begin to express tyrosine hydroxylase (TH) (the rate limiting enzyme in dopamine synthesis) by E12.5,26 and then migrate extensively from the rhombic isthmus in a rostro-ventral direction to their final positions in the ventral midbrain (the substantia nigra, ventral tegmental area and retrorubral field) (see ref. 24). Although these studies showed that DA neurons were born in the ventral portion of the midbrain (see ref. 22), the relationship of these neurons to the floor plate was not defined. Sections of embryonic rat midbrain marked with antibodies to a floor plate marker, FP4,27 and to three markers of midbrain dopaminergic neurons, dopamine (the neurotransmitter used by these neurons), TH and the retinoic acid converting enzyme AHD-2, showed that at E14, soon after DA neurons differentiate, TH+ neurons are found in close proximity to floor plate cells, and many appear to reside within the floor plate (Fig. 2.1B). These neurons did not simply migrate to the floor plate after they were specified, but instead appear to assume their identity in close proximity to the floor plate. This is evident from the finding that a narrow (~100 µ m wide) explant comprising the midline of the midbrain, isolated from E11 embryos, before DA neurons are born, give rise to many DA neurons after 3 days in culture. Similar midline explants derived from spinal cord levels never give rise to DA neurons.28 Thus, DA neurons appear to be born and differentiate near the midbrain floor plate. Using in vitro tissue recombination studies, it was further shown that an exogenous floor plate could induce DA neurons in vitro. The ventral aspect of the E9 rat embryo (3 somite) neural tube, devoid of the midline and endogenous floor plate, were grown in vitro in contact with E12 spinal cord floor plate, midbrain floor plate, or with control tissue for 5 days. Coculture with floor plate derived from spinal cord or midbrain caused the induction of many TH+, dopamine+, AHD-2+ DA neurons (more than 100 neurons/explant), whereas dorsal neural tube cultured with control tissue did not give rise to any TH+ neurons.28,29 Neuronal differentiation, identified with an antibody to intermediate filament, occurred in all the explants, irrespective of the presence of a floor plate. When the E9 explants were further divided along the longitudinal axis into dorsal (lateral) and ventral (medial) pieces, and placed in contact with exogenous floor plate, induction of DA neurons was detected in both the future dorsal and ventral explants.28 Thus, the floor plate not only restores development of DA neurons in the appropriate ventral location, but can induce them in dorsal midbrain regions where they are normally not found. A lower frequency of induction was observed in explants from E10-12 embryos,28 suggesting that the fate of cells along the dorsoventral axis of the midbrain becomes restricted soon after neural tube closure. These findings were consistent with previous evidence that the floor plate and notochord could influence the development of particular neuronal classes in the spinal cord and hindbrain. For example, spinal motoneurons, hindbrain serotonergic neurons and BMP-6/ DVR-6-expressing cells could be induced in dorsal locations by a contact-dependent signal emanating from an ectopic notochord or floor plate.30,31 Likewise, motoneurons were shown to be induced by a diffusible factor from the floor plate.32

Specification of Dopaminergic Neurons: Inductive Factors and Their Receptors

17

Fig. 2.1. DA Neurons Reside Near the Floor Plate, and are Induced by the Floor Plate and Shh-N.(A)Sagittal section through an E14 rat embryo hybridized with a probe to tyrosine hydroxylase (TH). TH is a synthetic enzyme for DA, and a marker of DA neurons. (B) Coronal section through the ventral midbrain of E14 rat embryo, stained for floor plate cells (FP), and DA neurons (TH). (C and D) sagittal sections of E14 WT (C) and En2-HNF-3β transgenic (D) embryos, stained with antibodies to dopamine. Dopamine expressing cells are found only in a ventral location in WT embryos (C), but in both the dorsal and ventral midline regions of transgenic embryos (D). (E,F) E9 neural tube explant grown at a distance (~40µ m) (E), or in contact (F), with an E12 spinal cord FP for 5 days, in vitro, in a collagen gel matrix. TH + DA neurons develop in the NT when it is grown in contact with FP (F). (G and H) E9 explants grown for 5 days with control CM (G) or Shh-N CM (230 nM) (H), and stained for TH.

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The Development of Dopaminergic Neurons

Induction of a Supernumerary Floor Plate In Vivo Results in the Appearance of Ectopic DA Neurons Further evidence for a physiological role of the floor plate in specification of DA neurons was provided by transgenic mice expressing the hepatocyte nuclear factor 3βHNF-3β) gene in ectopic, dorsal mid-hindbrain regions, under the control of the Engrailed 2 (En2) promoter enhancer. These mice develop a floor plate-like structure in the dorsal neural tube31, and a second group of dopamine+ neurons in the dorsal midbrain, where DA neurons normally do not develop28 (Fig. 2.1C,D). These dorsal, TH+ neurons reside adjacent to the induced floor plate which expresses Shh,28 a marker of floor plate.33,34 Although in these mice, dopamine+ neurons were found adjacent to Shh-expressing cells in both ventral and dorsal locations, they were still restricted to the same A-P level as DA neurons in wild type (WT) animals28 (Fig. 2.1E,F). Thus, the floor plate can control DA fate along the D-V, but not the A-P axis.

Induction of Midbrain DA Neurons is Mediated by Contact with the Floor Plate The developing ventral midbrain contains DA neurons, located near the floor plate, and motoneurons, located more dorsally. Experimental data indicated that both types of neurons could be specified by the floor plate,28-30,32 raising the question of how this is achieved. In the spinal cord, motoneurons were shown to develop at a distance from the ventral midline and to be induced by a diffusible factor secreted, e.g., from the floor plate.32 In contrast, DA neurons were found in close proximity to FP4+ floor plate cells both in vivo and in explant culture, suggesting that induction of these cells might require contact with floor plate cells. In fact, this is the case; E9 dorsal neural tube explants grown in contact with floor plate show induction of DA neurons, whereas explants grown at a distance from the floor plate do not28 (Fig. 2.1E,F). Thus, the induction of DA neurons by the floor plate is via a contact-dependent signal, or requires high concentrations of a diffusible signal.

Shh Mediates the Induction of DA Neurons by Floor Plate Candidate molecules involved in such a short-range induction of DA neurons initially included members of the Wnt, hedgehog (HH), transforming growth factor β(TGF-β)-like molecules, or ligands of tyrosine kinase receptors, since several of these molecules function as short-range inductive signals in other systems (for review see refs. 35-38). The fact that DA neurons differentiate adjacent to Shh-expressing cells,28,39 and that this molecule was implicated in patterning and growth of a variety of tissues in insects and vertebrates,37,40 including the contact-dependent induction of floor plate cells by notochord cells33,34,41-43 and the induction of motoneurons,42-44 suggested Shh as a particularly good candidate for the floor plate inducer of DA neurons. Consistent with this possibility, we found that agonists of cAMP which were shown to block the Hh response in Drosophila45-48 and vertebrates49 prevented the induction of DA neurons by an endogenous or exogenous floor plate.28 Shh is synthesized as a 45 kDa precursor protein that undergoes an autoproteolytic cleavage to yield an ~20 kDa amino-terminal cleavage product (Shh-N), which remains mostly cell-associated, and a carboxy-terminal cleavage product (Shh-C), which diffuses more freely.42,50-52 Only the amino-terminal products of the Hh (i.e., Shh-N) family members have been implicated in mediating signaling activities in Drosophila,51 and in vertebrates.42,49 To test the ability of Shh-N to specify DA neurons in vitro, dorsal midbrain explants were cultured for 5 days in the presence of Shh-N conditioned medium (Shh-N CM) made in 293 cells expressing residues 1-198 of Shh.42,51 The Shh-N CM was able to induce FP3/4+ floor plate cells, and TH+ DA neurons in a dose-dependent fashion (Fig. 2.1G,H and

Specification of Dopaminergic Neurons: Inductive Factors and Their Receptors

19

ref. 39). TH+ DA neurons could be induced by Shh-N CM in the absence of differentiated FP3/4+ floor plate cells, suggesting that Shh-N induces DA neurons directly, in the absence of differentiated floor plate cells. A similar induction was seen using recombinant Shh-N which was produced in bacteria, suggesting that Shh-N could substitute for the floor plate.28 Shh was not only sufficient to induce ectopic DA neurons in the dorsal midbrain, but it was also shown to be necessary for the development of endogenous DA neurons in the ventral midbrain, as illustrated by the fact that these neurons failed to appear in the presence of Shh blocking antibodies.53 A similar dependency on Shh was reported for DA neurons in the forebrain53, and motoneurons in the spinal cord.54 Taken together, these findings were consistent with the idea that Shh, derived from the floor plate and notochord, is necessary and sufficient for the induction of DA, and other neuronal cell types, along the D-V axis of the neural tube.

Shh Receptors and Intracellular Signaling Molecules Genetic studies in Drosophila implicated a number of molecules in the Hh receptor/ signaling cascade. Two transmembrane proteins served as candidate receptors: the 12 transmembrane (TM) protein Patched (Ptc),55-58 and the 7 TM protein, Smoothened (Smo).59-62 Genetic data suggested that Ptc was a negative regulator of the Hh signal, as the Hh signaling cascade is constitutively active in its absence. In contrast, Smo was suggested to be an essential component in the Hh pathway; Smo mutants display the same phenotype as Hh mutants62. Cloning of the vertebrate homologs of Ptc (vPtc and Ptc2)63-68 and Smo (vSmo)69 has allowed, first, localization of these molecules in the developing vertebrate embryo to address their suitability as candidate receptors, and, second, biochemical experiments to examine possible physical interactions between Hh family members and their putative receptors. In situ hybridization analyses in the rat revealed that vSmo and vPtc were found in all Shh-responsive tissues, such as the early neural folds and neural tube,28,33,34,41,42,69 pre-somitic mesoderm and somites,49,70 and developing limb bud,71 gut72 and eye.61 Transcripts for vSmo were also observed in tissues whose development is regulated by other members of the vertebrate Hh protein family,69 such as embryonic testes (Dhh),73 cartilage (Ihh)74 and muscle (the zebrafish echidna Hh).75 vPtc2 is expressed primarily in the testes, and is not detectable in the brain.67, 68 The expression of vSmo and vPtc shows considerable overlap; for example, in the embryonic nervous system vSmo and vPtc are initially expressed throughout the neural folds and early neural tube (vSmo mRNA is evenly distributed along the dorsal-ventral axis whereas vPtc mRNA is found at higher levels ventrally) and by E15 their mRNAs are restricted to cells which are in close proximity to the ventricular zone.69 vSmo and vPtc mRNAs are also found adjacent to Shh-expressing cells in the embryonic lung, epiglottis, thymus, tongue, jaw, taste buds, teeth and skin.69 Expression of these molecules continues in adults; vSmo mRNA can be found in multiple tissues including heart, brain, liver, lung, skeletal muscle, kidney and testis. In the brain, these molecules are concentrated in regions known for high plasticity, such as the hippocampus, cortex and cerebellum (MH and AR, unpublished observations and ref. 76). Consistent with the presence of Shh in adult tissues, this protein was found to be a survival factor for subpopulations of mature DA neurons,77 and a mitogen for granule cells in the cerebellum.78 To examine whether Shh could bind to vPtc or vSmo, in vitro, cell based binding studies were performed. Suprisingly, although Smo was deemed essential for Hh signaling in Drosophila, Shh could not bind to Cos-7 cells transfected with vSmo69 (Fig. 2.2A). In contrast, all three members of the vertebrate Hh family bound to cells overexpressing vPtc63,69 (Fig. 2.2A; Carpenter et al, submitted), and Shh could be coimmunoprecipitated with vPtc,

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The Development of Dopaminergic Neurons

Fig. 2.2. Shh-N binds to Patched but not to Smoothened (A) Shh-N binds to cells expressing vPtc but not to cells expressing vSmo. Staining of COS-7 cells expressing the Flag-tagged vSmo (left two panels), or (Myc-tagged vPtc (right two panels) with anti-Flag (Smo) antibody (Smo+) anti-Myc (vPtc) antibody (Ptc+), or with IgG-Shh-N. Only cells expressing vPtc bind Shh-N. No binding of other IgG fusion proteins to vPtc-expressing cells and no binding of the various tagged forms of Shh-N to untransfected cells were detected (data not shown). (B) Shh-N binds to vPtc but not to vSmo. (B; left), coimmunoprecipitation of epitope-tagged vPtc or epitope-tagged vSmo (Smo) with IgG-Shh-N (IgG-Shh), from cells expressing either vPtc or vSmo alone, as indicated. Only vPtc could be coimmunoprecipitated by the IgG-Shh-N protein. Immunoprecipitation of vSmo could be achieved only with antibodies to the Smo epitope tag (Smo Ab). (B; middle), crosslinking of 125I-Shh-N (125I-Shh) to cells expressing vPtc or vSmo in the absence or presence of excess unlabeled Shh-N (Cold Shh - or +). 125I-Shh-N could be crosslinked only to vPtc-expressing cells. (B; right), coimmunoprecipitation of 125I-Shh-N by antibodies to Myc-tagged vPtc (Patched) or to Flag-tagged vSmo (Smo). 125I-Shh-N could be coimmunoprecipitated with antibodies to the Myc-tagged vPtc, but not with antibodies to the Flag-tagged vSmo and only in cells that expressed vPtc. (C) Model describing the putative SHH receptor and its activation by Shh-N or following inactivation of Ptc. (It is important to note that the actual binding site of Shh-N to Ptc and the interaction sites between Ptc and Smo have not been determined at the molecular level. In addition, the illustrated secondary structures of Smo and Ptc are based only on the primary structure of these proteins and are therefore hypothetical.)

Specification of Dopaminergic Neurons: Inductive Factors and Their Receptors

21

but not with vSmo (Fig.2.2B). Shh binding to vPtc shows high affinity with a Kd of about 460 pM.69 Although Shh binds directly to vPtc, but not to vSmo, two lines of evidence suggest that vSmo, like its Drosophila counterpart, is involved in the Shh signaling cascade, and is a positive regulator of this signal. First, vPtc and vSmo reside together as a complex in the membrane, in the presence or the absence of Shh, and in fact the three molecules can be coimmunoprecipitated as a complex.69 Secondly, it has been shown that Shh, Ptc and Smo all play key roles in basal cell carcinoma (BCC), a syndrome comprised of skin cancer and developmental abnormalities. Thus, mutations leading to a truncated or unstable vPtc protein,65,66,79-82 as well as mutations which lead to a constitutively active Smo protein, were shown to be associated with familial and sporadic forms of BCC.83 Moreover, when the constitutively active vSmo mutant M2 (Smo-M2)83 or Shh84 is overexpressed in the skin of transgenic mice, under the control of the keratin promoter, skin lesions of the BCC type are induced. Likewise, mice which have reduced levels of Ptc develop medulloblastomas.85 Together, the genetic, biochemical and in vivo biological data suggest a model in which vPtc is a ligand-binding component, and vSmo a signaling component in a multisubunit Shh receptor. In addition, vPtc appears to be a ligand-regulated suppressor of the signaling unit vSmo69 (Fig. 2.2C). It remains to be determined whether Smo is constitutively active in the absence of Ptc or whether, under these circumstances, Smo would still require a specific ligand for activation. Of interest is the fact that expression of WT vSmo under the same Keratin promoter produced no detectable phenotype,83 suggesting that the endogenous Ptc, which is present in skin, is sufficient to downregulate the ectopically expressed WT vSmo, and to prevent aberrant Shh signaling. Alternatively, it is possible that Smo, by itself, is not capable of signal transduction in the skin and that the Smo-M2 mutant has assumed a novel activity. Finally, it remains possible that WT Smo requires an additional ligand for activation, and that release of inhibition by Ptc is not sufficient to activate this receptor.

Intracellular Mediators of the Shh Signal Genetic and biochemical studies in Drosophila have identified several intracellular molecules which appear to function as signaling components in the Shh pathway. These include the putative serine threonine kinase, Fused;86,87 a kinesin-like molecule designated Costal;88,89 a protein with novel structure, Suppressor of Fused;90 the transcription co-activator CBP;91 the zinc finger transcription factor Cubitus interruptus (Ci);92 and a protein, Slimb, which facilitates the degradation of Ci through the ubiquitin pathway.45 However, although several of these proteins were shown to form a large complex in the cell cytoplasm,88,89 no information is available as to the process by which this complex is activated by Smo, or on the mechanisms by which the signal is transduced to the cell nucleus. Within the nucleus, the zinc finger transcription factor Ci was shown to be necessary and sufficient to mediate many known functions of Hh in Drosophila.92-94 In vertebrates, therefore, attention has focused on the role of homologs of Ci, which include the three Gli genes Gli-1,95-99 Gli-2 and Gli-3,100 with the idea that at least one of the three GLI proteins, which are expressed in the neural tube and developing limbs101-103 (Fig. 2.3A), plays the role of Ci in vertebrates. Consistent with the hypothesis that the GLI proteins are involved in cell patterning in vertebrates, mutations in Gli-3 are associated with developmental disorders in humans (cephalopolydactyly)104 and mouse (extra toes).105,106 Moreover, Gli-1 expression is upregulated by Shh in vitro103 and in vivo in the developing chick limb74,102 and neural tube,107 and functional GLI binding sites were identified in the promoter region of two Hh-responsive genes.94,103 Upregulation of Gli-1 in response to Shh could contribute to amplification of the Shh signal. Evidence that the GLIs could mediate many of the known effects of Shh in the vertebrate limb and neural tube came from studies in which this gene was ectopically expressed. Thus,

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The Development of Dopaminergic Neurons

Fig. 2.3. Expression of the Gli genes in the normal and transgenic rat brain. (A) Coronal sections of E12, WT rat brain hybridized with probes to Gli-1, -2 and -3. D marks dorsal mid-hindbrain and V marks ventral mid-hindbrain. (B) Schematic drawing illustrating the regions of normal (WT) and ectopic dorsal (TG) expression in the brain (solid lines in embryos). Arrowheads mark region of overgrowth in the TG embryos, and the comparable region in the WT embryos.

ectopic expression of a Gli-1-VP16 hybrid molecule in the chick limb bud could induce the Shh-responsive gene Ptc,102 whereas ectopic expression of the three GLI proteins in the frog, led to ectopic induction of ventral cell markers and mature neurons.108,109 Finally, ectopic expression of human Gli-1 in the dorsal mid-hindbrain region of the neural tube in transgenic mice was shown to result in profound morphological changes due to overgrowth of the mid-hindbrain region of the brain107(Fig. 2.3B). Further analysis of these embryos showed that by embryonic day 12, there is suppression of dorsal gene expression, for example Pax-3 (Fig. 2.4A,B), and induction of ventral genes such as the Hh receptor Ptc (Fig.2. 4 C-F), and that, slightly later in embryogenesis, there is a robust induction of Shh itself, and the formation of ventral classes of neurons, including both DA and serotonergic (5HT) neurons.107 Gli-1 transgenic animals show cell fate changes and tissue patterning events that are similar to animals which overexpress Shh-N under the same promoter (see Fig. 2.5 and ref. 107), thus suggesting that GLI-1, or a GLI-related protein, can be a key mediator in the reception and transmission of the morphogenic signals exerted by Shh in the vertebrate neural tube. Further support for the role of GLIs in Hh signaling came from recent reports on mice with deletions in Gli-1, Gli-2 or both. Although Gli-1 deficient mice (Gli-1–/–) and Gli2 heterozygous mice (Gli-2+/–) appear normal,Gli-1–/–;Gli-2+/– mice die at birth and have no floor plate. In contrast, Gli-1–/–;Gli-3+/–mice show a Gli-3+/–phenotype, which resembles mice with an enhanced Shh signal.110 Thus, increasing evidence suggests that these proteins, which have overlapping patterns of expression, cooperate in mediation of the Shh signal.109,111

Specification of Dopaminergic Neurons: Inductive Factors and Their Receptors

23

Fig. 2.4. Gene expression is altered in Gli-1 transgenic animals. (A-F) Cryostat sections of E12 WT and Gli-1 TG mice, subjected to in situ hybridization using probes for Pax-3 (A,B) and Ptc (C-F). D marks dorsal mid-hindbrain and V marks ventral mid-hindbrain. Light arrow in (A) points to Pax-3 expression in WT mid-hindbrain, and dark arrowhead shows region of Pax-3 downregulation in TG animals (B). E and F are high powered views of C and D.

Induction Along the A-P Axis In order to identify signals that provide positional information for DA neurons along the A-P axis, an explant culture system was used. Initially, E9 (0-6 somite) rat embryos were dissected into seven pieces along the transverse axis, cultured for 5-7 days, and examined for the presence of endogenous DA neurons and endogenous serotonergic (5HT) neurons (Fig. 2.6D,E). A clear segregation of DA and 5HT neurons was observed, with DA neurons arising in the midbrain and forebrain (Fig. 2.6E; explants v1 and 3), and 5HT neurons arising in the hindbrain53 (Fig. 6E explants v4, 5 and 6). The close apposition, but marked segregation of these two neuronal classes was seen in vivo as well; midbrain DA neurons reside rostral to the rhombic isthmus and hindbrain 5HT neurons caudal to this boundary53,107 (Fig. 2.6C). The proximity of the DA neurons to the isthmus raised the possibility that this structure is a source of positional signals for these neuronal populations. Consistent with this possibility, we found that the isthmus could induce DA neurons in the ventral aspect of the caudal forebrain (v2), an area of the brain which does not normally make DA neurons and which constitutes an ectopic location, with respect to the normal position of these neurons along

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The Development of Dopaminergic Neurons

Fig. 2.5. DA and 5HT neurons are ectopically induced in Shh-N and Gli-1 TG mice (A-F) Sections of E14 WT (A,B), Shh-N TG (C,D) and Gli-1 TG (E,F) animals stained for DA (A,C,E) and 5HT (B,D,F) markers. Both types of neurons are ectopically induced dorsally in the Shh-N and Gli-1 (C-F) TG mice. D marks dorsal mid-hindbrain and V marks ventral mid-hindbrain. White arrowheads point to ventral expression and black arrowheads point to dorsal expression. Lower magnification is shown for serotonergic neurons in the Gli-1 TG animals to illustrate that several subgroups of serotonergic neurons appear to be induced along the extent of the hindbrain. the A-P axis.53 Since the signaling molecule FGF8 is discretely expressed in the isthmus both in vitro and in vivo112 (Figs. 2.6A,B,E and 2.7A,B), and was shown to be a patterning molecule in the mid-hindbrain region,15 it became a prime candidate as the mediator of isthmus activity, with regard to DA neurons. In agreement with a role for FGF8 in the specification of DA neurons, these neurons could be induced in ectopic ventral-caudal forebrain regions (v2) in the presence of FGF8 (FGF1, 2, 5, 7 and 9 could not induce DA neurons ectopically at the concentrations tested).53 Moreover, the development of endogenous DA neurons, in either the mid or forebrain, was prevented by blocking the FGF8 signal (Fig. 2.7B).53 Endogenous FGF8 was blocked by the addition of soluble FGFR3 IgG to the culture medium (FGFR3 IgG binds to FGF1, 2, 4, 6, 8, 17 and 18, and acts as an antagonist to these ligands53 (FGFs 9-18 have not been tested), which prevented the development of DA neurons in the midbrain explants. FGF4 and 6 were able to substitute for FGF8 in inducing DA neurons in the caudal forebrain, and are also blocked by FGFR3 IgG. However, FGFR1 IgG, which binds to FGF4 and 6, but not to FGF8 (FGFR1 IgG also binds FGF1, 2, and 5), does not prevent endogenous DA neurons from developing.53 Thus, endogenous FGF8 (but not FGF4 or 6), appears to be necessary for the specification of DA neurons. The idea that FGF8 plays a critical role of in DA neuron development is further supported by FGF8 gene deletion studies. Mice which have severely reduced levels of FGF8 suffer deficits in the caudal midbrain and rostral hindbrain,113 and show an absence of TH+, Ptx3+, DA neurons.53 Mice with a moderate reduction in the levels of FGF8113 do generate a population

Specification of Dopaminergic Neurons: Inductive Factors and Their Receptors

25

Fig. 2.6. Location of Early Gene Markers and Mature Neurons Along the A-P axis of the Rat Embryo. (A-C) Parasagittal sections of the E14 rat ventral mid/hindbrain stained for: (A) Fgf8 and Shh mRNA; (B) Fgf8 mRNA (arrow) and TH+ DA neurons (TH); (C) TH and serotonin (5HT). The arrows mark the mid-hindbrain boundary and the site of Fgf8 expression. DA neurons are confined to the rostral side of the isthmus and 5HT neurons to the caudal side. (D) 6 somite stage rat embryo in situ hybridized for Shh ( ventral view). The lines represent transection sites. The numbers represent the presumptive rostral forebrain (1), caudal forebrain (2), midbrain (3), and hindbrain (4-6). The boundary between 3 and 4 is the rhombic isthmus (or mid-hindbrain boundary); marked by an arrow (d = presumptive dorsal, v = presumptive ventral). (E) E9 explants dissected as outlined in (D) were in situ hybridized to Fgf8 (top row). Ventral explants were cultured in collagen gel for 36 hours and then stained for Shh (middle row), or for 6 days and then double immunostained for TH and 5HT; a positive signal is marked by TH+ or 5HT+ (bottom row). Broken white lines outline the explants. Anterior is to the left, posterior to the right. of midbrain dopaminergic neurons, but their numbers are dramatically reduced compared to normal littermates.53 Together, these data indicate that FGF8 fits the criteria for an endogenous inducer of DA neurons. Interestingly, FGF17 and 18 are expressed in the mid-hindbrain region close to where DA and 5HT neurons develop,114 can induce DA neurons in ectopic locations (Ye W, unpublished observations), and appear to be expressed early enough in development to act as endogenous inducers of DA neuron development.114 However, the Fgf8 gene deletion studies113 suggest that if these molecules are physiologically important in the specification of DA neurons, they must function downstream of FGF8.

Intersections of Shh and FGF8 Specify the Position and Identity of DA and 5HT Neurons The findings that Shh is responsible for the position of DA neurons along the D-V axis, whereas FGF8 can control the fate and location of these neurons along the A-P axis, raised the possibility that intersections of these two molecules specify the location of DA neurons

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The Development of Dopaminergic Neurons

Fig. 2.7. Shh and FGF8 are necessary for the development of DA and 5HT neurons (A) Ectopic induction of DA neurons by the isthmus. 6 somite rat ventro-caudal forebrain explant (v2) was cultured alone (A; left) or in combination with a 10 somite chick rhombic isthmus explant (A; right) for 6 days, and stained for TH and a chick-specific neuronal marker L1. TH+ DA neurons were ectopically induced in v2 by the chick isthmus. (B) Differentiation of DA and 5HT neurons requires Shh. Ventral, 6 somite midbrain explants v3, or rostral hindbrain explants v4, were cultured for 6 days either in control medium or with Shh function-blocking antibody (αShh) added at day 0, and stained for TH and 5HT. (C) FGFR3 IgG (this reagent binds to FGF1, 2, 4, 5, 6, 8 and acts as an antagonist to these ligands), blocks the appearance of midbrain DA and rostral hindbrain 5HT neurons. TH and 5HT neurons develop normally in 6 somite ventral mid-hindbrain explants v3/4 grown for 6 days in the presence of FGFR1 IgG (C; left) (this reagent binds to FGF1, 2, 4, 5, 6, but not 8) applied at day 0, but fail to develop in the presence of FGFR3 IgG (C; right) applied at day 0. β-tubulin+ neurons readily appear in explants v3/4 grown for 6 days in the presence of FGFR3 IgG (not shown). Broken white lines mark the explants.

in the neural tube. This idea is supported by the findings that DA neurons could be induced in the dorsal aspect of the caudal forebrain (d2), a tissue devoid of endogenous FGF8 or Shh, by a combination of purified Shh and FGF8; neither factor applied alone could reproduce this effect53 (Fig. 2.8A). 5HT neurons in the rostral hindbrain (v4, but not v5 and 6), were also found to critically depend on both Shh and FGF8 for their development 53 (Fig. 2.7B), but in addition, a third signal, FGF4, may participate in their genesis.53 When FGF4 is added to ventral midbrain explants, which contain endogenous FGF8 and Shh, ectopic 5HT neurons are induced. Surprisingly, under these conditions the endogenous midbrain DA neurons no longer develop53 (Fig. 2.8B). Thus, it appears that FGF4, in combination with Shh and FGF8, allows the development of 5HT neurons in midbrain explants, whereas midbrain tissue not exposed

Specification of Dopaminergic Neurons: Inductive Factors and Their Receptors

A

B

v2+ uncoated bead

d2+ uncoated bead + 100 nM Shh-N

d2 + FGF8 coated bead

d2+ FGF8 coated bead + 100 nM Shh-N

v3 control

27

v3 control + 10ng/ml FGF4

Fig. 2.8. Shh and FGF8 are sufficient to induce DA neurons ectopically (A) Control (uncoated) beads do not induce DA neurons in explants taken from 6 somite rats, and cultured for 6 days. DA neurons (TH+) are induced in the dorsal aspect of a 6 somite caudal forebrain explant (d2), in the presence of a combination of Shh and FGF8 proteins, but not in the presence of Shh , or FGF8 coated beads, alone. Dashed white lines mark the explants, solid white lines outline beads. (B) FGF4 induces 5HT neurons in ventral midbrain explants. 6 somite ventral midbrain explants (v3) were cultured for 6 days in control medium (E), or in the presence of 10 ng/ml FGF4 (F) and double stained for TH and 5HT.

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The Development of Dopaminergic Neurons

Fig. 2.9. Schematic representation of anterior DA and rostral 5HT neuronal specification in the early embryo. (A) Ventral view of a flat-mount presomitic embryo in situ hybridized with Fgf4 and Otx2. The expression domain of Otx2 encompasses the forebrain and midbrain. Extraembryonic tissues were removed. Anterior is to the left. (B) A model illustrating the mechanism by which the positions and fates of DA and 5HT neurons are controlled. Anterior is to the left, F: forebrain, M: midbrain, H: hindbrain, NE: neural epithelium; is: isthmus; PS: primitive streak. Top panel: presomitic embryo. Middle and bottom panel: late somitogenic embryo.

Specification of Dopaminergic Neurons: Inductive Factors and Their Receptors

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to FGF4, but which contains Shh and FGF8, allows the development of DA neurons. A summary of the specification of anterior DA and rostral 5HT neurons in the early embryo is presented in Figure 2.9B. In addition to FGF4, FGF2 (but not FGF1, 5, 6, 8, 9,17 or 18 at the concentrations tested), can also ectopically induce 5HT neurons in tissue that has endogenous sources of Shh and FGF8.53 Although neither FGF2 nor FGF4 are locally expressed in the hindbrain neural plate or mesoderm, FGF4 is expressed in the primitive streak, a region juxtaposed to the posterior neural plate53, 115 (Fig. 2.9A), and may prepattern the future 5HT progenitors from this location. Induction of 5HT neurons by FGF4 occurs only when it is added to the tissue before the 10 somite stage; thus, the FGF4 signal must precede Shh and FGF8.53 FGF2 and 4 ectopically induce 5HT neurons in tissue from which the paraxial mesoderm was removed, indicating that these factors may act directly on neural tissue, but may also be able to function through the axial mesoderm. Caudalization of the neural tube in response to FGF2 or FGF4 has been demonstrated in the chick12 and frog.116-118 In some cases FGF2 was shown to caudalize the neural tube indirectly by modifying the paraxial mesoderm.12 Storey et al119 also showed a direct effect of FGFs on neural tissue, but looked only at early general markers, and not at specific classes of neurons. Taken together, these findings suggest that FGF8 and FGF4 have different functions and are required at different times for the development of 5HT neurons. The possibility that FGF4 and 8 play distinct roles is further supported by the observation that FGFR1 IgG, which blocks the activity of FGF4, but not of FGF8, prevents neural development when added at the presomitic stage53 but, in contrast to FGFR3 IgG, fails to block the development of endogenous 5HT neurons when it is added between 0-6 somites.53 Nevertheless, it is important to note that the FGF family is comprised of a large number of ligands (at least 18), and mediates its actions through four distinct receptors. Multiple ligands bind each receptor, and each ligand binds multiple receptors; therefore the assignment of a particular biological event to a ligand-receptor pair is problematic, and it remains possible that the observed differences in biological responses are quantitative, rather then qualitative.

Conclusion This chapter highlights experiments that have investigated signaling centers and secreted factors which specify the two major groups of DA neurons, one in the midbrain and one in the forebrain. Significant progress has been made in identifying the floor plate, and the isthmus and ANR53 as critical signaling centers, and Sonic hedgehog and FGF8 as necessary signals in these specification events. For convenience, DA neurons in the mid or forebrain have been treated as a single, homogenous group, although anatomical, morphological and immunocytochemical studies of adult animals have shown that this is clearly not the case. Future experiments will hopefully shed light on additional mechanisms which participate in the control of DA cell fate and function. Specifically, it will be important to identify additional transcription factors and extracellular molecules which specify DA neurons as a population and as subgroups, as well as molecules which control the migration of DA neurons to the appropriate regions in the adult brain, and which control the extension and targeting of the DA axons. Some progress has been made along these lines. For example, the orphan nuclear receptor Nurr1 was found to be expressed in developing DA neurons, and gene deletion studies have shown that Nurr1 expression is necessary for the development of DA neurons in the midbrain.120 Another transcription factor, expressed exclusively in midbrain DA neurons, is Ptx3, a bicoid-related homeobox gene product and a member of the Ptx subfamily. Although uniquely expressed in midbrain DA neurons from early embryonic ages (E11.5 in the

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The Development of Dopaminergic Neurons

mouse),121 a functional role for Ptx3 in DA neuron specification, and/or maintenance has yet to be demonstrated. With further understanding of the molecular processes which direct the development of DA neurons, it may be possible to restore or modify their function in diseased states.

Acknowledgments We thank W. Anstine for help with preparation of the figures, E. Berry for help with the manuscript and W.L.Ye and D. Stone for critical reading of the manuscript.

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46. Lepage T, Cohen SM, Diaz-Benjumea FJ et al. Signal transduction by cAMP-dependent protein kinase A in Drosophila limb patterning. Nature 1995; 373:711-715. 47. Li W, Ohlmeyer JT, Lane ME et al. Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development. Cell 1995; 80:553-562. 48. Strutt DI, Wiersdorff V, Mlodzik M. Regulation of furrow progression in the Drosophila eye by cAMP-dependent protein kinase A. Nature 1995; 373:705-709. 49. Fan C-M, Porter JA, Chiang C et al. Long-range sclerotome induction by Sonic hedgehog: Direct role of the amino terminal cleavage product of modulation by the cyclic AMP signaling pathway. Cell 1995; 81:457-465. 50. Lee JJ, Ekker SC, von Kessler DP et al. Autoproteolysis in hedgehog progein biogenesis. Science 1994; 266:1528-1537. 51. Porter JA, Ekker SC, Young KE et al. The product of hedgehog autoproteolytic cleavage active in local and long-range signaling. Nature 1995; 374:363-366. 52. Bumcrot DA, Takada R, McMahon AP. Proteolytic processing yields two secreted forms of Sonic hedgehog. Mol Cell Biol 1995; 15:2294-2303. 53. Ye W, Shimamura K, Rubenstein JLR et al. FGF8 and Shh signals create inductive centers for dopaminergic and serotonergic neurons in the anterior neural plate. Cell 1998; 93:755-766. 54. Ericson J, Morton S, Kawakami A et al. Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity. Cell 1996; 87:661-673. 55. Hooper JE, Scott MP. The Drosophila patched gene enclodes a putative membrane protein required for segmental patterning. Cell 1989; 59:751-765. 56. Nakano Y, Guerrero I, Hidalgo A et al. A protein with several possible membrane spanning domains encoded by the Drosophila segment polarity gene patched. Nature 1989; 341:508-513. 57. Hidalgo A, Ingham P. Cell patterning in the Drosophila segment: Spatial regulation of the segment polarity gene patched. Development 1990; 110:291-301. 58. Ingham PW, Taylor AM, Nakano Y. Role of the Drosophila patched gene in positional signalling. Nature 1991; 353:184-187. 59. Nüsslein-Volhard C, Wieschaus E, Kluding H. Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. I. Zygotic loci on the second chromosome. Wilhelm Roux’s Arch Dev Biol 1984; 193:267-282. 60. Jürgens G, Wieschaus E, Nüsslein-Volhard C et al. Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. II. Zygotic loci on the third chromosome. Wilhelm Roux’s Arch Dev Biol 1984; 193:283-295. 61. Alcedo J, Ayzenzon M, Von Ohlen T et al. The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the Hedgehog signal. Cell 1996; 86:221-232. 62. van den Heuvel M, Ingham PW. Smoothened encodes a receptor-like serpentine protein required for hedgehog signalling. Nature 1996; 382:547-551. 63. Marigo V, Davey RA, Zuo Y et al. Biochemical evidence that Patched is the Hedgehog receptor. Nature 1996; 384:176-179. 64. Goodrich LV, Johnson RL, Milenkovic L et al. Conservation of the hedgehog/patched signaling pathway from flies to mice: Induction of a mouse patched gene by hedgehog. Genes Dev 1996; 10:301-312. 65. Hahn H, Wicking C, Zaphiropoulous PG et al. Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 1996; 85:841-851. 66. Johnson RL, Rothman AL, Xie J et al. Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 1996; 272:1668-1671. 67. Motoyama J, Takabatake T, Takeshima D et al. Ptch2, A second mouse Patched gene is co-expressed with Sonic Hedgehog. Nat Genet 1998; 18:104-106. 68. Carpenter D, Brush J, Frantz G et al. Characterization of the two patched receptors for the vertebrate hedgehog protein family. Proc Nat Acad Sci USA 1999; 95:13630-13634. 69. Stone DM, Hynes M, Armanini M et al. The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature 1996; 384:129-134.

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70. Johnson RL, Laufer E, Riddle RD et al. Ectopic expression of Sonic hedgehog alters dorsal-ventral patterning of somites. Cell 1994; 79:1165-1173. 71. Riddle RD, Johnson RL, Laufer E et al. Sonic hedgehog mediates the polarizing activity of the limb. Cell 1993; 75:1401-1416. 72. Roberts DJ, Johnson RL, Burke AC et al. Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut. Development 1995; 121:3163-3174. 73. Bitgood MJ, Shen L, McMahon AP. Sertoli cell signaling by Desert hedgehog regulates the male germline. Current Biology 1996; 6:298-304. 74. Vortkamp A, Lee K, Lanske B et al. Indian hedgehog and parathyroid hormone-related protein regulate the rate of cartilage differentiation. Science 1996; 273:613-622. 75. Currie PD, Ingham PW. Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish. Nature 1996; 382:452-455. 76. Traiffort E, Charytoniuk DA, Faure H et al. Regional distribution of sonic hedgehog, patched, and smoothened mRNA in the adult rat brain. J Neurochem 1998; 70:1327-1330. 77. Miao N, Wang M, Ott JA et al. Sonic hedgehog promotes the survival of specific CNS neuron populations and protects these cells from toxic insult in vitro. J Neurosci 1997; 17:5891-5899. 78. Wechsler-Rrya RJ, Roosa J, Scott MP. Sonic Hedgehog regulates proliferation of granule cell precursors in the developing cerebellum. Devel Biol 1998; 198:194. 79. Chidambaram A, Goldstein AM, Gailani MR et al. Mutations in the human homologue of the Drosophila patched gene in Caucasian and African-American nevoid basal cell carcinoma syndrome patients. Cancer Research 1996; 56:4599-601. 80. Unden AB, Kerstin B, Zaphiropulos PG et al. The gene for Gorlin’s syndrome, human patched, is consistently overexpressed in both hereditary and sporadic basal cell carcinomas. J Invest Dermatol 1997; 108:596. 81. Gailani MR, Stahle-Backdahl M, Leffell DJ et al. The role of the human homologue of Drosophila patched in sporadic basal cell carcinomas [see comments]. Nature Genetics 1996; 14:78-81. 82. Wicking C, Shanley S, Smyth I et al. Most germ-line mutations in the nevoid basal cell carcinoma syndrome lead to a premature termination of the PATCHED protein, and no genotype-phenotype correlations are evident. American Journal of Human Genetics 1997; 60:21-6. 83. Xie J, Murone M, Luoh S-M et al. Activating Smoothened mutations in sporadic basal-cell carcinoma. Nature 1998; 391:90-92. 84. Oro AE, Higgins KM, Hu Z et al. Basal cell carcinomas in mice overexpressing sonic hedgehog. Science 1997; 276:817-21. 85. Raffel C. Sporadic medulloblastomas contain PTCH mutations. Cancer Research 1997; 57:842-845. 86. Mariol M-C, Preat T, Limbourg-Bouchon B. Molecular cloning of fused, a gene required for normal segmentation in the Drosophila melanogaster embryo. Mol Cell Biol 1987; 7:3244-3251. 87. Préat T, Thérond P, Lamour-Isnard C et al. A putative serine/threonine protein kinase encoded by the segment-polarity fused gene of Drosophila. Nature 1990; 347:87-89. 88. Robbins DJ, Nybakken KE, Kobayashi R et al. Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein costal2. Cell 1997; 90:225-234. 89. Sisson JC, Ho KS, Suyama K et al. Costal2, a novel kinesin-related protein in the Hedgehog signaling pathway. Cell 1997; 90:235-245. 90. Pham A, Therond P, Alves G et al. The Suppressor of fused gene encodes a novel PEST protein involved in Drosophila segment polarity establishment. Genetics 1995; 140:587-598. 91. Akimaru H, Chen Y, Dai P et al. Drosophila CBP is a co-activator of cubitus interruptus in hedgehog signalling. Nature 1997; 386:735-738. 92. Domínguez M, Brunner M, Hafen E et al. Sending and receiving the Hedgehog signal: Control by the Drosophila Gli protein cubitus interruptus. Science 1996; 272:1621-1625.

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93. Orenic T, Slusarski DC, Kroll KL et al. Cloning and characterization of the segment polarity gene cubitus interruptus Dominant of Drosophila. Genes Dev 1990; 4:1053-1067. 94. Alexandre C, Jacinto A, Ingham WP. Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the Cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins. Genes Dev 1996; 10:2003-2013. 95. Kinzler KW, Bigner SH, Bigner DD et al. Identification of an amplified, highly expressed gene in a human glioma. Science 1987; 236:70-73. 96. Kinzler KW, Ruppert JM, Bigner SH et al. The GLI gene is a member of the Kruppel family of zinc finger proteins. Nature 1988; 332:371-374. 97. Kinzler KW, Vogelstein B. The GLI gene encodes a nuclear protein which binds specific sequences in the human genome. Mol Cell Biol 1990; 10:634-642. 98. Roberts WM, Douglass EC, Peiper SC et al. Amplification of the gli gene in childhood sarcomas. Cancer Res 1989; 49:5407-5413. 99. Ruppert JM, Vogelstein B, Kinzler KW. The zinc finger protein GLI transforms primary cells in cooperation with adenovirus E1A. Mol Cell Biol 1991; 11:1724-1728. 100. Ruppert JM, Vogelstein B, Arheden K et al. GLI-3 encodes a 190-kilodalton protein with multiple regions of GLI similarity. Mol Cell Biol 1990; 10:5408-5415. 101. Hui C-C, Slusarski D, Platt KA et al. Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm- and mesoderm-derived tissues suggests multiple roles during postimplantation development. Dev Biol 1994; 162:402-413. 102. Marigo V, Johnson RL, Vortkamp A et al. Sonic hedgehog differentially regulates expression of Gli and Gli3 during limb development. Develop Biol 1996; 180:35-40. 103. Sasaki H, Hui C-C, Nakafuku M et al. A binding site of Gli proteins is essential for HNF-3b floor plate enhancer activity in transgenics and can respond to Shh in vitro. Development 1997; 124:1313-1322. 104. Vortkamp A, Gessler M, Grzeschik H-H. GLI3 zinc finger gene interrupted by translocations in Greig syndrome families. Nature 1991; 352:539-540. 105. Vortkamp A, Franz T, Gessler M et al. Deletion of GLI3 supports the homology of the human Greig cephalopolysyndactyly syndrome (GCPS) and the mouse mutant extra-toes (Xt). Mamm Genome 1992; 3:461-463. 106. Hui C-C, Joyner AL. A mouse model of Greig cephalopolysyndatyly syndrome: The extra-toesJ mutation contains an intragenic deletion of the Gli3 gene. Nature Genet 1993; 3:241-246. 107. Hynes M, Stone DM, Dowd M et al. Control of cell pattern in the neural tube by the zinc finger transcription factor and oncogene Gli-1. Neuron 1997; 19:15-26. 108. Lee J, Platt KA, Censullo P et al. Gli1 is a target of Sonic hedgehog that induces ventral neural tube development. Development 1997; 124:2537-2552. 109. Brewster R, Lee J, Ruiz i Altaba A. Gli/Zic factors pattern the nerual plate by defining domains of cell differentiation. Nature 1998; 393:579-583. 110. Mo R, Freer AM, Zinyk DL et al. Specific and redundant functions of gli2 and gli3 zinc finger genes in skeletal patterning and development. Development 1997; 124:113-123. 111. Park H, Platt K, Matise M et al. Genetic analysis of the role of gli genes during embryogenesis in double mutant mice. Develop Biol 1998; 198:217-343. 112. Crossley PH, Martin GR. The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. De- velopment 1995; 121:439-451. 113. Meyers EN, Lewandowski M, Martin GR. An Fgf8 mutant allelic series generated by Creand Flp-mediated recombination. Nature Genetics 1998; 18:136-141. 114. Hoshikawa M, Ohbayashi N, Yonamine A et al. Structure and expression of a novel fibroblast growth factor, FGF-17, preferentially expressed in the embryonic brain. Biochem & Biophys Res Comm 1998; 243:187-191. 115. Niswander L, Martin GR. Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 1992; 114:755-768.

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116. Cox WG, Hemmati-Brivanlou A. Caudalization of neural fate by tissue recombination and bFGF. Development 1995; 121:4349-4358. 117. Kengaku M, Okamoto H. bFGF as a possible morphogen for the anteroposterior axis of the central nervous system in Xenopus. Development 1995; 121:3121-3130. 118. Lamb TM, Harland RM. Fibroblast growth factor is a direct neural inducer, which combined with noggin generates anterior-posterior neural pattern. Development 1995; 121:3627-3636. 119. Storey KG, Goriety A, Sargent CM et al. Early posterior neural tissue is induced by FGF in the chick embryo. Development 1998; 125:473-484. 120. Zetterström RH, Solomin L, Jansson L et al. Dopamine neuron agenesis in Nurr-1-deficient mice. Science 1997; 276:248-250. 121. Smidt MP, van Schaick HSA, Lanctôt C et al. A homeodomain gene Ptx 3 has highly restricted brain expression in mesencephalic dopaminergic neurons. Proc Natl Acad Sci USA 1997; 94:13305-13310.

CHAPTER 3

Development of Midbrain Dopaminergic Neurons Carla Perrone-Capano, Roberto Pernas-Alonso and Umberto di Porzio

T

he catecholamine dopamine (DA) plays a key role in the physiology of most vertebrate and invertebrate organisms. DA is an important regulator of many neural functions, including motor integration, neuroendocrine hormone release, cognition, emotive behaviors and reward.1 In mammals DA neurons are relatively few, when compared to the total number of brain neurons.2 They are located mainly in the ventral midbrain to form the retrorubral nucleus (A8), the substantia nigra (area A9) and the ventral tegmental area (area A10).3,4 In rodents, neurons arising from the substantia nigra project to the striatum (corresponding to the caudate-putamen in primates) and receive innervation from multiple structures in the diencephalon and telencephalon. The striatal development and organization is reviewed by Liu and Graybiel (this book). Cellularly, DA midbrain neurons can be distinguished according to the presence of various specific proteins such as parvalbumin, calbindin, cholecystokinin and calretinin, although no clear functional differences have been attributed to these different subpopulations.5,6 The ascending nigrostriatal pathway regulates motor control, and its degeneration in humans is associated with Parkinson’s disease, the syndrome described by the English neurologist in his “Essay on the shaking palsy” in 1817.7 Neurons from the ventral tegmental area project to the limbic system and cortex, and are involved in emotional and reward behavior and in motivation.1 Disturbances in this system have been associated with schizophrenia (although direct evidence is still elusive), addictive behavioral disorders and attention-deficit hyperactivity disorder (ADHD).8,9 DA also modulates interactions between prefrontal cortex and visual association areas, which are important in visual memory.10 In addition, dopaminergic neurotransmission is involved in learning and memory dysfunction associated with traumatic brain injury.11 All three dopaminergic mesencephalic nuclei (A8, A9 and A10 region) project towards the hippocampal formation, although the functional significance of the mesohippocampal DA system is largely unknown. It has been suggested that this projection could have a role in modulation of memory processes.12 In addition to its hippocampal innervation, the retrorubral A8 dopaminergic cell group projects to the substantia nigra and ventral tegmental area and possibly is involved in the coordination of the nigrostriatal and mesolimbic systems. In the mammalian forebrain smaller clusters of DA cells lie in the subparafascicular thalamic nucleus (area A11),13 the hypothalamic arcuate nucleus (area A12, see chapter 5), the incertohypothalamic nucleus (area A13) and the olfactory bulb (see chapter 9). At the biochemical level, DA is synthesized from tyrosine by tyrosine hydroxylase (TH); its metabolism and function is summarized in Figure 3.1. Upon release from the presynaptic The Development of Dopaminergic Neurons, edited by Umberto di Porzio, Roberto Pernas-Alonso and Carla Perrone-Capano. ©1999 R.G. Landes Company.

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Fig 3.1. Dopamine metabolism. Tyrosine is converted to L-3,4-dihydroxyphenylalanine (L-DOPA) by tyrosine hydroxylase (TH) and L-DOPA is converted to dopamine (DA, star) by aromatic L-amino acid decarboxylase (AAAD). DA can be further converted into noradrenaline by the dopamine-β-hydroxylase enzyme, and noradrenaline into adrenaline by phenyl-ethanolamine-N-methyltransferase (pathways not shown in the figure). DA is transported into vesicles by the synaptic vesicle transporter VMAT2 (small rods with arrows). When vesicles fuse with the presynaptic plasma membrane, DA is released into the synaptic cleft and interacts with postsynaptic D1-type or D2-type receptors (which modulate cAMP level). DA action at the synapse is terminated predominantly by re-uptake into the presynaptic terminal through the dopamine transporter DAT (large rods with arrows). DAT is blocked by cocaine and can transport the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP + ), which derives from 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), by the action of the glial monoamine oxidase, MAO. Once back into the DA terminals, the neurotransmitter can be repackaged into vesicles or catabolized by mitochondrial MAO into 3,4 dihydroxylphenylacetic acid (DOPAC).

Development of Midbrain Dopaminergic Neurons

39

terminal into the synaptic cleft, DA acts through D1-R (D1 and D5) and D2-R (D2, D3, D4) subfamilies of G-protein-coupled receptors (see chapter 7).14 DA neurotransmission is terminated by uptake of the released messenger into the presynaptic DA fibers. The physiological role and clinical relevance of dopaminergic neurons are well recognized. The mechanisms underlying their development have been the object of intense investigation and, while we begin to understand many fundamental details, the overall picture still eludes us.15 In this chapter, we will review recent studies that unravel the cellular and molecular events responsible for the differentiation and maturation of developing midbrain DA neurons.

The Birth of Mesencephalic DA Neurons When are DA neurons born during CNS development? To investigate their ontogeny, immunohistochemistry using antibodies against TH or DA, 3H-thymidine labeling of proliferating DA neuroblasts and sensitive molecular biology techniques have largely been employed. In the mesencephalon, TH, the rate-limiting enzyme in the biosynthetic pathway of catecholamines (i.e., dopamine, noradrenaline and adrenaline), is expressed early during ontogeny (see below). TH has been used as a marker of all catecholaminergic neuroblasts: TH+ cells are considered the precursors of dopaminergic neurons in the midbrain, noradrenergic neurons in the brain stem and adrenergic neurons in the ventral medulla oblungata. DA midbrain neuroblasts are generated near the midbrain-hindbrain junction16 and migrate radially to their final position in the ventral midbrain. In the mouse midbrain, rare and scattered TH+ cells and fibers have been detected by immunocytochemistry starting at embryonic day (E) 9.5 close to the ventricular ependymal layer, suggesting that DA differentiation can occur in early postmitotic neural precursors.17 TH+ clusters reminiscent of the areas A9 and A10 can be detected at E13 (Fig. 3.2).17,18, * Interestingly, TH gene expression in putative DA neuroblasts is maintained during migration from the ependymal layer toward the ventral mesencephalon. Many migrating TH+ cells show already distinct neuronal morphological features (Fig. 3.3). This appears to be a distinct feature of midbrain DA neurons. For instance, in the olfactory bulb TH is not expressed until the cells have reached their final destination, the glomerular layer.19 The sequence of developmental events for mesencephalic dopaminergic neurons is similar in humans and mice. The duration of the developmental period is, however, significantly protracted in humans: TH+ cells appear in the ventral mesencephalon at 6.5 weeks adjacent to the ventricular zone; their ventral migration begins at 6.7 weeks and TH+ neurites are seen initially in the developing putamen at 9.0 weeks.20 In vitro studies suggest that proliferation of DA neuroblasts is probably influenced by various growth factors. Fibroblast growth factor (FGF) 2, also known as basic FGF, and epidermal growth factor (EGF) act as mitogens for neuronal precursors in fetal rat mesencephalic cultures and delay their differentiation. Under these conditions, the number of TH+ precursors appears increased (Perrone Capano et al, unpublished observations).21,22 However the role played by FGFs or other growth factors in the genesis of DA neurons in vivo remains to be established.

*

Often in the specialized literature neuronal birthday in rodents may appear controversial due mostly to the use of different methods in assessing embryonic age.

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Fig 3.2. Early appearance of TH+ cells in mouse midbrain. Time course of appearance of TH+ cells is shown by anti-tyrosine hydroxylase immunoreactivity. Tissue were fixed in 5% acrolein.17 (A) shows an early cluster of TH+ cells at E9.5-E10. The cluster is on one side only due to difficulties in early embryo orientation during cutting;17 bar = 170 µm. In (B) TH+ cells in ventral nuclei are shown in E10.5-11 mesencephalon; bar = 212 µm. In (C) TH+ clusters at E13 appear in two distinct groups, reminiscent of A9 and A10 areas; bar = 212 µm

DA Cell Lineage Specification Inductive Signals The precise anatomical localization and functional differentiation of DA neurons in the mammalian brain is achieved through the action and gradient disposition of various diffusible factors. In the last few years significant progress has identified the signaling centers and secreted factors that play a key role in the induction of DA function. Experiments that have investigated the role of these inductive molecules and their receptors are reviewed by Hynes and Rosenthal (this book). Briefly, a wealth of data from tissue transplantation and explant culture studies and biochemical and genetic experiments has demonstrated that DA neurons develop at sites where the signals of two distinct molecules, Sonic hedgehog (SHH) and FGF8, intersect and that these two extracellular inducers are both necessary and sufficient for the induction of DA neurons. SHH, a secreted protein produced first by the notochord (a mesodermal structure underlying the neural plate) and then by the floor plate (a specialized group of neuroepithelial cells at the ventral midline of the neural tube), initially induces in the neural tube a ventral cell fate characterized by the expression of specific markers, including the SHH receptor Patched, and the transcription factors HNF-3β and Gli-1 (chapter 2). Subsequently, these ventralized cells further differentiate to assume distinct neuronal identities as a function of the duration, context or concentration of SHH that they encounter.23 SHH is necessary and sufficient for induction of the DA neurons along the dorso-ventral but not the antero-posterior axis.24 SHH interacts with FGF8, which in turn is responsible for DA neuron induction along the antero-posterior axis of the neural tube. FGF8 is locally produced in the isthmus (a known organizing center located at the mid/hindbrain boundary) and in the anterior neural ridge (in the rostral forebrain where the future hypothalamic DA neurons arise). The possibility that intersections of SHH and FGF8 signaling specify the location of DA neurons in the neural tube is supported by the findings that these factors can cooperate to induce DA neurons in ectopic locations. These experiments indicate that signaling centers and secreted signals establish a functional epigenetic Cartesian grid of positional information

Development of Midbrain Dopaminergic Neurons

41

Fig 3.3. Migrating TH+ precursors. Many TH+ cells appear immediately after leaving the ependymal layer and migrating radially toward the ventral mesencephalon at E13 (A). Migrating TH+ cells clearly show neurites and typical cytoplasmic immunoreactivity (B). (A) Bar = 100 µm. (B) Bar = 40 µm. in the neural tube, specifying cell fates along the two main axes of this system and inducing multiple classes of neurons according to their position.24

Transcription Factors The inductive secreted molecules, including SHH and FGF8, are thought to activate cascades of other signaling molecules and transcription factors which lead to the final differentiation of DA neurons. Two transcription factors, Nurr1 and Ptx3, expressed at crucial times in differentiating midbrain DA cells, have been recently identified.

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The Development of Dopaminergic Neurons

Nurr1, an “orphan” member of the steroid-thyroid hormone receptor superfamily, is expressed predominantly in the CNS, mainly in limbic areas and the ventral midbrain and, at a lower level, in the diencephalon and in the olfactory bulbs.25 In the mouse, the onset of Nurr1 expression in the ventral midbrain occurs in DA neuroblasts one day before the appearance of TH, and its expression continues in mature dopaminergic neurons during adulthood. Absence of Nurr1 in Nurr1 knockout mice leads to agenesis of midbrain DA neurons, as shown by absence of several dopaminergic cell markers (TH, the retinoic acid converting enzyme AHD2 and the receptor tyrosine kinase c-Ret) in the midbrain and the consequent lack of striatal dopamine innervation.26 Null mutant mice are born at the expected frequency without gross morphological brain abnormalities but die within the first two days after birth (perhaps due to the inability to suckle). Interestingly, DA cell groups in the diencephalon (areasA11/A13), as well as DA neurons of the olfactory bulb27 are not affected in Nurr1 null mutants and continue to express TH at the normal time, thus excluding the possibility that Nurr1 directly regulates TH gene expression. In Nurr1 heterozygous conditions, A9 and A10 DA neurons appear normally, although there is reduced striatal DA level. These data indicate that lowered Nurr1 gene dosage affects maintenance of the differentiated mesencephalic DA neurons but not their development.26 An additional feature of Nurr1 is that it can heterodimerize with the receptor for 9-cis-retinoic acid (RXR), activating transcription in response to RXR ligands.28,29 Nurr1 is an immediate early gene rapidly induced by electrical activity in cell lines and in the adult brain.30,31 These observations suggest that Nurr1 could be the point of convergence between retinoic and activity-dependent signaling pathways, which could thus be involved in DA neuron differentiation. Ptx3 is a bicoid related homeobox gene, selectively expressed in mesencephalic DA neurons shortly after Nurr1, in part under FGF8 control. The onset of Ptx3 expression coincides with that of TH. At later stages, Ptx3 expression remains restricted to the mesencephalic DA system and this association is conserved in the adult brain.24,32 In the absence of Nurr1, neuroepithelial cells that should give rise to DA neurons adopt a normal ventral localization marked by HNF-3β and Ptx3. However, these late precursors fail to differentiate into DA neurons, showing that Nurr1 is essential to commit Ptx3 positive ventral mesencephalic precursors towards DA differentiation. Further, as development progresses, these midbrain DA precursor cells degenerate in the absence of Nurr1, resulting in increased apoptosis of ventral midbrain neurons in newborn null mutant mice.27 Thus, while Nurr1 is not involved in the induction of Ptx3 expression, it is critically involved in maintenance of Ptx3-expressing cells. In this regard, it has been proposed that Ptx3 and Nurr1, although regulated independently, may function in a cooperative manner to regulate factors required for terminal differentiation of midbrain DA neurons. Taken together, these data indicate that Nurr1 is essential for both survival and final differentiation of ventral mesencephalic late DA precursors into fully functional DA neurons, whereas the role of Ptx3 in DA neuron differentiation remains to be clarified.27 Figure 3.4 summarizes the early events and the time course of specific gene onset in rodent DA neuron development (see below) as well as the role of putative trophic factors and striatal target cells (see below).

Specific Gene Expression During Differentiation Once ventral midbrain neurons have acquired a dopaminergic specification, a set of genes involved in the maturation of DA properties are activated before the establishment of DA neurotransmission, which in rodents occurs at around E15-E16. Amongst the various specific dopaminergic markers, TH appears early. How the expression of the TH gene is regulated at these developmental stages is still unknown. In the more mature CNS, regulation

Development of Midbrain Dopaminergic Neurons

43

Fig 3.4. Tentative model and time course for DA neuron development. The diagram summarizes the early events and the time course in rodent DA neuronal development. The onset of some key genes is outlined, as well as the putative intervention of trophic factors and the influence of striatal target cells. For details, see text. FGF8, fibroblast growth factor 8; SHH, sonic hedgehog; D2-R, D2 type dopamine receptors; AHD2, retinoic acid-generating enzyme aldehyde dehydrogenase; VMAT2, synaptic vesicle monoamine transporter; Nurr1 and Ptx3, transcription factors; ret, receptor tyrosine kinase c-Ret, which together with GFRα1 forms the receptor complex for the glial cell line-derived neurotrophic factor (GDNF); TH, tyrosine hydroxylase; DAT, dopamine transporter; GLURs, glutamate receptors; E, embryonic age in days. of TH takes place at the mRNA and protein levels. Various mechanisms intervene to regulate TH activity: feedback inhibition, allosteric regulation, enzyme phosphorylation and stability, transcriptional regulation, alternative RNA processing and translational regulation.33 DA neurons also express DA receptors (autoreceptors) before the functional onset of DA neurotransmission.34 Autoreceptors belong to the D2-R class.35 In the adult, they are distributed on the DA somata, dendrites and nerve terminals. The latter seem to modulate DA synthesis36 and DA release,37 while those localized at the cell body or dendrites seem to influence basal firing by modulating the rates of impulse activity.38 Binding studies and in situ hybridization data show that these autoreceptors appear at E13-E14 in the rat midbrain, two days after TH immunoreactivity and their number increase thereafter.34,39 The early prenatal appearance of D2 autoreceptors in the embryonic midbrain suggests that they may have a regulatory role in the development of DA neurons. Indeed, dopamine is accumulated in ventral midbrain neurons shortly after their initial differentiation, when DA pathways and functional neurotransmission are not yet established.40,41 In addition, DA is released

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The Development of Dopaminergic Neurons

spontaneously from developing midbrain neurons in cultures.42,43 An interesting hypothesis suggests that DA may influence cellular differentiation and circuitry formation early in development through receptor-mediated effects on process outgrowth, and that it can have a trophic effect during embryogenesis.44 However, in TH knockout mice in which the TH gene is expressed only in noradrenergic neurons under the dopamine-β-hydroxylase promoter, and therefore unable to synthesize DA exclusively in dopaminergic neurons, midbrain DA neurons can develop, differentiate and form appropriate projections and connections with the striatum. Also, neurogenesis in the striatum is essentially normal in the absence of DA, thus suggesting that catecholamines can in part substitute for one another. The striatum in fact receives a large noradrenergic innervation from the locus coeruleus. Mice unable to synthesize DA show, however, early postnatal lethality due to hypoactivity and impairment of feeding and are rescued by administration of the DA precursor L-DOPA.45 Like TH, the synaptic vesicle monoamine transporter gene (VMAT2 ) is also expressed early in the rat ventral midbrain (at least E12 in the rat),41 several days before the establishment of nigrostriatal DA neurotransmission. VMAT2 belongs to the vesicular neurotransmitter transporters family and allows transport and storage of monoamines into dense core vesicles in most aminergic neurons using the electrochemical gradient generated by a vesicular H+-ATPase.46 vmat2 null mutant mice are hypoactive, die within a few days after birth and manifest severe impaired monoamine storage and release. In addition, these mutants are supersensitive to the drugs of abuse cocaine and amphetamine, which act on catecholaminergic neurons. Electrical stimulation of striatal slices, as well as depolarization of midbrain cultures in the homozygous mutant mice, is unable to evoke DA release.47,48 These results show that loading of vesicles by VMAT2 is indispensable for the impulse-dependent DA neurotrasmission in the striatum. Interestingly, vmat2 heterozygous adult mice are more susceptible to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), suggesting that VMAT2-mediated sequestration of the neurotoxin into vesicles may play an important role in attenuating MPTP toxicity in vivo.49 In vmat2 knockout mice, monoamine synthetic rate is higher than normal, but their levels in the brain are extremely low. In addition, the higher toxicity of the MPTP metabolite 1-methyl-4-phenylpyridinium (MPP+) in vmat2 heterozygotes strongly suggests that VMAT2-mediated transport determines the rate of synaptic vesicle filling and the amount of monoamine released. Due to the early appearance of VMAT241 and DA40 during midbrain ontogeny and the presence of functional release in ventral mesencephalon in primary cultures,42 it seems highly plausible that vesicular storage also occurs in the embryonic brain. However, null mutant mice indicate that vesicular compartmentalization of monoamines is not absolutely required until birth.47,48 An additional role of VMAT2 could consist in clearing the cytoplasm of free DA, which readily oxidizes to produce toxic free radicals. An additional marker present only in midbrain DA neurons, and not in other CNS DA neurons, is the retinoic acid-generating enzyme aldehyde dehydrogenase (AHD2). This enzyme is expressed early in mouse development, one day after the appearance of TH, and it is mainly present in the somata of nigral neurons located in the pars compacta of the A9 nucleus and in their growing projections to the striatum. In the adult it is present in axons and terminals of the mesostriatal and mesolimbic system (particularly the nucleus accumbens).50 The retinoid-synthesizing action of AHD2 may play a role in development of DA neurons, since RXR can heterodimerize with Nurr1 to promote signaling (see previous section), which in turn could lead to differentiation of midbrain DA neurons. Moreover, since retinoic acid acts as a transcriptional regulator, it has been hypothesized that axonal transport of AHD2 is a way by which mesencephalic neurons could exert a non-receptor-mediated influence on gene transcription in the forebrain.50

Development of Midbrain Dopaminergic Neurons

45

Soon after the achievement of final commitment, developing midbrain DA neurons express the c-ret protooncogene and the GFRα1 gene (for the nomenclature, see ref. 51). These genes encode for components of a multireceptor complex interacting with the glial cell line-derived neurotrophic factor (GDNF), the most potent trophic factor yet described for midbrain dopaminergic neurons and spinal motoneurons (chapter 4).52 RET belongs to the receptor tyrosine kinase family and is the signaling component of the GDNF receptor complex, whereas GFRα1 is anchored to the cell surface via a glycosyl-phosphatidylinositol (GPI) linkage and is the ligand binding subunit. Their mRNAs are both clearly present in the A9 and A10 DA neurons from E12.5,53,54 as well as in other areas which are known targets of GDNF action. Developing DA midbrain neurons also express receptors for various neurotrophins which in vitro act as “dopaminotrophic factors”: trkB, the high affinity receptor for BDNF and NT-4/5, and trkC, the high affinity receptor for NT-3.55, 56 However, their precise role in vivo remains unclear.

Phenotypic Maturation As for other neuronal populations, it is plausible that autocrine, paracrine, glial-mediated and target-derived trophic factors are required to achieve differentiation, maturation, and survival/maintenance of postmitotic DA neurons. Putative “dopaminotrophic” factors are reviewed in chapter 4 and will not be discussed here. Besides the already known molecules, other still unidentified epigenetic factors must be involved in the maturation of the DA function. Among these environmental influences, target interactions appear to play an important role in modulating key aspects of midbrain DA neurotransmission. Data from our group show that target neurons have a pivotal influence on the maturation of midbrain DA neurons and modulate DA synthesis and DA uptake.57,58 The latter seems dependent on a direct influence of striatal neurons on the regulation of the dopamine transporter (DAT ) gene expression during development, at least in vitro.

Expression of the Dopamine Transporter

DAT is a member of the multigene family encoding Na+/Cl–-dependent neurotransmitter transporters, and its gene product mediates high-affinity uptake of the released DA into the presynaptic DA neuron (Fig. 3.1).59 In addition to its physiological function, DAT is the site of action of amphetamine and cocaine59,60 and it is responsible for the selective accumulation in DA neurons of MPP+.61 Thus, MPP+ enters DA neurons selectively by competing with DA,62 and is then accumulated into the synaptic vesicles by VMAT2 (Fig. 3.1). Cocaine blocks the dopamine transporter and increases synaptic availability of dopamine, while amphetamine redistributes DA from synaptic vesicles to the cytosol, promoting DAT-mediated reverse transport and calcium-independent DA release,63 as shown in dat and vmat2 null mutants.64,48 Chronic metamphetamine treatment has a neurotoxic effect on striatal DA terminals, increasing extracellular DA levels and free radical formation and reducing DA content and DAT binding sites in the striatum.65,66 The same treatment on mice lacking DAT does not produce significant changes, indicating that the transporter is required for metamphetamine-induced striatal dopaminergic toxicity.67 Intriguing recent observations indicate that DAT, as well as other neurotransmitter transporters, can function in ways similar to ion channels in addition to its role as DA carrier.68, 69 DAT may thus modulate membrane potential by ionic currents independently from its carrier action, contributing to signaling in the nervous system beyond a transporter’s canonical role. Immunohistochemical and in situ hybridization analyses on the adult rodent brain demonstrate that distribution of the transcript and the protein correspond quite closely

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The Development of Dopaminergic Neurons

with DA cell bodies and terminals, respectively.70, 71 Interestingly, the protein is also localized along nigral dendrites and cell bodies, suggesting that DA uptake is involved in the regulation of the extracellular concentration of dopamine in the substantia nigra,72,73 where it acts on DA autoreceptors to modulate DA synthesis and release (see above). DAT function can be controlled at various levels in order to modulate DA neurotransmission in the mature brain. The level of accumulation of both the protein72 and the mRNA varies among different DA areas.74,75 In addition, DAT activity can be modulated at the posttranslational level by phosphorylation76,77 and, perhaps, by glycosylation.78,79 The latter seems important for targeting DAT to appropriate membrane compartments. At the transcriptional level, DAT expression is downregulated in aging human substantia nigra,80 and in rat nigral neurons following repeated cocaine administration,81,75 whereas it is upregulated in the substantia nigra and the ventral tegmental area of amphetamine-sensitized rats.82 The human DAT gene contains a strong non-tissue-specific core promoter. Restriction of DAT gene expression to DA neurons seems dependent on negative regulatory sequences in the first intron, possibly like the neuron restrictive silencing elements.83 However, additional regulatory elements involved in defining cell-specific activity of DAT remain to be identified. DAT gene inactivation in transgenic mice has confirmed unequivocally the physiological role for DAT as the most critical component in terminating DA neurotransmission and its role as an obligatory target for the behavioral and the biochemical action of psychostimulants. Homozygote dat null mice show spontaneous hyperlocomotion due to protracted persistence of DA in the extracellular space and are insensitive to the action of amphetamine and cocaine.60 dat null mutant mice also show anterior pituitary hypoplasia, dwarfism, and an inability to lactate.84 Moreover, the absence of DAT produces extensive adaptive changes to control DA neurotransmission, such as a great decrease in the level of TH and in the content and release of DA.85 Thus DAT not only regulates the duration and intensity of DA neurotransmission but also plays a critical role in regulating presynaptic DA homeostasis, maintaining the delicate balance between DA synthesis, release, and degradation. Given the importance of neurotransmitter transport for DA function, defect in such a transport might well be expected to underlie some neurological diseases involving dysfunction in DA neurotransmission. Indeed, an association between polymorphism at the DAT locus and attention-deficit hyperactivity disorder,86 but not schizophrenia,87 has been shown. In addition, there is evidence that in the substantia nigra pars compacta of Parkinson patients the surviving DA neurons upregulate TH mRNA level and downregulate DAT mRNA level, suggesting that differential regulation of genes involved in neurotransmission can compensate for the reduced DA content.88

Interaction with Striatal Target Cells During embryonic development, DA synthesis, storage and high-affinity uptake appear to develop asynchronously, in a non-correlated fashion. In cells acutely dissociated from the embryonic rat ventral mesencephalon, measurable DA is detected as early as E12.5 and its concentration increases sharply at E16, reaching a plateau before birth. In the striatum, DA is first detected at E16, suggesting that DA nigral fibers reach their target tissue at this embryonic age,40 in accordance with morphological data showing the arrival of the first TH+ and AHD2+ axons to the striatum at that age.50 In contrast to the early appearance of endogenous DA levels in the mesencephalon, specific high-affinity DA uptake in rat mesencephalic cells is found only at E16, and increases sharply between E16 and E18, reaching a plateau before birth.40 Thus, the onset of DA uptake and its subsequent increase seem concomitant with the arrival of the first DA fibers to the striatum.

Development of Midbrain Dopaminergic Neurons

47

Consistent with the late appearance of the high-affinity DA uptake, DAT gene transcript is detected during the ontogeny of rat ventral mesencephalon only at around E15. On the contrary, the mRNAs for other genes involved in DA neurotransmission, namely TH and VMAT2, are already present at E12.41 Thus, the onset of DAT gene expression is delayed for several days when compared to that of the other transcripts examined and is concomitant with the establishment of the first contacts of the presynaptic DA fibers with the target striatal neurons. Striatal cells could be involved in the regulation, at a transcriptional level, of a key step in the maturation of DA neurotransmission in vivo. The level of DAT gene transcription and the corresponding uptake sites89,42 are selectively increased in rat E13 mesencephalic DA neurons in vitro after addition of E16 striatal cells in coculture (Fig. 3.5). More mature mesencephalic DA neuron cultures (E16) are not susceptible to the striatal influences on DAT mRNA and function. These observations suggest that mesencephalic DA neurons respond to target influences only within a restricted developmental window. Upregulation of DAT mRNA level by striatal cells in mesencephalic DA neurons in culture seems to require direct cell interactions, since target cells are ineffective when separated from mesencephalic cells by a barrier which allows diffusion of soluble molecules.89 Interestingly, the still unidentified “signals” derived from target striatal cells appear to be specific, since non-target cortical or cerebellar cells fail to stimulate DA uptake or DAT gene expression (Fig. 3.6). 89,90 Thus, DAT gene expression in developing mesencephalic DA neurons is conditioned by a specific cellular environment and probably requires continuous stimuli mediated by specific and direct cell interactions. Taken together, these results show that the maturation of DA neurotransmission follows a complex developmental pattern of activation of various genes, which can be selectively modulated by specific interaction with the developing target tissue. What is the physiological significance of the late appearance of DAT mRNA and its functional activity during prenatal midbrain development? The most obvious interpretation is that DA uptake is a critical step required only when synaptic transmission is established. This event probably occurs at around E15-E16 in the rodent brain, when developing DA neurons reach their target and when expression, or increased expression, of other DA1 pre- and postsynaptic markers (TH, DA receptors) takes place. The late appearance of DA uptake and its subsequent increase could explain why in early postnatal midbrain organotypic cultures there is an elevation in the extracellular concentration of DA due to spontaneous release, which is markedly more prolonged than in mature striatum.43 Cragg et al suggested that DA may have an action in developing circuits over spatial and temporal scales that vastly exceed those in mature synaptic transmission.43 This “morphogenic” role, although not essential for the development of brain DA circuits,45 could depend on the highly regulated temporal expression of DAT. Reduction of striatal target size induced by excitotoxic lesion in immature rats results in apoptosis of substantia nigra TH+ neurons, supporting the view that striatum is also necessary for survival and maintenance of postnatal nigral DA neurons.91 A large wealth of data demonstrate that specific influences derived from the target striatum are still required for the further postnatal development of the nigrostriatal pathway. In early postnatal organotypic slice cultures and cocultures of rat DA neurons of the ventral mesencephalon, TH+ neurites penetrate the striatal slice and thereafter stop their growth, whereas in cerebellar co-cultures no TH+ fibers enter the cerebellar tissue.92 On the other hand, a role of the cortex in midbrain DA fiber outgrowth in the striatum is suggested by the experiments of Plentz and Kitai. They show that in triple organotypic cultures, aimed at the reconstruction of the complex nigra-striatum-cortical network, TH+ fiber growth is blocked by the action of glutamate antagonists acting selectively on the metabotropic receptors.93

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The Development of Dopaminergic Neurons

Fig 3.5. TH+ neurons in midbrain primary cultures. Anti-tyrosine hydroxylase immunofluorescence in 4% paraformaldehyde-fixed 4 week old E13 ventral mesencephalic primary culture. A role of striatal cells in the regulation of DA fiber outgrowth was also observed in primary cocultures from early embryonic midbrain, where the growth of DA neurites is (temporarily) stopped when they encounter the target striatal cells, to subsequently restart, thus mimicking the onset of the well known phenomenon of “synapses en passant” so widely present in the striatum.94 The striatum thus seems to regulate, both in vivo and in vitro, the maturation of the dopaminergic function during early and late development and the survival of DA neurons in postnatal life. This hypothesis is also supported by a wealth of transplant studies showing that the striatum can sustain maturation, axonal growth and survival of grafted embryonic mesencephalic DA neurons (see chapter 8). Interestingly, only few DA neurons obtained from the embryonic hypothalamus, which do not normally innervate the striatum, survive if grafted into the lesioned mouse adult striatum,95 while fetal noradrenergic neuroblasts from the locus coeruleus are unable to restore striatal functional reinnervation.96 These findings are consistent with the hypothesis that in the nigrostriatal system a matching between proper targets and proper presynaptic elements is required for the maturation of embryonic dopaminergic neurons.

Acquisition of Polarized Shape In the substantia nigra DA neurons appear highly polarized, with axons directed rostrally toward the basal ganglia and the frontal cortex and dendrites confined caudally within the substantia nigra pars reticulata. Studies in vitro suggest that glial cells can differentially dictate the morphology of mesencephalic DA neurons. 97 The effect differs when mesencephalic or striatal glial cultures (or their conditioned media) are used.98 Glial

Development of Midbrain Dopaminergic Neurons

49

Fig 3.6. Target striatal cells increase dopamine transporter function and gene expression. In (A), 3H-DA uptake was measured as previously described40 in one week old E13 ventral mesencephalic primary cultures (M, ) and cocultures with target E16 striatal (M+ST, ) and non-target E16 cortical (M+CX, ) cells; striatal (ST, ) and cortical (CX, ) cultures show no uptake above background level. In (B), dopamine transporter (DAT) mRNA was measured in E13 mesencephalic ( ) primary cultures and cocultures with E16 striatal ( ) or cortical ( ) cells. Semi-quantitation of DAT transcripts was achieved by reverse transcriptase-PCR assay, and normalized to hypoxantine-phosphoribosyltransferase (HPRT) mRNA.41 Asterisks represent p<0.05 between cocultures and control mesencephalic cultures (ANOVA, Scheffé F -test).

The Development of Dopaminergic Neurons

50

monolayer from the mesencephalon (where DA cell bodies and their dendrites lie in vivo) allows development of highly branched DA neurons and induces dendritic and axonal elongation. In contrast, DA neurons grown on striatal glia (where, in vivo, DA axons sprout) develop a “linear” morphology with a single, thin axon. Thus, the acquisition of DA neuron polarity could be regulated by glia-neuron interactions. It has been proposed that these effects depend on differential adhesion properties of the two glial subpopulations which would preferentially allow axon elongation (low adhesion), or dendrite growth (high adhesion).98

Conclusion Key questions remain in developmental neurobiology regarding how neurons are specified, acquire their peculiar characteristics and find their final location within the CNS, elongate axons to find the right targets and establish functional synapses. Over the past few years, studies have begun to enlighten us on these questions. The development of the midbrain dopaminergic system has been studied extensively. It shows how complex and varied neural functions subserved by a small group of neurons can be achieved trough the correct interplay of an intrinsic program and extrinsic environmental influences. Recent findings indicate that midbrain neural plate progenitors differentiate into DA neurons by the combined action of two extracellular inducers, Sonic hedgehog and FGF8, and that these molecules are necessary and sufficient for the early induction of DA neurons along the ventral neuraxis. After this commitment, the function of two transcription factors is required in order to acquire the final determination and maintenance of the DA neurons in the midbrain. The orphan steroid nuclear receptor and transcription factor Nurr1 is essential for the final differentiation and survival of ventral mesencephalic DA precursors. Subsequently these neurons become positive for Ptx3, a homeodomain transcription factor that is uniquely expressed in midbrain DA neurons. Later, the maturation of DA neurotransmission follows a further complex developmental pattern of activation of various genes and modulation of their product activities. Several DA functions appear to be selectively modulated by specific interaction with the developing target tissue. Indeed, committed and determined DA neurons express the key genes involved in DA neurotransmission asynchronously, at different times in development. Synthesis and accumulation of DA is achieved shortly after expression of Nurr1 and Ptx3 , but the DAT gene remains repressed until DA axons begin to reach their target. Only at this time are DAT gene expression and functional uptake detectable. Cell contacts between the presynaptic DA neurons and target striatal neurons are apparently necessary for the fine modulation of DAT expression and uptake function, at least in primary cultures. Thus, cell-cell interaction, which plays a key role in the development of the entire nervous system from flies to man, has a crucial role in DA neuron maturation and maintenance.

Acknowledgments This work was supported by grants from UILDM-Telethon (621/95), Associazione Italiana Ricerca sul Cancro, Regione Campania (L.R. 1994 n. 41), PF Biotecnologie CNR. We thank Paola Da Pozzo for her contribution to some of the experiments here described.

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regions including the nigrostriatal dopamine system. Brain Res Mol Brain Res 1996; 41:111-120. 26. Zetterstrom RH, Solomin L, Jansson L et al. Dopamine neuron agenesis in Nurr1-deficient mice. Science 1997; 276:248-250. 27. Saucedo-Cardenas O, Quintana-Hau JD, Le WD et al. Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc Natl Acad Sci USA 1998; 95:4013-4018. 28. Perlman T, Jansson L. A novel pathway for vitamin A signaling mediated by RXR heterodimerization with NGFI-B and NURR1. Genes Dev. 1995; 9:769-782. 29. Forman BM, Umesono K, Chen J et al. Unique response pathways are established by allosteric interactions among nuclear hormone receptors. Cell 1995; 81:541-550. 30. Law SW, Conneely OM, DeMayo F et al. Identification of a new brain-specific transcription factor, NURR1. Mol Endocrinol 1992; 6:2129-2135. 31. Xing G, Zhang L, Zhang L et al. Rat nurr1 is prominently expressed in perirhinal cortex, and differentially induced in the hippocampal dentate gyrus by electroconvulsive vs. kindled seizures. Mol Brain Res 1997; 47:251-261. 32. Smidt MP, van Schaick HS, Lanctot C et al. A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons. Proc Natl Acad Sci USA 1997; 94:13305-13310. 33. Kumer SC, Vrana KE. Intricate regulation of tyrosine hydroxylase activity and gene expression. J. Neurochem 1996; 67:443-462. 34. Schambra UB, Duncan GE, Breese GR et al. Ontogeny of D1A and D2 dopamine receptor subtypes in rat brain using in situ hybridization and receptor binding. Neuroscience 1994; 62:65-85. 35. Koeltzow TE, Xu M, Cooper DC et al. Alterations in dopamine release but not dopamine autoreceptor function in dopamine D3 receptor mutant mice. J Neurosci 1998; 18:2231-2238. 36. Kehr W, Carlsson A, Lindqvist M et al. Evidence for a receptor-mediated feedback control of striatal tyrosine hydroxylase activity. J Pharm Pharmacol 1972; 24:744-747. 37. Farnebo LO, Hamberger B. Drug-induced changes in the release of 3 H-monoamines from field stimulated rat brain slices. Acta Physiol Scand Suppl 1971; 371:35-44. 38. Aghajanian GK, Bunney BS. Dopamine “autoreceptors”: Pharmacological characterization by microiontophoretic single unit recording studies. Naunyn Schmiedebergs Arch Pharmacol 1977; 297:1-7. 39. Sales N, Martres MP, Bouthenet ML et al. Ontogeny of dopaminergic D-2 receptors in the rat nervous system: Characterization and detailed autoradiographic mapping with [125I]iodosulpride. Neuroscience 1989; 28:673-700. 40. Fiszman ML, Zuddas A, Masana MI et al. Dopamine synthesis precedes dopamine uptake in embryonic rat mesencephalic neurones. J Neurochem 1991; 56:392-399. 41. Perrone-Capano C, Tino A, di Porzio U. Target cells modulate dopamine transporter gene expression during brain development. Neuroreport 1994; 5:1145-1148. 42. Daguet MC, Di Porzio U, Prochiantz A et al. Release of dopamine from dissociated mesencephalic dopaminergic neurons in primary cultures in absence or presence of striatal target cells. Brain Res 1980; 191:564-568. 43. Cragg SJ, Holmes C, Hawkey CR et al. Dopamine is released spontaneously from developing midbrain neurons in organotypic culture. Neuroscience 1998; 84:325-330. 44. Reinoso BS, Undie AS, Levitt P. Dopamine receptors mediate differential morphological effects on cerebral cortical neurons in vitro. J Neurosci Res 1996; 43:439-453. 45. Zhou Q-Y, Palmiter RD. Dopamine-deficient mice are severely hypoactive, adipsic, and aphagic. Cell 1995; 83:1197-1209. 46. Liu Y, Edwards RH. The role of vesicular transport proteins in synaptic transmission and neural degeneration. Annu Rev Neurosci 1997; 20:125-156. 47. Wang YM, Gainetdinov RR, Fumagalli F et al. Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivity to cocaine and amphetamine. Neuron 1997; 19:1285-1296.

Development of Midbrain Dopaminergic Neurons

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48. Fon EA, Pothos EN, Sun BC et al. Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action. Neuron 1997 19:1271-1293. 49. Gainetdinov RR, Fumagalli F, Wang YM et al. Increased MPTP neurotoxicity in vesicular monoamine transporter 2 heterozygote knockout mice. J Neurochem 1998; 70:1973-1978. 50. McCaffery P, Drager UC. High levels of a retinoic acid-generating dehydrogenase in the meso-telencephalic dopamine system. Proc Natl Acad Sci USA 1994; 91:7772-7776. 51. GFRα Nomenclature Committee. Nomenclature of GPI-linked receptors for the GDNF ligand family. Neuron 1997; 19:485. 52. Lindsay RM, Yancopoulos GD. GDNF in a bind with known orphan: Accessory implicated in new twist. Neuron 1996; 17:571-574. 53. Avantaggiato V, Dathan NA, Grieco M et al. Developmental expression of the ret protooncogene. Cell Growth Differ 1994; 5:305-311. 54. Nosrat CA, Tomac A, Hoffer BJ et al. Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mRNAs. Exp Brain Res 1997; 115:410-422. 55. Hyman C, Juhasz M, Jackson C et al. Overlapping and distinct actions of the neurotrophins BDNF, NT-3, and NT-4/5 on cultured dopaminergic and GABAergic neurons of the ventral mesencephalon. J Neurosci 1994; 14:335-347. 56. Merlio JP, Ernfors P, Jaber M et al. Molecular cloning of rat trkC and distribution of cells expressing messenger RNAs for members of the trk family in the rat central nervous system. Neuroscience, 1992; 51:513-532. 57. Prochiantz A, di Porzio U, Kato A et al. In vitro maturation of mesencephalic dopaminergic neurones from mouse embryos is enhanced in presence of their striatal target cells. Proc Natl Acad Sci U S A 1979; 76:5387-5391. 58. di Porzio U, Daguet MC, Glowinski J et al. Effect of striatal cells on in vitro maturation of mesencephalic dopaminergic neurones grown in serum-free conditions. Nature 1980; 288:370-373. 59. Amara SG, Arriza JL. Neurotransmitter transporters: Three distinct gene families. Curr Opin Neurobiol 1993; 3:337-344. 60. Giros B, Jaber M, Jones SR et al. Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 1996; 379:606-612. 61. Liu Y, Peter D, Roghani A et al. A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter. Cell 1992; 70:539-551. 62. Schinelli S, Zuddas A, Kopin IJ et al. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine metabolism and 1-methyl-4-phenylpyridinium uptake in dissociated cell cultures from the embryonic mesencephalon. J Neurochem 1988; 50:1900-1907. 63. Sulzer D, Chen TK, Lau YY et al. Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport. J Neurosci 1995; 15:4102-4108. 64. Jones SR, Gainetdinov RR, Wightman RM et al. Mechanisms of amphetamine action revealed in mice lacking the dopamine transporter. 1998; J. Neurosci. 18:1979-1986. 65. Frey K, Kilbourn M, Robinson T. Reduced striatal vesicular monoamine transporters after neurotoxic but not after behaviorally-sensitizing doses of methamphetamine. Eur J Pharmacol 1997; 334:273-279. 66. Bennett BA, Hollingsworth CK, Martin RS et al. Methamphetamine-induced alterations in dopamine transporter function. Brain Res 1998; 782:219-227. 67. Fumagalli F, Gainetdinov RR, Valenzano KJ et al. Role of dopamine transporter in methamphetamine-induced neurotoxicity: Evidence from mice lacking the transporter. J Neurosci 1998; 18:4861-4869. 68. Sonders MS and Amara SG. Channels in transporters. Curr Opin Neurobiol 1996; 6:294-302. 69. Lester HA, Cao Y, Mager S. Listening to neurotransmitter transporters. Neuron 1996; 17:807-810. 70. Revay R, Vaughan R, Grant S et al. Dopamine transporter immunohistochemistry in median eminence, amygdala, and other areas of the rat brain. Synapse 1996; 22:93-99. 71. Lorang D, Amara SG, Simerly RB. Cell-type-specific expression of catecholamine transporters in the rat brain. J Neurosci 1994; 14:4903-4914.

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The Development of Dopaminergic Neurons

72. Nirenberg MJ, Vaughan RA, Uhl GR et al. The dopamine transporter is localized to dendritic and axonal plasma membranes of nigrostriatal dopaminergic neurons. J Neurosci 1996; 16:436-447. 73. Hersch SM, Yi H, Heilman CJ, Edwards RH et al. Subcellular localization and molecular topology of the dopamine transporter in the striatum and substantia nigra. J Comp Neurol 1997; 388:211-227. 74. Blanchard V, Raisman-Vozari R, et al. Differential expression of tyrosine hydroxylase and membrane dopamine transporter genes in subpopulations of dopaminergic neurons of the rat mesencephalon. Mol Brain Res 1994; 22:29-38. 75. Burkett SA, Bannon MJ. Serotonin, dopamine and norepinephrine transporter mRNAs: Heterogeneity of distribution and response to ‘binge’ cocaine administration. Brain Res Mol Brain Res 1997; 49:95-102. 76. Huff RA, Vaughan RA, Kuhar MJ et al. Phorbol esters increase dopamine transporter phosphorylation and decrease transport Vmax. J Neurochem 1997; 68:225-232. 77. Reith ME, Xu C, Chen NH. Pharmacology and regulation of the neuronal dopamine transporter. Eur J Pharmacol 1997; 324:1-10. 78. Patel A, Uhl G, Kuhar MJ. Species differences in dopamine transporters: Postmortem changes and glycosylation differences. J Neurochem 1993; 61:496-500. 79. Vaughan RA, Brown VL, McCoy MT et al. Species- and brain region-specific dopamine transporters: Immunological and glycosylation characteristics. J Neurochem 1996; 66:2146-2152. 80. Bannon MJ, Poosch MS, Xia Y et al. Dopamine transporter mRNA content in human substantia nigra decreases precipitously with age. Proc Natl Acad Sci USA 1992; 89:7095-7099. 81. Xia Y, Goebel DJ, Kapatos G et al. Quantitation of rat dopamine transporter mRNA: Effects of cocaine treatment and withdrawal. J Neurochem 1992; 59:1179-1182. 82. Shilling PD, Kelsoe JR, Segal DS. Dopamine transporter mRNA is upregulated in the substantia nigra and the ventral tegmental area of amphetamine-sensitized rats. Neurosci Lett 1997; 236:131-134. 83. Kouzmenko AP, Pereira AM, Singh BS. Intronic sequences are involved in neural targeting of human dopamine transporter gene expression. Biochem Biophys Res Commun 1997; 240:807-811. 84. Bosse R, Fumagalli F, Jaber M et al. Anterior pituitary hypoplasia and dwarfism in mice lacking the dopamine transporter. Neuron 1997; 19:127-138. 85. Jones SR, Gainetdinov RR, Jaber M et al. Profound neuronal plasticity in response to inactivation of the dopamine transporter. Proc Natl Acad Sci USA 1998; 95:4029-4034. 86. Cook EH Jr, Stein MA, Krasowski MD et al. Association of attention-deficit disorder and the dopamine transporter gene. Am J Hum Genet 1995; 56:993-998. 87. Daniels J, Williams J, Asherson P et al. No association between schizophrenia and polymorphisms within the genes for debrisoquine 4-hydroxylase (CYP2D6) and the dopamine transporter (DAT). Am J Med Genet 1995; 60:85-87. 88. Joyce JN, Smutzer G, Whitty CJ et al. Differential modification of dopamine transporter and tyrosine hydroxylase mRNAs in midbrain of subjects with Parkinson’s, Alzheimer’s with parkinsonism, and Alzheimer’s disease. Mov Disord 1997; 12:885-897. 89. Perrone Capano C, Tino A, Amadoro G et al. Dopamine transporter gene expression in rat mesencephalic dopaminergic neurones is increased by direct interaction with target striatal cells in vitro. Mol Brain Res 1996; 39:160-166. 90. di Porzio U, Estenoz M. Positive control of target cerebellar cells on norepinephrine uptake in embryonic brainstem cultures in serum-free medium. Dev Brain Res 1984; 16:147-157. 91. Macaya A, Munell F, Gubits RM et al. Apoptosis in substantia nigra following developmental striatal excitotoxic injury. Proc Natl Acad Sci USA 1994; 91:8117-8121. 92. Holmes C, Jones SA, Greenfield SA. The influence of target and non-target brain regions on the development of mid-brain dopaminergic neurons in organotypic slice culture. Brain Res Dev Brain Res 1995; 88:212-219.

Development of Midbrain Dopaminergic Neurons

55

93. Plenz D, Kitai ST. Regulation of the nigrostriatal pathway by metabotropic glutamate receptors during development. J Neurosci 1998; 18:4133-4144. 94. Denis-Donini S, Glowinski J, Prochiantz A. Specific influence of striatal target neurons on the in vitro outgrowth of mesencephalic dopaminergic neurites: A morphological quantitative study. J Neurosci 1983; 3:2292-2299. 95. Zuddas A, Corsini GU, Barker JL et al. Specific reinnervation of lesioned mouse striatum by grafted mesencephalic dopaminergic neurones. Eur J Neurosci 1990; 3:72-85. 96. Hudson JL, Bickford P, Johansson M et al. Target and neurotransmitter specificity of fetal central nervous system transplants: Importance for functional reinnervation. J Neurosci 1994; 14:283-290. 97. Denis-Donini S, Glowinski J, Prochiantz A. Glial heterogeneity may define the threedimensional shape of mouse mesencephalic dopaminergic neurones. Nature 1984; 307:641-643. 98. Rousselet A, Autillo-Touati A, Araud D et al. In vitro regulation of neuronal morphogenesis and polarity by astrocyte-derived factors. Dev Biol 1990; 137:33-45.

CHAPTER 4

Growth Factor Actions on Developing Midbrain Dopaminergic Neurons J. Engele and N. Bayatti rowth factors* are polypeptide molecules that are believed to exert profound effects during both the development and regeneration of the nervous system. For example, they may rescue developing neurons from naturally occurring cell death, protect adult neurons from toxic insults and promote neuronal regeneration after brain lesions. From early on, these features have been considered as indicators that growth factors might also be of potential value in the therapy of certain neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and amytrophic lateral sclerosis.2,3 These ideas initiated a keen interest in the identification of growth factors affecting midbrain dopaminergic neurons, a neuronal population crucially involved in the etiology of Parkinson’s disease. A decade ago, fibroblast growth factor 2 (FGF2, originally known as basic fibroblast growth factor), was the first growth factor found to affect the in vitro development of embryonic dopaminergic neurons.4 The search was highlighted by the discovery of glial cell line-derived neurotrophic factor,5 which is currently regarded as the most promising growth factor for a possible therapy of Parkinson’s disease.6,7 Along the way, a puzzlingly large number of other growth factors belonging to different growth factor families have been identified which affect the in vitro development of dopaminergic neurons (see Table 4.1). In this chapter, we will review the known effects of growth factors on cultured dopaminergic neurons and then attempt to define the role that these growth factors play during the development of dopaminergic neurons in vivo.

G

Growth Factors Have Multiple Effects on Cultured Midbrain Dopaminergic Neurons The assay system routinely used by most investigators to identify growth factors affecting dopaminergic neurons consists of dissociated cell cultures of embryonic day (E) 14 to E16 rat mesencephalon. These cultures allow for the growth of postmitotic dopaminergic neurons, but not for the proliferation of dopaminergic precursors still present at this developmental *

Due to recent advances, the more general term growth factor instead of neurotrophic factor will be used. According to the original definition, (for a review see Korsching1) a neurotrophic factor has to fullfill the following criteria: Firstly, it must be present in limited concentrations in the target area of a given neuron. Secondly, it must be taken up by the responsive neuron and retrogradely transported to the soma to exert its effect. However, it has become apparent over the last few years that, in addition to this retrograde mode of action, various growth factors affect neurons in an autocrine or paracrine manner.1 Moreover, most growth factors originally found to affect neurons also act on non-neuronal cells such as glia. The Development of Dopaminergic Neurons, edited by Umberto di Porzio, Roberto Pernas-Alonso, and Carla Perrone-Capano. ©1999 R.G. Landes Company.

The Development of Dopaminergic Neurons

58

Table 4.1 Growth factor effects on cultured postmitotic dopaminergic neurons Growth Factor

S

M

T

N

References

BDNF

+

+

+

+

8,9,30,31,62,101-104

NT-3

+

+

+

?

9,103

NT-4/5

+

+

+

+

9,66,103

TGFβ-1,-2,-3

+

+

?

+

67,68,105

BMP-2,-4,-6,-7,-12

+

? + (BMP-6,-7) (BMP-2,-6)

106

Activin A

+

?

?

+

105

GDF-5

+

?

?

+

107

GDNF

+

+

+

?

5

Neurturin

+

?

?

?

25

Persephin

+

?

?

?

108

FGF-1, -2

+

+

+

+

4,8,42,48,109-112

EGF

+

?

+

+

48,53,111,113

TGFα

+

+

+

?

114

IGF-1,-2

+

-

+

+

8,48

CNTF

?

?

+


?

115

PDGF-BB

+

+

?

?

29,30

Midkine

+

?

+

?

11

HGF

+

+

+

?

117

IL-6,-7

+

?

?

?

118,119

Neurotrophins

TGFβ-superfamily

Others

S, survival; M, morpohology; T, transmitter turnover; N, neuroprotection; +, effects detectable; -, no detectable effects; ?, not tested;
, only in combination with dopamine.

Growth Factor Actions on Developing Midbrain Dopaminergic Neurons

59

stage. Growth factors that have been found to affect dopaminergic neurons under these assay conditions, and the specific effects exerted by these factors, are listed in Table 4.1. One prominent feature of these growth factors is their capability to rescue dopaminergic neurons from apoptotic cell death that usually occurs as a consequence of the culture conditions. Most of the identified growth factors are also capable of protecting dopaminergic neurons from insults induced by certain neurotoxins such as 6-OH-dopamine or N-methy-4-phenylpyridinium ion. In addition to their effects on dopaminergic cell survival, all identified growth factors seem to promote the morphological and functional maturation of dopaminergic neurons. These effects include increases in neurite length, number of primary neurites, number of branching points, soma size, dopamine (re)uptake level, tyrosine hydroxylase level/activity and dopamine content. Evidence exists that individual growth factors affect these morphological and functional parameters with different potencies.8,9 Some growth factors, e.g., insulin-like growth factor 1 (IGF-1)8 and insulin-like growth factor 2 (IGF-2) seem to specifically affect either the function or morphology of dopaminergic neurons. These differential effects could be caused by the culture conditions and/or reflect the mechanism by which these growth factors affect dopaminergic neurons (see following section) and thus have to be considered with some caution. Although none of these identified growth factors have been directly tested for effects on developing dopaminergic neurons in vivo, it is generally assumed that the growth factors affecting dopaminergic neurons in vitro also act on the intact brain. This assumption is essentially based on several observations made in adult animals. For example, many of the listed growth factors including brain-derived neurotrophic factor (BDNF), neurotrophin 4/5 (NT-4/5), growth/differentiation factor 5 (GDF5), GDNF, neurturin, fibroblast growth factor 1 (FGF1), FGF2, ciliary neurotrophic factor (CNTF), platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) rescue adult dopaminergic neurons from degenerative changes induced by either mechanical or chemical lesion of the nigrostrial fiber tract in vivo.6,10-25 Other studies have also observed that BDNF, GDNF or neurturin injected into the adult striatum stimulates dopamine turnover.25-28 To date, the only growth factors that have been tested on dopaminergic neurons in species other than rat are BDNF, GDNF, FGF2 and PDGF.29-35 The preliminary conclusion which can be drawn from these studies is that growth factors initially found to affect the development of rat dopaminergic neurons exert similar effects in other species, including monkey and human. In contrast to the large body of literature concerning the effects of growth factors on postmitotic dopaminergic neurons, little information is available about their role in the genesis of dopaminergic neurons. In the rat mesencephalon, postmitotic dopaminergic neurons are first detectable at E12.5.36 Bouvier and Mytilineou37 have observed that FGF2 is a mitogen for neuronal precursors in E12 mesencephalic cultures, leading to a marked increase in dopaminergic cell numbers and also in numbers of non-dopaminergic neurons. Other investigators have provided evidence that FGFs as well as the neurotrophins BDNF, neurotrophin 3 (NT-3) and NT-4/5 are capable of instructing neuronal precursors to differentiate into dopaminergic neurons.38-40 Intriguingly, all growth factors exert these instructive effects only in the presence of catecholamines. It has been suggested that in the developing brain such catecholamines might either derive from maternal sources or from neurons known to transiently express these neurotransmitters during brain development.

60

The Development of Dopaminergic Neurons

Some Growth Factors Affect Dopaminergic Neurons by an Indirect Glial-Mediated Mechanism Many of the growth factors that promote the survival and differentiation of cultured dopaminergic neurons also act as glial mitogens (see Table 4.2). In addition, it is well documented that glia as well as glial precursors secrete a factor(s) with potent effects on dopaminergic cell survival and differentiation41-46 (Engele, unpublished observations). The nature of this neurotrophic activity/activities is unknown, but is thought to represent a novel growth factor(s) for dopaminergic neurons.41,44,47 These findings indicate the possibility that the primary target for some of the identified growth factors are not dopaminergic neurons themselves but glia, which then, in a second step, would affect dopaminergic neurons. Initial support for the existence of such an indirect glial-mediated mode of action of growth factors on dopaminergic neurons came from studies with FGF1 and FGF2.42,48 These studies showed that the survival-promoting effects of FGF on cultured dopaminergic neurons are paralleled by a massive increase in glial numbers. Inhibition of this FGF-induced glial proliferation completely abolished the survival-promoting effects of this growth factor on cultured dopaminergic neurons. A subsequent and more detailed analysis of the effects of FGF2 on developing dopaminergic neurons revealed that this indirect glial-mediated mode of action of FGFs only occurs during early stages of embryonic development and switches to a direct effect at later developmental stages49 (see also below). The onset of these direct effects of FGFs on dopaminergic neurons seems to be species-specific and to occur much earlier in mouse development as compared to rat. Other growth factors which may be regarded as affecting dopaminergic neurons indirectly through glia are EGF and transforming growth factor αTGF-α both of which act through the same receptor, as well as PDGF and a number of bone morphogentic proteins (BMP-2, BMP-4, BMP-6, BMP-7 and BMP-12), see Table 4.2. Expression of the respective receptors of these growth factors by dopaminergic neurons has not been conclusively demonstrated. A few studies have reported that dopaminergic neurons express EGF and PDGF.30,50,51 However, these findings have not been supported by growth factor-induced expression of immediate early genes, e.g., c-fos, as a readout for functional signal transduction coupling, in dopaminergic neurons.52 Moreover, consistent with the proposed glial-mediated mode of action, EGF promotes dopaminergic cell survival and differentiation only in the presence of proliferating glia.53 Our recent findings indicate that the indirect glial-mediated growth factor effects are restricted to early embryonic stages of dopaminergic cell development. Only dopaminergic neurons cultured from the E15, but not those from E17, rat mesencephalon, respond to TGF-α and PDGF with an increase in cell survival. This correlates with loss of sensitivity of dopaminergic neurons for glial-derived neurotrophic activities between E15 and E17 of rat development49 (Engele, unpublished observations). The lack of sensitivity of dopaminergic neurons for glial-derived neurotrophic activities during late embryonic development does not necessarily mean that dopaminergic neurons remain insensitive to the influences of these indirectly acting growth factors during adulthood. In fact, some growth factors which we have classified as acting indirectly were found to rescue adult dopaminergic neurons from lesion-induced cell death.16,17 This raises the possibility that under certain pathological conditions dopaminergic neurons might be able to reexpress their sensitivity to glial-derived neurotrophic activities. Currently, several opinions exist with regard to the exact mechanism underlying the indirect glial-mediated mode of action of growth factors on dopaminergic neurons. Using cortical glia, Gaul and Lübbert41 provided evidence that growth factors exert their effects via the induction of neurotrophic activities within glia. Such inductive effects have not been observed in our laboratory using mesencephalic glia.54 We proposed that indirectly acting

Growth Factor Actions on Developing Midbrain Dopaminergic Neurons

61

growth factors increase the extracellular concentration of glial-derived neurotrophic activities by stimulating glial proliferation. It is not clear whether these contradictory outcomes reflect differences in the experimental set ups or differences in neurotrophic activities provided by cortical and mesencephalic glia.

Growth Factors with Direct Effects on Dopaminergic Neurons According to the criteria outlined in the previous section, a number of growth factors seem to exert direct effects on dopaminergic neurons. In the following, we will refer to this type of growth factor as a“dopaminergic growth factor”. GDNF, BDNF, NT-3, NT-4/5 and, under special circumstances, FGF1 and 2 are growth factors that act directly on dopaminergic neurons. We will not discuss NT-4/5 in detail, since this growth factor signals through the BDNF receptor.55 As shown in Table 4.2, all these growth factors do not require proliferating glia to affect dopaminergic cell survival and/or differentiation. Moreover, dopaminergic neurons within both the substantia nigra (SN) and the ventral tegmental area (VTA) seem to express the appropriate receptors for all these growth factors. This includes the high affinity receptor for BDNF, trkB; the high affinity receptor for NT-3, trkC;9,56 the GDNF receptor complex, consisting of the transducing subunit Ret and the binding subunit GFRα1 (also referred to as GDNFRα or TrnR1);57-59 as well as the FGF receptors FGFR1, FGFR2, and FGFR3.60,61 Finally, all these factors specifically activate growth factor-coupled signal transduction pathways in dopaminergic neurons.52 Studies on the time course of the survival-promoting action of GDNF and BDNF on cultured dopaminergic neurons have led to the surprising observation that both growth factors support dopaminergic cell survival only after a delay of at least four days after initial treatment62,63 (Engele, unpublished observations). This finding was quite unexpected, since cultured dopaminergic neurons showed signs of a functioning signal transduction machinery for all these growth factors long before the onset of survival promoting effects.52,63 We have reason to believe that this time lag reflects the initial absence of additional extracellular signals in mesencephalic cultures, required to initiate a survival promoting program. The dependence of growth factor-induced neuronal survival on such additional signals has been originally observed with retinal ganglion cells and ciliary ganglion neurons. The survival of these neurons is not promoted by growth factors unless they are depolarized and/or their intercellular cyclic AMP (cAMP) levels are increased pharmacologically.64,65 Recently, we obtained similar findings with midbrain dopaminergic neurons. Increasing intracellular cAMP levels by forskolin or dibutyryl cyclic AMP (dbcAMP), or depolarizing cells with potassium clearly accelerated the onset of the survival-promoting effects of BDNF and GDNF on cultured dopaminergic neurons63 (Engele, unpublished observations). A particular point of interest in these studies was to determine whether a similar cooperative mechanism applies for NT-3. Several studies have reported that NT-3 alone completely fails to affect dopaminergic cell survival,49,62,66 but see also Hyman et al.9 We have observed that in the presence of either dbcAMP or elevated potassium, NT-3 robustly promotes dopaminergic cell survival (Engele, unpublished observations). In summary, these findings suggest that growth factors are not, as often assumed, the masters of the game but just part of a multifactorial system necessary for proper neuronal development, maintenance and regeneration. It will be one of the future challenges to clearly define the other players and to understand how they interact with growth factors. At present, a number of growth factors remains which can not be classified with certainty as acting directly or indirectly. In this group of “ambiguous factors”, the most promising candidates for receiving the label “dopaminergic growth factor” are the transforming growth factor βs (TGF-β1, TGF-β2 and TGF-β3). While a direct action of TGF-βs on dopaminergic neurons has yet to be established, it is evident that TGF-βs do not affect dopaminergic

-

no49

acts through BDNF receptors

yes49

NT-3

NT-4/5

FGF-1,-2 (late rat development)

yes48,53 yes106 (BMP-6,-12) yes42,48 ?

yes48,53,114

yes106

yes42,48

yes49

EGF/TGFα

BMP-2,-4,-6,-7,-12

FGF-1,-2 (early rat development)

PDGF-BB

Indirectly acting

-

no49,101

BDNF

?

-

Effect dependent on proliferating glia

no5

Mitogen for mesencephalic glia

GDNF

Directly acting

Growth factor

Table 4.2. Classification of growth factors according to their mode of action

no52

yes52 (in mouse)

?

no52

yes52

yes52

yes52

yes52

Activates Fos in dopaminergic neurons

?

?

?

yes/no50,51,120

yes60,61

yes9,56

yes9,56

yes25,57,58

Receptor expressed by dopaminergic neurons

62 The Development of Dopaminergic Neurons

? ? ? ? ? -

yes49

?

yes49

?

yes107

no105

?

IGF-1,-2

HGF

IL-6

Il-7

GDF-5

Activin A

Persephin

-, does not apply; ?, not tested

?

?

Midkine

?

?

?

Neurturin

-

no68,105

TGFβ-1,-2,-3

Unknown mechanism

?

?

?

?

-

?

-

?

?

?

Table 4.2. cont., Classification of growth factors according to their mode of action (cont.)

?

?

?

?

?

?

?

?

yes25,57,58 (signaling subunit)

?

Growth Factor Actions on Developing Midbrain Dopaminergic Neurons 63

64

The Development of Dopaminergic Neurons

neurons via a glial-dependent mechanism.67,68 Another candidate is the recently identified GDNF homolog, neurturin.69 Under both in vitro and in vivo conditions, neurturin affects dopaminergic neurons with an efficacy that is indistinguishable from that of GDNF.25 It is interesting to note that dopaminergic neurons do not express genuine neurturin receptors25,70 (Hynes M, personal communication). Since both the neurturin and GDNF receptor complex show a close structural relationship,57,58,71 it is currently assumed that neurturin might affect dopaminergic neurons via GDNF receptors.25 The group of “ambiguous growth factors” also comprises midkine, IGF-1, IGF-2, hepatocyte growth factor (HGF), interleukin 6 (IL-6), interleukin 7 (IL-7), GDF5, activin A and persephin. Our functional knowledge of these growth factors is presently not at a stage which would allow any conclusion on their exact mode of action on dopaminergic neurons.

Growth Factor Sensitivity Defines Distinct Subpopulations of Dopaminergic Neurons Initial in situ hybridization studies indicated that not all dopaminergic neurons express receptors for all dopaminergic growth factors. For example, only a subpopulation of neurons in the SN and the VTA exhibit labeling for trkC as well as FGFR1, FGFR2 and FGFR3, whereas most neurons show labeling for trkB and ret.56,57,60,61 We have recently followed up these initial observations by using stimulated c-fos expression as an experimental paradigm to quantify dopaminergic neurons responsive to distinct growth factors. Our studies showed that in the embryonic mesencephalon about two-thirds of all midbrain dopaminergic neurons are responsive to GDNF; a similar number respond to BDNF, whereas only half and one-third of all dopaminergic neurons are responsive to FGF2 and NT-3 respectively.52 We have further observed that these four differently sized groups of growth factor-responsive dopaminergic neurons overlap. As illustrated in Figure 4.1, these observations lead to the definition of three subpopulations of dopaminergic neurons which respond to different sets of dopaminergic growth factors . One subpopulation is responsive to GDNF, BDNF, NT-3, FGF1 and FGF2, a second one to BDNF, GDNF, FGF1 and FGF2 and a third one to GDNF and BDNF. In addition, a fourth subpopulation seems to exist which is unresponsive to any of these four growth factors. It should be stressed, however, that this classification is preliminary in the sense that any additional dopaminergic growth factor identified in the future will lead to the definition of new subpopulations. At present, no additional information is available on the topographical localization of these various dopaminergic subtypes within the ventral mesencephalon, except for the finding that the action of NT-4/5 is confined to a subpopulation of calbindin-expressing dopaminergic neurons in the SN.72 It is currently thought that redundant growth factor systems exist for central nervous system neurons. This is essentially based on observations with various mouse lines carrying null mutations for either BDNF, NT-3, GDNF or the appropriate receptors.73-77 All these mutants showed unexpectedly few defects within the central nervous system, which contrasted with a massive impairment of specific components of the peripheral nervous system. Evidence for redundant growth factor effects on midbrain dopaminergic neurons also comes from the observations that BDNF, NT-3, GDNF, and FGF do not exert additive effects on dopaminergic cell survival63 (Engele, unpublished observations). However, the concept of such a redundant growth factor system does not fully explain the significance of the above defined subpopulations of growth factor-responsive dopaminergic neurons. In the case of a truly redundant mechanism, one should expect that all dopaminergic neurons are uniformly responsive to all dopaminergic growth factors. It has already been mentioned that, in addition to supporting neuronal survival, growth factors also affect other neuronal functions such as neurite length, neurotransmitter turnover and even electrical activity.78,79 It is also well documented that midbrain dopaminergic neurons are not uniform, but differ

Fig.4.1. Subpopulations of growth factor-responsive dopaminergic neurons. The bars on the left-hand side represent the four differently sized groups of dopaminergic neurons that are responsive to the indicated dopaminergic growth factors. Since these four groups overlap,52 it is possible to superimpose the bars (right-hand side). This allows the definition of three dopaminergic neuron subpopulations, each of which is sensitive to differing combinations of dopaminergic growth factors. A fourth subpopulation, comprising about one-fourth of all dopaminergic neurons and which is unresponsive to any of the listed dopaminergic growth factors, is not shown.

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with respect to their morphology and function.80 Examples for such functional differences are the rate of transmitter turnover and the spontaneous neuronal electrical activity. Thus, in addition to being a safety system, the diverse responsiveness of dopaminergic neurons might represent a means to individually shape cell morphology and function during development and/or to maintain these specific features during adulthood.

Fitting Growth Factors into the Developmental Schedule of Midbrain Dopaminergic Neurons In order to address the putative in vivo function of dopaminergic growth factors, it is necessary to first evaluate whether dopaminergic neurons have access to these factors within the intact brain. Growth factors capable of affecting dopaminergic cell development in vivo either derive from dopaminergic targets or are provided locally within the mesencephalon. During perinatal development, levels of BDNF, NT-3, GDNF, and FGF2 are very low within the cortical and striatal target areas and are only detectable by very sensitive methods (see, for example, refs. 81-84). In these brain areas, growth factor levels tend to decline further with ongoing maturation. At present, controversial opinions exist as to whether these small amounts of cortical and striatal growth factors are capable of affecting dopaminergic cell development85,86 (Engele, unpublished observations). Another and possibly more potent source for dopaminergic growth factors seem to be dopaminergic neurons themselves. In situ hybridization studies have demonstrated that in the brain of rat, monkey and human almost every dopaminergic neuron expresses mRNA encoding FGF2, and that a somewhat smaller number of dopaminergic neurons also expresses FGF1 mRNA.87 In addition, 10-50% of all dopaminergic neurons in the SN and VTA of adult rats were found to contain transcripts encoding BDNF and NT-3.88 More recently, Pochon and coworkers82 have demonstrated that most dopaminergic neurons in the adult rat mesencephalon also contain GDNF mRNA. Although the developmental role of these dopaminergic neuron-derived growth factors has not yet been experimentally addressed, an autocrine mechanism of growth factor support has been observed in the development of sensory and cortical neurons.89,90 It might, therefore, not be surprising to learn in the near future that distinct stages of dopaminergic cell development are predominantly affected by autocrine-acting dopaminergic growth factors. It should be mentioned, however, that we can not presently dismiss the existence of other local (mesencephalic) growth factor sources affecting developing dopaminergic neurons in a paracrine manner. In addition, dopaminergic neurons could be affected by anterogradely secreted growth factors.91 What are the implications of the known in vitro effects of growth factors on dopaminergic neurons in terms of their function in the developing brain? We have already pointed out that, due to our limited knowledge, no definitive conclusion can be made concerning the role growth factors play in the genesis of dopaminergic neurons, taking place between E12.5 and E16 in the rat.92 Future insights into the action of growth factors during this early generative phase might derive from studies on the orphan nuclear receptor, Nurr1,93 the homeodomain gene, Ptx394 and Sonic hedgehog95 or other factors that play a role in directing dopaminergic cell lineage. As outlined in this chapter, accumulating evidence suggests that the initial survival and differentiation of postmitotic dopaminergic neurons is predominantly affected by as yet unknown glial-derived neurotrophic activities. During this early stage of development a number of growth factors are further capable of affecting dopaminergic neurons indirectly via increasing the extracellular concentration of these glial-derived neurotrophic activities. After losing sensitivity for these glial influences around E17, dopaminergic neurons then seem to become dependent on the dopaminergic growth factors BDNF, NT-3, NT-4/5, GDNF, FGF1 and FGF2. When considering the putative in vivo action of these dopaminergic growth factors, it is necessary to keep in mind that at least

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BDNF, NT-3, and GDNF require additional signals to exert optimal survival effects on cultured dopaminergic neurons. It is not known whether this also applies to FGFs. Based on this cooperative mechanism, one could envision a scenario in which only dopaminergic neurons functionally integrated into a neuronal network would be rescued by dopaminergic growth factors, while others die. This is consistent with the fact that the proposed switch of growth factor dependence of dopaminergic neurons from glial-derived neurotrophic activities to BDNF, NT-3, NT-4/5, GDNF and FGFs is marked by the onset of a major dopaminergic cell death, occurring between E17 and postnatal day 7 of rat development.9699 Dopaminergic neurons surviving this phase of cell death then enter a second phase of differentiation which, in the rat, extends up to the third postnatal week.100 During this phase, the animal actively trains its motor skills. BDNF, NT-3, GDNF and FGFs could assist this fine tuning by individually sculpting the morphology and function of dopaminergic neurons. Whether or not this hypothesis will be supported by future experimental analysis, little doubt remains that dopaminergic cell development is affected by a complex network of growth factors.

Acknowledgments We would like to thank Prof. C. Pilgrim and Dr. G. Pezeshki for critical comments on the manuscript. Work in the author’s laboratory was supported by the Deutsche Forschungsgemeinschaft.

Note added in proof: A recent paper by Krieglstein et al (J Neurosci 1998; 18:9622-9634) demonstrated the presence of TGFb receptors in midbrain dopaminergic neurons. This paper further revealed that TGFb and GDNF cooperatively promote dopaminergic cell survival. A recently identified novel member of the GDNF family of growth factors, artemin, has been shown to have survival promoting effects on dopaminergic neurons (Baloh et al, Neuron 1998; 21:1291-1302).

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98. Janec EM, Burke RE. Naturally occurring cell death during postnatal development of the substantia nigra of the rat. Mol. Cell. Neurosci. 1993; 4:30-35. 99. Tepper JM, Damlama M, Trent F. Postnatal changes in the distribution and morphology of rat substantia nigra dopaminergic neurons. Neuroscience 1994; 60:469-477. 100. Voorn P, Kalsbeek A, Jorritsma-Byham B et al. The pre- and postnatal development of the dopaminergic cell groups in the ventral mesencephalon and the dopaminergic innervation of the striatum in the rat. Neuroscience 1988; 25:857-887. 101. Hyman C, Hofer M, Barde Y-A et al. BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Nature 1991; 350:230-232. 102. Beck KD, Knüsel B, Winslow JW et al. Pretreatment of dopaminergic neurons in culture with brain-derived neurotrophic factor attenuates toxicity of 1-methyl-4-phenylpyridinium. Neurodegeneration 1992; 1:27-36. 103. Studer L, Sprenger C, Seiler RW et al. Comparison of the effects of the neurotrophins on the morphological structure of dopaminergic neurons in cultures of rat substantia nigra. Eur. J. Neurosci. 1995; 7:223-233. 104. Spina MB, Squinto SP, Miller J et al. Brain-derived neurotrophic factor protects dopamine neurons against 6-hydroxydopamine and N-methyl-4-phenylpyridinium ion toxicity: Involvement of the glutathione system. J Neurochem 1992; 59:99-106. 105. Krieglstein K, Suter-Crazzolara C, Fischer WH et al. TGF-β superfamily members promote survival of midbrain dopaminergic neurons and protect them against MPP+ toxicity. EMBO J 1995; 14:736-742. 106. Jordan J, Böttner M, Schluesener HJ et al. Bone morphogenetic proteins: Neurotrophic roles for midbrain dopaminergic neurons and implications of astroglial cells. Eur J Neurosci 1997; 9:1699-1709. 107. Krieglstein K, Suter-Crazzolara C, Hötten G et al. Trophic and protective effects of growth/ differentiation factor 5, a member of the transforming growth factor-β superfamily, on midbrain dopaminergic neurons. J Neurosci Res 1995; 42:724-732. 108. Milbrandt J, de Sauvage FJ, Fahrner TJ et al. Persephin, a novel neurotrophic factor related to GDNF and neurturin. Neuron 1998; 20:245-253. 109. Giacobini MM, Almstrom S, Funa K et al. Differential effects of platelet-derived growth factor isoforms on dopamine neurons in vivo: -BB supports cell survival, -AA enhances fiber formation. Neuroscience 1993; 57:923-929. 110. Mayer E, Dunnett SB, Pellitteri R et al. Basic fibroblast growth factor promotes the survival of embryonic ventral mesencephalic dopaminergic neurons—I. Effects in vitro. Neuroscience 1993; 56:379-388. 111. Park TH, Mytilineou C. Protection from 1-methyl-4-phenylpyridinium (MPP+) toxicity and stimulation of regrowth of MPP(+)-damaged dopaminergic fibers by treatment of mesencephalic cultures with EGF and basic FGF. Brain Res 1992; 599:83-97. 112. Otto D, Unsicker K. FGF-2-mediated protection of cultured mesencephalic dopaminergic neurons against MPTP and MPP+: Specificity and impact of culture conditions, non-dopaminergic neurons, and astroglial cells. J Neurosci Res 1993; 34:382-393. 113. Ferrari G, Toffano G, Scaper SD. Epidermal growth factor exerts neuronotrophic effects on dopaminergic and GABAergic neurons: Comparison with basic fibroblast growth factor. J Neurosci Res 1991; 30:493-497. 114. Alexi T, Hefti F. Trophic actions of transforming growth factor α on mesencephalic dopaminergic neurons developing in culture. Neuroscience 1993; 55:903-918. 115. Magal E, Burnham P, Varon S et al. Convergent regulation by ciliary neurotrophic factor and dopamine of tyrosine hydroxylase expression in cultures of rat substantia nigra. Neuroscience 1993; 52:867-81. 116. Kikuchi S, Muramatsu H, Muramatsu T et al. Midkine, a novel neurotrophic factor, promotes survival of mesencephalic neurons in culture. Neurosci Lett 1993; 160:9-12. 117. Hamanoue M, Takemoto N, Matsumoto K et al. Neurotrophic effect of hepatocyte growth factor on central nervous system neurons in vitro. J Neurosci Res 1996; 43:554-564.

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118. Kushima Y, Hama T, Hatanaka H. Interleukin-6 is a neurotrophic factor for promoting the survival of cultured catecholaminergic neurons in a chemically defined medium from fetal and postnatal rat midbrain. Neurosci Res 1992 13:267-28. 119. von Coelln R, Unsicker K, Krieglstein K. Screening of interleukins for survival-promoting effects on cultured mesencephalic dopaminergic neurons from embryonic rat brain. Dev Brain Res 1995; 89:150-154. 120. Villares J, Faucheux B, Strada O et al. Autoradiographic study of [125I]epidermal growth factor-binding sites in the mesencephalon of control and parkinsonian brains post-mortem. Brain Res 1993; 628:72-76.

CHAPTER 5

The Effects of Sex and Sex Hormones on the Development of Dopaminergic Neurons Christof Pilgrim, Cordian Beyer, and Ingrid Reisert

Sex Differences of Adult Dopamine Systems

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ex differences in endocrine function and behavior, in particular reproductive behavior, occur in all vertebrates. It is believed that these differences are based on a sexually dimorphic neural circuitry. Evidence supporting this view is provided by sex differences in neuron numbers, synaptic density, and functional parameters of neurotransmission in certain brain areas and neural systems. In mammals, interest has traditionally focused on the hypothalamus, commonly seen as the center for control of reproductive behavior and integration of hormonal and neural responses of the organism. Notwithstanding this central role of the hypothalamus, it is important to note that more and more, often subtle, anatomical and/or functional sex differences have been and continue to be detected in all subdivisions of the mammalian brain. It is therefore not surprising that sex differences are not restricted to hypothalamic dopaminergic neurons known to be involved in neuroendocrine integration. Sex differences and effects of sex hormones have also been found in midbrain dopaminergic neurons and their projection areas. In order to familiarize the reader with the available evidence for dopamine-related sex differences, we begin with a brief survey about sex differences in hypothalamic and mesencephalic dopaminergic systems in the adult brain.

Hypothalamic Dopamine Systems

Hypothalamic control of pituitary secretion is sexually dimorphic1,2 and there is circumstantial evidence, mostly from observations made in the rat, that tubero-infundibular dopaminergic (TIDA) neurons play a role in mediating the effects of sex or sex hormones on the secretion of prolactin3 and gonadotropins.4,5 Early on, sex differences in dopamine levels were detected in the arcuate nucleus and median eminence, with male rats showing higher values than females.6 However, the turnover of dopamine in this region and its release into hypophysial portal blood is several-fold higher in females than in males.7,8 This corresponds well with the presence of higher levels of tyrosine hydroxylase (TH) mRNA and enzyme activity in the female than in the male arcuate nucleus and median eminence.9 Moreover, male and female TIDA neurons respond differently to hormonal signals. With respect to the feedback action of prolactin on DOPA synthesis, TIDA neurons of female rats are more sensitive than those of males.10 Downregulation of DOPAC levels by stress is seen in female animals only.11 In female TIDA neurons, estrogen stimulates, whereas in The Development of Dopaminergic Neurons, edited by Umberto di Porzio, Roberto Pernas-Alonso, and Carla Perrone-Capano. ©1999 R.G. Landes Company.

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males testosterone inhibits, immediate early gene expression.12 Sex differences can also be found in other hypothalamic dopaminergic cell groups. Similar to TIDA neurons, periventricular-hypophysial dopaminergic (tubero-hypophysial) neurons innervating the intermediate lobe exhibit higher transmitter synthesis and turnover rates than male neurons.13 The more dorsally situated dopaminergic cell groups appear to be more heterogeneous with regard to sex ratios. The rostral periventricular nucleus exhibits again a higher dopamine turnover14 and contains three times more TH mRNA-expressing neurons in the female than in the male15 whereas the dopaminergic neurons of the medial zona incerta were found to be more active in the male.14 The results obtained with the rat model suggest that—perhaps with the exception of tubero-hypophysial neurons—all of the above sex differences depend at least partially on circulating gonadal steroids. However, this is not necessarily true for other species, as the expression of nuclear estrogen receptor (ER) by adult hypothalamic dopaminergic neurons differs, e.g., between rats and primates.16,17

Mesostriatal and Mesolimbic Dopamine Systems Experimental as well as clinical data suggest that the extrapyramidal system is influenced by sex and sex hormones. Behavioral studies demonstrate sex differences in motor control. In rats, initiation of stereotype behavior by administration of amphetamine differs between sexes18,19 and in healthy humans, there are sex differences in capability of throwing and intercepting20 and in turning bias.21 Disturbances of motor control, such as Parkinson’s disease, Tourette’s syndrome, tardive dyskinesia, hereditary progressive dystonia, and attention-deficit/hyperactivity disorder show a sex-specific prevalence. 22-25 These observations may be related to sex differences observed in the mesostriatal dopamine system. Tissue levels,6 release26,27 and uptake28 of dopamine differ between male and female rat striatum. Recordings from neurons of the substantia nigra pars reticulata show a higher spontaneous activity in male than in female rats.29 Much less is known about sex differences in mesolimbic dopamine systems. Higher dopamine levels have been reported in male than in female rat diagonal band of Broca.6 The male rat nucleus accumbens is characterized by a higher density of D1 receptors as opposed to females.30 Interestingly, the latter sex difference extends into adulthood as opposed to the striatum, where the sex differences were found to be restricted to a transient, male-specific overproduction of dopaminergic receptors before the onset of puberty. Data contained in the majority of the above reports suggest that sex differences occuring in adult midbrain dopamine systems again depend on or are at least modulated by gonadal steroids.

Developmental Mechanisms The Role of Gonadal Steroids Sexual dimorphisms of the mammalian brain are generally ascribed to developmental effects of sex steroids during ontogenesis. Several mechanisms can be conceived to explain how sex steroids could bring about a sex-specific neural circuitry. Sex differences in proliferation of neuronal precursors or neuronal survival would result in sex differences in the number of cellular elements of a circuit. Sex differences in neuronal growth including soma, dendrites and axon would result in sex differences of receptive fields and/or synaptic input. All of these developmental events have been shown to be influenced by sex steroids (reviewed in ref. 31). Furthermore, there is ample evidence for the existence of a “critical” or “sensitive” period of brain development during which sex steroids must act to generate a sex-specific “blueprint”, which may become manifest only later in ontogenesis.32,33 In the rat, the earlier experimental data indicated the critical period to extend from embryonic day (E) 17/18 into the second postnatal week. Subsequently, Gorski and coworkers found

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the sensitivity for removal of endogenous sex steroids to last through at least postnatal day (P) 29.34 Their data appear to suggest the existence of multiple critical periods for different neural systems. Nevertheless, there is general agreement that the beginning of the critical period coincides with the peak of testosterone secretion from the fetal testis at E18.35 After entering the brain, testosterone may be aromatized to 17β-estradiol, the steroid thought to be responsible for the establishment of a male brain.36 It is believed that estrogens locally converted from androgens induce defeminization and masculinization of certain neural circuits as a prerequisite for sex-specific regulation of reproductive behavior and endocrine systems. According to this concept, development of a female brain is the “default pathway”, i.e., a passive event not requiring the presence of gonadal hormones. However, this view may be overly simplistic, as the developing female brain is also exposed to circulating estrogens. The difference between the hormonal environment of male and female brains may be a purely quantitative one in that the higher activity of the enzyme cytochrome P450 aromatase37 results in higher local levels of estrogen in the male brain. Evidence for a steroid-linked mechanism of sexual differentiation of the brain is provided by the effects of castration or hormonal treatment during development. Such experiments, when carried out in the critical period, may result in a sex-reversion of reproductive behavior and brain morphology.32,33 It is customary to distinguish such an “organizational” phase of steroid action, during which sex-specific neural circuits are laid down, from later ontogenetic periods, during which gonadal steroids are required to “activate” the sex-specific circuitry. While this is still a useful concept, it must be realized that considerable overlap may occur between organizational and activational effects of sex steroids with regard to timing as well as cellular mechanisms activated by the hormones.38 It appears that this concept of organizational and activational steroid action in the brain also applies to dopaminergic systems. As to the latter, we want to mention only briefly that both hypothalamic and nigrostriatal dopamine systems are subject to modulation by gonadal steroids during adulthood (cf. above). Since our present focus is on developmental mechanisms, we will not cover such activational effects of steroids in detail. Rather we will discuss the arguments that speak for the hypothesis that dopaminergic systems undergo a process of sex differentiation during brain development. The first argument is that sex differences in organization of dopaminergic systems and in functional indices of dopaminergic transmission begin to appear in the rat brain during the perinatal critical period. In different zones of the prenatal hypothalamus, there are sex differences in birthdates of TH-immunoreactive (TH-IR) neurons and in developmental time course of staining for TH. Zona incerta neurons are generated earlier in males than females, while the reverse is true for the arcuate nucleus.39 Between E17 and E20, the TH protein appears to accumulate more rapidly in males than in females.40 Specifically, TH-IR zona incerta neurons are larger in males than in females at E17 and E21.41 Another observation points to sex differences in transmitter metabolism in the perinatal rat hypothalamus. Higher activity of monoamine oxidase has been detected in the male than in the female anterior hypothalamus at P12.42 On the other hand, the function of dopaminergic neurons in the control of the pituitary appears to develop more rapidly in females, as an inhibition of LH secretion has been detected in two to three week old females but not males.5 Perinatal sex differences of dopaminergic systems are not restricted to the hypothalamus but have also been detected in the midbrain and projection areas of mesencephalic dopaminergic neurons. In the midbrain, sex ratios for numbers of TH-IR neurons at E17 and levels of dopamine at P12 are in favor of males.40,43 Beginning at E16 through E21, the female rat striatum contains higher densities of TH-IR axons than in the male.44 Likewise, in P10 and P21 cortex, higher levels of dopamine have been reported in female than in male rat.45

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The second argument for the occurrence of organizational effects of gonadal steroids on dopaminergic systems is that some of the sex differences seen in later life can be reversed in the rat by manipulation of the perinatal hormonal environment. Effects of perinatal androgen administration persisting into adulthood have been reported by Simerly and coworkers.4,15,46 They demonstrated sexual dimorphisms in numbers of TH-IR neurons and in TH-IR fiber density in the anterior periventricular nucleus of the adult rat hypothalamus which can be abolished by perinatal treatment of females with testosterone. Observations on ER “knockout” mice also underline the importance of steroid-mediated sex differentiation.47 Genetically male ER-deficient mice are characterized by an, albeit not complete, sex-reversal of the numbers of TH-IR neurons in the anteroventral periventricular hypothalamus. Developmental mechanisms are less clear as regards sex differences of midbrain dopaminergic neurons and their projection areas. Midbrain dopamine levels of P12 males were decreased to female levels by neonatal administration of diethylstilbestrol, a synthetic estrogen.43 Orchidectomy after birth and testosterone treatment of females reversed male to female and female to male monoamine oxidase activity values and dopamine levels in P10, P12 or P21 cortex.42,45 Because in these studies the follow-ups were confined to animals in the second and/or third postnatal week, the observations leave open the question of whether the sex reversal or the abolishment of sex differences was indeed permanent. However, even if the sex differences in dopaminergic transmission were of transient nature as has been observed by others,5,40,41,45 the observations would still be of interest. Transient changes in synaptic input are a frequently encountered phenomenon in neural development, which relates to the trophic action exerted by neurotransmitters during brain development. Dopamine is among the neurotransmitters that are presumed to participate in the shaping of the circuitry of target areas.48,49 With respect to sex differentiation of dopaminoceptive target cells, the possibility of an interplay between gonadal hormones and dopaminergic input needs to be considered. Provided that observations made in transfected cell lines50 can be reproduced in primary neurons or in vivo, the possibility arises that the ER may be activated, independently of the presence of its natural ligand, by stimulation of D1 receptors. Conversely, estrogens may activate signal transduction steps downstream of the D1 receptor (see below). It is thus quite probable that dopaminergic transmission and gonadal steroids interact in multiple ways to control the establishment of a sexually dimorphic circuitry in dopaminergic target areas in the forebrain. On the other hand, it must be taken into account that the sexual differentiation of dopaminergic systems themselves is mediated or modulated by input from other cells. The above in vivo experiments using manipulation of perinatal hormonal environment do not prove that developmental effects of sex steroids on dopaminergic neurons are direct. This is where the use of more simple experimental models, such as brain cell cultures, comes into play (see below).

Sex-Specific Vulnerability Dopaminergic systems appear to go through critical periods not only with respect to manipulation of systemic steroid levels but also to adverse effects of other neuroactive substances or environmental toxins. As mentioned above , there may be more than one critical period the timing of which might depend on condition, neural system, or species. Aside from the perinatal period, the prepubertal/adolescent period appears to be another vulnerable ontogenetic phase, during which dopaminergic transmission may be irreversibly altered.51 The salient point in our context is that the effects of interference with the development of dopaminergic systems have proved to be sex-specific or to interfere with the establishment of sex differences in several investigations. This holds for perinatal administration to rodents of dopamine itself,52 the dopaminergic antagonist haloperidol,5 nicotine,53,54 cocaine,55,56 ACTH,57 and stress.58 Male-specific neurotoxic effects of dopamine

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on developing dopaminergic neurons, presumably mediated by autoxidation of the transmitter, have even been shown to occur in primary cell culture.59 This latter observation, which has been made with male and female cells growing in identical hormonal environments, suggests that sex-specific vulnerability is an intrinsic property of dopaminergic neurons and is independent of the action of sex hormones. There is circumstantial evidence that sex-specific vulnerability of dopaminergic systems may also be found in humans and may be involved in pathophysiological mechanisms of neurological and affective disorders. Disorders related to alterations of dopaminergic function51,60 and presumed to have a neurodevelopmental cause, such as attention deficit/hyperactivity disorder and stuttering, Tourette’s syndrome and schizophrenia show a male-specific prevalence.23,25,61,62 Other indirect evidence argues for a female-specific susceptibility of developing mesolimbic dopamine systems to stress,63 which may in turn be related to the prevalence of major depressive disorder in female patients.64

Non-Classical Effects of Sex Steroids In the preceding chapters, we have dicussed the essential role that estrogens play in sex differentiation of dopaminergic neurons in that they modulate neuronal survival and growth. While it conforms to generally accepted views that sex differentiation and developmental effects of gonadal steroids pertain to hypothalamic dopaminergic neurons, we have emphasized that there is plenty of evidence that midbrain dopaminergic cells, too, are subject to organizational and activational effects of sex steroids. We have been able to demonstrate that estrogens are capable of stimulating outgrowth of TH-IR neurites,65 uptake of 3 H-dopamine,66 and TH mRNA expression67 in cultures of embryonic rat midbrain. Additive effects seen when 17β-estradiol and testosterone were applied simultaneously seem to be in line with the aromatization theory (see above). More recently, we have shown that the estrogen-forming enzyme aromatase is transiently expressed around birth in the ventral rat midbrain in vivo.68 However, interpretation of these results faces one major problem which will be discussed in this section. It is generally accepted that the effects of estrogen on target cells depend on the interaction of the steroid hormone with specific nuclear ERs, the subsequent activation of the ligand-receptor complex, and its binding to gene regulatory sequences in promoter regions of target genes, namely ER responsive elements and AP-1 sites.69-71 Such genomic effects involve transcriptional activation and protein synthesis, which usually require several hours for their expression.72 Aside from the well-known isoform of the ER (now termed ER-α), a novel isoform, ER-β, has recently been cloned73 which is widely expressed in the rat brain, especially in hypothalamus, preoptic area, and limbic brain.74,75 As to the midbrain, earlier observations had suggested that it was largely devoid of ER bearing cells except a few in the central gray.76-78 In contrast, the more recent studies employing probes specific for ER-β transcripts show that this subtype is expressed in the ventral tegmental area and substantia nigra.75,79 However, there is as yet no positive evidence that ER-β is expressed specifically by mesencephalic dopaminergic neurons. This lack of information raises the question of alternative mechanisms which could mediate estrogen effects on these neurons. In this context, we want to draw attention to a new concept of “non-genomic” cellular estrogen signaling that has been developed over the last years.80-82 This concept is based on findings which show that estrogens can change the physiology of forebrain neurons within seconds after application.83 The rapid onset, the time course, and the pharmacological profile, i.e., kinetics and insensitivity to transcriptional and translational inhibitors, clearly show that they the observations are not compatible with the classical genomic mechanism. Following a decade of intense research on this emerging new field, it seems now safe to conclude that estrogens may interact with specific binding sites/receptors on the plasma membrane of neurons,84-86

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thereby activating distinct intracellular signal transduction pathways (for a recent review see ref. 82) Among the signal cascades studied so far in the brain, estrogens have been shown to stimulate the formation of cyclic AMP (cAMP),87 the phosphorylation of the cAMP response element binding protein CREB88 and the formation of IP3,89 and to raise intracellular free Ca2+ levels.90 What then is the evidence that midbrain dopaminergic neurons are targets for such rapid “non-genomic” estrogen effects? In adult rats, estrogens elicit acute responses with respect to firing rate,91 dopamine synthesis,92 dopamine uptake,28 dopamine release,27 and amphetamine-induced stereotypic behavior.93 These effects largely occur within seconds to minutes after exposure. Based on these observations, we followed the hypothesis that such “non-genomic” mechanisms are activated not only in adulthood but also during development and that they are responsible for sex differentiation of midbrain dopaminergic neurons.65-67 Since activation of Ca2+-dependent signal cascades is required for stimulation of neuronal growth94 and dopamine synthesis,95 we monitored intracellular Ca2+ levels in cultured embryonic dopaminergic midbrain neurons. Briefly, we found that estrogens provoke a rapid (1-3 seconds) and transient Ca2+ release from intracellular stores via the IP3 signaling pathway. This effect was stereo- and steroid-specific, dose-dependent and mediated through membrane receptors.96 Interestingly, Ca2+ is not the only intracellular signal transduction system activated by estrogens in midbrain dopaminergic cells. Ongoing experiments revealed that estrogens also stimulate cAMP formation and CREB phosphorylation in these differentiating neurons (unpublished). By using different pharmacological approaches to inhibit Ca2+- and cAMP-dependent signaling cascades, we were able to demonstrate that the estrogen-stimulated Ca2+ increase stimulates adenylate cyclase, possibly via activation of a Ca2+/calmodulin protein kinase (CaMK).97 Finally, estrogen-dependent stimulation of neurite outgrowth in dopaminergic neurons65 was completely abolished by suppressing either the Ca2+ or the cAMP signaling pathways (unpublished). To our knowledge, this is the first evidence that “non-genomic” estrogen effects are involved in the control of differentiation of dopaminergic neurons. Future research on sex differentiation of midbrain dopaminergic neurons will have to clarify: 1. The nature of membrane-bound estrogen receptors; and 2. The signals activated downstream of CREB phosphorylation. Concerning the first question, it appears to be accepted that estrogens can interact, on the level of the plasma membrane, with distinct neurotransmitter receptors such as NMDA, dopamine, and GABA receptors, and voltage-sensitive ion channels (reviewed in ref. 82). However, at present, no data are available to show which of these interactions occur specifically in dopaminergic neurons. As to the activation of CREB by estrogens, it is known that control of neuronal differentiation by estrogen involves growth factor signaling pathways.98,99 CREB is a major mediator of neuronal neurotrophin responses.100 Development of midbrain dopaminergic neurons is controlled by members of the neurotrophin as well as the TGF-β family (for details see chapter 4). Future research strategies will, therefore, have to focus on crosscoupling of estrogen- and growth factor-dependent intracellular signaling cascades.

Steroid-Independent Sex Differentiation Thus far, the discussion has proceeded from the widely accepted view that sexual differentiation is an epigenetic phenomenon brought about by a hormonal signal from the developing male gonad, i.e., by cell-extrinsic cues in the environment of the developing dopaminergic neuron. This view has been contested by our group on the basis of experiments with primary cell cultures of embryonic rat brain. In order to distinguish effects of gonadal hormones from other sex-linked factors, a new experimental approach was adopted. Instead of raising cultures from pooled male and female rat embryos, we prepared sex-specific

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dissociated cell cultures after having separated male and female embryos by inspection of the gonads (for details see refs. 66, 101). The cultures, which were prepared from E14 midbrain and diencephalon, contain considerable numbers of dopaminergic neurons the phenotype of which closely resembles that found in vivo in the respective brain region.102,103 Surprisingly, these neurons develop region-specific morphological and functional sex differences despite the fact that male and female cells were raised in identical hormonal environments. The sex differences concern numbers and size of dopaminergic neurons, (re)uptake capacity of the transmitter, dopamine content, and DOPA synthesis. Diencephalic cultures of female rats were characterized by higher dopamine uptake101 as well as endogenous dopamine levels104 and DOPA synthesis rates105 than cultures derived from male tissue. Male diencephalic dopaminergic neurons of rat were larger in size41 but numbers of male and female dopaminergic neurons were not different.104 Male rat midbrain neurons exhibited a higher uptake capacity than female cells66 whereas dopamine content as well as numbers of dopaminergic neurons were higher in female cultures.104 DOPAC levels of midbrain cultures were not sexually dimorphic.104 Although it is obvious that most of the above results could not have been acquired if it were not for the availability of sex-specific neural cultures, the relevance to developmental processes taking place in vivo remains to be established. However, there are some clues that indicate that the observations made in vitro are specific and are not cultivation artifacts. The existence, albeit transient, of sex differences in dopaminergic cell numbers was confirmed in the mesencephalon of E17 intact rat embryos.40 Also, the sex differences in soma sizes of diencephalic dopaminergic neurons are qualitatively and quantitatively similar in vitro and in vivo.41 The salient point of the in vitro investigations on dopaminergic neurons developing in sex-specific cultures is that sexual dimorphisms of dopaminergic neurons may develop independently of the action of gonadal steroids. This is in contradiction to the generally accepted theory which holds that sexual differentiation of the brain is caused solely by the organizational effect of gonadal steroids present during a critical period of brain development. The brain tissue used to raise the above cultures was removed at E14, i.e., well before sex differences in hormonal environment of the embryo are known to develop.106,107 In order to cope with the remote possibility that sex steroid-dependent determinative events occur in utero before the brain tissue is taken into culture, additional cultures were prepared from embryos whose mothers had been treated with the estrogen antagonist tamoxifen or the androgen antagonist cyproterone acetate. These antisteroids competitively inhibit the effects of the gonadal hormones on target cells by interference with the uptake process, receptor binding, translocation to the nucleus, and/or DNA binding of the ligand-receptor complex. In spite of this pretreatment, DOPA synthesis rates were again higher in diencephalic cultures raised from females than from males.105 In conclusion, epigenetic control by the hormonal environment cannot be the only mechanism responsible for the generation of the sexual dimorphisms described above. Other mechanisms, such as cell-autonomous realization of a sex-specific genetic program, must be invoked to fully understand sexual differentiation of dopamine systems (cf. refs. 31, 108 for a detailed discussion of this issue). A list of genes that merit attention with respect to induction and/or maintenance of hormone-independent sex differences in the mammalian brain has recently been compiled by S.C. Maxson.109

Acknowledgments We gratefully acknowledge financial support by the Deutsche Forschungsgemeinschaft and the Biomed I program of the European Commission.

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20. Watson NV, Kimura D. Nontrivial sex differences in throwing and intercepting: Relation to psychometrically-defined spatial functions. Person individ Diff 1991; 12:375-385. 21. Mead LA, Hampson E. A sex difference in turning bias in humans. Behav Brain Res 1996; 78:73-79. 22. Kane JM, Smith JM. Tardive dyskinesia. Arch Gen Psychiatry 1982; 39:473-481. 23. Minde K. Hyperaktives syndrom. In: Remschmidt H, Schmidt MH, eds. Kinder- und Jugendpsychiatrie in Klinik und Praxis, Bd.3: Alterstypische, reaktive und neurotische Störungen. Stuttgart: Thieme, 1985:1-18. 24. Lilienfeld DE, Sekkor D, Simpson S et al. Parkinsonism death rates by race, sex and geography: A 1980s update. Neuroepidemiology 1990; 9:243-247. 25. Segawa M, Nomura Y. Hereditary progressive dystonia with marked diurnal fluctuation and dopa-responsive dystonia: Pathognomonic clinical features. In: Segawa M, Nomura Y, eds. Age-related dopamine-dependent disorders. Vol. 14, Monographs in Neural Sciences. Tokyo: Karger, 1995:10-24. 26. Becker JB, Ramirez VD. Experimental studies on the development of sex differences in the release of dopamine from striatal tissue fragments in vitro. Neuroendocrinology 1981; 32:168-173. 27. Becker JB. Direct effect of 17β-estradiol on striatum: Sex differences in dopamine release. Synapse 1990; 5:157-164. 28. Morissette M, Di Paolo T. Sex and estrous cycle variations of rat striatal dopamine uptake sites. Neuroendocrinology 1993; 58:16-22. 29. Wilson MA. Gonadectomy and sex modulate spontaneous activity of substantia nigra pars reticulata neurons without modifying GABA/benzodiazepine responsiveness. Life Sci 1993; 53:217-225. 30. Andersen SL, Rutstein M, Benzo JM et al. Sex differences in dopamine receptor overproduction and elimination. NeuroReport 1997; 8:1495-1498. 31. Pilgrim C, Hutchison JB. Developmental regulation of sex differences in the brain: Can the role of gonadal steroids be redefined? Neuroscience 1994; 60:843-855. 32. Dörner G. Sexual differentiation of the brain. Vitamins and Hormones 1980; 38:325-381. 33. Goy RW, McEwen BS. Sexual differentiation of the brain. Cambridge: MIT Press, 1980. 34. Davis EC, Shryne JE, Gorski RA. A revised critical period for the sexual differentiation of the sexually dimorphic nucleus of the preoptic area in the rat. Neuroendocrinology 1995; 62:579-585. 35. Ward OB, Wexler AM, Carlucci JR et al. Critical periods of sensitivity of sexually dimorphic spinal nuclei to prenatal testosterone exposure in female rats. Horm Behav 1996; 30:407-415. 36. Naftolin F, Ryan KJ, Davies IJ et al. The formation of estrogens by central neuroendocrine tissues. Recent Prog Horm Res 1975; 31:295-315. 37. Beyer C, Wozniak A, Hutchison JB. Sex-specific aromatization of testosterone in mouse hypothalamic neurons. Neuroendocrinology 1993; 58:673-681. 38. Arnold AP, Breedlove SM. Organizational and activational effects of sex steroids on brain and behavior: A reanalysis. Horm Behav 1985; 19:469-498. 39. Balan IS, Ugrumov MV, Borisova NA et al. Birthdates of the tyrosine hydroxylase immunoreactive neurons in the hypothalamus of male and female rats. Neuroendocrinology 1996; 64:405-411. 40. Reisert I, Schuster R, Zienecker R et al. Prenatal development of mesencephalic and diencephalic dopaminergic systems in the male and female rat. Dev Brain Res 1990; 53:222-229. 41. Kolbinger W, Trepel M, Beyer C et al. The influence of genetic sex on sexual differentiation of diencephalic dopaminergic neurons in vitro and in vivo. Brain Res 1991; 544:349-352. 42. Gaziri LCJ, Ladosky W. Monoamine oxidase variation during sexual differentiation. Neuroendocrinology 1973; 12:249-256. 43. Wilson WE, Agrawal AK. Brain regional levels of neurotransmitter amines as neurochemical correlates of sex-specific ontogenesis in the rat. Dev Neurosci 1979; 2:195-200.

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44. Ovtscharoff W, Eusterschulte B, Zienecker R et al. Sex differences in densities of dopaminergic fibers and GABAergic neurons in the prenatal rat striatum. J comp Neurol 1992; 323:299-304. 45. Stewart J, Kühnemann S, Rajabi H. Neonatal exposure to gonadal hormones affects the development of monoamine systems in rat cortex. J Neuroendocrinol 1991; 3:85-93. 46. Simerly RB, Swanson LW, Handa RJ et al. Influence of perinatal androgen on the sexually dimorphic distribution of tyrosine hydroxylase-immunoreactive cells and fibers in the anteroventral periventricular nucleus of the rat. Neuroendocrinology 1985; 40:501-510. 47. Simerly RB, Zee MC, Pendleton JW et al. Estrogen receptor-dependent sexual differentiation of dopaminergic neurons in the preoptic region of the mouse. Proc Natl Acad Sci USA 1997; 94:14077-14082. 48. Schmidt U, Beyer C, Oestreicher AB et al. Activation of dopaminergic D1 receptors promotes morphogenesis of developing striatal neurons. Neuroscience 1996; 74:453-460. 49. Schmidt U, Pilgrim C, Beyer C. Differentiative effects of dopamine on striatal neurons involve stimulation of the cAMP/PKA pathway. Mol Cell Neurosci 1998; 11:9-18. 50. Power RF, Mani SK, Codina J et al. Dopaminergic and ligand-independent activation of steroid hormone receptors. Science 1991; 254:1636-1639. 51. Lewis DA. Development of the prefrontal cortex during adolescence: Insights into vulnerable neural circuits in schizophrenia. Neuropsychopharmacology 1997; 16:385-398. 52. González MI, Leret ML. Role of monoamines in the male differentiation of the brain induced by androgen aromatization. Pharmacol Biochem Behav 1992; 41:733-737. 53. Lichtensteiger W, Ribary U, Schlumpf M et al. Prenatal adverse effects of nicotine on the developing brain. Prog Brain Res 1988; 73:137-157. 54. Fung YK, Lau Y-S. Effects of prenatal nicotine exposure on rat striatal dopaminergic and nicotinic systems. Pharmacol Biochem Behav 1989; 33:1-6. 55. Vathy I, Katay L, Mini KN. Sexually dimorphic effects of prenatal cocaine on adult sexual behavior and brain catecholamines in rats. Dev Brain Res 1993; 73:115-122. 56. Miller MW, Waziri R, Baruah S et al. Long-term consequences of prenatal cocaine exposure on biogenic amines in the brains of mice: The role of sex. Dev Brain Res 1995; 87:22-28. 57. Fameli M, Kitraki E, Stylianopoulou F. Effects of hyperactivity of the maternal hypothalamic-pituitary-adrenal (HPA) axis during pregnancy on the development of the HPA axis and brain monoamines of the offspring. Int J Dev Neurosci 1994; 12:651-659. 58. Reznikov AG, Nosenko ND. Early postnatal changes in sexual dimorphism of catecholamine and indoleamine content in the brain of prenatally stressed rats. Neuroscience 1996; 70:547-551. 59. Lieb K, Andrae J, Reisert I et al. Neurotoxicity of dopamine and protective effects of the NMDA receptor antagonist AP-5 differ between male and female dopaminergic neurons. Exp Neurol 1995; 134:222-229. 60. Comings DE, Wu S, Chiu C et al. Polygenic inheritance of Tourette syndrome, stuttering, attention deficit hyperactivity, conduct, and oppositional defiant disorder: The additive and subtractive effect of the three dopaminergic genes -DRD2, DβH, and DAT1. Am J Med Genet 1996; 67:264-288. 61. Andrews G, Harris M. The syndrome of stuttering. London: W.Heinemann Medical Books Ltd., 1964 62. Cowell PE, Kostianovsky DJ, Gur RC et al. Sex differences in neuroanatomical and clinical correlations in schizophrenia. Am J Psychiatry 1996; 153:799-805. 63. Alonso SJ, Navarro E, Rodriguez M. Permanent dopaminergic alterations in the n. accumbens after prenatal stress. Pharmacol Biochem Behav 1994; 49:353-358. 64. Young MA, Fogg LF, Scheftner WA et al. Sex differences in the lifetime prevalence of depression: Does varying the diagnostic criteria reduce the female/male ratio? J Affective Disord 1990; 18:187-192. 65. Reisert I, Han V, Lieth E et al. Sex steroids promote neurite outgrowth in mesencephalic tyrosine hydroxylase immunoreactive neurons in vitro. Int J Dev Neurosci 1987; 5:91-98. 66. Engele J, Pilgrim C, Reisert I. Sexual differentiation of mesencephalic neurons in vitro: Effects of sex and gonadal hormones. Int J Dev Neurosci 1989; 7:603-611.

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67. Raab H, Pilgrim C, Reisert I. Effects of sex and estrogen on tyrosine hydroxylase mRNA in cultured embryonic rat mesencephalon. Mol Brain Res 1995; 33:157-164. 68. Raab H, Beyer C, Wozniak A et al. Ontogeny of aromatase messenger ribonucleic acid and aromatase activity in the rat midbrain. Mol Brain Res 1995; 34:333-336. 69. Evans R. The steroid and thyroid receptor superfamily. Science 1988; 240:880-895. 70. Peale FV, Ludwig LB, Zain S et al. Properties of high affinity DNA binding site for estrogen receptor. Proc Natl Acad Sci USA 1988; 85:1038-1042. 71. Paech K, Webb P, Kuiper GGJM et al. Differential ligand activation of estrogen receptors ERα and ERβ at AP1 sites. Science 1997; 277:1508-1510. 72. Lee YL, Gorski J. Estrogen-induced transcription of the progesterone receptor gene does not parallel estrogen receptor occupancy. Proc Natl Acad Sci USA 1996; 93:15180-15184. 73. Kuiper GGJM, Enmark E, Pelto-Huikko M et al. Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 1996; 93:5925-5930. 74. Li X, Schwartz PE, Rissman EF. Distribution of estrogen receptor-β-like immunoreactivity in rat forebrain. Neuroendocrinology 1997; 66:63-67. 75. Shughrue PJ, Lane MV, Merchenthaler I. Comparative distribution of estrogen receptor-α and -β mRNA in the rat central nervous system. J comp Neurol 1997; 388:507-525. 76. Stumpf WE, Sar M. Steroid hormone target cells in the extrahypothalamic brain stem and cervical spinal cord: Neuroendocrine significance. J Steroid Biochem 1979; 11:801-807. 77. Blaustein JD. Cytoplasmic estrogen receptors in rat brain: Immunocytochemical evidence using three antibodies with distinct epitopes. Endocrinology 1992; 131:1336-1342. 78. Corodimas KP, Morrell JI. Estradiol-concentrating forebrain and midbrain neurons project directly to the medulla. J comp Neurol 1990; 291:609-620. 79. Kuiper GGJM, Carlsson B, Grandien K et al. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors α and β. Endocrinology 1997; 138:863-870. 80. Schumacher M. Rapid membrane effects of steroid hormones: An emerging concept in neuroendocrinology. Trends Neurosci 1990; 13:359-362. 81. McEwen BS. Non-genomic and genomic effects of steroids on neural activity. Trends Pharmacol Sci 1991; 12:141-147. 82. Wong M, Thompson TL, Moss RL. Nongenomic actions of estrogen in the brain: Physiological significance and cellular mechanisms. Crit Rev Neurobiol 1996; 10:89-203. 83. Nabekura J, Oomura Y, Minami T et al. Mechanism of the rapid effect of 17β-estradiol on medial amygdala neurons. Science 1986; 233:226-228. 84. Towle AC, Sze PY. Steroid binding to synaptic plasma membrane: Differential binding of glucocorticoids and gonadal steroids. J Steroid Biochem 1983; 18:135-143. 85. Pappas TC, Gametchu B, Watson CS. Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB J 1995; 9:404-410. 86. Ramirez VD, Zheng JB, Siddique KM. Membrane receptors for estrogen, progesterone, and testosterone in the rat brain: Fantasy or reality. Cell Mol Neurobiol 1996; 16:175-198. 87. Gu QG, Moss RL. 17β-estradiol potentiates kainate-induced currents via activation of the cAMP cascade. J Neurosci 1996; 16:3620-3629. 88. Zhou Y, Watters JJ, Dorsa DM. Estrogen rapidly induces the phosphorylation of the cAMP response element binding protein in rat brain. Endocrinology 1996; 137:2163-2166. 89. Favit A, Fiore L, Nicoletti F et al. Estrogen modulates stimulation of inositol phospholipid hydrolysis by norepinephrine in rat brain slices. Brain Res 1991; 555:65-69. 90. Nikezik G, Horvat A, Nedeljkovic N et al. 17b-estradiol in vitro affects Na+-dependent and depolarization-induced Ca2+ transport in brain synaptosomes. Experientia 1996; 52:217-220. 91. Chiodo LA, Caggiula AR. Alterations in basal firing rate and autoreceptor sensitivity of dopamine neurons in the substantia nigra following acute and extended exposure to estrogen. Eur J Pharmacol 1980; 67:165-166. 92. Pasqualini C, Olivier V, Guibert B et al. Acute stimulatory effect of estradiol on striatal dopamine synthesis. J Neurochem 1995; 65:1651-1657. 93. Becker JB. Estrogen rapidly potentiates amphetamine-induced striatal dopamine release and rotational behaviour during microdialysis. Neurosci Lett 1990; 118:169-171.

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94. Lipton SA, Kater SB. Neurotransmitter regulation of neuronal outgrowth, plasticity and survival. Trends Neurosci 1989; 12:265-270. 95. Zigmond RE, Schwarzschild MA, Rittenhouse AR. Acute regulation of tyrosine hydroxylase by nerve activity and by neurotransmitters via phosphorylation. Annu Rev Neurosci 1989; 12:415-461. 96. Beyer C, Raab H. Nongenomic effects of oestrogen: Embryonic mouse midbrain neurones respond with a rapid release of calcium from intracellular stores. Eur J Neurosci 1998; 10:255-262. 97. Cooper DMF, Mons N, Karpen JW. Adenylyl cyclases and the interaction between calcium and cAMP signalling. Nature 1995; 374:421-424. 98. Toran-Allerand CD. The estrogen/neurotrophin connection during neural development: Is co-localization of estrogen receptors with the neurotrophins and their receptors biologically relevant? Dev Neurosci 1996; 18:36-48. 99. Dueñas M, Luquín S, Chowen JA et al. Gonadal hormone regulation of insulin-like growth factor-I-like immunoreactivity in hypothalamic astroglia of developing and adult rats. Neuroendocrinology 1994; 59:528-538. 100. Finkbeiner S, Tavazoie SF, Maloratsky A, et al. CREB: A major mediator of neuronal neurotrophin responses. Neuron 1997; 19:1031-1047. 101. Reisert I, Engele J, Pilgrim C. Early sexual differentiation of diencephalic dopaminergic neurons of the rat in vitro. Cell Tissue Res 1989; 255:411-417. 102. Ahnert-Hilger G, Engele J, Reisert I et al. Different developmental schedules of dopaminergic and noradrenergic neurons in dissociation culture of fetal rat midbrain and hindbrain. Neuroscience 1986; 17:157-165. 103. Engele J, Pilgrim C, Kirsch M et al. Different developmental potentials of diencephalic and mesencephalic dopaminergic neurons in vitro. Brain Res 1989; 483:98-109. 104. Beyer C, Pilgrim C, Reisert I. Dopamine content and metabolism in mesencephalic and diencephalic cell cultures: Sex differences and effects of sex steroids. J Neurosci 1991; 11:1325-1333. 105. Beyer C, Eusterschulte B, Pilgrim C et al. Sex steroids do not alter sex differences in tyrosine hydroxylase activity of dopaminergic neurons in vitro. Cell Tissue Res 1992; 270:547-552. 106. Weisz J, Ward IL. Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses, and neonatal offspring. Endocrinology 1980; 106:306-316. 107. Baum MJ, Woutersen PJA, Slob AK. Sex difference in whole-body androgen content in rats on fetal days 18 and 19 without evidence that androgen passes from males to females. Biol Reprod 1991; 44:747-751. 108. Reisert I, Pilgrim C. Sexual differentiation of monoaminergic neurons—genetic or epigenetic? Trends Neurosci 1991; 14:468-473. 109. Maxson SC. Sex differences in genetic mechanisms for mammalian brain and behavior. Biomed Rev 1997; 7:85-90.

CHAPTER 6

Neural Development of the Striatal Dopamine System Fu-Chin Liu and Ann M. Graybiel

D

opamine is a functionally important neurotransmitter in the striatum that derives from the dopamine-containing mesostriatal and mesolimbic afferent systems.1,2 The dopamine-synthesizing neurons in the substantia nigra, pars compacta of the ventral mesencephalon constitute the mesostriatal afferents that extensively innervate the dorsal striatum (the caudate nucleus and putamen). By contrast, the ventral striatum (the nucleus accumbens and olfactory tubercle) receives dopamine-containing inputs mainly from neurons in the ventral tegmental area. Clinical study has unequivocally demonstrated that the striatal dopamine system is critical to the sensorimotor and psychomotor functions of the brain. The degeneration of dopamine-containing neurons in the substantia nigra is the main pathology of Parkinson’s disease, in which severe defects in movement control occur. The craving for abusive drugs such as cocaine and amphetamine is known to be dependent upon mesolimbic dopamine neurotransmission in the nucleus accumbens.3,4 Evidence has raised the possibility that abnormal function of dopamine receptors in the striatum is involved in pathogenesis of schizophrenia.5,6 The effects of dopamine neurotransmission in modulating striatal sensorimotor functions are thought to reflect a functional segregation of dopamine D1-class and D2-class receptors in striatal efferent systems.7,8 Dopamine D1-class receptors are strongly expressed in the direct striatopallidal and striatonigral pathways, whereas D2-class receptors are more strongly expressed in the indirect striatopallidal pathways. Activation of D1-class and D2-class receptors in these two sets of pathways can lead to antagonistic results in terms of movement control, but synergistic effects of these two systems are also critical7,8. Most current models of basal ganglia control emphasize that a balanced output of these two dopaminemodulated efferent systems is critical to appropriate motor control. The activity of D1-class and D2-class receptors in these two output pathways also differentially control neuropeptide expression in these efferent systems.8,9 Striatal expression of dynorphin and substance P is primarily associated with D1-enriched striatopallidal and striatonigral pathways, whereas enkephalin expression is associated with the D2-dominated striatopallidal pathway. Activation of D1-class dopamine receptors can enhance the expression of dynorphin and substance P in the striatonigral projection neurons. By contrast, inactivation of D2-class receptor results in up-regulation of enkephalin in striatopallidal projection neurons (for review see ref. 9). An impressive repertoire of dopamine-related signaling molecules is expressed at high levels in the striatum. These striatum-enriched signaling molecules include, aside from dopamine D1-class and D2-class receptors, the heterotrimeric G protein, Golf, the striatumspecific adenylyl cyclase type V (ACST), and dopamine- and cyclic adenosine 3':5'-monophosphate-regulated phosphoprotein (DARPP-32).10-12 The enrichment of these dopamine The Development of Dopaminergic Neurons, edited by Umberto di Porzio, Roberto Pernas-Alonso, and Carla Perrone-Capano. ©1999 R.G. Landes Company.

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signaling molecules in the striatum provides an anatomical and molecular basis for the high levels of dopamine signaling processing in the striatum. The striatal dopamine system is highly complex. This is because there are bidirectional axon-target interactions between the midbrain dopamine-containing neurons and striatal neurons. For the ascending mesostriatal system, striatal neurons are the targets of dopaminecontaining neurons in the midbrain. Conversely, the dopamine-containing neurons in the substantia nigra, pars compacta are also the targets of a subpopulation of descending striatonigral projection neurons (striosomal neurons, see below) that selectively project back to the dopamine-containing neurons in the substantia nigra, pars compacta.13,14 How these interconnected neuronal connections between midbrain dopaminergic neurons and neurons in the striatum are established, and how neurons in the ventral midbrain and the striatum interact during development, are the main questions addressed in this chapter.

Dopamine as a Potential Regulator of Neuronal Development: Expression of Dopamine Receptors in the Germinal Zones of Striatal Anglage The developmental time courses of striatal dopamine signaling molecules including dopamine, the synthetic enzyme of dopamine, tyrosine hydroxylase (TH), dopamine D1, D2 and D3 receptors, Golf, ACST and DARPP-32, are summarized in Table 6.1. Expression of dopamine D1, D2 and D3 receptors are first detected in the ganglionic eminence (striatal anlage) as early as embryonic day (E) 14, at which time the striatal primordium is starting to form. From E14-E18, there is a transient expression of D1 and D2 receptor mRNAs in the germinal zones of the striatum, the ventricular zone (VZ) and the subventricular zone (SVZ) of the ganglionic eminence.15-17 A remarkably high concentration of D3 receptor mRNA is detectable in the VZ of the ganglionic eminence as early as E14, and this expression persists throughout development.17 In adulthood, D3 receptor mRNA is still detectable in the SVZ of the striatum. The expression of dopamine D1, D2 and D3 receptor mRNAs in the germinal zones of the ganglionic eminence raises the possibility that activation of dopamine receptors may regulate neurogenetic events leading to the generation of striatal cells. A prerequisite for this developmental function for dopamine is that dopamine should be present in the ganglionic eminence. It is not yet technically feasible to determine with biochemical methods whether dopamine is present there. Immunohistochemical studies do indicate, however, that a few tyrosine hydroxylase- and dopamine-immunoreactive fibers are present in the germinal zones of ganglionic eminence as early as E12/E13 in the rat.18-21 These findings suggest that local release of dopamine may occur in the ganglionic eminence. An alternative source for dopamine is the cerebrospinal fluid of the ventricular system derived from the diencephalon and mesencephalon, but the reverse direction of flow holds at maturity. The possibility that very low levels of dopamine could be effective is reasonable, because D3 receptor mRNA is strongly and specifically expressed in the VZ, and dopamine has a high affinity for the D3 receptor.17,22 Despite this indirect evidence, it is not known whether dopamine-mediated activation of dopamine D1, D2 and D3 receptors takes place in the VZ and SVZ of the ganglionic eminence. Even if dopamine receptor activation occurred in the ganglionic eminence, it is not clear whether such putative receptor activation in the proliferative zones would lead to significant biological function. The cells in the VZ and SVZ of the ganglionic eminence are mostly undifferentiated precursor cells, and they have not yet expressed signaling molecules downstream from dopamine, including Golf and ACST (see Table 6.1). This does not rule out the possibility that the putative dopamine receptors are functional in the ganglionic eminence. It is conceivable that dopamine signal transduction in the VZ and SVZ is coupled to other cellular signaling molecules, and may lead to a biological action different from classical

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Table 6.1. Ontogeny of dopamine signaling molecules in the rat striatum Dopamine Signaling Molecule

VZ

SVZ

DZ

References

D1R mRNA

E14-E18

E14-E18

E14-adult

16,17

D1R IR

-

-

E15-adult

94

D2R mRNA

E14-E18

E14-E18

E14-adult

15-17

D3R mRNA

E14-adult

E19-adult

P0/P5-adult

17

Dopamine IR

-

E15-E16

E14-adult

20

TH IR

E12/E13-E19

-

E14.5-adult

18,19

Golf mRNA

-

-

E15-adult

95

ACST mRNA

-

-

E20-adult

95

DARPP-32 mRNA

E13-P0

E13-P0

E15*-adult

108-110

DARPP-32 IR

-

-

E14-adult

84

VZ, ventricular zone; SVZ, subventricular zone; DZ, differentiated zone; D1R, dopamine D1 receptor; D2R, dopamine D2 receptor; D3R, dopamine D3 receptor; Golf, G protein; ACST, striatum-specific adenylyl cyclase type V; DARPP-32, dopamine- and cyclic adenosine 3':5'monophosphate-regulated phosphoprotein; IR, immunoreactivity; E, embryonic day; P, postnatal day. *: data of E14 not available.

dopamine-mediated neurotransmission. For example, activation of recombinant dopamine D2 and D3 receptors in transfected cell lines has been reported to result in increase of mitogenesis.23,24 Activation of dopamine receptors also has been shown capable of regulating neurite outgrowth.25,26 The developmental effects of dopamine signaling may thus extend beyond neuronal communication.27 Despite the interesting possibility that dopamine may regulate the process of neuronal development, including cell proliferation and growth cone activity, genetic depletion of dopamine indicates no change in gross anatomy of the striatum aside from that accompanying a general reduction in brain size in the mutant mice. 28 Conversely, augmentation of dopamine synaptic action by knocking out the gene encoding the dopamine transporter did not appear to affect the striatal structure.29 Nor did genetic inactivation of the dopamine D2 receptor or D3 receptor seem to affect the development of the structure of the striatum.30-33 However, null mutation of the D1 receptor gene has been reported to result in reduced size of the striatum in the mutants,33,34 which suggests that the D1 receptor may be involved in the regulation of neuronal development. The lack of obvious effects on striatal neurogenesis in dopamine-deficient mutant mice28 nevertheless strongly argues

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against the possibility that activation of dopamine receptors by dopamine itself is critically involved in regulating cell proliferation, migration and differentiation. The gene knockout studies, however, collectively point out that dopamine signaling is important for regulating the expression of striatal chemical phenotypes, as expected from previous pharmacologic studies. For example, striatal expression of dynorphin is decreased in dopamine-depleted mutants, but is increased in dopamine transporter null mutants.28,29 In dopamine D1 receptor null mutant mice, striosomal expression of dynorphin, substance P and mu opioid receptor are dramatically reduced in the striatum without significant alteration in the formation of striosome-matrix compartments.34-36 Thus, dopamine signal transduction in the developing striatum may not be involved in regulation of neurogenetic events, but does have a crucial function in modulating the neurochemical phenotype of striatal neurons.

Developmental Regulationn of the Mesostriatal Dopamine-Containing Neurons by Striatal Target Cells A series of studies pioneered by Prochiantz’s group has shown that striatal cells have a profound influence on the differentiation of midbrain dopamine-synthesizing neurons.37 Coculture of ventral midbrain dopamine-containing neurons and striatal cells can significantly increase the synthesis of dopamine, dopamine uptake and expression of dopamine transporter gene in the cultured dopaminergic neurons.38-41 The enhanced neurochemical maturation of the dopamine-containing neurons could be mediated by their interactions with striatal target neurons or with striatal glia. Further studies have indicated that similar maturational effects could be obtained with striatal membranes and in culture containing serum-free medium in which few glia were present.42,43 Striatal astrocytes and culture medium conditioned with striatal glia also could promote the differentiation of midbrain dopaminergic neurons, and the effects appear to be region-specific.44-46 The striatal target cells not only can enhance differentiation of midbrain dopamine-containing neurons, but can also increase their survival.46-48 Such neurotrophic interactions between the midbrain dopaminergic neurons and striatal target cells also has been observed in grafting studies. The host striatum, following lesion of its dopamine-containing afferents, sustains selectively the growth and survival of implanted dopaminergic neurons from the ventral mesencephalon, but not hypothalamic dopaminergic neurons, which degenerate shortly after grafting.49 Co-grafting of ventral midbrain cells with striatal cells has been reported to enhance the survival and neurite outgrowth of dopaminergic neurons in the grafts.50-52 These data suggest that target-derived trophic factors from the developing striatum may regulate the differentiation of midbrain dopaminergic neurons. The glia-derived neurotrophic factor, GDNF, is a potent trophic factor for these neurons.53 As GDNF is transiently expressed in the developing striatum,54 it may serve as a target-derived retrograde trophic factor for the mesostriatal dopamine-containing neurons.

Developmental Regulation of Striatal Neurons by the Mesostriatal Dopamine Neurons Over 90% of striatal neurons are medium-sized projection neurons that give rise to projection systems leading to the external or internal segments of the globus pallidus or to the substantia nigra.55 Striosomal neurons project caudally to the substantia nigra earlier than do matrix neurons. Striatonigral afferents from striosomal neurons innervate the substantia nigra as early as E17 in the rat.56 The early projections into the substantia nigra of striosomal cells, and also of a small population of matrix cells, have been reported to be correlated with cell survival during the cell death period that occurs in the striatum during

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91

the first postnatal week.57 These data suggest that the early innervation of the substantia nigra by striatal neurons may be important for striatal cell survival. Study of cocultures of intermixed embryonic ventral midbrain cells and striatal cells has shown that the survival rates of both dopaminergic neurons and striatal neurons are increased in the cocultures.47,48 Similar trophic effects of midbrain cells on the survival of striatal cells also has been observed in a coculture system in which ventral midbrain explant tissue is physically separated from the cultured striatal cells by a membrane insert (F.-C. Liu et al., unpublished observations). These findings suggest that the ventral midbrain may release soluble factors that have neurotrophic effects on the development of striatal neurons. Two potential trophic factors that may be released from the mesostriatal dopamine neurons are the brain-derived neurotrophic factor and retinoic acid.

Brain-Derived Neurotrophic Factor Brain-derived neurotrophic factor (BDNF) is a strong trophic factor for striatal neurons. BDNF has been shown to increase the survival and differentiation of GABAergic neurons in striatal cell cultures as well as neurons in the cultures that are positive for calbindin-D28K, calretinin and DARPP-32.58-62 Null mutation of the BDNF gene also results in reduction of calbindin-D28K, parvalbumin and DARPP-32 expression in the striatum.62-64 It is of particular interest that in the adult, striatal neurons express little BDNF mRNA, but the striatum nevertheless contains high levels of BDNF immunoreactivity associated with nerve terminals.64 Moreover, lesions of striatal afferents in adults result in a dramatic decrease of striatal BDNF protein levels. As BDNF mRNA is expressed in the dopamine-containing neurons of substantia nigra, pars compacta,64 striatal BDNF is thus likely to be derived from afferents including dopamine-containing mesostriatal fibers. However, prenatal lesions of mesostriatal afferents made as early as E17-E18 do not appear to affect at least one striatal marker, calbindin-D28K,65 The effects of such early lesions on the striatal expression of DARPP-32, parvalbumin or other markers have not yet been reported. The possibility that BDNF anterogradely transported in mesostriatal afferents affects the development of striatal neurons remains to be clarified.

Retinoic Acid Another candidate for a mesostriatal afferent-associated factor that may regulate striatal development is retinoic acid (RA). RA is a well known morphogen that is important for regulating hindbrain development.66,67 It has been shown that a subpopulation of dopamine neurons in the substantia nigra contains the RA-generating dehydrogenase, murine class 1 (cytosolic) aldehyde dehydrogenase (AHD2).68 In the striatum, AHD2 immunoreactivity is present in the terminals of mesostriatal afferents. Retinoid receptors also are expressed in the developing striatum.70-71 These expression patterns raise the possibility that the mesostriatal afferents may release RA, which in turn regulates the development of striatal neurons. Recent studies show that RA can enhance DARPP-32 expression in striatal cell cultures.72,73 Moreover, cultured ventral midbrain explants are capable of releasing RA, as demonstrated by a positive response with a RA reporter cell line (F.-C. Liu et al, unpublished observations). Further studies indicate that co-culture of dissociated embryonic striatal cells with embryonic ventral midbrain explant tissue in the presence of RA precursor retinol can increase striatal DARPP-32 expression, and that this increase of DARPP-32 can be reduced by pretreatment with the AHD2 inhibitor, disulfiram (F.-C. Liu et al, unpublished observations). These findings suggest that RA may be generated in mesostriatal afferents and then regulate the differentiation of striatal target cells. In fact, RA not only can enhance striatal DARPP-32 expression, but also can coordinately up-regulate a set of other striatal dopamine signaling molecules, including the dopamine D1 receptor, Golf and ACST, in striatal

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explant cultures.74 It should be noted, however, that mesostriatal dopamine-containing neurons are not the only source of striatal RA. The striatal anlage itself also is capable of releasing RA (73,75; F.-C. Liu et al., unpublished observations).

Modulation of Mosaic Structure of Striatal Compartments by the Mesostriatal Afferents During Development The striatum is organized into a neurochemical mosaic made up of two neuronal compartments, striosomes (patches) and matrix.8,9 These two neuronal populations in the striatum were first defined by differential expression of neurochemical molecules in the two compartments. For example, in the mature striatum, striosomes contain low levels of acetylcholinesterase (AChE) and calbindin-D28K, whereas the matrix expresses high levels of AChE and calbindin-D28K. Subsequent studies showed that these two compartments also differ in their neuronal connections. Striosomes primarily interact with neural circuits of the limbic system, whereas the matrix compartment is primarily engaged in sensorimotor circuits. Studies of gene regulation further demonstrate that neurons in these two compartments have different patterns of gene expression in response to a variety of pharmacologic manipulations affecting dopamine receptors.76 The dopamine-containing mesostriatal afferents first innervate the differentiated zone of the ganglionic eminence with a homogeneous pattern.19-21 By E18 of rat embryogenesis, however, the mesostriatal innervation becomes heterogeneous. Patchy zones and a lateral rim of the developing striatum come to contain high densities of dopamine- and TH-immunoreactive fibers. These patchy dopamine-positive zones were named “dopamine islands”.77,78 The dopamine islands are developmentally regulated and are transient. Dopamine-positive, TH-positive fibers gradually increase in density in the matrix until, in species ranging from rat and mouse to cat, monkey and human,79 the fibers are almost homogeneously distributed at maturity. The “dopamine islands” are coincident with developing striosomes.20,80-84 This raises the interesting possibility that the dopaminecontaining mesostriatal afferents may attract the early-born striosomal neurons to form the modular organization of striosome-matrix compartments during striatal development. The dopamine-containing mesostriatal afferents may have an impact on the formation of compartments at two levels. One is that dopamine itself might signal the segregation of striosomes and matrix through an activity-dependent mechanism. The other is that non-dopamine factors associated with the mesostriatal afferents might regulate compartment formation (for example, BDNF and RA, see above). These two possibilities have been tested to determine whether compartment formation in the striatum is dependent on its dopamine-containing mesostriatal inputs. Pharmacologic blockade of these inputs did not influence the developmental segregation of striosome and matrix cells.83,85 Nor did embryonic lesions of the mesostriatal afferents affect the compartment formation.65,86 Other evidence from developmental mapping of the two immature striosomal markers, calcium/calmodulin-dependent protein kinase II (CaMK II) and DARPP-32, also shows that both CaMK II-positive cell clusters and DARPP-32-positive cell clusters (corresponding to striosomes) appear before the formation of “dopamine islands”.21,84 Taken together, these data suggest that neither dopamine-mediated activity nor the mesostriatal afferents themselves are required for the sorting out of striosomal cells from matrix cells during development. However, the maintenance of striosome-matrix compartmentation is affected by striatal interconnections with the midbrain. Early postnatal lesions of the substantia nigra result in disintegration of striatal compartmentation.87 Such lesions induced massive cell loss in the matrix compartment, but striosomal cell survival

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was not affected. As a consequence, surviving striosomal cells became evenly distributed in a shrunken striatum in the rats bearing early nigral lesions. Although the developmental segregation of striosomal and matrix cells does not seem to depend on the mesostriatal innervation, destruction of mesostriatal afferents can nonetheless strongly affect the development of chemical phenotypes of striatal compartments. For example, perinatal lesions of the mesostriatal afferent system and lesions of the substantia nigra in embryos result in loss of preferential expression of mu opioid receptors in striosomes.86,88,89 These findings suggest that the chemical phenotypes of striatal compartments are subject to regulation by the mesostriatal dopamine inputs (see below).

Dopamine Activity-Dependent Modulation of Compartmental Phenotypes During Striatal Development Since the groups of Olson and Tennyson discovered the dopamine islands of the developing striatum,77,78 a number of dopamine signaling-related molecules have been found to be heterogeneously distributed in the developing striatum. These molecules include dopamine, TH, the high affinity dopamine uptake sites (dopamine transporters), the dopamine D1 receptor, the dopamine D2 receptor, ACST and DARPP-32.16,20,77,78,80-84,89-95 As the dopamine island sites correspond to the sites of developing striosomes, these molecules are preferentially expressed in developing striosomes (Table 6.2). The high striosomal concentrations of signaling molecules related to dopamine suggests that dopamine-mediated activity might regulate the development of chemical phenotype of striatal compartments. We have previously proposed that differential regulation of the cAMP response element (CRE) binding protein (CREB) by dopamine and calcium may be part of the mechanism by which compartmental phenotypes are acquired in the striatum during development.96,97 Phosphorylation of CREB on ser133 is important for transactivation of its target genes.98,99 Stimulation of dopamine D1-class receptors and of L-type voltage-sensitive calcium channels induce transient phosphorylation of CREB in both compartments in organotypic cultures of neonatal striatum.96,100 However, the dynamics of the CREB phosphorylation that occurred in the two compartments was different. Sustained CREB phosphorylation occurred in striosomes upon D1 receptor stimulation, whereas sustained CREB phosphorylation occurred primarily in the matrix upon L-type calcium channel stimulation. Notably, the patterns of sustained CREB phosphorylation were tightly correlated with subsequent activation of the CRE-containing early response gene, c-fos, in the compartment showing sustained CREB phosphorylation. These data raise the possibility that activity-dependent sustained phosphorylation of CREB can trigger potential downstream genes during the critical period of phenotypic differentiation of striatal neurons. These experiments demonstrated further that the compartmental CREB phosphorylation patterns are themselves regulated by protein phosphatase activity. Inhibiting protein phosphatase-2B (calcineurin), which is expressed at high levels in developing striosomes, could change the matrix-predominant pattern of sustained CREB phosphorylation evoked in cultures treated with L-type calcium channel activator to one in which sustained CREB phosphorylation also occurred in striosomes. Inhibiting protein phosphatase-1 and/or 2A likewise led to sustained CREB phosphorylation in both compartments. Thus, regulation of sustained CREB phosphorylation by protein phosphatase activity may be one mechanism by which gene activation can be differentially targeted to developing striosomes and matrix by dopamine and calcium signals. Several genes expressed in the striatum, including those coding for prodynorphin, mu opioid receptors, proenkephalin and somatostatin, contain CRE motifs in their promoter regions.101-104 Dynorphin and mu opioid receptors are expressed at high levels in striosomes, whereas enkephalin and somatostatin are preferentially expressed in the matrix.9 These are

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Table 6.2. Preferential expression of dopamine signaling molecules in the developing striosomes Dopamine Signaling Molecule

Immature Striosome

Immature Matrix

References

TH and dopamine flourescence

High

Low

77,78,80-83,90

Dopamine

High

Low

20

High affinity dopamine uptake sites (dopamine transporter)

High

Low

91,92

Dopamine D1-class receptors

High

Low

16,89,93

Dopamine D2-class receptors

High

Low

91

Golf

High

High

95

ACST

High

Low

95

DARPP-32

High

Low

84,95,108,110

These dopamine signaling molecules are transiently expressed in striosomes from ca. E19-E20 to the second postnatal week in the rat except that Golf is expressed in both developing striosomes and matrix. The expression of these molecules in the matrix gradually increases during development, and eventually the molecules are nearly homogeneously distributed in the adult rat striatum.

good candidates for differential regulation by dynamic control of CREB phosphorylation in response to dopamine and calcium signals.

Specification of Topographic Projections of the Mesostriatal and Mesolimbic Afferents During Development The dopamine-containing neurons in the ventral mesencephalon are divided into two principal groups. The dopaminergic neurons in the substantia nigra, pars compacta are located in the lateral part of the ventral midbrain, and these neurons give rise to the mesostriatal afferent system that innervates the dorsal and lateral striatum (the caudoputamen). The dopaminergic neurons in the ventral tegmental area (VTA) are located in the ventromedial part of the midbrain, and they form the mesolimbic afferent system that projects to the medial and ventral striatum (the nucleus accumbens and olfactory tubercle) and to the prefrontal cortex.1,2 Thus, the midbrain dopamine inputs to their striatal targets are organized in a lateral to medial topography: the dopamine neurons in the lateral

Development of the Striatal Dopamine System

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midbrain (substantia nigra) project to the dorsolateral striatum whereas the dopamine neurons in the medial midbrain (VTA) project to the ventromedial striatum. An essential question for this topographic organization of dopaminergic axonal projections is how it is established during development. It is well known that the Eph family of receptor protein tyrosine kinases plays an important role in specification of retinotectal projection maps.105 An intriguing question is whether similar Eph-mediated molecular mechanisms underlie the specification of mesostriatal and mesolimbic topography. The EphB1 receptor and its ligand, Ephrin-B2, are expressed in a complementary pattern in the groups of midbrain dopamine neurons and their striatal target regions.106 EphB1 receptor is predominantly expressed in the substantia nigra (lateral midbrain), but its expression is low in the caudoputamen (lateral striatum). By contrast, the expression of Ephrin-B2 is low in the VTA (medial midbrain), but it is highly expressed in the nucleus accumbens and olfactory tubercle (ventromedial striatum). Moreover, Ephrin-B2 can inhibit neurite outgrowth and induce degeneration of substantia nigra neurons, but not VTA neurons in vitro. These data suggest that if the axons of dopaminergic neurons containing high levels of EphB2 receptors in the substantia nigra grew into the Ephrin-B2-rich ventromedial striatum, activation of EphB1 receptor signaling pathway would eventually eliminate these mistargeted neurons. This Ephrin-B2 induced cell death is quite different from the well known mechanism of establishing inhibitory domains of neurite outgrowth by Eph family in retinotectal projection systems.105 Nonetheless, it may be part of the molecular mechanisms by which the topography of midbrain dopamine inputs are specified during development. Other members of the Eph family have been shown to be differentially expressed in striatal compartments during development. For example, EphA4 receptor and its ligands Ephrin-A1 and Ephrin-A4 are preferentially expressed in the matrix compartment of postnatal striatum.107 However, it is not clear whether Ephrin-A1 and Ephrin-A4 are associated with mesostriatal afferents, nor how the co-expression of the Eph receptor and its ligands in the same matrix compartment would lead to sorting out of matrix cells from striosomal cells by an Eph-mediated repulsive mechanism.

Conclusion We have reviewed new lines of research on the cellular and molecular events controlling the development of the striatal dopamine system. This work emphasizes distinct phases in the developmental process and several key molecules that may help build the infrastructure of the striatum and its mesostriatal afferents.

Acknowledgments The preparation of this review was supported by National Health Research Institutes grant DOH87-HR-736, Taiwan, R.O.C. and by NIH 5 R01 HD28341.

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CHAPTER 7

The Involvement of Dopamine in Various Physiological Functions: From Drug Addiction to Cell Proliferation Daniela Vallone, Roberto Picetti and Emiliana Borrelli

D

opamine is an important regulator of different physiological functions in the central nervous system (CNS) as well as in other organs. Known dysfunctions of the dopaminergic system lead to diseases affecting the CNS such as Parkinson’s, Tourette’s syndrome, schizophrenia and the generation of pituitary tumors. In this review we center our attention on dopamine receptors, and the subsequent signal transduction cascades within the cell. Although many pharmacological and biochemical characteristics of the different dopamine receptors have been fully elucidated, a clear identification of the physiological functions in which each receptor is involved is still awaited. A great amount of information has come from the use of pharmacological tools and lesions of particular areas of the brain. However, since these studies are biased by the lack of specificity of these approaches, further investigations using genetically modified animal models will help to define the function of each component of the dopaminergic pathways. The cloning of the different dopamine receptors has given considerable insight for studies aimed at the characterization of their biochemical, pharmacological and physiological properties. The knowledge that dopaminergic signaling is mediated by five different type of receptors has opened new horizons to the development of pharmacological compounds specific for each receptor subtype. This will hopefully result in novel pharmacological treatments of neurological diseases in which identified components of the dopaminergic system are involved. In this regard a great advance in our studies has been the generation of genetically modified mice for different components of the dopaminergic pathways. These mice might be considered as models of dopaminergic deficiencies. The fine dissection of the phenotypes presented by these mutants is giving us new insights into specific functions of dopamine in vivo.

Pharmacological Profiles of Dopamine Receptors Dopamine receptors (DA-Rs) belong to the family of the seven transmembrane domain G-protein coupled receptors. At present, five different receptors have been isolated and named, from D1 to D5. The biochemical and pharmacological properties of these receptors still The Development of Dopaminergic Neurons, edited by Umberto di Porzio, Roberto Pernas-Alonso, and Carla Perrone-Capano. ©1999 R.G. Landes Company.

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The Development of Dopaminergic Neurons

allow for their classification into two subfamilies: the D1- and D2-like receptors. The D1-like subfamily comprises D1- and D5-R, while the D2-like includes D2-, D3- and D4-R. DA-Rs share a strong aminoacid (aa) homology within the transmembrane domain (TM) regions. The D1 and D5 receptors have 80% homology in the TM regions. Similarly, D2-R has 75% homology with the D3-R and 56% with D4-R.1 Whereas the N-terminal sequence of all the DA-Rs is similar in length, the size of the C-terminus and the third intracellular loop (il3) vary between the D1- and D2-like receptors.1,2 The C-terminus in both subfamilies contains phosphorylation sites which might be involved in agonist-dependent receptor desensitization.1,3,4 This region also contains a palmitoylation site which might induce it to form a putative fourth intracellular loop (il4). Palmitoylation of D1-R has been shown to be induced by agonist treatments.4 Whether this loop is present in DA-Rs in vivo and whether it might have a function as in the β2-adrenergic receptor5 is a matter of some controversy. Seven TM G-protein coupled receptors have several conserved aminoacid residues directly involved in DA binding. Indeed, the critical involvement of Asp80, Asp114, Ser193, 194 and 197 in DA binding has been shown for D2-R.1 These residues appear to be widely conserved and important for binding of DA in other DA-Rs.1 In addition, the third TM segment of D2-R can influence DA binding to the receptor.6,7 Dopaminergic ligands easily discriminate between the D1- and D2-like receptor subfamilies (Table 7.1). On the contrary, most of them do not clearly differentiate between members of the same subfamily. For example, the classical D1-R antagonist SCH-23390 or the agonist SKF-38393 have very similar affinities for both D1- and D5-R. However, some ligands have clearly different affinities. Indeed, DA is about 10-fold more potent at human D5- than at D1-R, and (+)butaclamol has a 10-fold higher affinity for D1- than for D5-R.8 A similar problem arises for the discrimination of the different D2-like receptors by pharmacological means, at least by using the classical D2-like specific agonists and antagonists. For example, the very well characterized D2 antagonist spiperone is 10-fold more selective for D2- and D4-R compared to D3-R. Domperidone has a 30-fold higher selectivity at D2- than at D3-R. Conversely, the antagonist (+)S-14297 has a 200-fold greater affinity at D3- than D2-R9. Interestingly, a recently developed D2-like antagonist, L-745,870, strongly discriminates between these receptors and has a 2000-fold higher affinity for D4-R.10 Dopamine has a higher affinity at D3-R than at D2- and D4-R (10 to 20-fold),11,12 and 7-OH-DPAT is 10-fold more specific for D3- over D2-R and 600-fold over D4-R.12 However, the pharmacological selectivity of these compounds has still to be determined in living animals. To this end, the use of animals lacking a particular receptor should help greatly in defining the selectivity of a particular compound for a specific receptor.

Distribution of Dopamine Receptors The distribution and abundance of the five DA-Rs is very different depending on which subtype is analyzed. Among the DA-Rs, D1- and D2-R are by far the most robustly and widely expressed. Conversely, D3-, D4- and D5-R have a more restricted pattern of expression. Whether there is a physiological significance for the expression of these receptors in particular areas is still under investigation. However, the restricted expression of some DA-Rs in the limbic system, as well as the higher affinity for antipsychotic drugs, has suggested a potential role of these receptors in neurological diseases. As shown in Table 7.2, D1-R is mainly expressed in the caudate putamen (CP), nucleus accubens (Acb), olfactory tubercle (OT), cerebral cortex (Cx) and amygdala.1 A high abundance of D1-R has been shown also in the island of Calleja and in the subthalamic nucleus.1 The binding of D1-R specific ligands could be observed in the substantia nigra (SN), in spite of the lack of mRNA expression. This seems to suggest that D1-R is synthetized in striatal neurons that send their projections to the SN via the direct striato-nigral pathway.1

0.9-2340

0.7-680

440-672

5000

21230

1-150

421

Dopamine

(-)Apomorphine

Bromocriptine

7-OH-DPAT

cis-8-OH-PBZI

SKF-38393

NPA

56-230

100-261

Clozapine

194-336

0.69-0.8

0.9-3

(+) Butaclamol

0.4-0.9

150-9560

2470

10-103

5.3-12.6

0.7-24

2.8-474

4.8-576

D2

80-270 5-27

187

0.5-100

15060

450

122-363

<0.9-261

D5

AJ-76

Antagonists

1900

Quinpriole

Agonists

D1

D1-like

Table 7.1. Pharmacological properties of dopamine receptors

83-620

4.1-11.2

35-91

5000

27

1-2

5-7.4

20-32

4-27

5.1-24

D3

D2-like

9-42

38-51

6.5

1000-1800

276

650

290-340

4

28-450

30-46

D4

The Involvement of Dopamine in Various Physiological Functions 103

18000

Raclopride

20400-4500

S(-)Sulpiride

1572 13-40

U-99194A

UH-232

2.9-9.2

78

13

8-206

314-800

6.7

969-1600

1.8-3.5

0.32-0.71

2.74-7.8

9.5

1.16-6.1

21-1000

3000-3650

7

2800-3690

237-2400

0.05-4

2.3-5.1

35

The ki values (nM) of several compounds are listed. For many ligands the lowest and the highest Ki found in the literature are given. Discrepancies among the values are due to differences among the cell types used for the binding experiments or to the affinity state of the receptor. 7-OH-DPAT: 7-hydroxy-N-N-di-n-propyl-2-aminotetralin; cis-8-OH-PBZI: cis-8-hydroxy-3-(n-propyl)1,2,3a.4.5.9b-hexahydro-1H-benz[e]indole; NPA: N-propylnorapomorphine

297

(+)S-14297

2.5-71

270-1100

0.11-0.35

SCH-23390 11000-77270

1.7-5

Risperidone 0.11-0.54

54-300

Remoxipride

1-5

0.06-0.37

99-350

Spiperone 135-4500

0.6-1.2

35-151

27-203

0.5-3

Haloperidol

130-314 0.3

73-90

Domperidone

Chlorpromazine

Table7.1. Pharmacological properties of dopamine receptors , (cont)

104 The Development of Dopaminergic Neurons

+

+

+

Olfactory tubercule (OT)

Cerebral cortex (Cx)

Amygdala

Ventral Tegmental Area (VTA)

Cerebellum +

+

+

+

Substantia nigra (SN) +

+

+

+

+

+

mRNA

D3-R protein

+

+

+

+

+

+

+

mRNA

Thalamus

+

+

+

+

+

+

protein

+

+

+

+

+

+

+

mRNA

D2-R

Hypothalamus

+

+

Nucleus accubens (Acb)

Hippocampus

+

Caudate putamen (CP)

Brain

protein

D1-R

Table 7.2. Anatomical localization of dopamine receptors

+

+

+

protein

+

+

+

mRNA

D4-R

+

+

+

+

+

+

+

protein

D5-R

+

+

mRNA

The Involvement of Dopamine in Various Physiological Functions 105

+

+

+

+

+ +

+

+

+

+

Localization of the five dopamine receptors proteins and mRNAs in several tissues. The localization of the DA-R products was assessed by radioligand binding or by immunohystochemistry. RNA localization was assessed by in situ hybridization or by Norther blot analysis. The "+" symbol indicates the presence of the protein/RNA in a given tissue. Since mice and rat behave similarly with respect to DA-Rs expression, the data from both model systems has been combined.

+

+

Kidney

Vascular system

+

Retina

+

+

+

+

+

Pituitary

Outside SNC

Island of Calleya

Table 7.2. Anatomical localization of dopamine receptors, (cont)

106 The Development of Dopaminergic Neurons

The Involvement of Dopamine in Various Physiological Functions

107

D2-R has a very similar distribution with respect to D1-R, especially in areas like the CP, Acb, OT and SN. Low quantities of D2-R are also present in Cx and in the ventral tegmental area (VTA).1,13 Outside the brain D2-R is localized in the retina, kidney, vascular system and pituitary gland.1,4,13 Examination of the neuroanatomic distribution of D3-R mRNA in rat brain indicates that it is distinct from that of D2-R mRNA and restricted to few brain regions such as the islands of Calleja, a few septal nuclei, hypothalamus, and distinct regions of the thalamus and cerebellum.1 Both mRNAs and proteins of D2- and D3-R are expressed by both dopaminergic and dopaminoceptive cells.1 Northern blot analysis revealed the presence of D4-R mRNA in the olfactory bulb, frontal cortex and hypothalamus in both rat and monkey brain.1 In rat brain, the expression of D5-R mRNA is very restricted to the hippocampus and thalamus1 and does not seem to overlap significantly with the distribution of mRNA of D1-R.1 Interestingly, D5-R mRNA seems to be much more widely distributed in the primate brain as compared to rodents, in particular in the Cx where it overlaps with the mRNA of D1- and D2-R.1 However, the D5-R protein seems to be present in the Cx, CP, OT, VTA, and SN in rat brain.1 Author: Table 7.1 does not show (+) for VTA under D5-R protein. Should text or table be changed?

The Dopamine Receptor Genes D1- and D2-like receptors genes differ in their genomic structure. Indeed, the coding regions of D1-like genes contain no introns, while the D2-like receptor subfamily is encoded by split genes. Among DA-Rs, the rat D2-R cDNA was the first to be isolated.14 The cloned cDNA coded for a protein of 415 aa, D2S (D2 short), and showed a typical D2 pharmacological profile. Several independent groups have succeeded in isolating a splice variant of this receptor, D2L (D2 long) from different species and tissues.13 The length of this cDNA variant is due to the insertion of 29 aa in the putative il3 of the receptor. The D2-R gene is a split gene composed of eight exons, seven of which are coding.2 The first exon is separated from the second by an intron which is at least 25 kilobases in length. The alternative splicing of the sixth exon, which encodes for the 29 aa insertion, generates the D2L and D2S isoforms.2 D3-R was cloned in 1990 by Sokoloff et al11 from a rat brain gene library. The gene codes for a 446 aa protein and also has several splice variants. For example, one variant contains a 21 aa insertion in the murine D3 il3,15 and another exists as a nonfunctional form composed of 428 aa.1 The D4-R gene, cloned by Van Tol et al16 from a human neuroblastoma cell line, is composed of five coding exons which generate a 387 aa protein. Gene variants have also been found in the case of the D4 gene. Indeed a 48 base pair sequence, which can be repeated up to seven times in the il3 of the D4-R, has been described.16 In contrast, the D5-R gene has some related pseudogenes that code for truncated and non functional receptors.1 During the last few years the study of the transcriptional regulation of DA-Rs has been receiving increased attention. These studies are of importance, since the elucidation of the factors involved in the regulation of the expression of DA-Rs might lead to alternative therapies in some dopaminergic diseases characterized by changes in DA-Rs expression. Interestingly, in spite of the very strict expression patterns of D1- and D2-R genes, their promoters show housekeeping characteristics. Both D1 and D2 gene promoters lack functional TATA and CAAT boxes.17-20 However, they contain binding sites for several transcription factors such as AP-1, AP-2 and Sp1.13,17,21

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The Development of Dopaminergic Neurons

The D1- and D2-R promoters show a different rate of transcription depending on the cell line in which they are expressed, indicating the binding of cell-specific nuclear factors.21,22 A negative modulator sequence, which binds a 130 kDa protein, has been identified in the D2-R promoter. This protein is present both in a neuroblastoma cell line and in nuclear extracts from rat striatum.23 More recently, we have characterized a functional retinoic acid responsive element (RARE) in the D2-R promoter.24 This sequence binds the heterodimer formed by retinoic acid receptors and retinoid X receptors (RAR/RXR), thus stimulating D2-R transcription several fold.24 Knockout (KO) mice lacking the expression of the isoform RXRγ, or double mutant mice lacking RARα/RXRγ, RXRγ/RXRβ, or RARβ/RXRγ show a severe decrease of the D2- and D1-R expression in the striatum.24,25 This indicates that retinoic acid plays an important role in the transcriptional regulation of both D1- and D2-R genes. In addition, the D1-R gene is transcribed by two promoters: one located upstream of the first non-coding exon and a second in a unique intron located upstream of the coding region. These two promoters are tissue specific and seem to influence transcript properties (e.g., abundance).22 Also, for the D2-R gene promoter two transcription start sites have been described.19 A polymorphism in the D2 promoter region which seems linked to schizophrenia26 has been reported. This might suggest that anomalies in the transcriptional regulation of DA-R genes could be associated with neurological diseases. Thus, besides the housekeeping features of the DA-R genes, their transcription seems to be tightly regulated spatially and temporally by a plethora of different cell type-specific nuclear factors. This might prevent abnormal and ectopic expression which could be detrimental to the physiology of the whole organism.

Signal Transduction As members of the seven TM receptor family, DA-Rs have neither intrinsic enzymatic activity nor the possibility to interact directly with cellular effectors. The stimulation of cellular effectors, upon ligand binding, is mediated through the interaction with GTP-binding proteins (G-proteins). These proteins belong to the family of heterotrimeric G-proteins, constituted by Gα, Gβ and Gγ subunits.27 Among different members of the seven TM receptor families, the il3 was proved to have a pivotal role in the interaction with heterotrimeric G-proteins.28 This region is the most variable among DA-Rs. D1/D2-R hybrids have shown that D2-R coupling to Gαi mainly involves regions of the il3, but also some of the il2.13,29 In our laboratory, we have shown that the 29 aa insertion, found in the il3 of the D2L isoform of D2-R, confers to this receptor a greater affinity for the binding to Gαi230,31 with respect to D2S. These findings were supported by a mutational analysis of the D2L-specific 29 aa insertion.32

D1 Receptor Since the early 1970s several reports have shown that DA increases adenylyl cyclase (AC) activity in neural tissues.1 Receptors of the D1-like subtype are positive regulators of cyclic AMP (cAMP)1,33,34 and their stimulation results in the activation of protein kinase A (PKA). PKA, in turn, phosphorylates cytoplasmic and nuclear proteins, regulates cellular metabolism, including ion channel function, and finally desensitizes seven TM G-protein coupled receptors.35,36 D1-R stimulation increases L-type Ca2+currents and leads to a reduction of N- and P-type Ca2+ currents in rat neostriatal neurons or in the pituitary-derived GH4C1 cells, via a direct or indirect action of PKA.37 Instead, in rat adrenal glomerulosa cells, this receptor

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109

inhibits T-type Ca2+ currents.38 Furthermore, D1-R can also mobilize intracellular Ca2+ stores by activating the cAMP pathway without activating phosphoinositide (PI) hydrolysis.37 However, D1-R also stimulates PI turnover by activating phospholipase Cγ (PLCγ). This might be possible through an indirect stimulation of the PLCγ via the cAMP pathway39 or via a yet uncharacterized member of the D1-like subfamily.40,41 Indeed, in D1-R KO mice the stimulation of PI is still present, whereas cAMP induction through D1-R is absent.42 The G-protein classically involved in D1-R stimulation is Gs,1 but PLCγ stimulation seems to be dependent upon Gq stimulation.41-43 In fibroblasts, it was reported that D1-R inhibits the activity of the Na+/K+- ATPase via the PKA pathway,1,37 while in striatal neurons a synergistic activity between D1- and D2-R is responsible for this inhibition.37 This inhibition leads to a transient membrane depolarization that affects cell excitability and consequently neurotransmitter release.

D2 Receptor D2-R activation affects many signal transduction pathways (Table 7.3). A DA-mediated inhibition of AC activity was shown in the anterior and intermediate lobes of the pituitary gland, and also in striatal cells.1 In the pituitary, DA inhibits the synthesis and release of hormones (prolactin, β-MSH), whereas in the striatum it regulates the firing of neurons as well as the synthesis of neuropeptides. D2-R triggers the inhibition of AC by coupling to signaling pathways blocked by pertussis toxin (PTX).1,2,13 This indicates that these receptors are associated to members of the Gi/Go-protein family.13The 29 aa insertion in the D2L il3 is also important for modifying the receptor specificity for G-proteins. Thus, the coupling of the D2-R isoforms to alternative G-proteins1 might differentially affect the inhibition of AC, depending on the cell type in which the receptor is expressed. D2-like receptors can directly modulate several different effectors by coupling to Gi/oα subunits.1,13 D2L and D2S differentially inhibit Ca2+ currents through coupling to Gαo in rat pituitary cells.1,37 In these cells D2-R inhibits two voltage-activated Ca2+ currents1,37 through coupling to Gαo. In addition, through a Gi/o protein, D2-R reduces N-type Ca2+ currents in rat neostriatal cholinergic interneurons44 and mediates the inhibition of Ca2+ channel activity in melanotrophs.45 D2-R influences also the PKC pathway that leads to the production of inositol-1,4, 5-trisphosphate (IP 3). 46 However, a direct modulation of the PLC by D2-R is still controversial. The activation of D2-R in pituitary lactotrophs inhibits AC, but also Ca2+ release from intracellular stores46 through the inhibition of IP3 synthesis. This could probably be due to a PLC inhibition, which is cAMP-dependent in the same way as described for D1-R.39 Interestingly, D2-R-transfected pituitary cells show a Ca2+ concentration decrease which is IP3-independent. However, in transfected fibroblasts both Ca2+ and IP3 concentrations are increased.1,37 These apparently contrasting results might be explained by the interaction of the D2-R with different G-proteins and/or by the presence of different PLC in various cell types. The D2-R modulation of intracellular Ca2+ concentrations might have an important role in DA biosynthesis, since D2-R are both pre- and post-synaptic. Indeed, tyrosine hydroxylase, the rate-limiting enzyme in DA production, is activated by the Ca2+/calmodulin dependent protein kinase (CaM kinase or CaM II).47 CaM II is subsequently activated by Ca2+-bound calmodulin.35 Another important effect of D2-R activation is the regulation of K+ currents in several neural tissues, such as the striatum and mesencephalic dopaminergic neurons, as well as in lactotrophs, melanotrophs and NG108-15 cells.1,48 D2L and D2S isoforms seem to

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110

Table 7.3. Signaling pathways induced by dopamine receptors Dopamine Receptor

G-protein

Direct Effector

D1

Gαs

Ac

cAMP




D5

Gαs

AC

cAMP




D2

Gαi, Gαo

AC

cAMP



Ca2+ channels

Ca2+



K+ channels

K+


or 

AC

cAMP



Ca2+ channels

Ca2+



K+ channels

K+



Gαi, Gαo

AC

cAMP



Gαt2

Ca2+ channels

Ca2+



K+ channels

K+



D3

D4

Gαi. Gαo

Intracellular Effect

This table presents a simplified view of the signal transduction cascades modulated by dopamine receptors in neuronal and pituitary cells. Arrows indicate the increase or decrease in the intracellular concentration of the second messenger molecules. In the case of D2-R, the effect on K+ concentration changes depending on the cells type used in the experiment.

differentially modulate outward K+ current through the interaction with different Gproteins.48 D2-R has also been hypothesized to control voltage-dependent cAMP-independent K+ channels and cAMP-dependent Ca2+-sensitive K+ channels.1,37 D2-R can affect the intracellular Ca2+ concentration through voltage-dependent Ca2+ channels by regulating K+ currents.1,13 In addition to the signal transduction pathways described, D2-R is also involved in modulating arachidonic acid (AA) synthesis. D2-R signaling can potentiate AA synthesis when Ca2+ concentration is raised, in CHO cells, but D1-R has no such effect.1,49 The effect on AA production is PTX-sensitive, strongly suggesting a principal role for D2-R.1,49 Instead, in rat striatal cells D1- and D2-R have opposing effects. Indeed, while D2-R stimulates AA synthesis, D1-R inhibits it,1,37 thus demonstrating a cell-specific effect of D1- and D2-R activation. Finally, several groups have reported an effect of D2-R activation on the Na+/H+ exchangers in several non-neural cell lines,13 as well as in rat anterior pituitary cells.37

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111

D3 Receptor Similarly to D2-R, the D3-R has also been described as an inhibitor of the endogenous cAMP level in several cell lines.1,37,50 However, D3-R seems to inhibit AC less efficiently than D2-R,37 with the smallest effect in a pituitary cell line.51 The D3-R decreased efficacy in AC inhibition might be due to a reduced ability to couple to G-proteins. In fact, it has been shown that the exchange of the il3 region of D3-R with that of D2-R results in a chimeric receptor as efficient as D2-R at AC inhibition.52 In a neuroblastoma cell line, D3-R inhibits calcium37 and potassium48 currents through a Gi/o protein. D3-R does not appear to be coupled to PI turnover in a mouse mesencephalic cell line.53 In C6 cells, D3-R stimulates the Na+/H+ exchanger through a PTX-independent G-protein pathway.54

D4 Receptor In addition to D2- and D3-R, D4-R also inhibits cAMP production in a variety of cell lines tested.55-57 D4-R cAMP inhibition is PTX-dependent. However, in MN9D cells it has been shown not to be due to the coupling to Gi2, GoA or GoB,53-55 but unexpectedly to the cone transducin Gαt2.55 Interestingly, some of the il3 reported variants of this receptor inhibit AC in a similar way in transfected cells.56,57 This suggests that the polymorphism present in the il3 does not affect the receptor’s potency at modulating AC activity. However, these variants could affect the activation of other signaling pathways not yet analyzed for this receptor. L-type Ca2+ currents in cerebellar granule cells have been reported to be inhibited in a cAMP-independent manner by D4-R through the activation of Gi/o proteins.58 Ca2+ currents and two K+ channels are also inhibited in GH4C1 pituitary cells59 and in nerve terminals of the neurohypophysis,60 respectively, by D4-R stimulation. In CHO cells D4-R upregulates AA release, through PKC activation and independently from cAMP levels, and stimulates the NaH-1 Na+/H+ exchanger.61

Dopamine and Locomotion The importance of DA in the control of movements is well demonstrated in pathological conditions such as Parkinson’s disease. Indeed, this disease is characterized by a strong reduction of circulating DA due to the degeneration of dopaminergic neurons. A very active research effort is aimed at understanding the molecular basis of the degeneration of dopaminergic neurons. Analyses of mouse mutants for a variety of genes, whose functions are required during development, has already given information regarding some early requirements necessary for dopaminergic neuronal survival.62 An important role has been attributed to some neurotrophic factors in the survival rate of these neurons. Pharmacological studies using D1- or D2-R antagonists have also clearly demonstrated the involvement of the dopaminergic system in the coordination of voluntary movements in rats or mice.63-65 In particular, treatments with D1-R antagonists result in a cataleptic response in mice66-68 and the administration of D2-R antisense oligonucleotides in rats and mice, reduces this spontaneous locomotor activity.69,70 The generation of mutant animals for four of the DA-Rs (D1-D4) and their respective behavioral analysis have shown that each of these mutants presents a locomotor phenotype. Unexpectedly, and in contrast to pharmacological analyses, locomotion in D1-R null mice is not affected,71 or its baseline increased, when compared to wild type littermates.72 These results confirm previous data suggesting a more complex interaction between the different types of DA-Rs in the regulation of voluntary movements, especially with respect to D1- and D2-R interactions. In the future it will be possible to better define these interactions, not only between the major types of receptors, but also with regard to the less expressed and least characterized dopaminergic receptors.

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The Development of Dopaminergic Neurons

We have analyzed the locomotor behavior of D2-R mutant mice73 using the open field, the ring, and the rotarod behavioral tests. The activity of homozygous mice was compared to that of heterozygous and wild type littermates. This comparative study clearly revealed a motor impairment in homozygous mice, which was also evident to a much lesser extent in heterozygous mice. This impairment is characterized by reduced movements and suppression of rearings. The rotarod test showed that homozygous mice have difficulties in coordinating movements. As for D1- and D2-R deficient mice, D3- and D4-R mutants also showed a locomotor phenotype. D3-R mutants present a hyperlocomotor phenotype which is in agreement with the results obtained by pharmacological studies using D3-R antagonists.74 Surprisingly, in D4-R mutants locomotion is also affected, and in particular is reduced in spite of the anatomical expression of this receptor.75,76 It will now be interesting to combine these mutations and try to unravel the different synergistic/antagonistic interactions between the different receptors of the dopaminergic family. The results obtained by the analysis of D1-R mutants, which contrast with previous studies using D1-R antagonists, underline the importance of the genetic approach for understanding the function of each receptor.

Dopamine and Drugs of Abuse The mesolimbic dopaminergic system consists of neurons originating in the VTA which have axonal projection to the limbic system. In particular, dopaminergic projections to the Acb have been implicated in the control of the reward mechanisms and in the psychomotor effects generated by drugs of abuse, including opiates, cocaine, amphetamine and alcohol.77,78 Cocaine and amphetamine increase DA levels into the synaptic cleft by blocking the activity of the DA transporter (DAT), and by reversing DA transport via DAT.77 On the other hand, opiates have an inhibitory effect on DA release in a variety of brain areas like striatum, frontal cortex, OT, Acb, mediobasal hypothalamus and amygdala.79 It has been established that DA plays a primary role in the behavioral effects caused by the intake of drugs of abuse.80,81 These effects are mediated by DA interaction with membrane receptors. The contributions of the different DA-R subtypes in response to drugs of abuse was initially investigated using animal models in which lesions of particular brain areas and/or pharmacological approaches were employed. From these studies it has been possible to show that selective lesions of the Acb,81 or DA-R blockade by D1- or D2-like receptor antagonists, attenuate hyperlocomotion and the reward effects caused by morphine, cocaine and amphetamine in rats or mice.81-83 On the contrary, the administration of D2-like receptor agonists such as quinpirole and bromocriptine completely mimics the effects of cocaine, or enhance these effects if administered in combination with cocaine.81 One of the limitations of these studies has been the lack of adequately selective antagonists or agonists directed at the five known DA-Rs. The generation of mice deficient for one of the five DA-Rs, obtained by homologous recombination, hopefully will shed some light on their specific involvement in this drug response. Indeed, in the last few years a remarkable amount of data has already been generated in this respect. A critical role for D1-R has been demonstrated in the locomotor behavior and stereotyped activities mediated by drugs of abuse in mice lacking this receptor type.84 These mice failed to exhibit locomotor hyperactivity and stereotyped behaviors following cocaine treatments when compared to wild type littermates. A possible explanation for this phenotype was given by the enabling role of D1-R on D2-R. Thus, the absence of D1-R also alters D2-R function. These animals, however, retain the capacity to show a place preference to cocaine, indicating that the rewarding properties of this drug are not affected in these mice.85

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In our laboratory, the study of D2-R null mice has demonstrated the crucial role of this receptor in the rewarding effects of opiates.86 In the Place Conditioning paradigm the D2–/– mice failed to show a preference for opiates, since they spent the same time in morphine- and in saline-associated compartments. The wild type littermates, however, showed a significant increase in the time spent in the drug-associated compartment during the testing phase. Interestingly, we also demonstrated that the D2-R is not required for the development of physical opiate dependence nor for the locomotor response to acute administration of morphine. In addition, D2-R activity seems to be specifically involved in the motivational component of opioid dependence. Indeed, when these mice were tested in a similar behavioral paradigm in their response to a natural reinforcer such as food, no differences were observed between D2-R-deficient and wild type mice. The behavioral phenotype of D2-R deficient mice in response to cocaine is presently under investigation, although preliminary studies performed in our laboratory suggest an impaired response to the administration of this drug (unpublished observations). More recently, D3-R KO mice have also been generated.76 These mice present a hyperlocomotor behavior and when challanged with cocaine they display a hypersensitivity to the effect of this drug. These interesting results have been explained by a possible modulatory role of D3-R on D1- and D2-R activities. Conversely, D4-R KO mice, in spite of the observed hypoactive locomotor phenotype, seem to be more sensitive to cocaine treatment.75 However, a clear explanation for this phenotype is still awaited. The behavior of these different mutants in response to various drugs is still under investigation in a number of different laboratories. Hopefully, it will be possible in the very near future to gain deeper insights into the role of each DA-R in the development of drugrelated effects, both at the molecular and behavioral levels.

Molecular Responses toTreatments with Drugs of Abuse

In the mesolimbic dopaminergic system, decreased levels of Giα and Goα87 and an upregulation of the cAMP pathway88 in response to chronic opiate and cocaine treatments have been observed. The behavioral modifications induced by the intake of drugs of abuse has generated growing interest in deciphering the molecular mechanisms underlying such behaviors. There is a general agreement about the fact that drugs of differing nature invariably provoke an increased release of DA in the Acb. At a molecular level, this leads to an overstimulation of DA-Rs. The signal transduction pathways activated by DA-Rs are very numerous. However, the best described effects mediated by DA are the activation or inhibition of the cAMP pathway and modulation of Ca2+ signaling. Stimulation of these pathways leads to the activation of nuclear factors such as the cAMP response element-binding protein (CREB) and members of the Jun/Fos families. Interestingly, these factors are also involved in the regulation of the expression of genes encoding several neuropeptides, including enkephalin89 and prodynorphin,90 whose expression is also altered after drug treatment. Several laboratories have demostrated that acute or chronic administration of cocaine increases CREB phosphorylation91 and the expression of specific members of Jun/Fos families92-97 in the Acb and in the CP. c-Jun, c-Fos and related proteins can then heterodimerize to constitute the AP-1 transcription factor complex,98 whose binding activity to DNA is also increased upon cocaine treatment.92-97 Blockade of c-fos gene expression in Acb, by local application of a c-fos antisense oligonucleotide, was recently reported to virtually abolish the locomotor stimulatory effect of intraperitoneal injections of cocaine.99 Psychostimulant-induced CREB phosphorylation and expression of the immediate early gene transcription factors (c-Fos, and Zif 268) after acute cocaine and amphetamine treatment are blocked in striatal neurons by D1-R91 and partially by D2-R antagonists.95

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These results were confirmed in mice treated with MPTP, in which dopaminergic neurons degenerate,100 and in D1-R deficient (-/-) mice.84 However, a clear definition of the different steps leading to the stimulation of these transcription factors is still unclear. A lot of work is still needed to clearly identify the cytoplasmic mediators that constitute the signaling pathway started by the binding of DA to its receptors, and finally leading to CREB and AP-1 activation. More interestingly, it will be of great importance to identify the genes which are regulated by these transcription factors and which are responsible for the behaviors induced by the intake of drugs of abuse.

Proliferative Role of Dopamine In Vitro Evidence exists of a mitogenic effect of D2-R activation in cultured cells. This effect was shown to be inhibited by PTX treatment, whereas cAMP analogs did not affect proliferation.101 Interestingly, D2-R mediated induction of mitogenesis in CHO cells could also be blocked by genistein, a tyrosine kinase inhibitor, thus revealing the ability of DA to induce tyrosine phosphorylation through the activation of protein kinases.101 A mitogenic response was also demonstrated upon activation of D3-R in a neuroblastoma/glioma hybrid cell line. This effect could also be abolished by PTX treatment.102 D2L, D3-, or D4-R activation induces a morphogenic effect in the MN9D cell line. D2L activation increased neurite number and branching, but had little effect on neurite length. D3-R activation increased both neurite branching and length, while D4-R augmented neurite branching and dramatically extended neurite length.103 However, in primary cultures of fetal rat ventral mesencephalic neurons, the chronic stimulation of D2-R impaired neither survival nor differentiation of these cells.104 Other reports show, instead, an effect of D2-R stimulation on apoptosis and differentiation of an olfactory neuronal cell line.105 In a lactotroph-derived cell line, GH4C1, DA inhibits cell growth through the D2S isoform. At this time, contrasting results have been reported on the inhibitory effect of PTX.106,107 Activation of D2-R in these cells seems to affect a phosphotyrosine phosphatase through PKC stimulation. In a corticotroph-derived cell line, AtT-20, bromocriptine, a D2-R agonist, induces apoptosis.108 These results might be in line with the effectivness of bromocriptine in reducing GH-secreting and ACTH-secreting adenomas. Thus it is clear, depending on the cell type analyzed, that differences can be found regarding the possible mitogenic effects of DA, acting through D2-like receptors. Indeed, in some cell lines, D2-R activation stimulates and in some others inhibits cell proliferation. This suggests the presence of different mechanisms and possibly the existence of different cell-specific interactions in the regulation of this function.

In Vivo In addition to its presence in the CNS, D2-R is also strongly expressed in the anterior (ALp) and intermediate lobes (ILp) of the pituitary gland.1,13,14 More precisely, it is found in the lactotroph and the melanotroph cells which produce the pituitary hormones prolactin (PRL),109-111 α-melanocyte stimulating hormone (α-MSH) and β-endorphin, 112,113 respectively. The dopaminergic regulation of these cells leads to the inhibiton of the synthesis and release of these hormones. Both D2L and D2S receptors are co-expressed by these cells and, as elsewhere, the ratio D2L/D2S always favors D2L expression. Interestingly, Kukstas et al114 reported the existance of two populations of lactotrophs with different D2L/D2S ratios, dependent upon progesterone and testosterone regulation. Besides the inhibitory control on prolactin and proopiomelanocortin (POMC) gene transcription, DA was also thought to control lactotroph proliferation by inhibiting the

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cAMP pathway.115 This key control of lactotroph proliferation seems to be counteracted by estrogens. Indeed, estrogens positively regulate lactotroph proliferation after birth.116,117 Thus, DA and estrogens exert an opposite effect on lactotroph growth. An additional demonstration of the negative dopaminergic control of this function is the efficacy of bromocriptine treatment in the regression of lactotroph-derived pituitary tumors (prolactinomas).118 A very recent analysis conducted in mice, in which components of the dopaminergic pathway have been genetically modified, has given some insights into the molecular mechanisms controlling these events.

Dopamine Acts as an Antiproliferative Factor in Pituitary Cells Dopamine is also an important regulator of pituitary functions, of which the best characterized is the control of PRL synthesis and release. Indeed, while hypothalamic factors such as thyrotropin-releasing hormone (TRH), vasoactive intestinal peptide (VIP) and ovarian hormones such as estrogens are known to positively control this hormone level, DA is the only negative modulator of its synthesis and release. Interestingly, mice lacking D2-R develop pituitary tumors of lactotroph origin. This effect arises earlier in females than in males, where these tumors appear very late in life.119 Interestingly, the analysis of TRH and VIP expression showed that their levels are unaffected in D2R–/– mice. In addition, we showed that D2-R–/– mice are also hypoestrogenic. Estrogen levels in D2R–/– females were as low as in males. These findings have led to important conclusions, since they define that the pituitary tumors arising in D2R–/– mice are not due to unbalanced estrogens or VIP or TRH activities, but to the absence of dopaminergic control. In the absence of dopaminergic control we have described a robust rise of PRL levels. We have shown that PRL receptors are present on lactotrophs. Thus, PRL acts as an autocrine modulator of lactotroph proliferation. A continuous stimulation of PRL receptors is mitogenic,120 which is likely to be the cause of pituitary tumors in these mice. Therefore, the inhibitory dopaminergic tone, by regulating PRL synthesis and release, is pivotal in the control of the rate of lactotroph proliferation. This report was the first showing an antiproliferative effect of a neuromodulator in the control of cell growth and cell differentiation. Lactotrophs are in fact the last cellular type to differentiate from a common precursor during pituitary ontogenesis. In temporal order, this lineage starts initially with thyrotrophs, then somatotrophs and finally lactotrophs. Interestingly, we found that in D2-R–/– mice the number of somatotrophs is diminished. One possibility is that the lineage is driven to produce lactotrophs, as a consequence of strong PRL stimulation on these cells’ proliferation. This is done at the expense of the somatotrophs which are consequently converted into this cell type. To compensate, a higher number of thyrotrophs are produced from the precursor cells. Thus, in addition to proliferation, DA also controls the differentiation of this lineage. The antiproliferative role of DA in the pituitary is supported by results obtained with the DA transporter (DAT) KO mice. These mice, in contrast, show a hypoplastic pituitary.121 In these mice, released DA is not removed,122 leading to an overstimulation of DA-Rs. Abnormal stimulation of D2-R in the pituitary leads to a decreased production of PRL and to a reduced number of lactotrophs.121 In DAT KO mice the number of somatotrophs is also reduced;121 this is because the expression of the hypothalamic growth hormone-releasing hormone (GHRH) is reduced.121 The novel concept of DA as a regulator of cell growth and differentiation was also confirmed by the effects of D2R absence on the other pituitary cell types which normally express D2-R, the melanotrophs. Indeed, we observed the hypertrophy of the intermediate lobe in D2R–/– mice, with a 40% increase in melanotroph number.123 We also observed an upregulation of the POMC gene, which is corticotropin-releasing factor (CRF)-

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independent.123 More strikingly, the products of the cleavage of the POMC genes were altered. POMC is normally processed by two prohormone convertases, PC1 and PC2, which are expressed in a cell-specific manner by two pituitary cell types, the corticotrophs in the ALp and the melanotrophs in the ILp. Only melanotrophs express D2-R and, therefore, we expected only the expression of the peptides specifically cleaved in this cell type to be affected. POMC processing gives rise to ACTH in corticotrophs and to α-MSH and β-endorphin in melanotrophs. Strikingly, in D2-R KO mice, a significant increase of the circulating levels of ACTH was observed. However, POMC expression in the corticotrophs was not altered. Previous reports had shown that PC1 and PC2 are differentially expressed in the ALp and ILp.124,125 Indeed, while the first is strongly expressed in the corticotrophs, the second is expressed in the melanotrophs. The analysis of the expression of these convertases showed that PC1 was abnormally upregulated in D2-R KO mice.123 This result adds even more to our knowledge of DA effects in vivo. Indeed, it illustrates a novel function of this meuromodulator in the control of pituitary cell identity. Thus, in the absence of dopaminergic control, melanotrophs lose their cellular specialization and these cells start to produce ACTH. Importantly, the overproduction of ACTH leads to an overstimulation of the adrenal cortex, a higher production of glucocorticoids and a hypertrophy of the adrenal cortex, leading to a phenotype that resembles Cushing’s syndrome.123 This result establishes an unprecedented link between the dopaminergic system and the etiology of Cushing’s syndrome. Thus, we believe that in future years many more still unknown functions will be attributed to dopamine by a careful analysis of the phenotype of the different mutants that have been generated. This, together with the biochemical, pharmacological and anatomical studies, will hopefully give us a clear picture of the function of the dopaminergic system.

Acknowledgments The authors thank Dr. D. Whitmore for discussions. This work was supported by grants from the Institut de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Centre Hospitalier Universitaire Régional, The Association pour la Recherce sur le Cancer, and fellowships from European Union (TMR Program, Marie Curie) to D.V., and Association France Parkinson to R.P.

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99. Ruskin DN, Marshall JF. Amphetamine- and cocaine-induced fos in the rat striatum depends on D2 dopamine receptor activation. Synapse 1994; 18:233-240. 100. Kano T, Suzuki Y, Shibuya M et al. Cocaine-induced CREB phosphorylation and c-Fos expression are suppressed in parkinsonism model mice. Neuro Report 1995; 6:2197-2200. 101. Lajiness ME, Chio CL, Huff RM. D2 dopamine receptor stimulation of mitogenesis in transfected Chinese hamster ovary cells: Relationship to dopamine stimulation of throsine phosphorylations. J Pharmacol Exp Ther 1993; 267:1573-1581. 102. Pilon C, Levesque D, Dimitriadou V et al. Functional coupling of the human D3 receptor in a transfected NG 108-15 neuroblastoma-glioma hybrid cell line. Eur J Pharmacol 1994; 268:129-139. 103. Swarzenski BC, Tang L, Oh YJ et al. Morphogenic potentials of D2, D3, and D4 dopamine receptors revealed in transfected neuronal cell lines. Proc Natl Acad Sci USA 1994; 91:649-653. 104. Van Muiswinkel FL, Drukarch B, Steinbusch HW et al. Chronic dopamine D2 receptor activation does not affect survival and differentiation of cultured dopaminergic neurons: Morphological and neurochemical observations. J Neurochem 1993; 60:83-92. 105. Coronas V, Feron F, Hen R et al. In vitro induction of apoptosis or differentiation by dopamine in an immortalized olfactory neuronal cell line. J Neurochem 1997; 69:1870-1881. 106. Florio T, Pan MG, Newman B et al. Dopaminergic inhibition of DNA synthesis in pituitary tumor cells is associated with phosphotyrosine phosphatase activity. J Biol Chem 1992; 267:24169-24172. 107. Senogles SE. The D2 dopamine receptor mediates inhibition of growth in GH4ZR7 cells:Involvement of protein kinase-C epsilon. Endocrinology 1994; 134:783-789. 108. Yin D, Kondo S, Takeuchi J et al. Induction of apoptosis in murine ACTH-secreting pituitary adenoma cells by bromocriptine. FEBS Lett 1994; 339:73-75. 109. Elsholtz HP, Lew AM, Albert PR et al. Inhibitory control of prolactin and Pit-1 gene promoters by dopamine. Dual signaling pathways required for D2 receptor-regulated expression of the prolactin gene. J Biol Chem 1991; 266:22919-22925. 110. Lew AM, Yao H, Elsoltz HP. G(i) alpha 2- and G(o) alpha-mediated signaling in the pit-1dependent inhibition of the prolactin gene promoter. Control of transcription by dopamine D2 receptors. J Biol Chem 1994; 269:12007-12013. 111. Lew AM, Elsholtz HP. A dopamine-responsive domain in the N-terminal sequence of Pit1. Transcriptional inhibition in endocrine cell types. J Biol Chem 1995; 270:7156-7160. 112. Chen CL, Dionne FT, Roberts JL. Regulation of the pro-opiomelanocortin mRNA levels in rat pituitary by dopaminergic compounds. Proc Natl Acad Sci. USA 1983; 80:2211-2215. 113. Cote TE, Felder R, Kebabian JW et al. D-2 dopamine receptor-mediated inhibition of pro-opiomelanocortin synthesis in rat intermediate lobe. Abolition by pertossis toxin or activators of adenylate cyclase.J Biol Chem 1986; 261:4555-4561. 114. Kukstas LA, Domec C, Bascles L et al. Different expression of the two dopaminergic D2 receptors, D2415 and D2444, in two types of lactotroph each characterised by their response to dopamine, and modification of expression by sex steroids. Endocrinology 1991; 129:1101-1103. 115. Weiner RI, Findell PR, Kordon C. Role of classic and peptide neuromediators in the neuroendocrine regulation of LH and prolactin. In: Knobil E. and Neill J, 1st ed. The Physiology of Reproduction. New York: Ravel Press, Ltd, 1988:1235-1281. 116. Lieberman M, Slabaugh M, Rutledge JJ et al. Steroids and differentiation. The role of estrogen in the differentiation of prolactin producing cells. J Steroid Biochem Mol Biol 1983; 19:275-281. 117. Elias KA,Weiner RI. Inhibition of estrogen-induced anterior pituitary enlargement and arteriogenesis by bromocriptine in Fisher 344 rats. Endocrinology 1987; 120:617-621. 118. Bansal S, Lee LA and Woolf PD. Abnormal prolactin responsivity to dopaminergic suppression in hyperprolactinemic patients. Am J Med 1981; 71:961-970. 119. Saiardi A, Bozzi Y, Baik JH et al. Antiproliferative role of dopamine: Loss of D2 receptors causes hormonal dysfunction and pituitary hyperplasia. Neuron 1997; 19:115-126.

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120. Mershon J, Sall W, Mitchner N et al. Prolactin is a local growth factor in rat mammary tumors. Endocrinology 1995; 136:3619-3623. 121. Bossé R, Fumagalli F, Jaber M et al. Anterior pituitary hypoplasia and dwarfism in mice lacking the dopamine transporter. Neuron 1997; 19:127-138. 122. Giros B, Jaber M, Jones SR et al. Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 1996; 379:606-612. 123. Saiardi A, Borrelli E. Absence of dopaminergic control on melanotrophs leads to Cushing’s syndrome in mice. Molecular Endocrinology 1998; 12:1133-1139. 124. Bloomquist BT, Eipper BA, Mains RE. Prohormone-converting enzyme: Regulation and evaluation of function using antisense RNA. Mol Endocrinol 1991; 5:2014-2024. 125. Day R, Schafer MK, Watson SJ et al. Distribution and regulation of the prohormone convertates PC1 and PC2 in the rat pituitary. Mol Endocrinol 1992; 6:485-497.

CHAPTER 8

Dopamine Neuron Grafts: Development and Molecular Biology Lauren C. Costantini and Ole Isacson

T

he introduction of grafting dopaminergic (DA) neurons into rodent host brains illustrated the potential of this technique for both experimental and clinical applications.1,2 Basic research utilizing these transplants has revealed information regarding development and connectivity of CNS neurons, while studies aimed at therapeutic strategies for Parkinson’s disease (PD) have shown the potential of this procedure for “biological replacements”, reconstructing the circuits within a degenerated brain.3 The current experiments involving fetal neural grafts provide information about mechanisms and processes involved in phenotypic DA neuron development, and serve as a guide to alternative cell sources for clinical neural transplantation.

Establishment of Surviving Dopamine Neuron Grafts

Studies have demonstrated that embryonic day (E) 14 for rodent tissue4 and 6.5-9 weeks post-conception for human tissue5 are the optimal ages of ventral mesencephalic (VM) donor tissue for DA neuronal survival and functional effects when transplanted into the DA-denervated striatum. The minimum number of surviving transplanted DA neurons required for functional effects to be revealed in animal models is approximately 100-200.6 Since only 10% of the transplanted VM cells are DA, and only 1-10% of these DA neurons survive,7-9 as many as 10-15 fetal VM per patient may be required for sufficient survival and reinnervation.10 Strategies to improve the survival of the DA neurons within these grafts are being considered, including treatment with growth factors, antioxidants, cotransplantation, and modified implantation procedures.11-15

Enhancing Cell Survival with Growth Factors and Target Tissue Methods to sustain the development and function of embryonic VM DA cells after transplantation into DA-depleted striatum are currently under investigation. Elements which are crucial for the maturation and connectivity of neurons during normal development of the brain may also play a role in the development and integration of grafted embryonic tissue. Based upon the observations that several neurotrophic factors affect the innervation of targets and the survival of neurons during development, administration of these factors along with transplanted “developing” fetal DA neurons has been examined. Among the trophic factors that can enhance development of DA neurons after grafting into rodent are brain-derived neurotrophic factor,11,16 basic fibroblast growth factor,17 and glial cell line-derived neurotrophic factor.18-20 Technical strategies have included exposure of the fetal cells to neurotrophic factors prior to transplantation16,17,21 or by administration after transplantation.11,18-20 The Development of Dopaminergic Neurons, edited by Umberto di Porzio, Roberto Pernas-Alonso, and Carla Perrone-Capano. ©1999 R.G. Landes Company.

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The target-derived neurotrophic factor hypothesis predicts the presence of DA-trophic activity in striatal tissue, and several studies support this hypothesis. Based on in vitro and in vivo observations of the enhancing effects of striatal tissue on nigral DA cell development and survival,22-24 we have demonstrated that inclusion of embryonic striatal cells,14 specifically from the lateral ganglionic eminence, enhanced the survival of DA neurons in VM transplants, and also required the transplantation of fewer nigral cells to produce a marked behavioral effect.13

Protecting Transplanted Cells with Antioxidants The poor survival of DA neurons after transplantation may be caused by damage to the VM tissue during preparation, or via cell death after transplantation such as that seen during development.25 A large proportion of DA cell death occurs during the first two weeks after transplantation due to these factors, and/or due to suboptimal conditions in the host brain during the early phases after transplantation.26 One hypothesis for the mechanism of this acute cell death is formation of free radicals during the process of dissociation. This is supported by evidence that VM from transgenic mice overexpressing Cu/Zn superoxide dismutase, the major free radical scavenging enzyme, produces transplants with four-fold greater survival of DA neurons and more extensive functional recovery.12 Treatment of transplanted cells with antioxidants increased the yield of surviving DA neurons, which correlated with an earlier onset of graft-induced functional effects.12,27 These findings have recently been taken into the clinical setting: One inhibitor of lipid peroxidation, tirilazad, has been included in the solutions for storage and preparation of donor tissue in the clinic; in addition, the patients are treated with this lazaroid for 72 hours after transplantation.28

Utilizing Medial VM for Dopamine Grafts Since several studies investigating optimal donor age have concluded that E13/14 (rat) yields most successful transplants,29 other issues seem critical. The selection of the most appropriate region for dissection is therefore of great importance. Presently, the embryonic dissections utilized for transplantation typically contain 2-10% DA neurons while the remainder are of other neuronal phenotypes, such as GABAergic.7,29 Other neurons within a developing transplant may hinder the survival and growth of the small proportion of DA neurons, since neurons compete for the limited supplies of trophic support for survival during development.30 Therefore, obtaining an initially enriched suspension of DA neurons may enhance the development of the transplants. Cells generated from the ventricular zone at E11 migrate ventrally and then move laterally to form the substantia nigra pars compacta (SNc) and ventral tegmental area. Tyrosine hydroxylase (TH; a synthetic enzyme involved in production of DA) immunoreactivity can be detected at E12.5 in the medio-basal region of the mesencephalon. By E14, TH+ cells are located laterally along the ventral surface to form the primordia of the SN.31,32 In order to enhance the relative proportion of DA neurons, we compared numbers of DA neurons from tissue dissected from the medial portion of the VM versus the lateral VM (Fig. 8.1). A higher proportion of TH+ neurons were observed in primary cultures of medial VM when compared with lateral or whole VM.33 Evaluation of E16 solid VM transplants revealed a larger number of surviving TH+ cells in grafts from medial region of VM compared to lateral VM.34 This higher proportion of DA neurons in medial VM can be attributed to the presence of a higher number of DA neurons in the medial region of the VM at E14. Alternatively, the higher proportion of TH+ neurons in medial VM may represent enhanced survival of DA neurons in medial VM. Dopamine neurons are responsive to a variety of trophic factors such as GDNF,35 bFGF,36 TGF,37 and BDNF.38 A more pure population of DA neurons present

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Fig 8.1. Dissection of E14 medial and lateral ventral mesencephalon (VM). Dashed lines through cartoon of entire brain show dissection of VM; dashed lines in enlarged VM region show differential dissection of medial VM (MVM) and lateral VM (LVM). Reprinted with permission from Neuroreport 1997; 8:2253-2257, ©Rapid Sciences. in medial VM may yield less competition from other cell types for trophic factors, and thus enhance the survival of the relatively small proportion of DA neurons present, since receptors for some growth factors have been detected throughout the SN.39 If this is the case, the development of DA neurons after transplantation would be hindered by the presence of other cells. This hypothesis is consistent with findings in nigro-striatal cotransplantation studies: When equal numbers of nigral and striatal cells are cotransplanted, enhanced DA cell survival is obtained.13 However, low DA cell survival results when a relatively low number

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of embryonic striatal cells are cotransplanted, possibly due to competition among the DA neurons for the limited amount of trophic support provided by the cotransplanted striatal cells.13 In addition to a higher proportion of TH+ neurons in medial dissections of VM, we also found that a higher proportion of these DA neurons expressed aldehyde dehydrogenase (AHD), a retinoic acid-generating enzyme.33,34 Dopamine neurons of the midbrain can be divided into subpopulations based upon expression of neuropeptides and enzymes,40 and AHD is expressed in this subpopulation of DA neurons early in development,41,42 shortly after the appearance of TH.32,43 The retinoid-synthesizing actions of AHD may play a role in development of DA neurons: Mice deficient in the nuclear receptor Nurr-1, which promotes signaling through heterodimerization with a 9-cis-retinoic acid receptor,44 failed to produce midbrain DA neurons.42 In addition, there is a prevalence of AHD-containing neurons among the SNc DA population which project to the dorsal-lateral and rostral regions of the striatum, previously shown to be regions involved in functional recovery after grafting of VM tissue (see below: Regional and phenotypic specification of dopamine neurons).45

Inducing Stem Cells and Other Progenitor Cells The quest for an unlimited cell source for DA transplantation has yielded not only a wide array of potential cells for this use, but also has provided information on the development of DA phenotypes. Totipotent stem cells, adrenal medulla and peripheral nerve cotransplants, carotid body cell aggregates, testis-derived sertoli cells, and cells obtained from transgenic animals are currently being analyzed both for future clinical use and for probing developmental questions. Pluripotent cells, used both in studies of neural differentiation and as future therapeutic tools, consist of growth factor-expanded neural progenitors, immortalized cell lines, embryonal carcinoma cells, and embryonic stem cells. Growth factor-expanded cells have been transplanted into the adult brain, forming small grafts which exhibit some migration of cells away from the implantation site.46-48 After growth-factor expanded stem cells isolated from developing CS were transplanted into adult striatum, Svendsen et al observed low cell survival within small grafts, with few differentiated cells expressing neuronal markers.47 Immortalized cell lines also exhibit a capacity to differentiate into a number of region-specific neuronal morphologies when transplanted into brain (for review, see ref. 49). The transplantation of these cells into neonatal brain resulted in differentiation into neurons and glia with region-specific morphology.50-53 However, when transplanted into adult brain, Lundberg et al observed that the plasticity of immortalized cells (generated from embryonic striatum or hippocampus) was more restricted: A majority of these cells differentiated into glia in the adult environment.54 Embryonic carcinoma cell lines can differentiate into terminal, nonproliferating neural phenotypes after pretreatment with retinoic acid and subsequent intracerebral transplantation, 55-58 and in some cases produce TH+ cells.56,57 Kleppner et al55 observed differentiated neuron-like cells which exhibited different patterns of innervation into the host brain depending on the region of the mouse brain in which they were implanted. Our laboratory has utilized transplantation as a means to investigate DA neuron development: We tested the potential of blastocyst-derived embryonic stem (ES) cells to differentiate into DA neurons.59 These totipotent cells were transplanted into adult mouse striatum and adult DA-lesioned striatum. The grafts developed large numbers of cells exhibiting neuronal morphology and immunoreactivity for neurofilament, neuron specific enolase, TH, and 5-hydroxytryptamine (Fig. 8.2). Though graft size and histology were variable, typical grafts of 5-10 mm 3 contained 10-20,000 TH + neurons, whereas dopamine-β-hydroxylase+ cells were rare. Most grafts also included non-neuronal regions,

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Fig. 8.2. Phenotype of blastocyst-derived embryonic stem cells after transplantation to rat striatum. The grafts develop large numbers of cells exhibiting neuronal morphology and immunoreactivity for THand 5-hydroxytryptamine (5HT). Dopamine-β-hydroxylase+ cells are rare. Scale bar, 200µm. Reprinted with permission fromExperimental Neurology 1998; 149:28-41, ©Academic Press.

immunoreactive for glial fibrillary acidic protein. Both monoaminergic neuronal cell types extended axons into the graft and into the surrounding host brain: TH+ graft axons grew preferentially into gray matter of the DA-denervated rat striatum, as is typical of endogenous striatal DA innervation. This specific innervation pattern has also been observed for DA axons growing into the host striatum from fetal ventral mesencephalic grafts, but is not exhibited by non-DA fibers from the same grafts,60,61 nor by axons from fetal cortical or striatal grafts to adult striatum.60 In contrast, 5-HT+ axons from ES cell grafts extended equally into white and gray matter regions of the host striatum. Thus, the difference between

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growth patterns of TH+ and 5-HT+ axons reflects characteristics that are typical of these mature CNS cell types. In contrast to results with other cell lines, the ES cells in our study did not seem to be dependent upon the site of transplantation for differentiation into neurons: We transplanted ES cells into mouse kidney capsule to determine the influence that brain-specific environment may have on the differentiation of these totipotent cells. Kidney capsule grafts also developed large numbers of cells exhibiting neuronal morphology and immunoreactivity for neurofilament, neuron specific enolase, TH, 5-HT, and glial fibrillary acidic protein. Neural induction regardless of transplant site in our paradigm is consistent with recent evidence suggesting that neuralization is a default pathway, and occurs spontaneously if pregastrula cells do not receive other inducing signals to form epidermal, mesodermal, or endodermal cells.62 This was first suggested by experiments showing that cells of the early gastrula ectodermal animal cap, that normally develop into epidermal tissue, all form neural tissue if dissociated.63 Bone morphogenic protein (BMP-4) and activin have been implicated as the major inducers of epidermal differentiation during gastrulation. Ectopic application of BMP-4 is sufficient to induce epiderm formation in dissociated animal pole cap cells,64 and homozygous knockout mice lacking functional BMP receptor (BMPR1) die in gastrulation,65 a time when epidermis would otherwise form. Also, antagonists of BMP-4 or activin signaling, such as noggin, follistatin, and chordin, which are produced in the Spemann organizer region, can induce the ectopic formation of neural tissue.66-68 Thus, any manipulation that disrupts these epidermis-inducing signals results in neural differentiation. In our experiments, transplanting cells that have been dissociated and expanded at the pre-gastrula stage may disrupt the localized cell-cell communications which otherwise inhibit neuralization. Alternatively, the kidney has been shown to contain many neurotrophic factors and also to enhance the development of TH+ neurons when cografted with VM.69 The lack of kidney structure in GDNF knockout mice also suggests the presence of dopaminotrophic factors present in the kidney which may have induced the totipotent ES cells to differentiate along the DA phenotype.70 These findings demonstrate that transplantation to the brain or kidney capsule results in a significant fraction of totipotent ES cells developing into putative DA or serotonergic neurons and that, when transplanted to the brain, these neurons are capable of innervating the adult host striatum.

Other Cell Sources Recent explorations of alternative cell sources have also contributed to the list of cells utilized for the study of DA neuron transplantation. Donor tissue from other species is an attractive alternative to human fetal tissue, particularly from a donor species that breeds in large litters, such as pig. The porcine DA system contains cell groups resembling A8, A9, and A10 of the rat, and differentiate into the homologous cell groups of human.71 In pig embryos of 28 days, cells of the VM are committed DA neurons expressing TH, yet have not extended processes.71 When a suspension of fetal pig VM was transplanted into the striatum of immunosuppressed DA-lesioned rats, these animals showed significant reduction in amphetamine-induced rotation, whereas animals not immunosuppressed showed transient behavioral recovery.6 Behavioral recovery was reversed after animals were removed from cyclosporin, suggesting that the grafts were rejected upon cessation of cyclosporin A treatment.72 TH+ neurons were observed within grafts in animals displaying a high degree of rotational correction. In addition to its obvious potential for use in the clinical setting (see below: “Recent clinical progress and developments”), xenotransplantation also provides a tool to analyze the development of various components of transplanted cells, to be discussed below (see below: “The inhibitory environment of adult brain”).

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Recently, somatic cell cloning, specifically the production of transgenic bovine embryos, has produced an alternative supply of embryonic VM tissue; these VM cells have improved motor function when transplanted into immunosuppressed parkinsonian rats.73 In order to circumvent the need for immunosuppression after transplantation, several cell sources have been introduced. Testis-derived sertoli cells have been shown to secrete trophic, tropic, and immunosuppressive factors; culturing these cells with embryonic neurons increased DA neuron survival and outgrowth.74 Transplantation of these cells produced behavioral recovery in hemiparkinsonian rats, with an increase in TH+ immunoreactivity in the zone around the transplant.74 A two month survival period of xenotransplanted porcine sertoli cells into rat brain in the absence of systemic immunosuppression indicates production of sufficient local immunosuppression at the site of transplantation, and may be an alternative method for protecting xenotransplanted cells. 75 Transplantation of chromaffin-like carotid body glomus cells into DA-denervated rat striatum developed into clusters of TH+ cells with neuronal morphology which extended fibers out into the host striatum, and produced behavioral recovery in turning behavior and sensorimotor orientation three months post-transplantation.76 And finally, revisiting the adrenal medulla grafts, animal studies utilizing co-grafts of adrenal medulla and peripheral nerve have indicated that this procedure can overcome the major problems encountered with adrenal grafts alone, such as limited survival and transient behavioral effects.77 Attachment of human fetal VM cells to microcarriers (Cytodex) allowed xenotransplantation into rat host without the need for immunosuppression; however, there was no evidence of TH fiber outgrowth into host striatum, and no functional results were reported.78

Regulation of Axonal Outgrowth from Dopamine Grafts The ability of fetal neurons to be placed into an ectopic region of an adult brain, survive, and extend neurites within this region is remarkable. The functional effects of VM transplants into DA-depleted striatum is often correlated with degree of striatal reinnervation.4, 13 However, there is some limitation in the ability of the transplanted neurons to extend neurites in the mature brain. Even though the graft-induced elevations in tissue DA concentrations are substantial, 79 values taken distant from the graft suggest that reinnervation of the whole striatum does not occur. The hypothesis for this sharp decline in density of TH+ fiber outgrowth is that age-dependent characteristics within the host brain alter outgrowth, since extensive outgrowth can be achieved when transplanted into immature (neonatal) host brain. Expression levels and patterns of adhesion molecules expressed by mature host brain are thought to be the culprits of this innervation-inhibitory effect.

The Inhibitory Environment of Adult Brain The limited regeneration in adult CNS and limited ability of nigral neurons to extend neurites in the mature host brain is also thought to be related to suboptimal properties of the mature striatum as a substrate for the extension of DA neurites.80 Allografts into immature host brain show robust neuronal and glial migration away from the transplant site, and a high degree of integration and target-directed neurite outgrowth (Fig. 8.3).3 Fetal cells transplanted into mature brains show neuronal reaggregation around the implant site and limited axonal outgrowth into host brain, suggesting an age-dependent increase in inhibitory or decrease in growth-promoting processes. Since both promoting and repulsive activities influence axonal guidance and extension, alteration of the host brain “substrate” has been examined to obtain more extensive outgrowth from grafts. The cell-adhesion molecules (CAMs) are involved in promoting neurite extension, by their incorporation into the extracellular matrix and subsequent binding to cell surfaces.81 Neurite outgrowth from fetal VM cells in culture is enhanced when plated on various cell

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Fig. 8.3. Integration and axonal outgrowth from fetal grafts. Fetal cells transplanted into immature host brain show robust neuronal (black) and glial (grey) migration away from the transplant site, and a high degree of integration and target-directed neurite outgrowth. Fetal cells transplanted into mature brain remain around the implant site and exhibit limited axonal outgrowth into host brain, suggesting an age-dependent increase in inhibitory or decrease in growth-promoting processes. Reprinted with permission from Trends in Neurosciences 1997; 20:477-482, ©Elsevier.

adhesion molecules.80 Intranigral transplants of VM do not successfully reinnervate striatum unless axonal growth is provoked by bridging the striatum and transplanted tissue via peripheral nerve tissue,82 laminin track,83 striatal cells,84 or grafts of fibroblast growth factor-transfected schwannoma cells.85 The transient expression of these molecules (GAP-43, NCAM and L1) within VM grafts only during the phase of axon elongation further suggests their contribution to outgrowth of developing neurons.86,87 Studies utilizing antibodies against growth-inhibiting factors, such as the IN-1 antibody raised against myelin-associated neurite growth inhibitor NT-35/250, have shown enhanced innervation of fetal neocortical innervation into adult host brain.88 The argument against outgrowth-inhibitory properties of adult brain stems from studies showing long-distance and target-specific axonal growth from human embryonic transplants into adult rat brain,89 as well as from porcine embryonic transplants into adult rat brain.61 The species-specific markers used in our studies of fetal porcine transplants into adult immunosuppressed rat brain allowed comparison of donor glial fiber and donor axonal growth in different host brain regions, demonstrating their distinct trophic characteristics. Target zones in adult host gray matter were selectively innervated by embryonic donor axons

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normally destined to form synapses there, whereas donor glial fibers grew irrespective of any target orientation within white matter tracts (Fig. 8.4A).61 Neuronal axons branched profusely in gray matter target region and only rarely penetrated or crossed white matter tracts. TH+ fibers from transplants placed into the SN were found coursing up toward the striatum through myelinated fiber bundles, then branching into host gray matter, as also shown by Wictorin from human xenotransplants into rat SN.89 Interestingly, the non-DA VM cells also grew toward distant gray matter target zones, such as medio-dorsal and ventral anterior thalamus. These data suggest that directional cues for axons, whether diffusible or substrate-bound, are provided by adult host target regions. Since porcine neural development continues four to five times longer than mouse, these axons may develop over a longer time course than that seen in rat-to-rat studies, as illustrated in the development of functional recovery in porcine-transplant recipients (8 weeks post-transplantation) as compared with allografts (6 weeks post-transplantation), Another aspect of host brain environment which can influence outgrowth from DA grafts is the lesion status: Denervation of the host does seem to promote fiber outgrowth, but has little effect on their survival. Two hypotheses have evolved to explain the apparent increase in outgrowth from grafted cells when transplanted into lesioned versus intact brain: First, outgrowth may be limited due to availability of sites for synaptic contacts, which are increased soon after lesion;90 alternatively, injury-induced neurotrophic factors have also been suggested,91 since graft development is enhanced after administration of extracts from injured brain into the implant site.92 Zhou and Chang93 showed a bridging method created by a “trophic track” formed by low-dose injection of excitatory amino acids; they observed TH+ fibers streaming along this lesion track from the intranigral VM graft into the striatum, and hypothesize that the lesion induces trophic molecules, extracellular matrices, and vasculature which support reinnervation by TH+ fibers.

Regional and Phenotrypic Specification of Dopamine Neurons Different regions of the striatum are associated with specific behaviors in rat; the dorsal striatum receives primary afferents from the motor areas of neocortex, and has been shown to be preferentially involved in rotational recovery after DA neuron transplantation.45 In the intact rat, the subpopulation of nigral DA neurons from A9 SNc which co-express AHD project their axons to the dorsal-lateral and rostral regions of the striatum (Fig. 8.4B). As described above (see above: “Utilizing medial VM for dopamine grafts”), the enriched population of DA neurons obtained from a medial versus lateral VM dissection also preferentially expresses AHD; when transplanted into adult DA-denervated rat striatum, these AHD/TH neurons innervate this region of the DA-depleted striatum3,34 (Fig. 8.4C), showing a preferential reinnervation of the dorso-lateral striatum corresponding to the normal projection pattern of AHD/TH neurons. Specific innervation by subsets of transplanted DA neurons was also demonstrated by Schultzberg, revealing reinnervation of the DA-depleted striatum by the population of grafted VM neurons lacking cholecystokinin (CCK).40 The CCK+ fibers were found in a narrow zone immediately adjoining the graft. These data suggest the presence of mechanisms which selectively favor the ingrowth of fibers from the appropriate DA neuronal subset. Thus, enrichment of the DA neuron subpopulation which specifically expresses AHD may allow more appropriate reinnervation of striatum after transplantation, and influence the degree of functional recovery in PD, possibly defined by tropic mechanisms intrinsic to the host brain.

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Reconstructing Synaptic Connections with Dopamine Grafts Functional effects of intrastriatal grafts of fetal DA cells have been illustrated in a range of animal behavioral tests.94-96 The behavioral effects observed are dependent on the survival of DA neurons within the striatum, since grafting of other tissue produces no behavioral effects97,98 and removal of transplanted tissue99 or immune rejection of transplanted functional analyses of DA grafts. Many groups have used unilateral, intraparenchymal injection of 6-hydroxydopamine (6-OHDA) as the means of producing a unilateral DA denervation of the striatum, then transplanting DA neurons into this denervated striatum. The spontaneous behavior induced by lesion is improved as tested in several parameters, and depends on graft placement, cell number, and density of reinnervation. Due to the imbalance in DA after unilateral lesion, the animal begins to rotate in response to DA-releasing drugs such as amphetamine.101 The transplantation of DA cells and subsequent reinnervation of the denervated striatum causes the animal to decrease its rotations in response to amphetamine,102 thus reversing the lesion-induced behavioral abnormalities. Compensation of other lesion-induced changes, such as lesion-induced increases in DA receptor binding,103 increased levels of enkephalin, and decreased levels of substance P104 demonstrate the capacity of these DA cells to affect postsynaptic and presynaptic mechanisms.4,102 However, more complex movements (such as food pellet retrieval, stair case and stepping tests) have exhibited limited responses to DA transplants.105,106 A microtransplantation procedure which increases the area of striatal reinnervation has shown improved paw reaching in addition to greater striatal reinnervation,15 suggesting that the limited behavioral recovery of some complex movements so often seen in previous studies may be due to inadequate striatal reinnervation.

Regulated DA Release from Fetal DA Grafts Methods to improve the number of DA cells that survive transplantation, and enhance the area of the striatum which becomes reinnervated by these cells, are continually being tested; however, the most important factor in obtaining complete and sustained functional effects is the successful formation of synapses between the transplanted cells and the host brain. The use of autologous adrenal cells, fibroblasts transfected with DA-producing enzymes, and other non-neuronal cell types which can secrete DA can perhaps circumvent the problems of limited availability and ethical issues associated with the use of fetal DA neurons. However, functional analyses from these studies indicate that placing a “DA pump” into the striatum may not be as effective in ameliorating the motor symptom of PD as the regulated, synaptic release obtained with transplanted DA neurons;10 in fact, when DA is directly administered into the ventricle of PD patients, serious psychoses develop,107 and recent data from differential display has shown the abnormal upregulation of over 10 genes within the striatum.108 Complications associated with unregulated DA levels are obvious when observing effects of long term L-dopa administration: As PD progresses and the DA neuron degeneration continues, the unregulated formation of DA within the striatum can lead to motor abnormalities such as dyskinesias. The physiological incorporation and regulation of DA release can only be achieved by DA neurons themselves, or by cells which express the complete set of feedback elements required to regulate the release and uptake of DA. Embryonic DA neurons are not “designed” to produce new connections with mature, established striatal neurons. However, synaptic connections between transplanted VM cells and host cells, as well as afferents from host neurons to transplanted cells, have been illustrated.109,110 The inclusion of fetal striatal tissue, specifically lateral ganglionic eminence, within VM transplants not only produced increased DA cell survival and extent of reinnervation into the DA-depleted host striatum, but also showed an increased number of

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Fig. 8.4. Target-specific innervation by grafted fetal cells. (A) Target zones in adult host gray matter are selectively innervated by embryonic pig donor DA axons normally destined to form synapses there, whereas non-DA donor fibers grow into host myelinated bundles. (B) In the intact rat, the subpopulation of nigral DA neurons from A9 SNc, which coexpress AHD, project their axons to the gray matter of dorso-lateral regions of the striatum. The ventral tegmental area (VTA) neurons from A10 coexpress CCK, and project to ventromedial striatum, nucleus accumbens, neocortex and limbic regions. (C) When the enriched population of TH/ AHD neurons obtained from a medial (versus lateral) VM dissection is transplanted into DA-lesioned adult rat striatum, these neurons preferentially reinnervate their normal dorso-lateral striatal target, shown to be involved in rotational recovery after DA neuron transplantation. TH/CCK neurons from VM show different patterns of outgrowth when placed into cortex.40 Reprinted with permission from Trends in Neurosciences 1997; 20:477-482, ©Elsevier.

host striatal cells which induced the immediate-early gene c-fos, indicating a higher degree of host cell activation.13 Several studies have shown normalized activity throughout the basal ganglia after transplantation. Nakao et al111 utilized cytochrome oxidase histochemistry to quantify neuronal activity in the 6-OHDA-lesioned rat; the lesion-induced increases in activity of the entopeduncular nucleus and SN reticulata were reversed by intrastriatal VM grafts, whereas the lesion-induced increases in globus pallidus and subthalamic nucleus were not affected by grafting.111 The same technique has been used in MPTP-treated monkey receiving VM transplants as well. Dopaminergic grafts increased the metabolic activity of the implanted striatum, particularly in the region of grafts containing greater numbers of DA neurons.112 Also of interest is the finding that the DA neurons exhibit the highest rate of metabolic activity among all cell types contained in the VM grafts.112 Positron emission tomography (PET) and carbon-11 labeled 2B-carbomethoxy-3B-(4-fluorophenyl)tropane (11C-CFT) have been utilized as markers for striatal presynaptic DA transporters in a unilateral lesion model in rat. In the lesioned striatum, the binding ratio was reduced to 15% to 35% of the intact side. After DA neuronal transplantation, behavioral recovery occurred only after the 11C-CFT binding ratio had increased to 75% to 85% of the intact side, revealing a threshold for functional recovery in the lesioned nigrostriatal system after neural transplantation.98

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Autoregulation of DA release and metabolism by intrastriatal grafts has been shown by microdialysis. Treatment with the DA receptor agonist apomorphine produced a decrease in DA in the grafted striatum.6,113 Correct regulation of DA levels by transplanted striatum is also suggested by the observation that behavioral recovery plateaus at high levels of cell survival, with further neuronal survival providing no additional behavioral effects.113 Further evidence for the formation of functional synapses and autoregulation from transplanted fetal neurons comes from the observation that dyskinesias, expressed either as contraversive circling after repeated L-dopa injections in rodents114 or L-dopa-induced dyskinesias in monkey (Widner H, personal communication) are reduced after transplantation. In patients, however, more variable results have been observed.115,116 These data suggest that even a high amount of extracellular DA within the grafted striatum (either through improved cell survival, increased DA release, or addition of L-dopa) will be regulated in a physiological manner by the transplanted DA neurons. A more recent approach manipulates the striatal cells themselves to produce L-dopa or DA. Various vector systems have been used to deliver TH, GTP-cyclohydrolase I (the rate-limiting enzyme for tetrahydrobiopterin synthesis), and aromatic L-amino acid decarboxylase to striatal neurons and glia, essentially turning them into DA-producing cells.117-121 While these approaches bypass the requirement for synapse formation between transplanted VM neurons and striatal neurons, and apomorphine-induced rotation has been decreased in unilaterally-lesioned animals in some studies, the release of DA from these striatal neurons has not been shown to be regulated, and the normal phenotype of these striatal neurons may also be altered. In addition, the drawbacks typically encountered with gene transfer (such as low transduction rates and limited gene expression) also arise.

Clinical Relevance The Urgency for an Improved Therapy for Parkinson’s Disease The simple concept of replacing lost neurons by inserting new cells was introduced as a new therapeutic strategy for treatment of PD two decades ago. The progressive loss of DA neurons in the SN and resultant decrease of DA levels in its targets produce the signs of PD: tremors, akinesias, muscle rigidity, and postural instability. Although the cause of this cell loss is not understood, therapeutic strategies to correct the impaired motor function due to the unbalanced basal ganglia circuitry have included pharmacological (L-dopa) as well as surgical (pallidotomy, 122,123 thalamotomy, 124 subthalamic nucleus stimulation 125) approaches. A major obstacle with long term L-dopa treatment is the appearance of severe side effects such as “on-off ” phenomena and dyskinesias. Although capable of relieving some parkinsonian motor symptoms (notably tremor and dyskinesias), the surgical approaches each have potential complications, such as cognitive disorders after thalamotomy126 and hemiparesis, frontal lobe syndromes and hemorrhage after pallidotomy.122,123 The specificity of cellular degeneration which occurs in PD (DA neurons of the SN), as well as the well defined target of these degenerating cells (the caudate and putamen), have contributed to the direct application of neural transplantation for this disorder. Early clinical transplantation studies involved the use of catecholamine-secreting adrenal medulla cells.127,128 The variable and transient alleviation of symptoms, as well as the poor adrenal medulla graft survival and high morbidity of patients, contributed to a transition to fetal cells for PD transplantation. Yet this cell source is not without its drawbacks, specifically the requirement for several fetuses per patient, all within the desired gestation age. However, more recent animal studies utilizing cografts of adrenal medulla and peripheral nerve have indicated that this procedure can overcome the major problems encountered with adrenal grafts alone as discussed above.77 Several PD patients have received these grafts, and

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studies report their success in relieving motor symptoms, through 24 months post-transplantation.129,130

Application of Dopamine Transplants to Parkinson’s Patients The successful data from rodent studies with fetal DA cells, as discussed throughout this chapter, were extended to nonhuman primate models of parkinsonism, induced either by the selective DA neurotoxin MPTP or by 6-OHDA.131 By demonstrating the capacity of these grafts to improve more complex movements and behavioral tasks, and correlating these improvements with histology and PET imaging of the grafts, these studies then led to the first clinical trials in humans. The initial (and in some cases ongoing) issues regarding the technical aspects of the procedure were assessed, such as patient monitoring, implantation technique, donor tissue properties, immunology of allografts, risk assessment of disease transmission, and ethical considerations. The first two patients to be transplanted were middle-aged females each with about a 14 year history of PD, and symptoms consisting of bradykinesia, rigidity, and severe “off ” phases.132 Each patient received four VM regions from aborted fetuses, implanted unilaterally into the caudate and putamen, and were immunosuppressed with cyclosporin. Each showed modest clinical improvement, including improved gait, which lasted several years in one patient and which was lost in the other after 11-13 months,132 suggesting an immunological rejection of the graft (immunosuppression was withdrawn after 24 months). At nine years, no functional effects persisted, suggesting that cell survival or development was lower than expected. Examination of the implantation procedure yielded the following improvements: decreasing time between abortion and dissection, and dissection and implantation; buffering of storage/dissection media; adding DNase to final step; reduced cannula size; more tissue. Encouraging results were found with the subsequent two patients receiving fetal VM suspensions: decreasing rigidity, bradykinesia, and number and length of daily “off ” periods, which were apparent 6-12 weeks post-transplantation.133 Three years later, both patients showed near normal 18F-fluordopa uptake in the grafted region, while the contralateral striatum showed decreased uptake when compared with that one year post-transplantation, indicative of progressive degeneration associated with the underlying disease process.134 A three year study has shown therapeutically valuable improvement in four out of six patients: Rigidity and hypokinesia improved bilaterally; however, no consistent changes in dyskinesias were observed116 One of these patients was without L-dopa from 32 months and had normal fluorodopa uptake in the grafted putamen at six years.116 Two additional patients were transplanted utilizing the same protocol, with similar clinical improvements, as well as positive PET results.135 Bilateral caudate/putamen grafts into two MPTP-exposed patients have produced marked motor improvement in both patients, correlating with increased uptake of fluorodopa.136 The transplantation of solid pieces of VM have also produced clinical improvement with increased fluorodopa in many (but not all) patients 6 and 46 months post-transplantation.5,137 Two instructive clinical studies have provided information on basic parameters. One compared clinical improvements with graft volume: One group of PD patients was transplanted with VM from one to two donors (volume of approximately 20 mm3), while a second group received tissue from three or more donors (approximate volume of 24 mm3).138 Both groups demonstrated significant improvement over presurgical baseline scores; however, the high volume group had significantly greater improvement on all UPDRS scores, suggesting that amount of donor tissue may influence clinical outcome. The second study correlated clinical improvement with immunosuppression: After over two years of immunosuppressive treatment, withdrawal from the cyclosporin treatment produced a decline in

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the graft-induced motor improvements, implicating a rejection-induced decline in function.139

Recent Clinical Progress and Developments As mentioned previously (see above: “Other cell sources”), xenotransplantation allows the acquisition of large quantities of accurately aged fetal tissue. The T cell-mediated rejection of xenografts can be inhibited by immune suppression,140 and studies have shown survival, function, and afferent/efferent connections of xenogeneic cells when transplanted into animal hosts,6,61 (and see reviews in refs. 141,142). The transplantation of E27 porcine VM unilaterally into the caudate and putamen of twelve immunosuppressed PD patients has produced clinical improvements: UPDRS “off ” scores improved 16.9 points in ten evaluable patients at 12 months.143 One patient from this study died seven months after surgery from a pulmonary embolism; histological analyses using species-specific markers revealed porcine cells and axonal projections from the grafts into host brain. All three identified grafts contained TH+ neurons (630 TH+ neurons in all), and non-TH+ neurons expressing pig-specific neurofilament protein were also observed within, and extending axons out of, the grafts.8 Microglial and T cell markers showed low reactivity in and around the pig cell graft perimeter. In addition to this histological study, autopsy data has been published from one other laboratory, who bilaterally transplanted 6.5-9 week human fetal VM into postcommisural putamen of several PD patients. Details from two of these patients who died 18-19 months after surgery of events unrelated to the grafting procedure have been reported.9,144-146 Both patients showed improved motor function and increases in fluorodopa uptake in the putamen on PET scanning. Histological analysis has shown over 200,000 surviving TH+ neurons in the male patient (12 sites) which reinnervated over 53% of the right putamen and 23% of the left putamen in a patch-matrix pattern.9 Electron microscopy revealed axo-dendritic and occasional axo-axonic synapses between graft and host, and analysis of TH mRNA revealed higher expression within the fetal neurons than within the residual host nigral cells.9 Autopsy of the second patient showed over 130,000 surviving TH+ neurons, reinnervating 78% of the putamen.146 Even in these healthy-appearing grafts and a six month regimen of cyclosporin treatment, pan macrophages and T and B cells were observed within the graft sites.145 These results have demonstrated the potential usefulness of neuronal replacement therapy for PD, relieving rigidity, akinesia, peak-dose dyskinesias, gait stability, speech, and swallowing. There is no indication that the disease process is negatively affecting the transplanted cells, although the endogenous DA system continues its progressive degeneration.116,134 The striatum has a remarkable capacity to compensate for very low levels of DA, as evidenced by the lack of parkinsonian symptomology until 80% of DA is lost. Thus a substantial, though perhaps incomplete, reinnervation may allow maximal functional outcome. However, the basic mechanistic problems with these grafts as outlined in the above review, specifically the limited development and reafferentation of host brain by these grafts, require the continued efforts of investigators in this field. Other issues such as graft location,147 immunologic questions,145,148 further progression of the disease, and continued exposure of fetal cells to L-dopa, remain under intense investigation.

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Conclusion The current understanding of the normal in situ maturation and phenotypic specializations of DA neurons located in the adult substantia nigra parallels the observations made of the development of committed fetal dopamine neurons placed as grafts into the adult CNS. The molecular signaling necessary for the final morphological specializations and connectivity of the nigro-striatal DA system must therefore be largely intrinsic to the developing DA neurons, or, alternatively, present in significant detail in the adult brain for this process to be completed in a normal way. These findings may be clinically applied to further improvements in DA neuron “replacement” in the PD brain, and provide functional restitution to patients with neurodegenerative diseases.

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100. Carder R, Snyder-Keller A, Lund R. Behavioral and anatomical correlates of immunologically induced rejection of nigral xenografts. Journal of Comparative Neurology 1988; 277:391-402. 101. Ungerstedt U, Arbuthnott G. Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system. Brain Research 1970; 24:485-493. 102. Dunnett SB, Whishaw IQ, Jones GH et al. Effects of dopamine-rich grafts on conditioned rotation in rats with unilateral 6-hydroxydopamine lesions. Neuroscience Letters 1986; 68:127-135. 103. Freed W, Ko G, Niehoff D et al. Normalization of spiroperidol binding in the denervated rat striatum by homologous grafts of substantia nigra. Science 1983; 222:937-939. 104. Cenci M, Campbell K, Bjorklund A. Neuropeptide messenger RNA expression in the 6-hydroxydopamine-lesioned rat striatum reinnervated by fetal dopaminergic transplants: Differential effects of the grafts on preproenkephalin, preprotachykinin and prodynorphin messenger RNA levels. Neuroscience 1993; 57:275-296. 105. Abrous D, Shaltot A, Torres E, et al. Dopamine-rich grafts into the neostriatum and/or nucleus accumbens: Effects on drug-induced behaviors and skilled paw reaching. Neuroscience 1993; 53:187-197. 106. Dunnett S, Wishaw I, Rogers D et al. Dopamine-rich grafts ameliorate whole body motor assymmetry and sensory neglect but not independent limb use in rats with 6-OHDA lesions. Brain Research 1987; 75:63-78. 107. Venna N, Sabin T, Ordia J et al. Treatment of severe Parkinson’s disease by intraventricular injection of dopamine. Applied Neurophysiology 1984; 47:62-64. 108. Gerfen C, Keefe K, Steiner H. Dopamine-mediated gene regulation in the striatum. Adv Pharmacol 1998; 42:670-673. 109. Mahalik T, Finger T, Stromberg I et al. Substantia nigra transplants into denervated striatum of the rat: Ultrastructure of graft and host interconnections. Journal of Comparative Neurology 1985; 240:60-70. 110. Doucet G, Murata Y, Brundin P et al. Host afferents into intrastriatal transplants of fetal ventral mesencephalon. Experimental Neurology 1989; 106:1-19. 111. Nakao N, Ogura M, Nakai K et al. Intrastriatal mesencephalic grafts affect neuronal activity in basal ganglia nuclei and their target structures in a rat model of Parkinson’s disease. Journal of Neuroscience 1998; 18:1806-1817. 112. Collier T, Redmond DJ, Roth R et al. Metabolic energy capacity of dopaminergic grafts and the implanted striatum in parkinsonian nonhuman primates as visualized with cytochrome oxidase histochemistry. Cell Transplantation 1997; 6:135-140. 113. Strecker R, Sharp T, Brundin P et al. Autoregulation of dopamine release and metabolism by intrastriatal nigral grafts as revealed by intracerebral dialysis. Neuroscience 1987; 22:169-178. 114. Gaudin D, Rioux L, Bedard P. Fetal dopamine neuron transplants prevent behavioral supersensitivity induced by repeated administration of L-dopa in the rat. Brain Research 1990;506:166-168. 115. Defer G, Geny C, Ricolfi F et al. Long-term outcome of unilaterally transplanted parkinsonian patients. I. Clinical approach. Brain 1996; 119:41-50. 116. Wenning G, Odin P, Morrish P et al. Short- and long-term survival and function of unilateral intrastriatal dopaminergic grafts in Parkinson’s disease. Annals of Neurology 1997; 42:95-107. 117. Imaoka T, Date I, Ohmoto T et al. Significant behavioral recovery in Parkinson’s disease model by direct intracerebral gene transfer using continuous injection of a plasmid DNA-liposome complex. Human Gene Therapy 1998; 9:1093-1102. 118. Mandel R, Rendahl K, Spratt S et al. Characterization of intrastriatal recombinant adeno-associated virus-mediated gene transfer of human tyrosine hydroxylase and human

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GTP-cyclohydrolase I in a rat model of Parkinson’s disease. Journal of Neuroscience 1998; 18:4271-4284. 119. Horellou P, Vigne E, Castel M et al. Direct intracerebral gene transfer of an adenoviral vector expressing tyrosine hydroxylase in a rat model of Parkinson’s disease. Neuroreport 1994; 6:49-53. 120. During M, Naegele J, O’Malley K et al. Long-term behavioral recovery in parkinsonian rats by an HSV vector expressing tyrosine hydroxylase. Science 1994; 266:1399-1403. 121. Kaplitt M, Leone P, Samulski R et al. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nature Medicine 1994; 8:148-154. 122. Samuel M, Caputo E, Brooks D et al. A study of medial pallidotomy for Parkinson’s disease: Clinical outcome, MRI location and complications. Brain 1998; 121:59-75. 123. Shannon K, Penn R, Kroin J et al. Stereotactic pallidotomy for the treatment of Parkinson’s disease. Efficacy and adverse effects at 6 months in 26 patients. Neurology 1998; 50:434-438. 124. Tasker R. Ablative therapy for movement disorders. Does thalamotomy alter the course of Parkinson’s disease? Neurosurg Clin N Am 1998; 9:375-380. 125. Benabid A, Pollak P, Gervason C et al. Long-term suppression of tremor by chronic stimulation of the ventral intermediate thalamic nucleus. Lancet 1991; 337:403-406. 126. Tasker R. Thalamotomy. Neurosurg Clin N Am 1990; 1:841-864. 127. Madrazo I, Leon V, Torres C. Transplantation of fetal substantia nigra and adrenal medulla to the caudate putamen in two patients with Parkinson’s disease. New England Journal of Medicine 1988; 318:51. 128. Backlund E, Granberg P, Hamberger B. Transplantation of adrenal medullary tissue to striatum in parkinsonism. Journal of Neurosurgery 1985; 62:169-173. 129. Date I, Asari S, Ohmoto T. Two-year follow-up study of a patient with Parkinson’s disease and severe motor fluctuations treated by co-grafts of adrenal medulla and peripheral nerve into bilateral caudate nuclei: Case report. Neurosurgery 1995; 37:515-518. 130. Watts R, Subramanian T, Freeman A et al. Effect of stereotaxic intrastriatal cografts of autologous adrenal medulla and peripheral nerve in Parkinson’s disease: Two-year follow-up study. Experimental Neurology 1997; 147:510-517. 131. Dunnett S, Annett L, Lindvall O. Nigral transplants in primate models of parkinsonism. In: Bjorklund A, Widner H, eds. Intracerebral Transplantation in Movement Disorders. Amsterdam:Elsevier, 1991:27-51. 132. Lindvall O, Rehncrona S, Brundin P et al. Human fetal dopamine neurons grafted into the striatum in two patients with severe Parkinson’s disease: A detailed account of methodology and a 6-month follow-up. Arch Neurol 1989; 46:615-631. 133. Lindvall O, Brundin P, Widner H et al. Grafts of fetal dopamine neurons survive and improve motor function in Parkinson’s disease. Science 1990; 247:574-577. 134. Lindvall O, Sawle G, Widner H et al. Evidence for long-term survival and function of dopaminergic grafts in progressive Parkinson’s disease. Annals of Neurology 1994; 2:172-180. 135. Peschanski M, Defer G, N’Guyen J et al. Bilateral motor improvement and alteration of L-dopa effect in two patients with Parkinson’s disease following intrastriatal transplantation of foetal ventral mesencephalon. Brain 1994; 117:487-499. 136. Widner H, Tetrud J, Rehncrona S et al. Bilateral fetal mesencephalic grafting in two patients with severe parkinsonism induced by MPTP. New England Journal of Medicine 1992; 327:1556-1563. 137. Freed CR, Breezr RE, Rosenberg NL et al. Survival of implanted fetal dopamine cells and neurologic improvement 12 and 46 months after transplantation for Parkinson’s disease. New England Journal of Medicine 1992; 327:1549-1555. 138. Kopyov O, Jacques D, Lieberman A et al. Outcome following intrastriatal fetal mesencephalic grafts for Parkinson’s patients is directly related to the volume of grafted tissue. Experimental Neurology 1997; 146:536-545. 139. Lopez-Lozano J, Bravo G, Brera B et al. Long-term improvement in patients with severe Parkinson’s disease after implantation of fetal ventral mesencephalic tissue in a cavity of

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the caudate nucleus: 5-year follow up in 10 patients. Journal of Neurosurgery 1997; 86:931-942. 140. Pedersen E, Poulsen F, Zimmer J et al. Prevention of mouse-rat brain xenograft rejection by a combination therapy of cyclosporin A, prednisolone and azathioprine. Experimental Brain Research 1995; 106:181-186. 141. Pakzaban P, Isacson O. Neural xenotransplantation: Reconstruction of neuronal circuitry across species barriers. Neuroscience 1994; 62:989-1001. 142. Isacson O, Breakefield XO. Benefits and risks of hosting animal cells in the human brain. Nature Medicine 1997; 3:964-969. 143. Ellias S, Palmer E, Kott S et al. Transplantation of fetal porcine ventral mesencephalic cells for treatment of Parkinson’s disease: One year safety and efficacy results. AAN Abstract 1998; 1:13. 144. Kordower JH, Freeman TB, Snow BJ et al. Neuropathological evidence of graft survival and striatal reinnervation after the transplantation of fetal mesencephalic tissue in a patient with Parkinson’s disease. New England Journal of Medicine 1995; 332:1118-24. 145. Kordower J, Styren S, Clarke M, et al. Fetal grafting for Parkinson’s disease: Expression of immune markers in two patients with functional fetal nigral implants. Cell Transplantation 1997; 6:213-219. 146. Kordower J, Freeman T, Chen E et al. Fetal nigral grafts survive and mediate clinical benefit in a patient with Parkinson’s disease. Movement Disorders 1998; 13:383-393. 147. Palfi S, Nguyen J, Brugieres P et al. MRI-stereotactical approach for neural grafting in basal ganglia disorders. Experimental Neurology 1998; 150:272-281. 148. Bakay R, Boyer K, Freed C et al. Immunological responses to injury and grafting in the central nervous system of nonhuman primates. Cell Transplantation 1998; 7:109-120.

CHAPTER 9

Dopaminergic Neurons in the Olfactory Bulb S. Denis-Donini

T

he presence of dopaminergic neurons in the olfactory bulb was described about thirty years ago. Even though new insights into their physiological role are starting to emerge, their function in olfactory processing is still far from being understood. Two subpopulations of neurons express dopaminergic properties in the bulb: they belong to the so-called periglomerular neurons and the external tufted cells. These neurons present a great interest from a developmental standpoint for two main reasons. First, they exhibit during development a great plasticity in neurotransmitter phenotype, in that the expression of dopaminergic properties is regulated through afferent inputs. Second, the periglomerular and glomerular neurons continue to be generated postnatally from neuroblasts that reside in the subependymal layer at the margin of the lateral ventricles and migrate into the bulb through a highly stereotyped pathway. For these reasons, such neurons have become a favorite subject of investigations aimed at unraveling the control of cell identity and, therefore, the signals that regulate the acquisition of a neurotransmitter status. In addition, they represent a choice subject for studying neuroblast ontogeny and the molecular cues underlying oriented migration, settlement and differentiation. In this review, the present knowledge on such neurons will be summarized with the hope of delineating a framework for future investigations. For an ampler view of the olfactory system, the reader is referred to the comprehensive reviews of Halasz and Shepherd,1 Macrides and Davis2 and Brunjes and Frazier.3

Anatomy and Circuitry of the Olfactory System The basic construction of olfactory circuits is remarkably conserved, even across distant phyla like mollusks, arthropods, chordates and vertebrates.4 However, even among rodents only, there is more than one difference in the number and types of dopaminergic cells and in their time of appearance during development. Enumerating these differences is beyond the scope of this review, which will be limited with few exceptions to the mouse and the rat (Fig. 9.1), where most functional studies concerning the role of dopamine have been performed. The olfactory system can recognize and discriminate a vast number of different odors. Odor discrimination is accomplished through a series of information processing steps at distinct anatomical sites of the olfactory system. The initial recognition occurs in olfactory receptor neurons in the olfactory epithelium. They present at the epithelial surface a specialized dendritic knob displaying ten or more cilia which are the primary site of olfactory reception. Sensory information is transmitted to the bulb via the olfactory axons that The Development of Dopaminergic Neurons, edited by Umberto di Porzio, Roberto Pernas-Alonso, and Carla Perrone-Capano. ©1999 R.G. Landes Company.

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Fig 9.1. Schematic diagram of the synaptic organization of the rodent olfactory bulb in a much simplified form. For details of synaptic connections see ref. 14. Cell bodies of inhibitory interneurons are represented in black, those of principal neurons in white. All interneurons, with the exception of hte dopaminergic periglomerular cells, are GABAergic. Although drawn separately, mitral and tufted dendrites may converge into the same glomerulus. Perliglomerular cells have presynmaptic dendrites and a true axon that may extend as far as five glomeruli away. The presynaptic dendrites may provide local inhibition, white the axons mediate lateral inhibition. Mirral and tufted cells secondary dendrites (horizontal branches) are involved in reciprocal synaptic interactions with granule cells. Granule cells are axonless and their only synaptic output is through the spines to the mitral dendrite branches, providing powerful inhibition. ON, olfactory nerve; GL, glomeruli; PG, periglomerular cell; M, mitral cell; T, tufted cells; G, granule cell. (Adapted from Shepherd, ref. 14)

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terminate within discrete anatomical units called glomeruli where they form synapses on the apical dendrites of the mitral and tufted cells (M/Tcells), the primary output neurons in the bulb. Glomeruli are spheroid or ellipsoid clusters of neuropil. Each sensory neuron projects to only a single glomerulus, but forms multiple synaptic contacts within the glomerulus. Since there are 2-4 million olfactory sensory neurons in the nasal cavity of the rat, and the olfactory bulb contains about 2000 glomeruli, each glomerulus receives input from about 1000-2000 olfactory sensory neurons. In the rabbit, the convergence ratio is about 10 times higher. The convergence provides a means for enhancing the detection of weak signals5 and may involve axons predominantly from olfactory receptor neurons (ORN) with similar or related types of receptors, implying that odor stimulation should give rise to spatial patterns of glomerular activation. Remarkably, exposure to odors produces odor-specific spatial patterns of focal 2-deoxyglucose (2-DG) uptake in the glomerular layer of the bulb6-8 suggesting that a spatial map is at the basis of odor coding. A similar conclusion has been reached with the cloning of a large family of olfactory receptors. Imaging of olfactory neurons expressing a given receptor show that they are scattered across a large region of the sensory epithelium, but the axons expressing one receptor gene converge on a few target regions in the bulb, the glomeruli.9,10 The activity of mitral cells is regulated essentially at two levels. The first occurs at the level of glomeruli through the periglomerular cells (PG) whose short bushy dendrites arborize within a glomerulus and receive and give off synapses of a reciprocal nature on the primary dendrites of M/T cells. In view of this dual role, the periglomerular cell dendrites, as well as the granule cell dendrites, are processes intermediate between axons and dendrites. However, PG cells have a true axon that distributes laterally and extends as far as five glomeruli away. Through PG cells, activity in one glomerulus can affect other glomeruli, suggesting that one of their functions is to mediate lateral inhibition between glomeruli and therefore to enhance contrast in odor discrimination.11,12 Most PG cells are GABAergic but a subpopulation has been shown to synthesize, in addition to GABA, dopamine.13 The second level of control is effected through the granule cells which are mainly GABAergic also. These cells lack an axon and give off only dendrites. Granule cells and mitral cells form reciprocal pairs of dendrodendritic synapses. Granule cells are depolarized by an excitatory input from the mitral cells and in turn provide a feedback inhibition that is believed to be the basis for the generation of rhythmic activity in the neuronal population. Such rhythmic potentials are a prominent characteristic of the olfactory bulb in the resting state as well as during olfactory induced activity (for a review see ref. 14). Because each granule cell also shows divergent synaptic contacts with a large number of neighboring M/Tcells, they are thought to induce lateral inhibition in neighboring M/T cells.15

Evidence and Possible Role for Dopamine in the Olfactory Bulb If the olfactory bulb circuitry seems rather simple, odor transduction is extremely complex. Olfactory receptor neurons seem to employ a variety of different transduction pathways that modulate the excitability of the receptor neuron. ORNs can be excited by one odor and inhibited by another and the same odor can excite some olfactory receptor neurons and inhibit others. This diversity allows the initial screening of odor information to occur in the receptor neurons (for review see refs. 16-20). At the glomerular level, it appears likely that the PG cells are excited by ORN and by mitral and tufted cells and that they mediate lateral and recurrent inhibition in the glomerular layer.11,21 Earlier studies indicated that small cells in the glomerular layer (GL) contain a catecholamine and can accumulate exogenous norepinephrine.22,23 Immunofluorescence studies demonstrated that neurons in the GL and in superficial regions of the external plexiform layer contain tyrosine hydroxylase (TH) and DOPA decarboxylase

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immunoreactivities but do not contain dopamine β-hydroxylase. These findings indicate that the neurons can synthesize DOPA and convert it to dopamine but cannot convert dopamine to norepinephrine. Electron microscopy analysis has demonstrated that the TH immunoreactivity is present in PG cells and in superficially situated tufted cells.24,25 Thus, it appears that at least two distinct classes of intrinsic neurons synthesize dopamine. Autoradiographic studies similarly have shown that both PG cells and superficially situated tufted cells accumulate catecholamine and precursors.26,27 What could be the function of dopamine in the bulb? The mere fact that dopaminergic periglomerular neurons also synthesize GABA, and that they are largely outnumbered by the vast majority of PG cells that only express GABA, has made them receive little attention, most probably because the question is difficult to tackle. In general, little is known about the functional organization and synaptic physiology of the GL of the bulb. This is attributable in large part to the relatively small size of most neurons in this layer that renders electrophysiological investigations particularly difficult. So there is not yet direct evidence about the role of dopaminergic neurons in the mammalian bulb, but a few astute studies have given hints and opened new perspectives about their function. Recent studies have shown that the dopaminergic system in the olfactory bulb can be affected by olfactory experience.28 As in most sensory systems which are strongly influenced by the sensory environment encountered during perinatal development and where early sensory deprivation results in marked changes in the structure and function of the deprived system, the mammalian olfactory system is also extremely sensitive to postnatal deprivation. Unilateral naris occlusion performed during the first three postnatal weeks results in both structural and functional changes in the deprived ipsilateral bulb. In the rat, there is a 25% reduction in the size of the ipsilateral olfactory bulb and a decrease in the size and number of most types of bulb neurons.29-32 Most importantly, olfactory deprivation reduces dopamine content by as much as 75%33,34 and reduces immunoreactivity for TH,35 without modifying GABA expression.36 In the deprived bulb, the basal activity rate of M/T cells was decreased compared to the normal bulb. However, with the restoration of odor stimulation, both laminar 2-DG uptake and the incidence of odor-reponsive M/T cells were significantly increased.28 Upon odor stimulus, M/T cells typically exhibit either an increase or a decrease in firing rate, or no change.37,38 Each of these response types could be identified in cells of the deprived bulb and in similar proportions to controls.39 These results suggest that dopamine does not detectably affect the occurence or general characteristics of M/T cells’ suppresive responses to odors. The odor-specific spatial pattern of local 2-DG uptake in the GL was largely retained in the deprived bulb, but the odor responsive foci often appeared to encompass somewhat larger, less discrete regions of the GL compared to foci in normal bulbs, suggesting that dopaminergic PG neurons contribute to lateral inhibition in the GL. The same type of experiment has been performed on older pups (naris closure at postnatal day (P) 30 and lasting two months) when deprivation has less drastic effects but still results in a significant decrease in dopamine synthesis.40 In such conditions also, deprivation increases responsiveness to olfactory stimulation and the proportion of M/T single units responding to a single odor was enhanced. Furthermore, the proportion of M/T cells responding to more than one odor was increased, suggesting a decrease in discrimination. Remarkably, the dopamine D2 receptor antagonist spiperone mimicked the effects of deprivation, confirming in mammals previous observations on the turtle olfactory bulb in vitro indicating that blocking dopaminergic receptor activity directly enhances olfactory responses.41 Such increased responsiveness has led to the view that olfactory stimulation modulates olfactory system function through controlling dopaminergic inhibition. A decrease in olfactory stimulation reduces dopamine levels and releases afferent input from both feedback and lateral inhibition.40 Such a conclusion is strenghtened by the observation that

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D2 receptors have been found essentially in the GL and in the olfactory nerve layer, suggesting a possible modulation of the afferent olfactory input by dopamine.42,43 Even though there is a deprivation-associated upregulation of dopaminergic D2 receptors in the bulb,44 this upregulation is apparently insufficient to completely compensate for the loss of dopamine. The modulation of afferent input by dopamine has been confirmed in another experimental paradigm. Odor learning has been shown to increase dopamine levels in the olfactory bulb.45 Young rats normally learn to become attracted to the odor of their mother, and the pairing of odor and tactile stimulation induces several changes including an increase in 2-DG uptake in responsive regions of the bulb GL when exposed to learned odors.46 At the same time, a suppression of the M/T cell response to the learned odor is registered.47 In trained pups, there is also an increase in the number of juxtaglomerular cells within such responsive focal regions when compared to their controls.48 In addition, the extracellular dopamine concentration in the juxtaglomerular region, monitored through microdialysis, substantially increases. It seems that the time course of the learning-associated effects may reflect a continued transmitter release and/or a relatively inefficient clearance of released DA from the extracellular space. DA released into the extracellular space might eventually reach the olfactory nerve layer and activate presynaptic D2 receptors. Activation of these receptors may modulate the activity of the ORN to incoming olfactory stimulation. Such an extrasynaptic mechanism could underlie sensory adaptation in the olfactory system by suppressing the activity of olfactory receptor neurons. A proposed alternative explanation, but difficult to prove, would be that the dopamine increase may be specific to the rewarding aspects of the stimulation. An interesting question raised by these studies concerns the high affinity uptake of dopamine. While 3H-DA can be transported in PG cells in vivo27 and in vitro,49 quantitative autoradiography reveals that the density of the 3H-mazindol labeled transport sites in the bulb is very low.44 Most DA neurons possess two or three amine transporters, a plasma membrane carrier that acts to terminate dopaminergic transmission by Na and Cl-dependent reaccumulation of DA into presynaptic neurons50 and vesicular transporters that use the pH gradient across the vesicle membrane generated by the vacuolar H+-ATPase to drive uptake and so act as a proton exchanger.51 Two vesicular monoamine transporters, VMAT1 and VMAT2, have been cloned52 and antibodies have been raised.53 Quite surprisingly, periglomerular neurons do not appear to express either VMAT1 or 2. So they either express extremely low levels of transporters, an unrelated transporter or no vesicular amine transporter. In the absence of vesicular amine transport activity, the regulated release of monoamine could involve reversal of the plasma membrane dopamine transporter54,55 that has been shown to exist in the DA neurons of the bulb.56 A reduced expression of vesicular amine transport by olfactory bulb interneurons could account for their apparent vulnerability in Parkinson’s patients who show reduced olfactory performances.57-59 Further insight into dopamine function has been provided by recent evidence demonstrating that the dopamine receptor agonist apomorphine blocks an odor-specific glomerular activity, as revealed by 2-DG uptake.60 Injection of apomorphine prior to odor stimulation completely abolishes the selective pattern of glomerular activation. Furthermore, the effects of apomorphine are prevented by pretreatment with the dopamine receptor antagonist haloperidol. These observations emphasize the probable involvement of dopamine and dopamine receptors in the bulbar processing of olfactory information. Although more work is required to clearly define a function for dopamine, the results of these studies and the presence of D2 receptors on olfactory nerve terminals suggest a neuromodulatory action of dopamine on afferent sensory input in the bulb. The role of dopamine in tufted cells has to my knowledge not been investigated; it would be like searching for a needle in a haystack. Dopamine release is often associated

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with the notion of reward. The existence of relatively few dopaminergic tufted cells suggest that they do not play a major role in olfactory transduction. Perhaps, they add just a little note of pleasure when evoked by a promising scent.

Ontogeny and Differentiation: Neurotransmitter Plasticity In the rodent olfactory bulb, neurons are generated in a strict sequential order from progenitor cells of the ventricular zone and subventricular zone.61 The principal projection neurons of the olfactory bulb, the mitral and tufted cells, are generated prenatally from the ventricular zone.62,63 While some interneurons arise locally within the bulb, most interneurons arise postnatally from the subventricular zone and then migrate into the olfactory bulb.64 Retrovirus-mediated gene transfer was used to mark populations of progenitor cells and their progeny at various times during development.65 These studies have demonstrated that a particular zone of the anterior edge of the lateral ventricle gives rise to neurons that traverse a highly restricted course, the rostral migratory stream, toward their final destination in the olfactory bulb. The bulk of this migration occurs during early postnatal stages but still proceeds, at a minor extent, in the adult.66 The molecular cues at the basis of the migration of neuroblasts to the olfactory bulb are still far from being understood. This tangentially oriented migration, at variance from radial migration, occurs without the guidance of radial glia (for reviews see refs. 67, 68). The neuroblasts appear to migrate as chains held together through homotypic interactions that are mediated in part by polysialic residues on NCAM. Once in the bulb, the neuroblasts change orientation and migrate radially to occupy positions in the granule cell layer and in the glomerular layer. From their position and typical morphology in the various layers of the bulb, it has been possible to identify them as PG cells and granule cells and to show that they synthesize dopamine or GABA.69 A decision confronting all the migrating cells concerns their choice of identity. It is not known whether the progenitor cells are themselves determined or whether the migrating cells acquire their phenotype in response to environmental influences. In the section that follows we will summarize what is known about neurotransmitter choice in the bulb. This is a more general problem in the nervous system, since individual neurons simultaneously synthesize, store and secrete one or more classical neurotransmitters in addition to three or more peptides. Transmitters and peptides are expressed in various combinations and an important question concerns how the particular combinations produced in each neurons are specified during development. The olfactory system presents some advantages to approaching this issue, since a series of investigations has shown that reciprocal interactions take place between ORN and the bulb during development and because afferent and target cells exist in two separate compartments. Afferent cells not only sustain the proper growth and differentiation of the bulb (for review see ref. 3) but they seem to exert a real morphogenetic effect, since they induce the formation of glomeruli in the bulb and even in ectopic regions of the brain.70,71 In addition, afferent cells have been shown to regulate both in vivo and in vitro neurotransmitter phenotypic expression in the two presumptive dopaminergic neuronal subpopulations of the bulb, the periglomerular and external tufted cells.72-74 That olfactory nerve fibers regulate during development the expression of TH in target cells has been suggested by the developmental pattern of TH immunoreactivity in the bulb. TH, the rate-limiting enzyme in the biosynthesis of catecholamines, is expressed in populations of cells that already express GABA around the time of birth when the axons of olfactory epithelial neurons have formed synapses in the bulb.75 The number of TH immunoreactive periglomerular neurons continues to increase after birth when olfactory nerve input into the bulb is more robust.76 In addition, if the nerve is given the possibility to enter

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into ectopic regions in the brain, as after bulbectomy, and penetrate into portions of the subependymal layer, structures resembling glomeruli can be observed. Furthermore, TH expression is induced in small surrounding neurons, similarly to bulb periglomerular cells.77 Removal of this input in the neonate prevents the developmental increase in TH-immunoreactive cells33 but has no effect on GABA expression.35,36 Therefore, afferent cells are indispensable for inducing the expression of dopaminergic phenotypes. They also seem to be necessary to maintain it, because upon mechanical or chemical deafferentation of the adult bulb, there is a marked decrease in the content of TH, dopamine and its metabolite DOPAC, suggesting that the expression of TH is regulated transsynaptically.72 This decrease in dopaminergic properties seems not to be due to cell death, since immunoreactivity for L-aromatic amino acid decarboxylase, the second enzyme in the catecholamine biosynthetic pathway, persists in juxtaglomerular cells during denervation.35,78 Another indication that the dopaminergic cells do not die is that the level of TH and dopamine return to normal after regeneration of the primary afferents.79-81 One cannot exclude, however, that new contingents of cells from the rostral migratory stream invade the bulb following axotomy. What is the nature of the environmental stimulus that causes the interneurons to adopt a dual neurotransmitter function (dopamine and GABA) during development? The molecular signaling involved in this induction has been investigated in tissue cultures.73 In olfactory bulb cultures from the mouse embryo (E15-16) the number of cells expressing a dopaminergic phenotype (assessed by TH expression and high affinity uptake of dopamine) is extremely low . If olfactory receptor neurons are added to the culture, about a five-fold increase in the number of dopaminergic cells is observed. This suggests that, already at E16, some bulb neurons susceptible to respond to signals from the afferent cells are present in the bulb, and that one can anticipate in vitro the timing of appearance of DA properties by exposing them to signals from afferent cells. These results also suggest that the signals that operate in vivo are the same in vitro. Three lines of evidence have indicated that the calcitonin gene-related peptide (CGRP) acts as a dopaminergic differentiating factor during development, at least in vitro: 1. It is synthesized in many olfactory receptor neurons; 2. When added at nanomolar concentration to olfactory bulb cultures, it mimics the effect of olfactory receptor neurons; 3. The induction of dopaminergic phenotypes brought about by olfactory receptor neurons is abolished by an antiserum to CGRP. Taken together, these results in vitro demonstrate that CGRP is a strong candidate for an anterograde dopaminergic differentiation factor whereby olfactory receptor neurons influence the neurotransmitter identity of its target by regulating in concert two traits of dopaminergic phenotypes.73 However, whether CGRP directly acts on the presumptive dopaminergic cells or stimulates other cells to produce other unknown differentiation factors remains to be determined. In order for CGRP to be a bona fide differentiation factor in vivo, its timing of expression in the embryo should coincide or precede the appearance of dopaminergic cells that, as we mentioned previously, occurs around the time of birth75 or even slightly earlier.74 Through in situ hybridization and immunohistochemistry experiments it has been shown that the expression of CGRP in the olfactory pathway is regulated both temporally and spatially.82 The neuropeptide starts to be expressed at E15, reaches a peak of expression between E16-E18 and slowly declines around birth. Its location also seems to be tightly regulated, since it is mostly concentrated in olfactory axons during fetal life but becomes confined in some stem cells and some olfactory receptor neurons after birth. CGRP mRNA appears two days before the peptide (E13). Surprisingly, from E13 to E19, CGRP-encoding mRNA can only be detected at the level of olfactory axons, mainly in

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their most distal segment in the olfactory nerve layer surrounding the bulb and at the level of glomeruli. This unsuspected localization of CGRP mRNA in the axons has been confirmed through in situ hybridization at the EM level.83 The fact that olfactory axons contain ribosomes and even polysomes83 suggests that CGRP mRNA translation could be regulated locally either in response to odor-induced activity or through incoming signals from the bulb. The timing of expression of CGRP and the localization of both peptide and transcripts in the olfactory axons are highly suggestive of a role for CGRP as a dopaminergic differentiation factor during development in vivo. Whether CGRP plays a role in the regulation of TH in the adult is not known. How does CGRP mediate its effect? The cloning of the CGRP receptor has been really difficult 84 but has just revealed a new strategy for creating receptor diversity.85 CGRP acts through seven transmembrane domain G-protein-coupled receptors that activate adenylate cyclase. Forskolin, an activator of the cyclase, induces TH immunoreactivity in some neurons in embryonic olfactory bulb cultures. This is not surprising, since TH expression is modulated by nearly every form of regulation and since the TH gene promoter harbors, among other regulatory sequences, a CRE element (for review see ref. 86). Nevertheless, forskolin does not elicit the capacity to take up dopamine in such neurons, indicating that induction of TH and of the amine carrier are distinct events that do not necessarily occur in concert (Denis-Donini, Busse, unpublished observations). We do not know either whether under such conditions the neurons synthesize dopamine. So, the question of the second messengers used by CGRP to regulate DA phenotypes remains unsettled. In smooth musle cells, CGRP induces relaxation by increasing cAMP levels and NO pathways.87 Interestingly, NO seems to mediate the formation of synaptic connections in the developing and regenerating olfactory pathway88 and there is a striking similarity in the timing of expression of nitric oxide synthase (NOS) and CGRP. Both are transiently expressed in the developing olfactory epithelium and are mostly concentrated in olfactory axon bundles reaching the bulb. It would be very interesting to understand if the expression of CGRP and NOS are somehow correlated. The possibility that activity could regulate expression of TH has been investigated. Odor stimulation induces the immediate early gene c-fos in intrinsic neurons of the bulb. c-fos could potentially bind an AP-1 consensus site that has been identified in the TH gene promoter (for review see ref. 86). Odor stimulates c-fos expression in multiple neuronal ensembles, including neurons surrounding individual glomeruli and cells distributed within spatially limited regions of the underlying external plexiform, mitral and granule cell layers.89 However, with anti-Fos antibodies, only a fraction of the TH+ cells are also positive for Fos, while the vast majority of TH+ cells are negative.90 These experiments do not permit us to draw definitive conclusions on the role of such activity in inducing dopaminergic phenotypes, but for the time being make it highly unlikely.

Conclusion Periglomerular dopaminergic cells are exceptional neurons. They have dendrites that have characteristics of both axons and dendrites, since they give and receive synapses even though they posess a true axon. Like a good “concierge” at the entrance to the bulb, they know everything about the comings and goings and refer it to the neighborhood. Like spies, they change identity according to conditions. Like soldiers, they arrive from distant places recalled by still unknown signals. We have understood some of their strategies but many are still elusive, let us hope for not too long.

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References 1. Halasz N, Shepherd GM. Neurochemistry of the vertebrate olfactory bulb. Neurosci 1983; 10:579-619. 2. Macrides F, Davis BJ. The olfactory bulb.In: Emson PC, ed. Chemical Neuroanatomy. New York: Raven Press,1983:391-425. 3. Brunjes PC, Frazier LL. Maturation and plasticity in the olfactory system of vertebrates. Brain Res Rev 1986; 11:1-45. 4. Hildebrand JG. Analysis of chemical signals by nervous systems. Proc Natl Acad Sci USA 1995; 92:67-74. 5. Van Drongelen W, Holley A, Doving KB. Convergence in the olfactory system: Qualitative aspects of odor sensitivity. J Theor Biol 1978; 71:39-48. 6. Sharp FR, Kauer JS, Shepherd GM. Functional organization of rat olfactory bulb analyzed by the 2-deoxyglucose method. J Comp Neurol 1979; 185: 715-734. 7. Jastreboff PJ, Pedersen PE, Greer CA et al. Specific olfactory receptor populations projecting to identified glomeruli in the rat olfactory bulb. Proc Natl Acad Sci USA 1994; 81:5250-5254. 8. Jourdan F, Duveau A, Astic L, Holley A. Spatial distribution of 2-deoxyglucose uptake in the olfactory bulb of rats stimulated with two different odors. Brain Res 1980; 188:139-154. 9. Ressler KJ, Sullivan SL, Buck LB. Information coding in the olfactory system: Evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 1994; 79:1245-1255. 10. Vassar R, Chao SK, Sitcheran R et al. Topographic organization of sensory projections to the olfactory bulb. Cell 1994; 79:981-991. 11. Pinching AJ, Powell TPS. The neuropil of the periglomerular region of the olfactory bulb. J Cell Sci 1971; 9:379-409. 12. Mori K. Membrane and synaptic properties of identified neurons in the olfactory bulb. Prog. Neurobiol 1987; 29:275-320. 13. Kosaka T, Hataguchi Y, Hama K et al. Coexistence of immunoreactivities for glutamate decarboxylase and tyrosine hydroxylase in some neurons in the periglomerular region of the rat main olfactory bulb: Possible coexistence of GABA and dopamine. Brain Res 1985; 343:166-171. 14. Shepherd GM, Greer CA. Olfactory bulb. In: Shepherd, GM, ed. The Synaptic organization of the brain, 3rd ed. New York: Oxford University Press,1990:133-169. 15. Yokoi M, Mori K, Nakanishi S. Refinement of odor molecule tuning by dendrodendritic synaptic inhibition in the olfactory bulb. Proc Natl Acad Sci USA 1995; 92:3371-3375. 16. Firestein S. Electrical signals in olfactory transduction. Curr Opin Neurobiol 1992; 2:444-448. 17. Reed RR. Signaling pathways in odorant detection. Neuron 1992; 8:205-209. 18. Dionne VC. Emerging complexity of odor transduction. Proc Natl Acad Sci USA 1994; 91:6253-6254. 19. Shepherd GM. Discrimination of molecular signals by the olfactory receptor neuron. Neuron 1994; 13:771-790. 20. Laurent G. Odor images and tunes. Neuron 1996; 16:473-476 21. Pinching AJ, Powell TPS. The neuron types of the glomerular layer of the olfactory bulb. J Cell Sci 1971; 9:305-345. 22. Dahlstrom A, Fuxe K, Olson L et al. On the distribution and possible function of monoamine nerve terminals in the olfactory bulb of the rabbit. Life Science 1965; 4:2071-2074. 23. Lichtensteiger, W. Uptake of norepinephrine in periglomerular cells of the olfactory bulb of the mouse. Nature 1966; 210:955-956. 24. Halasz N, Johansson O, Hokfelt T et al. Immunohistochemical identification of two types of dopamine neurons in the rat olfactory bulb as seen by serial sectioning. J Neurocytol 1981; 10:251-259. 25. Halasz N, Ljundhahl A., Hokfelt T et al. Transmitter histochemistry of the rat olfactory bulb. I. Immunohistochemical localization of monoamine synthetizing enzymes. Support for intrabulbar, periglomerular dopamine neurons. Brain Res 1977; 126:455-474.

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26. Halasz N, Ljundahl A, Hokfelt T. Transmitter histochemistry of the rat olfactory bulb. II. Fluorescence, histochemical, autoradiographic and electron microscopic localization of monoamines. Brain Res 1978; 154:253-271. 27. Priestly JV, Kelly JS, Cuello AC. Uptake of 3H dopamine in periglomerular cells of the rat olfactory bulb: An autoradiographic study. Brain Res. 1979; 165:149-155. 28. Guthrie KM, Wilson DA, Leon M. Early unilateral deprivation modifies olfactory bulb function. J Neurosci. 1990; 10:3402-3412. 29. Meisami E, Safari L. A quantitative study of the effects of early unilateral olfactory deprivation on the number and distribution of mitral and tufted cells and on glomeruli in the rat olfactory bulb. Brain Res 1981; 221:81-107. 30. Benson TE, Ryugo DK, Hinds JW. Effects of sensory deprivation on the developing mouse olfactory system: a light and electron microscopic, morphometric analysis. J Neurosci 1984; 4:638-653. 31. Skeen LC, Due BR, Douglas FE. Neonatal sensory deprivation reduces granule cell number in mouse olfactory bulbs. Neurosci. Letter 1986; 63:5-10. 32. Frazier LL and Brunjes PC. Unilateral odor deprivation: Early postnatal changes in olfactory bulb cell density and number. J Comp Neurol 1988; 269:355-370. 33. Brunjes PC, Smith-Crafts LK, McCarthy R. Unilateral odor deprivation: Effects on the development of olfactory bulb catecholamines and behavior. Dev Brain Res 1985; 22:1-6. 34. Wilson DA and Wood JG. Functional consequences of unilateral olfactory deprivation: Time course and age sensitivity. Neurosci 1992; 49:183-192. 35. Baker H. Unilateral, neonatal olfactory deprivation alters tyrosine hydroxylase expression but not aromatic amino acid decarboxylase or GABA immunoreactivity. Neurosci. 1990; 36:761-771. 36. Kosaka T, Kosaka K, Hama K et al. 1987 Differential effect of functional olfactory deprivation on the GABAergic and catecholaminergic traits in the main olfactory bulb. Brain Res 1987; 413:197-203. 37. Mair RG. Response properties of rat olfactory bulb neurons. J. Physiol 1982; 326:341-359. 38. Mair RG, Gesteland RC. Response properties of mitral cells in the olfactory bulb of the neonatal rat. Neurosci 1982; 77:3117-3125. 39. Wilson DA, Sullivan RM, Leon M. Single unit analysis of postnatal learning: Modified olfactory bulb output response patterns to learned attractive odors. J Neurosci 1987; 7:3154-3162. 40. Wilson D, Sullivan RM. The D2 antagonist spiperone mimics the effects of olfactory deprivation on mitral/tufted cell odor response patterns. J. Neurosci. 1995; 15:5574-5581. 41. Nowycky MC, Halasz N, Shepherd GM. Evoked field potential analysis of dopaminergic mechanisms in the isolated turtle olfactory bulb. Neurosci 1983; 8:717-722. 42. Nickell W, Norman AB, Wyatt LM, Shipley MT. Olfactory bulb DA receptors may be located on terminals of the olfactory nerve. Neuroreport 1991; 2:9-12 43. Coronas V, Srivastava LK, Liang JJ, Jourdan F, Moyse E. Identification and localization of dopamine receptor subtypes in rat olfactory mucosa and bulb: A combined in situ hybridization and ligand binding radioautographic approach. J Chem Neuroanat 1997; 12:243-257 44. Guthrie KM, Pullara JM, Marshall JF et al. Olfactory deprivation increases dopamine D2 receptor density in the rat olfactory bulb. Synapse 1991; 8:61-70. 45. Coopersmith R, Weihmuller FB, Kirstein CI et al. Dopamine increases in the neonatal olfactory bulb during odor preference training. Brain Res 1991; 564:149-153. 46. Coopersmith R, Leon M. Enhanced neural response to familiar olfactory cues. Science 1984; 225:849-851. 47. Wilson DA, Leon M. Spatial patterns of olfactory bulb single-unit responses to learned olfactory cues in young rats. J Neurophysiol 1988; 59:1770-1782. 48. Woo CC, Leon, M. Increase in a focal population of juxtaglomerular cells associated with early learning. J Comp Neurol 1991; 305:49-56. 49. Denis-Donini S, Estenoz M. Interneurons versus efferent neurons: Heterogeneity in their neurite outgrowth response to glia from several brain regions. Dev Biol 1988; 130:237-249

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50. Amara SG, Kuhar MJ. Neurotransmitter transporters: Recent progress. Ann Rev Neurosci 1993; 16:73-93. 51. Johnson RG. Accumulation of biological amines into chromaffin granules: A model for hormone and neurotransmitter transport. Physiol Rev 1988 68:232-307 52. Liu Y, Peter D, Roghani A et al. A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter. Cell 1992; 70:539- 551. 53. Peter D, Liu Y, Sternini C et al. Differential expression of two vesicular monoamine transporters. J Neurosci 1995; 15:6179-6188. 54. Raiteri M, Cerrito F, Levi G. Dopamine can be released by two mechanisms differentially affected by the dopamine transport inhibitor nomifensine. J Pharmac Exp Ther 1979; 208:195-202 55. Sulzer D, Maidment NT, Rayport S. Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons. J Neurochem 1993; 60:527-535. 56. Cerruti C, Walther DM, Kuhar MJ et al. Dopamine transporter mRNA expression is intense in rat midbrain neurons and modest outside midbrain. Molec Brain Res 1993; 18:181-186. 57. Daniel SE, Hawkes CH. Preliminary diagnosis of Parkinson’s disease by olfactory bulb pathology. Lancet 1992; 340:186. 58. Stern MB, Doty RL, Dotti M et al. Olfactory function in Parkinson’s disease subtypes. Neurology 1994; 44:266-268: 59. Gainetdinov RR, Fumagalli F, Wang YM et al. Increased MPTP neurotoxicity in vesicular monoamine transporter 2 heterozygote knockout mice. J. Neurochem 1998; 70:1973-1978 60. Sallaz M, Jourdan F. Apomorphine disrupts odour-induced patterns of glomerular activation in the olfactory bulb. NeuroReport 1992; 3:833-836. 61. Farbman AI. Developmental neurobiology of the olfactory system. In: Getchel TV, Doty RL,Bartoshuk LM and Snow JB Jr, eds. Smell and Taste in Health and Disease. New York: Raven Press, 1991:19-33. 62. Hinds JW. Autoradiographic study of histogenesis in the mouse olfactory bulb. I. Time of origin of neurons and neuroglia. J. Comp Neurol 1968; 134:287-304. 63. Bayer SA. 3H-thymidine-radiographic studies of neurogenesis in the rat olfactory bulb. Exp Brain Res 1983; 50:329-340. 64. Altman J. Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J Comp Neurol 1969; 137:433-458 65. Luskin MB. Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 1993; 11:173-189. 66. Lois C, Alvarez-Buylla A. Long distance migration in the adult mammalian brain. Science 1994; 264:145-1148. 67. O’Rourke NA. Neuronal chain gangs: Homotypic contacts support migration into the olfactory bulb. Neuron 1996;16:1061-1064. 68. Goldman SA, Luskin MB. Strategies utilized by migrating neurons of the postnatal vertebrate forebrain. TINS 1998; 21:107-114. 69. Betarbet R, Zigova T, Bakay RA, et al. Dopaminergic and GABAergic interneurons of the olfactory bulb are derived from the neonatal subventricular zone. Int J Dev Neurosci 1996; 14:921-930. 70. Graziadei PPC, Levine RR, Monti Graziadei GA: Regeneration of olfactory axons and synapse formation in the forebrain after bulbectomy in neonatal mice. Proc Natl Acad Sci USA 1978; 75:5230-5234. 71. Graziadei PPC, Monti Graziadei GA. Neuronal changes in the forebrain of mice following penetration by regenerating axons. J Comp Neurol 1986; 247:344-356. 72. Kawano T, Margolis FL. Trans-synaptic regulation of olfactory bulb catecholamines in mice and rats. J Neurochem 1982; 39:342-348. 73. Denis-Donini S. Expression of dopaminergic phenotypes in the mouse olfactory bulb induced by the calcitonin gene-related peptide. Nature 1989; 339:701-703.

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74. Baker H, Farbman AI. Olfactory afferent regulation of the dopamine phenotype in the fetal rat olfactory system. Neurosci 1993; 52:115-134. 75. Specht LA, Pickel VM, Joh TH et al. 1981. Light microscopic immunohistochemical localization of tyrosine hydroxylase in prenatal brain. II Late ontogeny. J Comp Neurol 1981; 199:255-276. 76. McLean JH, Shipley MT. Postmitotic, postmigrational expression of tyrosine hydroxylase in olfactory bulb dopaminergic neurons. J Neurosci 1988; 8:3658-3669. 77. Guthrie KM, Leon M. Induction of tyrosine hydroxylase expression in rat forebrain neurons. Brain Res. 1989; 497:117-131. 78. Stone DM, Wessel T, Joh TH, et al. Decrease in tyrosine hydroxylase, but not aromatic L-amino acid decarboxylase, messenger RNA in rat olfactory bulb following neonatal, unilateral odor deprivation. Molec Brain Res 1990; 8:291-300 79. Nadi NS, Head R, Grillo M et al. Chemical deafferentation of the olfactory bulb: Plasticity of the levels of tyrosine hydroxylase, dopamine and norepinephrine. Brain Res 1981; 213:365-371. 80. Baker H, Kawano T, Margolis FL et al. Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat. J Neurosci 1983; 1:69-78 81. Baker H, Kawano T , Albert V et al. Olfactory bulb dopamine neurons survive differentation induced loss of tyrosine hydroxylase. Neuroscience 1984; 11:605-615 82. Denis-Donini S, Chini, B, Vitadello M. Developmentally regulated expression of CGRP in the mouse olfactory pathway. Eur J Neurosci 1993; 5:648-656. 83. Denis-Donini S, Branduardi P, Campiglio S, Candia-Carnevali D. Localization of CGRP mRNA into developing olfactory axons. Cell Tissue Res 1998; 294:81-91. 84. Van Valen F, Piechot G, Jurgens, H. Calcitonin gene-related peptide (CGRP) receptors are linked to cyclic adenosine- monophosphate production in SK-N-MC human neuroblastoma cells. Neurosci Lett 1990; 119:195-198. 85. McLatchie LM, Fraser NJ, Main MJ et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 1998; 393:333 86. Kumer SC, Vrana KE. Intricate regulation of tyrosine hydroxylase activity and gene expression. J Neurochem 1996; 67:443-462 87. Rekik M, Delvaux M, Frexinos J et al. The calcitonin gene-related peptide activates both cAMP and NO pathways to induce relaxation of circular smooth muscle cells of guineapig ileum. Peptides 1997; 18:1517-1522. 88. Roskama AJ, Bredt DS, Dawson TM et al. Nitric oxide mediates the formation of synaptic connections in developing and regenerating olfactory receptor neurons. Neuron 1994; 13:289-299. 89. Guthrie MK, Anderson AJ, Leon M et al. Odor-induced increases in c-fos mRNA expression reveal an anatomical “unit” for odor processing in olfactory bulb. Proc Natl Acad Sci USA 1993; 90:3329-3333. 90. Guthrie KM, Gall CM. Odor increase fos in olfactory bulb neurons including dopaminergic cells. Neuroreport 1995; 6:2145-2149.

CHAPTER 10

Dopamine in Drosophila: Neuronal and Developmental Roles Wendi S. Neckameyer

P

erturbations in dopaminergic signaling pathways in nervous tissue have been implicated in the etiology of schizophrenia, Alzheimer’s disease, and some depressive disorders.1,2 Dopamine is also present in the central nervous systems (CNSs) of arthropod and cniderian species. Studies of the fruit fly Drosophila melanogaster have demonstrated that dopamine not only functions as a neurotransmitter, but that it plays multiple and novel roles in different organ systems, and is absolutely required for normal development. Drosophila may be considered to be two animals in one: the juvenile larval stage, and the adult fly. The female deposits an egg, and the larva hatches from the egg after approximately 22 hours (at 25°C). There are three larval instars, or stages: the first and second last 24 hours, and the third three days. During the initial stages of larval life, the animals are negatively phototactic and their prime directive is merely to burrow in a food source, eat, and grow. The larval tissues increase in size but do not proliferate; the only proliferating tissues are the imaginal discs, which will form the adult fly during metamorphosis. Near the end of the third larval instar, the animal becomes positively phototactic, leaves the food source, and pupates. During pupariation, essentially all the larval tissues are histolyzed, and the adult tissues form. When metamorphosis is complete, the animal ecloses from the pupal case, and is sexually mature within a day. After fertilization, the female stores a male’s sperm within a spermatheca, and fertilizes each egg as it is extruded. There is no zygotic expression within the embryo until a few hours after egg-laying, and those proteins that are expressed in the newly deposited egg are maternally contributed. In mammalian tissues, dopamine modulates “simple” behaviors such as locomotor activity, and more complex behaviors such as sexual receptivity and cognition. Is it possible that at least some of the signaling machinery responsible for dopaminergic modulation has been maintained throughout evolution? Conversely, are the developmental requirements for dopamine in Drosophila conserved, at least in part, in mammalian development? We propose that dopamine is an ancient signaling molecule that has been adapted for use in many physiological contexts, and that the requirements for dopamine in diverse tissues have been conserved.

Biosynthetic Pathways and Evolutionary Considerations Not only is dopamine found in such evolutionarily diverged phyla as arthropods and mammals, but the transmitter biosynthetic machinery and G-protein signaling mechanisms also appear to be highly conserved (Table 10.1). Significant progress in the molecular-genetic analyses of these pathways has been accomplished in both rodents and in Drosophila. The Development of Dopaminergic Neurons, edited by Umberto di Porzio, Roberto Pernas-Alonso, and Carla Perrone-Capano. ©1999 R.G. Landes Company.

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Table 10.1. Gene

Mutant

Locus

Reference

tyrosine hydroxylase

pale(ple)

65B

Neckameyer and Quinn, 1989

GTP cyclohydrolase I

Punch (Pu)

57C

O'Donnel et al, 1989

14-3-3

leonardo

46E

Skoulakis and Davis, 1996

dopa decarboxylase

ddc

37C1-2

Hirsh and Davidson, 1981

35EF

Gotzes et al, 1994; Sugamori, et al, 1995

D1 dopamine receptor

D1 dopamine receptor

DAMB/ DopR99B

99B

Feng, et al, 1996; Han et al, 1996

adenylyl cyclase

rutabaga (rut)

12F5-7

Levin, et al, 1992

Gsα

60A

Quan et al, 1989

protein kinase A RI

77F

Kalderon and Rubin, 1988

protein kinase A DCO

30C1-6

Kalderon and Rubin, 1988 Foster et al, 1988

Unlike what is found in mammalian systems, dopamine is apparently the major, if not the only, catecholamine in the Drosophila central nervous system, and thus only tyrosine hydroxylase and dopa decarboxylase enzymatic activities are present (to synthesize L-DOPA and dopamine). There is no substantial evidence for the presence of either epinephrine or norepinephrine, nor has activity been detected for the biosynthetic enzymes phenylenthanolamine-N-methyl transferase and dopamine-β-hydroxylase. Although material crossreacting with antibodies raised against dopamine-β-hydroxylase can be detected in Drosophila, this is apparently due to the homologous enzyme, tyramineβ-hydroxylase, and which is required for the synthesis of octopamine.3 Nassel and Elekes4 have reported that in two species of blowflies there is an exact correspondence between the dopamine-immunoreactive and tyrosine hydroxylase-immunoreactive neurons, suggesting that in these insects as well, dopamine accounts for all catecholaminergic neurotransmission. Drosophila tyrosine hydroxylase (DTH65B) is a single copy gene whose 508 residue deduced amino acid sequence predicts 50% identity with rat tyrosine hydroxylase.5 The strong conservation of this enzyme across a great evolutionary distance is evidenced not only by the nucleic acid and amino acid homology between tyrosine hydroxylases from different species, but also by the conservation of regulatory mechanisms. Using an enzymatic

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assay developed for mammalian tyrosine hydroxylase, activity was detected in Drosophila head.5 The serine residues in mammalian tyrosine hydroxylase which are phosphorylated by cAMP-dependent protein kinase and by Ca2+/calmodulin-dependent protein kinase II are conserved in the Drosophila enzyme; although DTH can be phosphorylated in vitro by these two kinases (Neckameyer, unpublished results), the specific phosphorylated residues remain to be formally identified. Similar to its mammalian counterparts, DTH requires a pterin cofactor; Punch, a Drosophila locus encoding guanosine triphosphate (GTP) cyclohydrolase I, catalyzes the first reaction in pteridine biosynthesis.6 Drosophila GTP cyclohydrolase I is a complex locus: several transcripts arise from alternative splicing that result in different protein isoforms which share approximately 60% identity with the rat liver and human enzymes.7 Null Punch alleles have a phenotype similar to that of the DTH mutant pale (ple), an embryonic lethal mutation with perturbed catecholamine levels and an unpigmented pharate cuticle.8-10 As in mammals, L-DOPA is a precursor to melanin, and defects in L-DOPA production thus lead to underpigmentation. Tyrosine hydroxylase is activated by the phosphorylation of specific serine residues within the protein; in some cases, activation after phosphorylation requires the activity of an additional protein, brain protein 14-3-3. A homolog for the mammalian 14-3-3 protein, LEONARDO, also exists in Drosophila, although it is not yet known whether DTH is activated by the product of the leonardo gene after phosphorylation by Ca2+/calmodulin-dependent protein kinase II, as has been proposed to occur in mammals.11 The single gene encoding the Drosophila 14-3-3 counterpart shares 88% identity with a mammalian isoform.12,13 Dopa decarboxylase, the second enzyme in the biosynthesis of both dopamine and serotonin, has also been cloned in Drosophila.14-15 Although it is a single copy gene, it is part of a much larger phenol oxidase gene cluster on the second chromosome. Deletions of ddc also result in unpigmented embryos that die before the first larval instar.16 However, neither epidermal nor nervous system expression of DDC is required for viability,17 suggesting that either there is no vital requirement for dopamine in these tissues, or that another decarboxylase can serve to generate dopamine from L-DOPA. The focus for viability for dopamine in early Drosophila development has not yet been precisely defined, but likely resides outside the cuticle and CNS. Dudai and colleagues18 have previously demonstrated a dopaminergic activation of adenylyl cyclase activity in vitro, which was distinct from the effects of both octopamine and serotonin. To date, only two dopamine receptors have been cloned in Drosophila (DopR35EF, and DopR99B or DAMB).19-21 Both appear to be D1-like in nature, since they stimulate adenylyl cyclase when activated, although their pharmacology is distinct from that of the mammalian D1 receptors. The DopR99B receptor has been shown (when expressed in Xenopus oocytes) to be directly coupled to two different G-protein-mediated pathways: one that stimulates adenylyl cyclase, and which is pertussis toxin sensitive, and one which requires an intracellular calcium signal, a pathway which is pertussis-toxin insensitive.22 It is likely that the entire family of dopamine receptors has not yet been molecularly characterized, and it remains possible that D2-like receptors exist in Drosophila. There is only a single locus in Drosophila encoding the α subunit of G-proteins; the α subunit determines the specificity of interaction with both the receptor and effector molecules. This single locus, however, apparently encodes two gene products via alternative splicing: DGsα S and DGsα L, which differ only by three amino acid residues near the carboxy terminus.23 The Drosophila G-proteins share over 70% identity with the bovine homolog and are sensitive to cholera but not pertussis toxin.24 The Drosophila Gsα proteins are able to activate mammalian adenylyl cyclase with comparable efficiency relative to the mammalian G-protein subunits.25

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As in mammalian systems, there appear to be two types of adenylyl cyclase activities: one stimulated by, and one unresponsive to, the addition of calcium and calmodulin.26 Thus far, only a calcium/calmodulin-responsive adenylyl cyclase has been cloned; this is the product of the rutabaga (rut) gene. rut flies have been shown to be defective when tested in learning and memory paradigms.27 When transfected into human embryonic kidney 293 cells, the rut cDNA was shown to be capable of coupling to the endogenous Gs protein in these cells, again demonstrating the strong evolutionary conservation of signaling machinery. The catalytic and regulatory subunits of protein A kinase in Drosophila are also each encoded by a single genetic locus.28 The predicted amino acid sequence of the Drosophila RI regulatory homolog is 71% identical to its mammalian counterpart; at least three gene products arise from the single locus via alternative splicing. The catalytic subunit, DC0, shares 82% identity with its mammalian homolog.28,29 At least four transcripts arise from this locus, but it is likely these are redundant; only DC0 is essential.30 Although DC0 is widely distributed in diverse tissues, its expression is greatest in neuronal tissues. Nearly all of the above-mentioned genes have been shown to be deficient in at least some aspect of learning and/or memory, and almost all are expressed in the mushroom body structures within the adult Drosophila brain (see Table 10.1). The mushroom bodies are believed to be the focus for learning acquisition and memory retention in Drosophila.31 This provides further evidence that dopaminergic mechanisms underlie aspects of learning and memory. L-DOPA, the product of the tyrosine hydroxylation reaction, is further metabolized to melanin, and derivatives of L-DOPA are also used to crosslink proteins to chitin within the cuticle, especially within the adult fly (see below). Regulation of dopamine levels in Drosophila is likely to be controlled by the enzyme N-acetyltransferase rather than by monoamine oxidases or catecholamine-O-methyltransferase.32 This enzyme is present in nervous tissue as well as in the hypoderm; it appears to inactivate dopamine in the CNS and functions in the cuticle by generating the sclerotizing agent N-acetyldopamine. Enzymatic activity of N-acetyltransferase has not been examined in other tissues where tyrosine hydroxylase is expressed.

The Role of Dopamine in Central and Peripheral Nervous Tissues Catceholamine Localization in the CNS The stereotypic pattern of catecholamine and tyrosine hydroxylase immunoreactivity in Drosophila has been well established. The approximately 80 larval and 220 adult neurons (excluding those in the optic lobe) are widely distributed within the Drosophila CNS. The larval pattern is established by 18-20 hours of embryogenesis (at 25°C, the Drosophila melanogaster embryo hatches at 22 hours), as determined by glyoxylic acid-induced histofluorescence, dopamine-like immunoreactivity, and tyrosine hydroxylase immunoreactivity.33 Consistent with this pattern of expression, dopa decarboxylase levels show a dramatic rise nearing the end of embryogenesis, which is maintained through the first larval instar.34 Glyoxylic acid-induced histofluorescence of larval brains reveals three bilaterally symmetrical clusters comprised of 4-6 neurons within the brain lobes (Fig. 10.1B). Within the fused ventral ganglion there are three subgroups of catecholamine-fluorescing neurons: In the subesophageal region, there are 4 unpaired and 2 paired sets of medial neurons within the ventral cellular rind; there are a row of 7-8 single unpaired neurons along the ventral midline and 10 bilaterally symmetric lateral neurons which are dorsal to the medial unpaired neurons (Fig. 10.1B, see also ref. 35). The developing optic lobes do not detectably express

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Fig. 10.1. Localization of catecholamine fluorescence and DTH expression in the larval CNS. (A) Schematic drawing of the larval brain. ol, optic lobe; bl, brain lobe, vg, ventral ganglion. (B) Glyoxylic acid-induced histofluorescence, showing localization of catecholaminergic cell bodies. (C) Staining of a larval CNS from a transgenic line containing the core DTH promoter fused to the E. coli β-galactosidase reporter gene. mb, mushroom bodies. The dorsal lateral dopaminergic neurons may also be visualized in this plane of focus.

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dopamine immunoreactivity.33 This stereotypic pattern in larval CNSs is apparently conserved, as a similar pattern is detected in the hemipteran Rhodnius prolixus.36 During metamorphosis, the larval pattern of dopaminergic neurons persists, even though the nervous tissue undergoes substantial reorganization. The dopaminergic cell clusters in the larval brain lobes are maintained in the adult, and three additional clusters can be observed during metamorphosis. These are the anteromedial (AM), posteromedial (PM), and medulla clusters (MC); the AM cells can be detected within 48 hours after pupariation, the AM cluster by 72 hours, and the MC neurons by 96 hours.33 The MC neurons are smaller cells found clustered within the adult optic lobe. Similar to what has been observed for the brain lobes, the dopaminergic pattern in the larval ganglion is maintained in the adult thoracic ganglion.33 The localization of the TH-immunoreactive neurons visualized in adult Drosophila brains is also apparently conserved in other insect species: The clusters appear homologous with those detected in the blowflies Phormia terraenovae and Calliphora erythrocephal,4 and these neurons appear to be interneurons which are excluded from the antennal lobes and the mushroom body calyces. All dopaminergic processes also appear confined to the CNS. There is widespread DA- and TH-immunoreactivity found within all regions of the central complex that defines populations of intrinsic neurons as well as processes. The mushroom bodies, believed to be the “seat” of learning and memory,31 are also densely innervated by dopaminergic neurons. Work done by Menzel and colleagues37 in honeybees suggests that this function for mushroom bodies is conserved. As mentioned previously, several of the genes within the proposed dopaminergic signal transduction pathway are also expressed in the mushroom bodies (see Table 10.1); mutations in many of these genes affect learning in different behavioral paradigms. The core DTH promoter, when fused to the E. coli β-galactosidase reporter gene and integrated into the Drosophila genome, drives expression of the reporter in catacholaminergic neurons as well as in the mushroom bodies (Figs. 10.1C, 10.2B; see also ref. 38). Immunocytochemical analysis reveals that both the DTH protein and dopamine are clearly localized to cell bodies within the mushroom bodies (and elsewhere in the brain). This pattern of expression is consistent with a role for dopamine in learning in Drosophila. A role for dopamine in the development of the nervous system cannot be excluded, although dopaminergic neurons are present even in the absence of dopamine.39 However, it is not known if the fine organization and function of the CNS is normal since ple is lethal at the embryonic-first larval instar boundary, and thus, these animals cannot be tested in behavioral paradigms. Similarly, in TH- transgenic mice, catecholaminergic neurons appear to develop.40 Again, normal CNS function cannot be ascertained due to the lethality of these mice in a late prenatal stage.

Dopamine as a Neurotransmitter Dopamine has now been shown to act as a neurotransmitter to modulate different behaviors in Drosophila. An early reference demonstrated biogenic amine modulation of learning in both positively and negatively reinforced tasks using temperature-sensitive mutations in the dopa decarboxylase gene to deplete Drosophila of both dopamine and serotonin.41 However, these experiments could not distinguish whether one or both neurotransmitters modulated these types of learning; in addition, these results have not been replicated, presumably because of the accumulation of genetic modifiers in the mutant strains. Recently, Neckameyer38 has established that both dopamine and functional mushroom tissue are required for normal habituation in adult Drosophila. Mature males, when first exposed to immature males, will perform courtship rituals; the intensity and duration of this behavior rapidly diminishes with time. Depletion of dopamine in adult

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Fig.10. 2. Staining of an adult brain from a transgenic line containing the core DTH promoter fused to the E. coli β-galactosidase reporter gene. (A) Schematic drawing of the mushroom body structures in the adult brain. ol, optic lobe; ca, calyx; p, peduncle; γ and β, gamma and beta lobes within the mushroom body; m, median bundle; eb, ellipsoid body, fb, fan-shaped body. The latter two are part of the central complex. (B) Transgenic adult brain showing lacZ staining in the mushroom body. The dopaminergic cell bodies are not visible in this staining pattern.

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males, via the systemic introduction of the tyrosine hydroxylase inhibitor 3-iodotyrosine, severely impaired the ability of these flies to cease their courtship of immature males. Pharmacological treatment of flies with both 3-iodotyrosine and L-DOPA rescued the learning behavior, demonstrating that dopamine is responsible for modulating this response. Ablation of the mushroom bodies by treatment of newly hatched larva with hydroxyurea also resulted in the inability of treated mature males to cease courtship when placed with untreated immature males. These data are the first demonstration that dopamine acts as a signaling molecule within the mushroom bodies to mediate a form of learning. This is consistent with the localization of tyrosine hydroxylase to this tissue; anatomical studies have demonstrated that all subdivisions of the central body complex are highly innervated by dopaminergic processes.4,33 Dopamine also modulates an experience-independent sexual behavior in Drosophila, that of female sexual receptivity.42 Although the male Drosophila enacts highly ritualized courtship behavior, the female signals her receptivity in response to the male’s cues by merely decreasing her locomotor activity, and spreading her vaginal plates, so that the male may copulate and intromission may occur (usually within five minutes in flies 4-5 days old). When newly eclosed females are systemically depleted of dopamine, they are significantly less receptive to males than are untreated females; this effect is reversed by the addition of L-DOPA (Fig. 10.3). These females provide the correct visual and pheromonal cues, as males court them as insistently as they do untreated females,42 evidence that dopamine is not required for this aspect of pheromonal production. Dopamine-depleted females behave similarly to that of newly eclosed virgin females, as males court them vigorously but they reject his attempts. Fertilized females will also reject a male’s overtures, but these females will extrude their ovipositors and send olfactory cues to the male that she is unavailable, leading to his eventual relinquishment of all courtship attempts. There are two foci for female sexual behavior in Drosophila, the abdomen (for the pheromone production), and the brain. Sexual receptivity in female Drosophila has been mapped to neurons in the dorsal anterior region of the brain;43 it is presumed these neurons are dopaminergic or receive dopaminergic input. Female receptivity may be regulated via interactions with hormonal pathways, since depletion of dopamine levels in adult Drosophila females is also required for normal ovarian maturation and fecundity. Thus, there are possibly interacting roles for dopamine in the modulation of female reproduction in both the brain and ovaries. In mammals, female sexual receptivity is dependent on the hormones estrogen and progesterone, which interact with specific receptors in the brain to regulate behavior. Presynaptic actions of dopamine within the female rodent brain stimulate lordosis;44 it has been suggested that dopamine may activate these steroid hormone receptors in a ligand-independent manner.45 It is quite possible that dopaminergic modulation of female sexual receptivity in Drosophila also occurs via a steroid hormone receptor-signaling pathway. Dopamine also modulates innate behaviors such as locomotor activity (Fig. 10.4; Walzer M, Neckameyer W, unpublished observations). When adult male Drosophila are placed in an observation chamber marked with grid lines, dopamine-depleted males cross the lines a significantly greater number of times relative to controls; this effect can be rescued by L-DOPA, the product of the tyrosine hydroxylation reaction. This is consistent with an earlier study of fly populations selectively bred for spontaneous locomotor activity, which demonstrated that in the inactive strain, dopamine levels were maximal, compared to the active strain.46 These analyses were done using females only, but suggest that lowered dopamine levels result in greater activity.

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Fig. 10.3. Dopamine-depleted females are significantly less sexually receptive to males. Control and treated 4-6 day old virgin females were placed in a courtship chamber with a 4-6 day old male. 3IY, treatment with 10 mg/ml of the tyrosine hydroxylase inhibitor 3-iodotyrosine from eclosion until use in the experiment. 3IY + L-DOPA, females treated with 10 mg/ml 3IY plus 10 mg/ml L-DOPA. n = 28, 28 and 20 for control, 3IY and 3IY + L-DOPA, respectively. Normal 4-6 day old adult virgin flies will generally copulate within seven minutes when introduced to each other in a courtship observation chamber.

Dopamine apparently modulates few, if any, behaviors during the larval stage (see ref. 47; also Walzer M, Neckameyer W, unpublished observations). Animals depleted of dopamine or given excess L-DOPA demonstrate normal locomotive, phototactic, and feeding behaviors, and display the same odor preference for heptanol and aversion to salt as do untreated larvae. However, within a few hours after dopamine depletion, the treated larvae become aphagic and lethargic, and eventually die. This behavior is strikingly similar to that displayed by dopamine-deficient transgenic mice:48 Although apparently normal at birth, within a few weeks these mice become listless, cease eating and drinking, and die. Whether this is a consequence of dopaminergic modulation of exploratory behavior and/or a physiological consequence of the requirement for dopamine during development (see below) is not yet clear. However, larvae with mutations in zygotic DC0 (the catalytic subunit of protein kinase A), exhibit delayed larval growth and lethargic behavior, and die within a few days.49 It is conceivable that this behavior may therefore be modulated by dopaminergic activation of a cAMP-signaling cascade.

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Fig. 10.4. Dopamine levels affect locomotor activity in Drosophila. Four day old male flies were maintained on either 2% yeast/5% sucrose (control, n = 48), or yeast-sucrose plus 10 mg/ml of the tyrosine hydroxylase inhibitor 3-iodotyrosine (3IY, n = 47), or 10 mg/ml 3IY plus 10 mg/ml L-DOPA (3IY + L-DOPA, n = 43). Individual flies were gently aspirated into a 60 mm petri dish marked with 1 cm grid lines; locomotor activity was quantitated by counting the number of times a fly crossed a grid line in 2 minutes after a 30 second acclimation period. * p<.01.

Physiological Effects of Dopamine The effects of application of dopamine to either the intact larval CNS or to isolated neuromuscular preparations results in altered activity (Cooper R, Neckameyer W, unpublished observations). The filleted third larval instar preparation has provided an effective system in which to investigate the influence of neuromodulators on CNS activity as well as in the periphery at the neuromuscular junctions.50 The segmental architecture is consistent from animal to animal, and each muscle can be distinctly identified. While recording from a small length of the intact fourth segmental nerve in a suction electrode, the activity of the root may be monitored (Fig. 10.5), during which time the activity profile is readily recorded over a few hours. This preparation thus allows the effects of various neuromodulators on CNS activity to motor commands to be tested. Normally, the activity

Fig. 10.5. Spontaneous activity in a segmental root. The activity measured in the root is a function of the intact CNS command to that particular segment. (A) While the preparation is bathed in physiological saline, there are rhythmic bursts of activity at an enhanced frequency over baseline activity. The frequency of the bursts reach approximately 30 Hz (background frequency is 8 Hz). (B) After exchanging the bathing medium with a solution containing 10 µM dopamine, the bursting frequency rapidly becomes prolonged in duration, with a longer interval between bursts. (C) After 10 minutes of continued exposured to 10 µM dopamine, the bursts of activity cease, and the firing frequency remains relatively constant (~10 Hz). Time scale: Each trace is 1 minute, 20 seconds in duration with frequency plots on the right. The frequency plots, in Hz, are an average of every 5 events.

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Fig. 10.6. The effects of dopamine on the excitatory junctional potentials (EJPs). Segmental nerves 3 or 4 were stimulated while the EJPs were recorded with an intracellular electrode in muscle 6 of the stimulated segment. Isolated responses from stimulation of both the Is and Ib motor neurons are shown while the preparations are bathed in saline and during exposure to dopamine at either 1 mM (A) or at 10 µM (B). The reduction in the EJP amplitude is more rapid at the higher dose. Note the slight enhancement of the EJP amplitude, followed by a depression, after the addition of 10 µM dopamine. is in the form of rhythmic bursts, which can be altered by the application of dopamine. In all cases, the bursting pattern is reduced and eventually results in the tonic firing of the motor neurons. In examining the influence of dopamine on synaptic transmission at neuromuscular junctions, the motor neurons are severed from the CNS and the axons are stimulated with a suction electrode while monitoring the postsynaptic responses in the muscle fibers with an intracellular electrode. The amplitude of the excitatory junction potentials (EJPs) may be used as an indication of synaptic efficacy in these preparations. Applications of a low

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concentration of dopamine (10 µM) result in a transient increase in the EJP amplitude, followed by depression. Higher concentrations of dopamine (1 µM) result in a rapid decline in the EJP amplitude (Fig. 10.6). In most cases, washing and exchanging the bathing medium back to physiological saline can reverse the depression caused by dopamine.

Sensory Systems Dopamine has not been considered to play a role in signaling systems outside the central nervous system. However, Hall and colleagues have shown that the dopamine receptor DopR99B is also found in peripheral targets, since transcripts are expressed in appendages (legs and antennae), although not in bodies.20 Other molecules within the G-proteincAMP signaling cascade are also found in peripheral tissues; e.g., Gsα in the eye.24 Preliminary work on DTH suggests that it is expressed in specific domains within the developing imaginal discs (Neckameyer W, Mayer A manuscript in preparation), and that depletion of dopamine in larval life results in behavioral perturbations in the adult fly. These initial results suggest that dopamine may also play a role in development of normal sensory tissues.

Dopamine and Fertility There is substantial evidence that dopamine is required for normal female gonadal development. Drosophila tyrosine hydroxylase mRNA is expressed in the nurse cell cytoplasm and within follicle cells through at least the first 12 stages of egg chamber development.47 Since DDC, DTH and PU are coexpressed in the developing ovarian egg chambers, it seems highly probable that dopamine is produced in this tissue in situ. No tyrosine hydroxylase mRNA expression is detected in the mature eggs; this reflects the likelihood that the protein rather than the mRNA is transported into the oocyte cytoplasm, which is what has been found for the product of the Pu gene.51 Evidence that dopamine is required for normal gonadal development (beyond the expression patterns of the biosynthetic genes) is two-fold: Larvae depleted of dopamine for one day during the second instar larval stage were significantly less fertile relative to control animals; and reduction of dopamine levels in newly eclosed females, when the ovaries are not yet mature, yielded small ovaries with shrunken oocytes (Fig. 10.7; see also ref. 47). Females so treated are significantly less fertile than controls.42 The catalytic subunit for the cAMP-dependent protein kinase, DC0, is required for normal fertility as well as for viability (the maternal DC0 contribution persists through at least the first half of embryogenesis; zygotic mutations result in lethality during the first larval instar).30 At least part of the DC0 mutant ovarian phenotype resembles that seen for ovaries from dopamine-depleted females: the mature eggs appear reduced in size, with apparent degeneration of the nurse cells.49 It has been suggested that these defects in oogenesis arise from a single PKA signaling pathway. It is interesting to speculate that the reduction in fecundity in dopamine-depleted females may occur via perturbations in this specific cAMP-mediated transduction cascade. It is also possible that the reduced female sexual receptivity seen in dopamine-depleted females may be directly linked to the ovarian phenotype. Newly eclosed females, although very attractive to males, are not sexually mature and reject the male’s overtures; the behavior of the dopamine-depleted females resembles that of newly eclosed virgins. Perhaps the behaviors signaling sexual receptivity are initiated only after sexual maturity is complete. Dopa decarboxylase is apparently not required for fertility, although the gene is expressed and protein activity can be detected in ovaries;16 again, it is possible that another decarboxylase functions in this tissue to generate dopamine.

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Fig. 10. 7. Feulgen staining of ovaries from control and dopamine-depleted females. Newly eclosed females were maintained for 48 hours on 2% yeast, 5% sucrose (control, A) or yeast/ sucrose plus 10 mg/ml of the tyrosine hydroxylase inhibitor 3-iodotyrosine (B). The ovaries were then hand-dissected and subjected to Feulgen staining. Samples were photographed and enlarged at the same magnification. Although tyrosine hydroxylase is expressed in male gonadal tissues (Neckameyer W, Mayer A, manuscript in preparation), there is no requirement for dopamine in male fertility.42

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Dopamine as a Developmental Signal in Other Tissues It seems likely that dopamine is required for early embryonic development in Drosophila since many components of the dopamine biosynthetic pathway and signaling machinery are present as maternal contributions in the early embryo. As mentioned above, tyrosine hydroxylase, dopa decarboxylase and GTP cyclohydrolase I (the products of the ple, ddc and Pu genes, respectively) are found in the Drosophila embryo previous to the start of zygotic transcription. Ovaries contain the same ddc transcript as do 2 hour old embryos; this mRNA is undetectable by four hours of embryogenesis, suggesting a maternal role.34 The dopamine receptor DopR35EF is apparently also expressed as a maternal transcript since the mRNA is detected in the syncytial blastoderm.19 A functional role for dopamine in early development is suggested by the observation that adult females depleted of dopamine as a consequence of exposure to the tyrosine hydroxylase inhibitor 3-iodotyrosine produce large numbers of inviable eggs;47 treatment with α-methyl-p-tyrosine revealed that almost half of the fertilized embryos were arrested in embryonic stages ranging from 4 to 11 hours after egg laying (the beginning stages of organogenesis).52 Since zygotic expression of tyrosine hydroxylase is not detected until 10 hours of embryogenesis, either the maternal biosynthetic machinery for dopamine synthesis is maintained until that time, or there is no requirement for dopamine until later in embryonic development.47 This is consistent with previous studies showing that DA can be detected in ovaries prior to egg laying, and then not until after 15 hours of embryogenesis (see ref. 16 for review). This is similar to the transgenic tyrosine hydroxylase-knockout mice, where TH function is required for viability during late embryonic development and just after birth.40,48 Additional evidence suggesting that dopamine is critical during Drosophila development comes from mosaic studies done with Ddc+-Ring X chromosome-bearing flies. In this study, 45% of flies with some proportion of Ddc-null tissues die before the adults eclose from the pupal case.17 Reduced larval dopamine levels result in significant lethality and an increase in developmental time until eclosion.47 The surviving flies display no obvious defects in cuticular structure or in pigmentation, and larvae with reduced (or no detectable) dopamine levels appear normal in all tested behavioral paradigms.47 These results strongly suggest that dopamine is required for the normal development of specific non-neuronal, non-cuticular tissues. In late stage embryos, DTH is expressed in a subset of ectodermally-derived tissues, only some of which secrete cuticle, including the CNS, the foregut and the hindgut. However, ple embryos die as unhatched larva, suggesting a vital requirement beyond a role in cuticle formation for dopamine. The exact nature of this requirement remains to be elucidated. L-DOPA is metabolized to other aminergic derivatives that are used to crosslink proteins and chitin during cuticular development. This is termed sclerotization, and is essential for cuticular integrity. In general, the biogenic amine (either L-DOPA or dopamine) is oxidized to form a quinone, which then interacts covalently to both chitin as well as to the amino groups of cuticular proteins.16

Conclusion In addition to its fundamental role as a transmitter in nervous tissue, there is growing evidence to suggest a vital requirement for dopamine in development, as well as an involvement for dopamine in the physiology of peripheral structures. These multiple roles for dopamine, some first described in Drosophila, appear to be evolutionarily conserved across species. Although dopamine may modulate diverse aspects of learning acquisition in different species, its role in the modulation of this kind of behavior may arise from an ancient signaling pathway. Similarly, the varied roles for dopamine in Drosophila reproduction

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(female sexual receptivity and ovarian development) have also been described in diverged species. This is not to imply that every requirement for dopamine has been conserved. In insects, dopamine is crosslinked into the external cuticle to help provide structural integrity; there is no described analagous role in vertebrate species. However, cuticular sclerotization is a structural rather than a signaling role for dopamine, and likely reflects a specific adaptative use. Therefore, we suggest that across species, dopamine has been recruited as a signal in many of the same diverse physiological contexts, including behavior, fertility, reproduction, and in the development of neuronal and non-neuronal tissues.

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19. Sugamori K, Demchyshyn L, McConkey F et al. A primordial dopamine D1-like andenylyl cyclase-linked receptor from Drosophila melanogaster displaying poor affinity for benzaepines. FEBS Lett 1995; 362:131-138. 20. Feng G, Hannan F, Reale V et al. Cloning and functional characterization of a novel dopamine receptor from Drosophila melanogaster. J Neurosci 1996; 16:3925-3933. 21. Han K, Millar N, Grotewiel et al. DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies. Neuron 1996; 16:1127-1135. 22. Reale V, Hannan, F, Hall L et al. Agonist-specific coupling of a cloned Drosophila melanogaster D1-like dopamine receptor to multiple second messenger pathways by synthetic agonists. J Neurosci 1997; 17:6545-6553. 23. Quan F, Forte M. Two forms of Drosophila melanogaster Gsα are produced by alternative splicing involving an usual splice site. Mol Cell Biol 1990; 10:910-917. 24. Quan F, Wolfgang W, Forte M. The Drosophila gene coding for the a subunit of a stimulatory G protein is preferentially expressed in the nervous system. Proc Natl Acad Sci USA 1989; 86:4321-4325. 25. Quan F, Thomas L, Forte M. Drosophila stimulatory G protein a subunit activates mammalian adenylyl cyclase but interacts poorly with mammalian receptors: Implications for receptor-G protein interactions. Proc Natl Acad Sci USA 1991; 88:1898-1902. 26. Livingstone M, Sziber P, Quinn W. Loss of calcium/calmodulin responsiveness in adenylate cyclase of rutabaga, a Drosophila learning mutant. Cell 1984; 37:205-215. 27. Levin L, Han P. Hwang P et al. The Drosophila learning and memory gene rutabaga encodes a Ca2+/calmodukin-responsive adenylyl cyclase. Cell 1992; 68: 479-489. 28. Kalderon D, Rubin G. Isolation and characterization of Drosophila cAMP-dependent protein kinase genes. Genes Dev 1988; 2:1539-1556. 29. Foster J, Higgins G, Jackson F. Cloning, sequence and expression of the Drosophila cAMP-dependent protein kinase catalytic subunit gene. J Biol Chem 1988; 236:1676-1681. 30. Melendez A, Li W, Kalderon D. Activity, expression and function of a second Drosophila protein kinase A catalytic subunit gene. Genetics 1995; 141:1507-1520. 31. de Belle S, Heisenberg M. Associative odor learning in Drosophila abolished by chemical ablation of mushroom bodies. Science 1994; 263:692-695. 32. Dewhurst S, Croker S, Ikeda K et al. Metabolism of biogenic amines in Drosophila nervous tissue. Comp Biochem Physiol 1972; 43B: 975-981. 33. Budnik V, White K. Catecholamine-containing neurons in Drosophila melanogaster: Distribution and development. J Comp Neurol 1988; 268:400-413. 34. Gietz R, Hodgetts R. An analysis of dopa decarboxylase expression during embryogenesis in Drosophila melanogaster. Dev Biol 1985; 107: 142-155. 35. Budnik V, Martin-Morris L, White K. Perturbed pattern of catecholamine-containing neurons in mutant Drosophila deficient in the enzyme dopa decarboxylase. J Neurosci 1986; 6:3682-3691. 36. Flanagan T. Wholemount histofluorescence of catecholamine-containing neurons in a hemipteran brain. J Insect Physiol 1984; 30:796-804. 37. Bicker G, Menzel R. Chemical codes for the control of behavior in arthropods. Nature 1989; 337:33-39. 38. Neckameyer W. Dopamine and mushroom bodies in Drosophila melanogaster: Experiencedependent and -independent aspects of sexual behavior. Learning Memory 1998: 5:157165. 39. Budnik V, White K. Genetic dissection of dopamine and serotonin synthesis in the nervous system of Drosophila melanogaster. J Neurogenet 1987; 4:309-314. 40. Kobayashi K, Morita S, Sawada H et al. Targeted disruption of the tyrosine hydroxylase locus results in severe catecholamine depletion and perinatal lethality in mice. J Biol Chem 1995; 270:27235-27243. 41. Tempel B, Livingstone M, Quinn W. Mutations in the dopa decarboxylase gene affect learning in Drosophila. Proc Natl Acad Sci USA 1984; 81:3577-3581. 42. Neckameyer W. Dopamine modulates female sexual receptivity in Drosophila melanogaster. J Neurogenet, 1998; 12:101-114.

43. Tompkins L, Hall J. Identification of the brain site controlling receptivity in mosaics of Drosophila melanogaster. Genetics 1983; 103:179-195. 44. Wilson C, Thody A, Hole D et al. Interaction of estradiol, α melanocyte stimulating hormone and dopamine in the regulation of sexual receptivity in the female rat. Neuroendocrinol 1991; 54:14-22. 45. Mani S, Allen J, Clark J et al. Modulation of ligand independent activation of the human estrogen receptor by hormone and antihormone. Science 1994; 265:1246-1250. 46. Tunnicliff G, Rick J, Connolly K. Locomotor activity in Drosophila—V. A comparative biochemical study of selectively bred populations. Comp Biochem Physiol 1969; 29:1239-1245. 47. Neckameyer W. Multiple roles for dopamine in Drosophila development. Dev Biol 1996; 176:209-219. 48. Zhou Q, Palmiter R. Dopamine-deficient mice are severely hypoactive, adipsic and aphagic. Cell 1995; 83:1197-1209. 49. Lane M, Kalderon D. Genetic investigation of cAMP-dependent protein kinase function in Drosophila development. Genes Dev 1993; 7:1229-1243. 50. Cooper R, Stewart B, Wojtowicz J, Wang S, Atwood H. Quantal measurement and analysis methods compared for crayfish and Drosophila neuromuscular junctions and rat hippocampus. J Neurosci 1995; 61:67-79. 51. Chen X, Reynolds E, Ranganakulu G et al. A maternal product of the Punch locus of Drosophila melanogaster is required for precellular blastoderm nuclear divisions. J Cell Science 1994; 107:3501-3513. 52. Pendelton R, Rasheed A, Hillman R. The initial site of catecholamine action during embryogenesis in Drosophila melanogaster. Pharmacologist 1997; 39:43.

CHAPTER 11

Genetic Analysis of Dopaminergic Neurons in the Nematode Caenorhabditis elegans Robyn Lints and Scott W. Emmons

W

hy does a cell become a dopaminergic neuron? More generally, why do cells express certain differentiated characteristics and not others? This broad question can be approached in the nematode C. elegans because all the cells are visually identifiable, and the cell lineages and patterning events that lead to their differentiation can be probed genetically. In C. elegans, 14 of 381 neurons plus 4 non-neuronal cells express dopamine. Six of the dopaminergic neurons, all sensory neurons, arise during embryogenesis. While the cell fate specification process occurring during early C. elegans embryonic blastomere formation has been studied extensively,1 the more complex late embryonic cell lineages giving rise to neurons are less well understood. More information is available for the eight dopaminergic sensory neurons and four dopamine-containing non-neuronal cells that arise postembryonically. For these cells, genes required for specification of cell fate, as well as for defining specific neuronal characteristics, have been identified by mutant screens. The patterning processes that have been uncovered so far are similar to those known to pattern much larger nervous systems in insects and vertebrates. Hence, studies in C. elegans may shed light on how dopaminergic neurons arise in more complex animals. We have recently initiated studies focused specifically on development of the C. elegans dopaminergic neurons. In this article, we first give a general description of these neurons, their role in C. elegans behavior, and some general aspects of their development. We next describe a “bottom up” approach to isolate the genes involved in biosynthesis and metabolism of dopamine and study their regulation. How these genes are turned on only in specific neurons, and how their regulation is integrated with other neuronal characteristics, should provide insight into how the dopaminergic fate is determined. Finally, we discuss the regulatory pathways known to define cell fates, including the dopaminergic fate and expression of the genes of dopamine metabolism, in the cell lineages leading to dopaminergic neurons. Progress in understanding cell fate specification in the lineages leading to one class of dopaminergic neurons, the ray neurons, has recently been reviewed.2 The development of two other types of C. elegans neurons have been extensively studied: the serotonergic hermaphrodite-specific neurons (HSN neurons), and the mechanosensory neurons sensitive to light touch.3,4 A general description of the C. elegans nervous system may be found in ref. 5. More recent reviews of various aspects of C. elegans nervous system function and development may be found in ref. 6. The Development of Dopaminergic neurons, edited by Umberto di Porzio, Roberto Pernas-Alonso, and Carla Perrone-Capano. ©1999 R.G. Landes Company.

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C. elegans Dopaminergic Cells: Structure and Function The 14 C. elegans neurons containing dopamine are illustrated in Figure 11.1. Eight are members of a single class of putative mechanosensory neurons present in both sexes, and six are members of a second class of sensory neurons found only in the adult male. Four additional non-neuronal cells are a component of copulatory structures in the male tail. Dopamine is detected in all these cells in whole worms by a formaldehyde-induced fluorescence (FIF) assay,7 as well as by expression of a reporter gene for the key dopamine biosynthetic enzyme tyrosine hydroxylase (see below) (unpublished results). The eight putative mechanosensory neurons containing dopamine are present in 4 sensilla in the head and in 2 pairs of lateral sensilla (Fig. 11.1). (Sensillum is the term used with nematodes for a simple sensory structure consisting of one or a few sensory neurons and support cells8). The head sensilla, known as cephalic sensilla, contain the dopaminergic CEP neurons. The dorsal left/right (L/R) pair of cephalic sensilla contains the CEPd(L/R) neurons, and the ventral pair contains the CEPv(L/R) neurons. The anterior pair of lateral sensilla are known as the deirids and contain the dopaminergic ADE(L/R) neurons. The posterior pair are the postdeirids and contain the dopaminergic PDE(L/R) neurons. The CEP and ADE neurons arise embryonically, whereas the PDE neurons develop from postembryonic cell lineages. CEP, ADE, and PDE are thought to represent a single type of mechanosensory neuron because they have similar sensory endings containing arrays of microtubules lying underneath the cuticle (Fig. 11.2). These neurons may directly or indirectly allow the animal to mechanically detect the presence of food. When crawling on an agar surface, nematodes slow when they encounter a bacterial lawn and begin to feed. This behavioral change is lost in cat-2 mutants lacking dopamine (see below), and in animals from which the CEP, ADE, and PDE neurons have been destroyed by laser ablation.9 Thus, as in higher systems, dopamine appears to be involved in modulation of behavioral circuits and may even implement a kind of primitive reward function. CEP, ADE, and PDE make connections to command interneurons that control locomotion; thus, sensory function is linked to control of motor behavior.10,5 However, how these circuits operate is not known in detail. The second class of dopaminergic cells comprises three bilateral pairs of sensory neurons in the male tail, R5A(L/R), R7A(L/R), and R9A(L/R) (Fig. 11.1). These sensory neurons are contained within the rays, which are male-specific sensilla required for mating.11 There are nine pairs of rays, each containing the endings of two sensory neurons held by a support cell at an opening through the cuticle (Fig. 11.2). The two ray neurons are of two types, distinguished by their ultrastructure.12 The A-type neuron (RnA, n = 1-9 for the nine rays) ends inside the ray tip with a blunt dendritic ending. The dendritic process contains a long striated rootlet. The B-type neuron (RnB) has an electron-dense, pointed ending situated within the opening of the ray to the outside. The cell bodies of the B-type neurons contain dilated cisternae. Rays are numbered from anterior to posterior and can be discriminated by their stereotypical positions in an acellular fan (Fig. 11.1). It is the A-type neurons of rays 5, 7, and 9 that contain dopamine.12 One of the two neurons in rays 1, 3, and 9 contains serotonin;13 whether it is the A- or B-type is not known. The neurotransmitters expressed by the remaining ray neurons are also not known. The rays allow the male to sense contact of his tail with the hermaphrodite. Because the B-type neurons end at an opening to the outside, it is assumed that they are chemosensory, probably signaling the presence of moeities on the hermaphrodite cuticle. The A-type neurons, on the other hand, might be mechanosensory and detect movement of the acellular fan in which the rays are held. Axonal targets of the ray neurons include male-specific interneurons, but the wiring of the male has not been fully determined as it has been for the hermaphrodite.12

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Fig 11.1. Dopaminergic neurons of C. elegans. Shown is a lateral view of an adult male. The head is on the left, the tail carries the copulatory fan containing rays. All of the 14 dopaminergic neurons are sensory neurons present in bilateral pairs (L/R). The sensory endings of the CEPd(L/R) and CEPv(L/R) lie in cephalic sensilla just outside the lips that surround the mouth. Axonal output is in the nerve ring. The ADE(L/R) and PDE(L/R) are respectively in the deirid and postdeirid sensilla. Cephalic, deirid, and postdeirid neurons are present in both sexes. Sensory endings of the male-specific neurons R5A(L/R), R7A(L/R), and R9A(L/R) extend into the corresponding rays; axon output is in the preanal ganglion.

On sensing a hermaphrodite with its tail, a male stops swimming forward and commences execution of a copulatory behavioral sequence.14 This response requires the rays. Copulation starts with the male pressing his tail against the hermaphrodite and commencing to swim backwards. As he does so, he slides his tail along the hermaphrodite body in a search for the vulval opening. Liu and Sternberg14 studied the individual functions of the rays during male mating by selective laser ablations. They reported that ablation of the dopaminergic ray neurons resulted in the males executing sloppy turns if they reached the end of the hermaphrodite without finding the vulva. Thus dopamine may play a role in proper execution of one of the steps of the mating program, the turn around the end of the hermaphrodite. Loer and Kenyon,13 on the other hand, reported that cat-2 males, which lack dopamine, made relatively normal turns, but had a different defect. They tended not to stop on initially encountering the hermaphrodite, but continued moving forward, appearing disinterested. This behavioral defect suggests a role for dopaminergic ray neurons in inhibiting competing behavioral repertoires in a reward function similar to that postulated for the CEP, ADE, and PDE neurons when the worm encounters food. Most likely, the dopaminergic ray neurons function in several steps of the copulatory program. The third class of dopaminergic cells is a component of specialized male mating structures known as spicules. The spicules, normally held within the proctodeal cavity, are inserted by the male into the hermaphrodite vulva to anchor him there during sperm transfer. We have recently found that the spicule socket cells stain positively for dopamine in the FIF assay, and also express a tyrosine hydroxylase reporter gene (unpublished results; Jiang L, Sternberg P, personal communication). The function of dopamine in these cells is unknown. Specification of cell fates in the lineages leading to the spicules has been studied both genetically and by cell ablations.15,16 However, their expression of dopamine has not been investigated, and these cells are not treated further in this article.

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Fig. 11.2. Sensory endings of dopaminergic sensory neurons. A: Sensory ending of CEP neuron. The putatively mechanosensory CEP neurons have ciliated endings lying outside the hypodermis, beneath the cuticle. The opening through the hypodermis is created by a socket (so) and sheath (sh) cell. CN, a small nubbin extending into the cuticle; TAM, microtubule-associated material. Bar = 1.0 µm. B: Cutaway view of the tip of a ray, showing how the endings of the RnA and RnB neurons are held by the structural cell, Rnst, at an opening to the exterior. A reprinted with permission from Perkins LA, Hedgecock EM, Thomson JN et al. Dev Biol 1986; 117:456-487. ©Academic Press, 1986.

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Sex-Specific Development Because the rays and spicules are male-specific structures, analysis of their development is relevant to the question of how a nervous system becomes sexually dimorphic. C. elegans males differ from hermaphrodites not only in morphology, but also in their ability to execute copulatory behavior. This behavior arises in part from 87 male-specific neurons and 36 neuronal support cells, including the dopaminergic ray neurons and spicule cells. At hatching, males and hermaphrodites are nearly identical. Male-specific cells arise from precursor cells that divide postembryonically only in males, or that divide following male-specific division patterns.11,17 Therefore, the nervous system becomes dimorphic because additional cell divisions give rise to new neurons, although rewiring is also known to occur, and additional differences will undoubtedly be found when the male nervous system is reconstructed. Male-specific cell lineages are under the control of the global sex determination pathway. The terminal regulator is the Zn-finger transcription factor TRA-1.18 In animals with two X chromosomes, TRA-1 is active and promotes hermaphrodite development while repressing male development. In animals having a single X, TRA-1 is inactive and development follows the male pathway. Downstream of the global factor TRA-1 are expected to be regulatory factors acting in subsets of tissues. One of these, the putative transcription factor MAB-3, is necessary for development of the rays.19 MAB-3 is similar in sequence and in function to Drosophila doublesex, and there is also a conserved human gene expressed in testes.20 Thus, the sexual dimorphism of the C. elegans nervous system may be governed by an ancient mechanism which allows for development of new neurons, including dopaminergic neurons, and new behavioral circuits.

Hierarchical Specification of Neuronal Properties and the Role of bHLH Transcription Factors How many different developmental pathways contribute, possibly independently of one another, to the definition of the various properties of a neuron? Some cellular functions, such as the extension of processes, are shared by virtually all neurons. Others may be specific to a class, such as motor neurons or sensory neurons of a particular modality, and still others, such as axon targeting, may by unique to a single neuron. The ray dopaminergic neurons illustrate the hierarchical and independent nature of such properties. All the A-type ray sensory neurons have a similar ultrastructure and clearly are sensory neurons of a single type. Yet only a subset within this class express the neurotransmitter dopamine. Thus we may investigate independently what genes define the neuronal fate, what genes define the neuron type, and what genes define selection and expression of neurotransmitter. The genetic instructions that define neurons may reflect this hierarchical nature of neuronal properties. A high level function may direct a cell to express generic neuronal properties. More restricted pathways may then be expressed to define specific aspects of cellular phenotype. In C. elegans, as in other animals, bHLH (basic helix-loop-helix) transcription factors related to the achaete/scute gene family of Drosophila appear to act at a high level in a genetic hierarchy leading to neuronal development. A gene of this type in C. elegans is the bHLH gene lin-32. In lin-32 mutants, many neurons, including all of the dopaminergic neurons, can be absent (Kenyon C, Hedgecock E, personal communication).4,21 The effects of lin-32 mutations are incompletely penetrant and are enhanced by mutations in a second bHLH protein, the product of the gene hlh-2 (D. Portman and S.W. Emmons, unpublished). HLH-2 is a homolog of Drosophila daughterless and mammalian E proteins.22 Thus LIN-32 may have a heterodimerization partner of the da/E class as do achaete-scute genes of other species.23

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Fig. 11.3. Lineage transformations in lin-32 mutants. The achaete/scute family gene lin-32 is necessary for development of many neurons, including dopaminergic neurons. Shown are the wild type (left) and mutant (right) cell lineages. Ray precursor cells, Rn (n = 1-9), give rise to the ray sublineage (top left). The V5.pa neuroblast gives rise to the postdeirid (middle left). The Q neuroblast gives rise to mechanosensory neurons (bottom left). The lineal relationships of Rn cells and V5.pa are shown in Figure 11.6. Q is the sister cell of V5. In lin-32 loss-of-function mutants, the neuroblasts give rise to hypodermal cells (right-hand lineages, open circles). The scales on the left show the hours of postembryonic development and the corresponding postembryonic stages.

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The postembryonic PDE and RnA neurons are absent in lin-32 mutants because the neuroblast that generates them adopts a hypodermal fate (Fig. 11.3). Abnormalities have also been observed in the lineages leading to CEP neurons, but these abnormalities and the defects in pathways leading to other neurons have not been fully described (Kenyon C, Hedgecock E, personal communication). lin-32 is widely expressed in the developing nervous system.24 Because many neuron types are affected, and because the known cell fate transformations in lin-32 mutants affect cells two generations before the birth and differentiation of neurons, lin-32 and hlh-2 appear to act at a high level in a neurogenic hierarchy and are candidates for genes that initiate neurogenic programs in non-neuronal cells. The similarity between C. elegans and Drosophila in the nature of the affected cell lineages, the structures of the sensilla affected, and the fate transformations in mutants, all suggest the programs initiated by achaete/scute genes may be ancient ones common to many animals.21,23

Regulatory Genes Specifying Neuronal Differentiation Little is known about the genes that comprise the genetic subprograms that are initiated by achaete/scute transcription factors. Two genes that act downstream of lin-32 in C. elegans are unc-86 and vab-3. Both of these genes encode transcription factors with conserved homologs in other organisms that function during nervous system development. unc-86 is necessary to direct neuronal differentiation away from pathways that lead to dopaminergic neurons, while vab-3 is necessary for assembly of sensory structures containing dopaminergic neurons. unc-86 encodes a transcription factor of the POU class. It is related to genes of the mammalian Brn-3 family known to act in neuronal differentiation pathways in the mammalian CNS.25,26 In unc-86 loss-of-function mutants, there are extra dopaminergic cells. This defect was traced to lineage reiterations resulting when daughter cells in certain lineage branches failed to assume their correct new states, remaining instead in the state characteristic of their mother and repeating their mother’s division pattern.27 UNC-86 is expressed in the sister cells or lineage branches of CEPd(L/R), ADE(L/R), and PDE(L/R) neurons, and in the aunts of CEPv(L/R).28,29 In unc-86 loss-of-function mutants, these non-dopaminergic lineage branches or cells assume the characteristics of the related branches or cells that express dopamine and generate extra dopaminergic neurons. Thus unc-86 blocks development of dopaminergic neurons. unc-86 also functions downstream of lin-32 in neuronal lineages that do not contain dopaminergic branches, while UNC-86 is expressed widely in the developing nervous system and in differentiated neurons.4,25 Thus unc-86 function is not confined to the differentiation of any particular type of neuron. Its role is most fully understood in the development of mechanosensory neurons sensitive to light touch. In touch neurons, UNC-86 acts as a cofactor of a LIM-type transcription factor in activation of neuronal differentiation genes specific to this class of cells.30 It seems likely that similar transcription factors, possibly of the POU family, that act combinatorially to define neuronal properties will be identified in the pathways leading to the dopaminergic and other types of neurons. vab-3 mutations define one isoform or set of isoforms of the C. elegans homolog of the conserved Pax-6 transcription factor.31,32 Pax-6 is involved in many patterning events in development of the nervous systems of vertebrates and insects, and in particular plays an important role in development of the eye and olfactory epithelium.33 In vab-3 mutants, the sensory structures of the head, including the cephalic sensilla that contain the CEP neurons, are disorganized.34 Sensory neurons and support cells are present and differentiated, yet they are unassembled or assemble abnormally. PAX-6 protein is present in head neurons as they differentiate and in some neurons throughout adulthood.35 Thus, in these neurons, Pax-6 may act at a late stage of the neuronal hierarchy as a differentiation transcription

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factor. Its role appears to be to regulate expression of cell recognition or adhesion functions that guide each neuron and support cell to make the correct heterotypic cell contacts necessary for the assembly of a sensilla.

Heterochronic Genes Define Types of Neuronal Subprograms How is a particular neurogenic program selected when a proneural gene such as lin-32 causes an epidermal cell to become a neuroblast? Expression of lin-32 during the second (L2) larval stage in the postembryonic epidermal cell lineage initiated by the V5 neuroectoblast gives rise to a postdeirid containing a PDE dopaminergic neuron (see Fig. 11.6). lin-32 expression in the same lineage later during the third (L3) larval stage results in generation of rays, containing neurons of types different from the postdeirid neurons, including RnA dopaminergic neurons. How is the decision to make a postdeirid or a ray made? The distinction between postdeirid and ray depends, in part, on the action of heterochronic genes. Heterochronic genes define a regulatory pathway that records the stages of postembryonic development cells have already executed and determines which stage they adopt.36 Mutations of genes in this pathway result in misspecification of developmental time and thus cause cells either to repeat or skip over normal developmental stages. In heterochronic mutants that skip the second larval stage, the neuroblast that normally generates the postdeirid still expresses a neuronal sublineage at the appropriate time, but it is the ray rather than postdeirid sublineage that it expresses.37 Thus, the action of the heterochronic pathway determines the type of neuronal sublineage that is expressed. Whether this pathway alters the subset of implementation genes that are activated by lin-32, or instead alters the activities of the implementation genes by altering their targets, is an interesting question for future studies.

Genes of Dopamine Biosynthesis, Metabolism and Utilization One way to approach these complex questions of neuronal specification is to start with the differentiation genes themselves and work backwards through the regulatory hierarchy. We have initiated such an approach to understand dopaminergic neuron specification in C. elegans. Differentiation genes expected to be expressed by all dopaminergic neurons include genes encoding enzymes or structural proteins required for dopamine biosynthesis, packaging, transport, reuptake and possibly degradation (Fig. 11.4). Among these, the gene for tyrosine hydroxylase, which converts tyrosine to L-DOPA, is expected to be unique to cells containing catecholamines. The gene for this enzyme in C. elegans is cat-2. A mutation in cat-2 causes loss of dopamine in all cells.7 We have shown that this mutation is a nonsense mutation expected to result in an inactive, truncated polypeptide (Lints R, Emmons SW, submitted). Other enzymes of the dopamine biosynthetic pathway may be expressed in dopaminergic cells and in cells containing other biogenic amines, such as serotonergic cells. These enzymes include aromatic L-amino acid decarboxylase (AAAD), which converts L-DOPA to dopamine and 5-hydroxytryptophan to 5-hydroxytryptamine (serotonin), as well as enzymes of the pterin cofactor biosynthetic pathway, required to synthesize the essential pterin cofactor of amino acid hydroxylating enzymes. The gene for AAAD in C. elegans is bas-1 (Loer C, personal communication). In bas-1 mutants both dopamine and serotonin are absent (Garriga G, personal communication).13 A gene possibly in the pterin-cofactor pathway is cat-4. In a cat-4 mutant, not only are dopamine and serotonin lost, but there are also cuticle defects, possibly resulting from loss of a cuticle crosslinking reaction that also utilizes this cofactor. The defect in serotonin biosynthesis in cat-4 mutants can be rescued

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Fig. 11.4. Biosynthetic pathway of dopamine. Enzymes are shown on the left, the corresponding C. elegans genes on the right. AAAD, aromatic L-amino acid decarboxylase; VMAT, vesicular monoamine transporter.

by exogenous administration of 5-hydroxytryptophan, indicating that the block in cat-4 occurs before the decarboxylation step carried out by AAAD.13 Transportation of dopamine from its site of synthesis to the nerve terminals requires the function of a vesicle monoamine transporter protein (VMAT). The gene for this protein is cat-1.38 Loss-of-function mutations in cat-1 result in reduced levels of dopamine in axons and nerve terminals but not in cell bodies.7 It should be possible to identify additional genes expected to be expressed in dopaminergic cells, such as genes for dopamine reuptake and possibly degradation, as the full C. elegans genomic sequence becomes available. Loss-of-function mutations in none of the known genes results in loss of the affected neurons themselves.7,13 Therefore, expression of neurotransmitter and neuronal activity via these transmitters are not necessary for neuronal differentiation. Transgenes we constructed to express GFP (green fluorescent protein) under the control of cat-2 regulatory sequences are expressed in the 18 known dopaminergic cells and in no other cells (Lints R, Emmons SW, submitted). Gene expression in C. elegans is conveniently assayed in living nematodes by means of GFP reporter genes because the cuticle and body are transparent. We conclude that expression of dopamine by a cell is regulated in part by transcriptional activation of the gene for the key biosynthetic enzyme tyrosine hydroxylase. Expression of VMAT (cat-1) has been studied by both antibody staining and reporter

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gene expression.38 Expression is found in neurons that contain dopamine, serotonin and possibly octopamine, the known monoamine neurotransmitters in C. elegans.39 Expression of the other genes expected in dopaminergic cells has not yet been studied. It seems most likely that a combination of transcription factors found uniquely in dopaminergic cells is responsible for expression of the suite of genes required for utilization of dopamine as a neurotransmitter. Comparisons of the promoter sequences of these genes may make it possible to identify the target sequences for this unique combination.

β Signal Induces Expression of Dopamine by Ray Neurons A TGF-β Expression of dopamine by only three out of the nine pairs of A-type ray neurons shows that specification of neurotransmitter can be made independently of other neuronal characteristics. How is the choice of neurotransmitter made? Our recent results show the dopaminergic ray subset is established by the combined action of a TGF-β signal and Hox genes. Both the TGF-β pathway and Hox genes were implicated in specifying ray properties because of the effects of mutations in the TGF-β pathway and in Hox genes on ray morphogenesis.40,41 The TGF-β pathway in the male tail has been defined by mutations in several of the pathway components (Fig. 11.5). We found that mutations in any of these components result in loss of dopamine expression by R5A, R7A and R9A, as assayed by the FIF assay (Lints R, Emmons SW, submitted). Expression of dopamine by the remaining dopaminergic cells is unaffected. Consistent with this, expression of the cat-2::GFP reporter is lost in the ray neurons but not in other dopaminergic cells (Lints R, Emmons SW, submitted). Therefore, assuming that the reporter faithfully reflects activity of the endogenous gene (a relatively safe assumption in C. elegans), we conclude that a TGF-β signal acts, directly or indirectly, at the level of transcriptional regulation of cat-2, the gene for the tyrosine hydroxylase biosynthetic enzyme, to cause expression of dopamine in a subset of the rays. When does this TGF-β signal act? Figure 11.6 shows the postembryonic cell lineages leading to the rays. We showed earlier, employing a cell ablation technique by means of laser microsurgery, that ray morphological characteristics are established by the time ray precursor cells (Rn cells) divide at the beginning of the third larval stage.40 Temperature-shift studies with a temperature-sensitive allele of daf-4, the gene for the type II TGF-β receptor, have likewise established that ray morphological identity is defined by action of the TGF-β pathway in the Rn cell (Baird SE, personal communication). Similar temperature shift studies to define the time of action in establishment of neurotransmitter identity are in progress.

β Signal Specifies Dopaminergic Cells within an The TGF-β Equivalence Group We imagine that the TGF-β signal required for expression of dopamine by R5A, R7A and R9A acts in one of two ways. It might act instructively to specify which neurons are to use this neurotransmitter. That is, A-type neurons in several or all rays may be capable of being induced to transcribe cat-2 by a TGF-β signal, but only a subset receive the signal and hence express this fate. By this model, spatially patterned expression of the TGF-β ligand accounts for the restricted expression of dopamine among the ray A-type neurons. Alternatively, the rays may be prepatterned by an independent mechanism to express dopamine. Possibly only R5A, R7A and R9A are capable of expressing cat-2 in response to a TGF-β signal. By this model, expression of the ligand need not be spatially restricted, and could be ubiquitous. It acts only as a trigger, or permissive signal, to allow rays to develop along predetermined pathways. In order to discriminate between the instructive and permissive models, we studied the effects of ubiquitous expression of the TGF-β ligand, DBL-1 (Fig. 11.5). Ubiquitous

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Fig. 11.5. The TGF-β pathway in the C. elegans male tail. The role of each of the known genes is shown below the pathway. The C. elegans ligand DBL-1 is a homolog of Drosophila decapentaplegic (dpp) and vertebrate bone morphogenetic proteins (BMP). Smad proteins transduce the signal from the cytoplasm into the nucleus.46 Thanks to Y. Suzuki and W. Wood and to R. Padgett for personal communications regarding dbl-1 and sma-6 respectively.

expression was brought about by heat shock of a strain carrying a DBL-1 transgene in which the endogenous DBL-1 promoter was replaced by a heat shock-inducible promoter. Results indicate that after heat shock, rays 3, 4, 6 and 8, in addition to rays 5, 7, and 9, express both dopamine (by FIF assay) and TH (by cat-2::GFP reporter gene expression) (Lints R, Emmons SW, submitted). Hence, additional rays activate the dopaminergic pathway in response to a TGF-β signal. The simplest interpretation of this result is that such rays do not receive ligand in wild type. Hence we conclude that expression of the ligand is spatially patterned. This conclusion is tentative because we have not measured the level of DBL-1 after heat shock and do not know the effect of high levels of expression. It is possible that, contrary to the above conclusion, the rays are pre-patterned and are normally permissively induced by a low and spatially uniform level of ligand, but that high levels of ligand in the heat shock experiment override this prepatterning. The remaining rays, that is, rays 1 and 2, have not so far been observed to express dopamine or the cat-2 reporter gene after heat shock induction of DBL-1 expression. Assuming the transgene provides DBL-1 uniformly in the region of the rays, this result indicates that some rays are incapable of expressing dopamine in response to a TGF-β signal and hence that the rays are not all equivalent. Thus, in addition to possible spatial patterning of the ligand, there is also a prepatterning of the rays. Those rays capable of expressing dopamine in response to a TGF-β signal we may term a dopaminergic equivalence group. Equivalence groups have been defined in C. elegans as groups of cells that are capable of assuming the same developmental pathway or fate, but which are directed into different pathways by signals.42 Perhaps the best known of these equivalence groups is that which gives rise to the vulva.43 This equivalence group consists of six epidermal cells lying on the ventral midline in the center of the body. Each of these six cells is capable of contributing to vulval development, yet in wild type only three of them do. Expression of vulval fates by these three is selectively induced by the patterned expression of an EGF ligand.44 Thus, we see that vulval induction may be similar to induction of the dopaminergic ray neurons. Equivalence groups raise two questions: What defines the equivalence group, and what is the source of the signal? In the case of the vulva, the EGF ligand is generated by a cell called the anchor cell in the developing gonad, which underlies the region of the presumptive

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Fig. 11.6. The postembryonic cell lineages leading to the rays. A TGF-β signal is required for R5A, R7A and R9A to express dopamine (arrows). The dopaminergic PDE neuron is also indicated by an arrow. At the top is shown an L1 larval worm immediately after hatching. The hypodermal seam cells divide at the times shown on the left-hand scale (hours post-hatching). All the cells are hypodermal except for the differentiating neurons and support cells of the phasmid, postdeirid, and rays. lin-32 is necessary for V5.pa to express the postdeirid lineage and for ray precursor cells R1-R9 to express the ray lineage (see Fig. 11.3). vulva. Examination of the positions of the cells of the three dopaminergic rays shows that they lie on the dorsal side of the cluster of developing ray cells, separated from the cells of the other rays by a row of epidermal cells (Fig. 11.7). A possible source of the TGF-β signal may be sought in this region.

A Hox Gene May Define the Dopaminergic Equivalence Group The answer to the second question raised by equivalence groups, how the group is specified, may also be answered in a similar way for both the vulval equivalence group and the dopaminergic ray equivalence group. The vulval equivalence group is defined by expression of the gene lin-39. lin-39, encoding a homeodomain transcription factor, is a member of the C. elegans Hox gene cluster. In C. elegans there are four genes related to the Hox-cluster genes of insects and vertebrates.29 As in other organisms, these regulatory transcription factor genes are expressed in partially-overlapping domains along the anteroposterior body axis and act to define the region-specific differentiation of serially-repeated cells.

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Fig. 11.7. Positions of the ray cells in the posterior epidermis of an L4 larval male. At the time illustrated, the cell lineages (Fig. 11.6) are completed, but the cells have not yet differentiated. R5A, R7A, and R9A are filled. Their unique dorsal location in the group of cells suggests the inducing TGF-β ligand may come from a dorsal source. Cells were visualized by immunohistochemical staining with an antibody to a component of the cell adherens junction that joins each cell to its neighbors. Most of the nematode surface is covered by a hypodermal syncytium (hyp7). Cells labeled R1.p-R9.p are the posterior daughters of the ray precursor cells (R1-R9, Fig. 11.6). The three unlabelled cells next to each Rn.p cell will differentiate into the A-type neuron, the B-type sensory neuron, and the ray structural cell. In the male tail, the dopaminergic equivalence group may be defined in part by expression of a member of the Hox gene cluster, egl-5. egl-5 encodes an ortholog of Drosophila Abdominal-B. EGL-5 is expressed in ray 5 and in those lineage branches descending from the postembryonic blast cell V6 that can be induced to express dopamine by ectopic expression of TGF-β (Fig. 11.8).45 In an egl-5 mutant, ray 5 fails to express dopamine or the cat-2 reporter gene (Lints R, Emmons SW, submitted).* Hence, we propose that egl-5 may act to define the cells that are capable of responding to TGF-β. What gene or genes allow rays 7, 8 and 9, descended from the T blast cell, to respond to TGF-β are not known.

Conclusion The C. elegans genomic sequence makes it potentially possible by analysis of sequence similarities to identify the genes for all of the components a neuron needs to synthesize and utilize dopamine. Comparisons of the promoters of these genes to each other may allow identification of the cis-acting sequences that respond to the combination of transcriptional determinants that define the dopaminergic state. How these determinants come to be expressed in only certain cells in the nervous system is the challenge for future studies. In C. elegans, there is the possibility of a powerful combination of genetic, molecular, and cellular approaches to identify all the components of the developmental pathway leading to dopaminergic neurons.

* Furthermore, egl-5 is necessary for rays 3-5 to respond to ectopic TGF-β by expressing dopamine.

Fig. 11. 8. Expression patterns of the Hox genes mab-5 (black) and egl-5 (grey) in the cell lineages leading to rays 1-6.45,47 The expression of these two genes determines the morphological properties of the rays.40, 47 Hox genes may also determine neurotransmitter: Lineage branches expressing EGL-5 are the ones that express dopamine in response to TGF-β induction.

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Acknowledgment We thank J. Lipton for his editorial comments and members of the laboratory for discussions. This work was supported by NIH grant GM39353 to S.W.E. S.W.E. is the Siegfried Ullmann Professor of Molecular Genetics.

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Index A

E

Aldehyde dehydrogenase (AHD) 16, 42-44, 46, 126, 131, 133 Amine transporter 149 Anterior visceral endoderm 3, 9

Epiblast 3, 5 Estrogen 75-82, 115, 164

C

Female sexual receptivity 164, 169, 172 FGF8 7, 15, 24-27, 29, 40-43, 50 Fibroblast growth factor (FGF) 29, 39, 57, 59-61, 64, 67, 70, 123, 124, 130 Floor plate 2, 5, 15, 16, 18, 19, 22, 29, 40

Caenorhaditis elegans 175, 176, 179, 181-187 Cell identity 116, 145 Cell proliferation 114 Calcitonin gene-related peptide (CGRP) 151, 152, 156 Critical period 76-78, 81

D D2L 107-109, 114 D2S 107-109, 114, 118 DAT 38, 43, 45-47, 49, 50, 54, 112, 115 Dopamine (DA) 15-19, 22-30, 37-50, 59, 68, 75-81 Dopamine (DA) receptor 43, 47, 76, 83, 8792, 101, 106, 132, 134, 149, 159, 169, 171 Dopamine (DA) release 43-45, 80, 112, 132, 134, 149 Dopamine (DA) synthesis 16, 43, 45, 46, 80, 148, 171 Dopamine (DAT) transporter 38, 43, 45-47, 49, 50, 54, 112, 115, 149 Dopamine (DA) uptake 45-47, 49, 52, 80, 81 Dopaminergic cell development 60, 66, 67 Dopaminergic cell survival 59-61, 64, 73 Dopaminergic growth factor 61, 64-66 Dopaminergic (DA) neuron 15-19, 23-25, 27, 29, 30, 39-48, 50, 57, 59-61, 64-67, 73, 75-81, 109, 111, 114, 123-126, 128, 131-134, 137, 138, 148, 150, 161, 162, 175, 177, 179, 180-182, 187 Dopaminergic neuron specification 29 Dopaminergic neurotransmission 39, 42-47, 50 Dopaminergic specification 42 Dopaminergic (DA) system 37, 42, 50, 52, 53, 75-79, 81, 101, 111-113, 116, 128, 136, 137,148 Dopaminergic transmission 78, 149 Drosophila melanogaster 157, 160

F

G Ganglionic eminence 88, 92, 124, 132 Gbx2 4, 7 Glia 50, 60, 61, 126, 134, 150 Gonadal steroids 76-79, 81

H Habituation 162 hox 2, 10, 133, 184, 186-188, 190

I Induction 1, 4, 6, 16, 18, 19, 22, 26, 40, 42, 50, 81, 109, 114, 128, 151, 152, 185, 188 Integration 37, 75, 123, 129, 130 Isthmic organizer 7

K Knockout mice 42, 44, 53, 118, 128, 171

L Lateral ganglionic eminence 124, 132 Locomotion 46, 53, 111, 112, 176

The Development of Dopaminergic Neurons

192

M

S

Mesencephalon 1, 39, 41, 44, 46, 47, 50, 57, 59, 60, 64, 66, 67, 81, 124, 125 Midbrain 2, 3, 4, 7, 11, 15-19, 23-29, 31, 37, 39, 41-45, 47, 48, 50, 57, 61, 64, 75-81, 126 Midbrain development 47 Midbrain dopaminergic neurons 16, 25, 45, 57, 61, 64, 73, 75, 78, 80 Midbrain explants 18, 24, 26, 27 Mmidbrain neurons 37, 42-45, 80, 81 Migratory stream 150, 151 Mushroom bodies 160, 162, 164

Sclerotization 171, 172 Serotonergic 16, 22-24, 128, 175, 182 Serotonergic cell 182 Serotonergic (SN) neurons 61, 64, 66, 102, 107, 124-126, 128, 131, 133, 134,140, 189 Sex differences 75-78, 81 Sexual dimorphism 76, 78, 81, 179 SHH 20, 40, 41, 43 Shh 5, 15, 17-22, 24-27, 29 Signal transduction 21, 60, 61, 78, 80, 101, 109, 110, 113, 162 Stem cell 126, 151 Striosome 90, 92, 93, 95 Substantia nigra 16, 37, 46-48, 61, 68, 69, 76, 79, 102, 124, 137 Synaptic vesicle monoamine transporter 43, 44

N Nematode 175, 176, 183, 187 Neurotransmitter status 145 Neurotrophic factor 45, 57, 59, 123, 124 Neurotrophins 45, 59, 69, 72, 86 Nurr1 29, 41-44, 50, 66

O Otx2 3-5, 7, 28 Outgrowth 44, 47, 48, 55, 79, 80, 129-131, 133 Ovarian development 172

P Periglomerular neurons 145, 148-150 Physiology 37, 79, 108, 148, 171 Pituitary 15, 46, 75, 77, 101, 107, 109-111, 114 Pituitary gland 107, 109, 114 Pituitary tumors 101, 115 Porcine 128-131, 136, 137 Ptx3 24, 29, 41-43, 50, 66

R Reproduction 164, 171, 172 Rostral 1, 2, 3, 4, 7, 12, 23-26, 28, 29, 40, 48, 76, 126, 131, 150, 151

T Transforming growth factor (TGF) 18, 60, 67, 69, 72, 124, 184-188 Transforming growth factor β (TGFβ) 18, 73, 187 Transgenic 17, 18, 21-23, 46, 124, 126, 129, 161-163, 165, 171 Transplant 2, 3, 15, 48, 55, 123-137 Tufted cells 14, 145-150 Tyrosine hydroxylase (TH) 16-19, 24-27, 37-44, 46-48, 59, 75-79, 109, 124, 126-129, 131, 133, 134, 136, 147, 148, 150-152, 158-160, 162, 164-166, 169-171, 176, 177, 182, 183-185

V Ventral mesencephalon 31, 39, 41, 44, 46, 47, 51, 64, 125 VMAT2 38, 43-45, 47, 149 Vulnerability 79

X Xenotransplant 128, 129, 131, 136, 137