International Review of Neurobiology Volume 59 Disorders of Synaptic Plasticity and Schizophrenia

International REVIEW OF Neurobiology Volume 59 International REVIEW OF Neurobiology Volume 59 SERIES EDITORS RONALD...

Author: John Smythies

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International REVIEW OF

Neurobiology Volume 59

International REVIEW OF

Neurobiology Volume 59 SERIES EDITORS RONALD J. BRADLEY Department of Psychiatry, School of Medicine Louisiana State University Medical Center Shreveport, Louisiana, USA

R. ADRON HARRIS Waggoner Center for Alcohol and Drug Addiction Research The University of Texas at Austin Austin, Texas, USA

PETER JENNER Division of Pharmacology and Therapeutics GKT School of Biomedical Sciences King’s College, London, UK

EDITORIAL BOARD PHILIPPE ASCHER TAMAS BARTFAI FLOYD E. BLOOM MATTHEW J. DURING PAUL GREENGARD KINYA KURIYAMA HERBERT Y. MELTZER SALVADOR MONCADA SOLOMON H. SNYDER CHEN-PING WU

ROSS BALDESSARINI COLIN BLAKEMORE DAVID A. BROWN KJELL FUXE SUSAN D. IVERSEN BRUCE S. MCEWEN NOBORU MIZUNO TREVOR W. ROBBINS STEPHEN G. WAXMAN RICHARD J. WYATT

International REVIEW OF

Neurobiology Volume 59 EDITED BY

JOHN SMYTHIES Center for Brain and Cognition University of California, San Diego La Jolla, California 92093 Department of Neuropsychiatry Institute of Neurology Queen Square, London

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ISBN: 0-12-366860-3 PRINTED IN THE UNITED STATES OF AMERICA 04 05 06 07 08 9 8 7 6 5 4 3 2 1

CONTENTS

Contributors............................................................................ Introduction ............................................................................

xi xv

Loss of Spines and Neuropil Liesl B. Jones I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Introduction . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Dendrites, Spines, and Normal Plasticity. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Effects of Estrogen on Plasticity . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Long-Term Potentiation and Learning. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Alterations in Neuropil Components . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Alzheimer’s Disease. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Human Immunodeficiency Virus Encephalitis . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Huntington’s Chorea. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Schizophrenia . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Possible Link between Calcium and Dendritic Alterations. . . . . . . . . . . . . . . . .. Conclusion . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. References . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ..

1 2 3 4 6 6 7 8 9 11 12 13

Schizophrenia as a Disorder of Neuroplasticity Robert E. McCullumsmith, Sarah M. Clinton, and James H. Meador-Woodruff I. II. III. IV. V. VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . Clinical Features of Schizophrenia. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . Synaptic Plasticity and Schizophrenia . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . Substrates of Neuroplasticity . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . Glutamatergic Dysfunction in Schizophrenia . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .

v

19 20 21 23 25 39 39

vi

CONTENTS

The Synaptic Pathology of Schizophrenia: Is Aberrant Neurodevelopment and Plasticity to Blame? Sharon L. Eastwood I. Introduction . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. II. Studies of Proteins Associated with Synaptic Plasticity in Schizophrenia . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. III. Can the Synaptic Pathology of Schizophrenia Be Related to Changes in the Expression of Genes Involved in Development and Plasticity? . . .. IV. Discussion and Future Directions . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. References. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ..

48 49 57 64 65

Neurochemical Basis for an Epigenetic Vision of Synaptic Organization E. Costa, D. R. Grayson, M. Veldic, and A. Guidotti I. II. III. IV. V. VI. VII. VIII.

Introduction. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Conceptual Background for the Definition of Phenotypes and Genotypes Epigenetics and Evolution . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Biochemical Processes Included in Epigenetic Phenomena. . . . . . . . . . . . .. . . Epigenetics and Synaptic Plasticity . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Epigenetics Today . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . The Epigenetic Concept in Psychiatry . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Conclusions . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . References . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . .

73 74 75 75 78 85 86 87 87

Muscarinic Receptors in Schizophrenia: Is There a Role for Synaptic Plasticity? Thomas J. Raedler I. II. III. IV.

Introduction . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Muscarinic Receptors . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Schizophrenia. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Synaptic Plasticity . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. References. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ..

93 94 97 100 103

Serotonin and Brain Development Monsheel S. K. Sodhi and Elaine Sanders-Bush I. II. III. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. The Discovery of Serotonin and Classification of Serotonin Receptors .. The Role of Serotonin in Developmental Plasticity . . . . . . . . . . . . . . . . . . . . . . . .. Manipulation of the Serotonergic System Alters Synaptic Plasticity. . . . . .. Does Dysfunction of Serotonergic Signaling Result in Impaired Brain Development? . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. VI. Conclusions . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. References. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ..

112 113 114 128 132 149 150

CONTENTS

vii

Presynaptic Proteins and Schizophrenia William G. Honer and Clint E. Young I. Introduction . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . II. Presynaptic Proteins . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . III. Are All Presynaptic Proteins Affected Equally within a Single Brain Region? . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . IV. Are Different Brain Regions Affected Equally for a Given Presynaptic Protein? . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . V. What Are the Relationships between mRNA and Protein Findings When Both Are Measured in the Same Study? . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . VI. Microarray Studies . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . VII. Summary .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .

175 176 179 185 185 195 195 196

Mitogen-Activated Protein Kinase Signaling Svetlana V. Kyosseva I. II. III. IV. V. VI. VII.

Introduction . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . Mitogen-Activated Protein (MAP) Kinase Cascades .. . . . . . . . . . . . . . . . . . . . . . . . . Role of MAP Kinases in the Central Nervous System . . . . . . . . . . . . . . . . . . . . . . . . MAP Kinases of Schizophrenia . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . MAP Kinases in the Phencyclidine Rat Model of Schizophrenia. . . . . . . . . . . MAP Kinases and Psychiatric Disorders . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .

202 203 207 209 211 213 214 214

Postsynaptic Density Scaffolding Proteins at Excitatory Synapse and Disorders of Synaptic Plasticity: Implications for Human Behavior Pathologies Andrea de Bartolomeis and Germano Fiore I. Introduction . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . II. Structural and Functional Organization of Postsynaptic Density (PSD) Proteins: An Overview. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . III. PSD-95/SAP90. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . IV. Shank/ProSAP Proteins. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . V. SAP97. . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . VI. Homer Proteins . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . VII. Conclusive Remarks . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .

222 223 227 232 234 237 244 244

viii

CONTENTS

Prostaglandin-Mediated Signaling in Schizophrenia S. Smesny I. II. III. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Prostaglandin Hypothesis of Schizophrenia . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Membrane Phospholipid Hypothesis of Schizophrenia . . . . . . . . . . . . . . . . . . . .. Niacin Tests in the Field of Schizophrenia . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Summary.. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. References. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ..

255 256 257 260 265 266

Mitochondria, Synaptic Plasticity, and Schizophrenia Dorit Ben-Shachar and Daphna Laifenfeld I. II. III. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Plasticity in Schizophrenia . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Mitochondrial Dysfunction in Schizophrenia. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Mitochondria and Neuroplasticity. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Conclusion . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. References. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ..

274 275 279 282 287 288

Membrane Phospholipids and Cytokine Interaction in Schizophrenia Jeffrey K. Yao and Daniel P. van Kammen I. II. III. IV. V.

Abnormal Membrane Phospholipids. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Disturbed Immune Function .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Polyunsaturated Fatty Acids and Cytokines . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Stress and Immune Response . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Conclusion . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. References. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ..

298 306 309 310 311 314

Neurotensin, Schizophrenia, and Antipsychotic Drug Action Becky Kinkead and Charles B. Nemeroff I. Introduction . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. II. Preclinical Evidence Supporting the Role of Neurotensin in the Effects of Antipsychotic Drugs . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. III. Clinical Evidence Supporting the Role of Neurotensin Neurotransmission in the Pathophysiology of Schizophrenia. . . . . . . . . . . . .. IV. NTergic Compounds as Novel Antipsychotic Drugs . . . . . . . . . . . . . . . . . . . . . . . .. References. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ..

328 333 338 339 342

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ix

Schizophrenia, Vitamin D, and Brain Development Alan Mackay-Sim, FranC¸ois FE´ron, Darryl Eyles, Thomas Burne, and John McGrath I. II. III. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . Gene–Environment Interactions . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . Schizophrenia Susceptibility Genes . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . Gene–Environment Models of Schizophrenia . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . Conclusion. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .

352 353 355 360 369 370

Possible Contributions of Myelin and Oligodendrocyte Dysfunction to Schizophrenia Daniel G. Stewart and Kenneth L. Davis I. II. III. IV. V. VI. VII. VIII. IX.

Introduction . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. The Changing Role of Glia . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Background on Oligodendroglia and Myelin . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Disconnectivity in Schizophrenia . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Imaging Evidence for White Matter Involvement in Schizophrenia . . . . . .. Demyelinating Diseases and the Symptoms of Schizophrenia . . . . . . . . . . . . .. Age-Related Changes in Normal Aging . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. Expression of Myelin-Related Genes in Schizophrenia . . . . . . . . . . . . . . . . . . . . .. Direct Examinations of Myelin and Oligodendroglia in Schizophrenia . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. X. Mechanistic Considerations . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. XI. Summary and Future Directions . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. References . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ..

381 382 383 385 388 392 395 396 402 404 407 408

Brain-Derived Neurotrophic Factor and the Plasticity of the Mesolimbic Dopamine Pathway Oliver Guillin, Nathalie Griffon, Jorge Diaz, Bernard Le Foll, Erwan Bezard, Christian Gross, Chris Lammers, Holger Stark, Patrick Carroll, Jean-Charles Schwartz, and Pierre Sokoloff I. Introduction . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . II. Brain-Derived Neurotrophic Factor (BDNF) Control D3 Receptor Expression during Development . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . III. BDNF Triggers Ectopic D3 Receptor Expression and Behavioral Sensitization in Denervated Rats . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . IV. Normalization of Dopamine D3 Receptor Function Attenuates Dyskinesia Induced by Levodopa. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . V. BDNF and Dopamine D3 Receptor in Reactivity to Drug Cues . . . . . . . . . . . . VI. BDNF and Dopamine D3 Receptor in Stress, Depression, and Schizophrenia. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .

425 427 428 431 434 437 438 439

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S100B in Schizophrenic Psychosis Matthias Rothermundt, Gerald Ponath, and Volker Arolt I. II. III. IV. V. VI. VII. VIII.

Background and Hypotheses . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Origin and Molecular Structure of S100B. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . In Vitro/Cell Culture Experiments. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Morphology Studies (Animal Experiments) . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Serotonergic Regulation. . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Functional Studies. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Clinical Studies in Schizophrenic Patients . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Conclusions . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . References . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . .

445 448 449 454 456 457 458 461 463

Oct-6 Transcription Factor Maria Ilia I. POU Domain Proteins . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. II. Oct-6.... . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. III. Conclusions . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. References. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ..

471 475 485 486

NMDA Receptor Function, Neuroplasticity, and the Pathophysiology of Schizophrenia Joseph T. Coyle and Guochuan Tsai I. II. III. IV. V. VI. VII. VIII. IX.

Introduction. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . NMDA Receptors . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Effects of Dissociative Anesthetics . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Postmortem Studies of Glutamatergic Markers in Schizophrenia . . . . . .. . . Glutamate Receptor-Associated Genes . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Preclinical Studies on NMDA Receptor Hypofunction . . . . . . . . . . . . . . . . . . .. . . Clinical Trials of NMDAR Modulators in Schizophrenia. . . . . . . . . . . . . . . . .. . . NMDA Receptor and Neuroplasticity . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . Conclusion . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . References . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . .

Index ........................................................................................ Contents of Recent Volumes .....................................................

491 492 494 495 497 499 500 505 506 507 517 531

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Volker Arolt (445), Department of Psychiatry, University of Muenster, Muenster D-48149, Germany Andrea de Bartolomeis (221), Department of Neuroscience and Behavioural Science, University Medical School of Maples Frederico II, Naples 80131, Italy Dorit Ben-Shachar (273), Laboratory of Psychobiology, Department of Psychiatry, Rambam Medical Center-Technion IIT, Haifa 31096, Israel Erwan Bezard (425), CNRS UMR 5543, Basal Gang, Laboratoire de Neurophysiologie, Bourdeaux 33076, France Thomas Burne (351), School for Biomolecular and Biomedical Science, GriViths University, Brisbane, Queensland 4111, Australia Patrick Carroll (425), INSERM U 382, IBDM, Unite de Developpement et Pathologie du Montoneurone Spinal, Marseille 13228, France Sarah M. Clinton (19), Mental Health Research and Department of Psychiatry, University of Michigan, Ann Arbor, Michigan 48109 Erminio Costa (73), Psychiatric Institute, University of Illinois at Chicago, Chicago, Illinois 60612 Joseph T. Coyle (491), Department of Psychiatry, Harvard Medical School, MacLean Hospital, Belmont, Massachusetts 02478 Ken L. Davis (381), Molecular Neurobiology Branch, NIDA, National Institutes of Health (BPS), Baltimore, Maryland 21224 Jorge Diaz (425), Laboratoire de Physiologie, University Rene Descartesq, Paris 75006, France Sharon Eastwood (47), Department of Psychiatry, Warneford Hospital, Headington, Oxford OX3 7JF, United Kingdom Darryl Eyles (351), The Park Centre for Mental Health, Queensland Centre for Mental Health Research, Wacol, Queensland 4111, Australia Francois Fe´ron (351), School for Biomolecular and Biomedical Science, GriViths University, Brisbane, Queensland 4111, Australia Germano Fiore (221), Department of Neuroscience and Behavioural Science, University Medical School of Maples Frederico II, Naples 80131, Italy

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CONTRIBUTORS

Dennis Grayson (73), Psychiatric Institute, University of Illinois at Chicago, Chicago, Illinois 60612 Nathalie GriVon (425), INSERM U 573, Centre Paul Broca, Unite de Neurobiologie et Pharmacologie Moleculaire, Paris 75014, France Christian Gross (425), CNRS UMR 5543, Basal Gang, Laboratoire de Neurophysiologie, Bourdeaux 33076, France Alessandro Guidotti (73), Psychiatric Institute, University of Illinois at Chicago, Chicago, Illinois 60612 Olivier Guilin (425), INSERM U 573, Centre Paul Broca, Unite de Neurobiologie et Pharmacologie Moleculaire, Paris 75014, France William G. Honer (175), Centre for Complex Disorders, University of British Columbia, Vancouver, British Columbia V5Z 1L8, Canada Maria Ilia (471), Department of Neuroscience, Institute of Psychiatry, London SE5 8AF, United Kingdom Liesl B. Jones (1), Department of Biological Science, Lehman College, CUNY Bronx, New York 10468 Becky Kinkead (327), Department of Psychiatry and Behavioral Medicine, Emory University School of Medicine, Atlanta, Georgia 30322 Svetlana V. Kyosseva (201), Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 Daphna Laifenfeld (273), Laboratory of Psychobiology, Department of Psychiatry, Rambam Medical Center-Technion IIT, Haifa 31096, Israel Chris Lammers (425), INSERM U 573, Centre Paul Broca, Unite de Neurobiologie et Pharmacologie Moleculaire, Paris 75014, France Bernard Le Foll (425), INSERM U 573, Centre Paul Broca, Unite de Neurobiologie et Pharmacologie Moleculaire, Paris 75014, France Alan Mackay-Sim (351), School for Biomolecular and Biomedical Science, GriViths University, Brisbane, Queensland 4111, Australia James H. Meador-WoodruV (19), Mental Health Research and Department of Psychiatry, University of Michigan, Ann Arbor, Michigan 48109 Robert E. McCullumsmith (19), Mental Health Research and Department of Psychiatry, University of Michigan, Ann Arbor, Michigan 48109 John McGrath (351), The Park Centre for Mental Health, Queensland Centre for Mental Health Research, Wacol, Queensland 4111, Australia Charles B. NemeroV (327), Department of Psychiatry and Behavioral Medicine, Emory University School of Medicine, Atlanta, Georgia 30322 Gerald Ponath (445), Department of Psychiatry, University of Muenster, Muenster D-48149, Germany Thomas J. Raedler (93), Klinik fur Psychiatrie und Psychotherapie, Universitatsklinikum Hamburg-Eppendorf, Hamburg 20246, Germany

CONTRIBUTORS

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Matthias Rothermundt (445), Department of Psychiatry, University of Muenster, Muenster D-48149, Germany Elaine Sanders-Bush (111), Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232 Jean-Charles Schwartz (425), INSERM U 573, Centre Paul Broca, Unite de Neurobiologie et Pharmacologie Moleculaire, Paris 75014, France Stefan Smesny (255), Department of Psychiatry, University of Jena, Jena D-07743, Germany Monsheel Sodhi (111), Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232 Pierre Sokolo (425), Service des Prs Loo et Olie, Service Hospilato Universitaire, Paris 75014, France Holger Stark (425), Institute fur Pharmakologie un Toxikologie, Universitat Bonn, Bonn 53113, Germany Daniel Stewart (381), Department of Psychiatry, Mount Sinai Medical Center, New York, New York 10029 Guochuan Tsai (491), Department of Psychiatry, Harvard Medical School, MacLean Hospital, Belmont, Massachusetts 02478 Daniel P. van Kammen (297), Aventis, Inc., Bridgewater, New Jersey 08807; Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania 19104; Department of Psychiatry, Columbia University, New York, New York 10032 JeVrey K. Yao (297), VA Pittsburgh Healthcare System, Pittsburgh, Pennsylvania 15206; Department of Psychiatry, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213; Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 Clint E. Young (175), Centre for Complex Disorders, University of British Columbia, Vancouver, British Columbia V5Z 1L8, Canada

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INTRODUCTION

Over the last ﬁfty years the search for the biochemical basis of schizophrenia has focused on diVerent subjects at diVerent times. The ﬁrst speciﬁc biochemical hypothesis was put forward in 1952 by Osmond and Smythies. This was the transmethylation hypothesis that was based on the chemical similarity between the psychotomimetic drug mescaline and the neurotransmitter adrenaline. Later similar hypotheses were advanced based on the chemical similarity between other psychotomimetic agents, such as dimethyltryptamine and o-methylbufotenin, and the neurotransmitter serotonin. It was supposed that a simple genetically induced metabolic fault might result in the synthesis of some neurotoxic compound, as in the case of phenylketonuria. However, as no such compounds were detected in either the normal or the schizophrenic brain in any meaningful quantity, interest in these hypotheses lapsed. The next candidate hypothesis was the adrenochrome theory of HoVer, Osmond and Smythies (1954). Adrenochrome is the oxidative product of adrenaline and is a psychotomimetic compound (HoVer et al., 1954; Grof et al., 1963). But, as adrenochrome was not detected in vivo, interest in this theory also lapsed. However, recently interest has revived as it is now apparent that adrenochrome may be formed normally in the C2 adrenergic group in the medulla. Some adrenergic neurons in this nucleus are pigmented and the pigment is probably neuromelanin (Gai et al., 1993). In the synthesis of neuromelanin from adrenaline adrenochrome is an essential intermediary metabolite. Furthermore, catecholamine o-quinones (including adrenochrome) are, in part, detoxiﬁed by 5-conjugation with glutathione S-transferase. Harada et al. (2001) report that schizophrenics have an increased frequency of deletion of the gene for this enzyme (p ¼ 0.0075) and an even higher rate in the subgroup of disorganized schizophrenics (p ¼ 0.0008). Neurotoxic catecholamine o-quinones may play a normal role in synapse deletion. There is also now strong evidence to support a role for such o-quinones derived from dopamine in Parkinson’s disease (Smythies 2002). So this hypothesis merits further research. The next hypothesis was the dopamine hypothesis advanced by Arvid Carlsson that was based on the serendipitous discovery that the anti-psychotic drug chlorpromazine is a dopamine-receptor blocker. So it seemed natural to suppose that schizophrenia might be associated with an over-active dopamine system. This was followed by the glutamate xv

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hypothesis. NMDA receptor antagonists such as ketamine induce a syndrome not unlike type 2 schizophrenia. So it seemed plausible that an underactive glutamate system might be involved. Another possibility suggested was an overactive neurotoxic glutamate system early in life resulting in an damaged underactive system later on. Other hypotheses linked to further neurotransmitters such as serotonin have also entered the list. The present book looks at a wider ﬁeld. The seminal ﬁnding has been the discovery that many schizophrenics show a diminution in the number of dendritic spines on neurons in the cortex and striatum, and in the density of the associated neuropil. Therefore, it would be logical to look for abnormalities in the extremely complex biochemical mechanisms that control the development and pruning of dendritic spines (see Smythies 2002 for a review) and in neuroplasticity in general. The authors of the chapters in this book each present an informed review of the latest data in a series of disparate but interlocked individual ﬁelds relevant to this problem. Each chapter presents an up-to-date account in the state of our knowledge in that particular ﬁeld. It is, moreover, of particular interest to note that the 19 diVerent ﬁelds covered in this book range very widely over the extent of brain neurochemistry, yet they are united in that these disparate ﬁelds are all relevant to synaptic plasticity and all show robust abnormal ﬁndings in schizophrenia. It is becoming clear that schizophrenia is not due to a single metabolic fault, as the earlier hypotheses supposed, but to a most complex interaction of a number of interlocked genetic factors and environmental insults. Each of these new understandings oVers fresh promise of quite new and exciting therapeutic advances in the treatment of this disease. John Smythies References Gai, W. P., Geffen, L. B., Denoroy, L., and Blessing, W. W. (1993). Loss of C1 and C3 epinephrine-synthesizing neurons in the medulla oblongata in Parkinson’s disease. Ann. Neurol. 33, 357–367. Grof, S., Vojtechovsky, M., Vitek, V., and Prankova, S. (1963). Clinical and experimental study of central effects of adrenochrome. J. Neuropsychiatr. 5, 33–50. Harada, S., Tachikawa, H., and Kawanishi, Y. (2001). Glutathione S-transferase M1 gene deletion may be associated with susceptibility to certain forms of schizophrenia. Biochem. Biophys. Res. Comm. 281, 267–271. Hoffer, A., Osmond, H., and Smythies, J. (1954). Schizophrenia: a new approach. Part II. J. Ment. Sci. 100, 29–45. Osmond, H., and Smythies, J. (1952). Schizophrenia: a new approach. J. Ment. Sci. 98, 309–320. Smythies, J. The Dynamic Neuron. Cambridge Mass. MIT Press, 2002.

LOSS OF SPINES AND NEUROPIL

Liesl B. Jones Lehman College, CUNY Bronx, New York 10468

I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Introduction Dendrites, Spines, and Normal Plasticity EVects of Estrogen on Plasticity Long-Term Potentiation and Learning Alterations in Neuropil Components Alzheimer’s Disease Human Immunodeficiency Virus Encephalitis Huntington’s Chorea Schizophrenia Possible Link between Calcium and Dendritic Alterations Conclusion References

I. Introduction

It was once thought that the brain was in a static state in adulthood and that neurons were incapable of changing their morphology with changing environments. It is now known that many regions of the brain are actually dynamic and that neurons show a great propensity for plasticity, much of which occurs through changes in neuropil elements in response to incoming information. The neuropil in the cortex is made up of axons, dendrites, and spines, each of which plays a role in neuronal communication. A loss of neuropil could alter the number of synapses made or synaptic surface area in the cortex, leading to changes in communication. A variety of disorders are associated with loss of one or more elements of the neuropil. Additionally, there are a variety of normal functions that lead to changes in structures, primarily spines, in the neuropil. The review focuses on the normal plasticity of dendrites and spines, as well as disorders that have shown alterations in the number of dendrites and spines. Within the cortex, spines are the primary site for incoming excitatory information and local inhibitory synapses. Dendritic spines are found on pyramidal cells and spiny stellate neurons. During development, spines appear following axonal in growth (Zhang and Benson, 2002). Spine density increases to their peak concurrent with the height of synaptogenesis and then decrease to adult levels (Petit et al., 1988). The developmental timing of this event depends on the cortical INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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region (Lubke and Albus, 1989), cortical laminae (Lund and Holbach, 1991), and varies among species (Munoz-Cuento et al., 1990; Munoz-Cuento et al., 1991; Zhang and Benson, 2002). In hippocampal cultures, synapses begin to develop on or around day 3 and spines are ﬁrst seen on or around day 9 (Petit et al., 1988). Spines appear to develop from dendritic ﬁlopodia and some appear to develop de novo from the dendrite shaft (Zhang and Benson, 2002). Depending on the cortical area, the pyramidal cells will have more dendrites with a greater number of spines or a fewer number of dendrites and fewer spines ( Duan et al., 2002; Jacobs et al., 2001). Cortical areas that integrate multiple forms of information, such as associational areas, tend to have pyramidal cells with many dendrites and spines as compared to primary sensory areas that receive one form of information ( Duan et al., 2002; Jacobs et al., 2001). It is thought that the more complex the integration of information is, the greater the need for synaptic surface area to receive the incoming information. In summary, there are more synapses being made in cortical areas that integrate multifunctional information.

II. Dendrites, Spines, and Normal Plasticity

Dendritic spines are subject to use-dependent plasticity. Spines have the ability to alter their shape, for example, the diameter or length of their necks. Because their necks are in general narrow or thin, this causes a large input resistance that results in large voltage changes no matter the size of the synaptic input (Gazzaley and Benson, 2002; Nikonenko et al., 2002). Spines are designed to isolate changes in calcium dynamics as well as other biochemical cascades; therefore, changes in the spines will not necessarily aVect the rest of the parent dendrite. Additionally, because the spines can isolate themselves, biochemical cascades occurring in the dendrites will not aVect the spines. There are several instances in normal brain development and function that involve either the addition of or the removal of spines. In general, spine addition occurs if the environment of an animal becomes more complex or during learning, for example, during long-term potentiation and in response to circulating steroid hormones (LieshoV and Bischof, 2003). As mentioned earlier, spines are designed to compartmentalize and regulate calcium. Increases in calcium levels in the spines have been correlated with long-term potentiation. Spines are very small so that a slight increase in calcium inﬂux generates a large increase in intracellular stores, leading to a triggering of calcium-mediated events within the spine. It is thought that this process somehow marks the spine so that newly generated proteins can ﬁnd their targets; in addition, it is thought that the cascades can also cause protein synthesis within the dendrite and the spines, therefore, would serve as a

LOSS OF SPINES AND NEUROPIL

3

means to retain the new proteins (Gazzaley and Benson, 2002). Both processes are activated through the N-methyl-d-aspartate (NMDA) receptor. As examples of normally occurring spine plasticity, we will examine long-term potentiation and the eVects of estrogen on spine plasticity. We will also examine an exception to the rule, which occurs in songbirds where learning has been examined extensively.

III. Effects of Estrogen on Plasticity

The example of estrogen is important when examining the eVects of the estrous cycle in females. During the estrous cycle, spine density decreases on apical dendrites in the 24-h period between proestrus and estrus (Gazzaley and Benson, 2002; Woolley et al., 1990). This time period correlates with estrogen levels going from their peak to their lowest level of expression. With increasing estrogen levels, spine levels increase as well. The increase in spine density is not speciﬁc to cells or synapse and therefore may likely have a more global aVect on the existing network of neurons (Gazzaley and Benson, 2002). Studies have shown that the estrogen eVect on spines is dependent on NMDA receptor activation. If the receptor is blocked, the increase in spine density due to exposure to estrogen is eliminated (Woolley and McEwen, 1992). Interestingly, the mechanisms that mediate these eVects appear to be similar in developing animals as young adults. Blocking cAMP-dependent protein kinase A and/or disrupting cAMP response elementbinding protein (CREB) phosphorylation blocks spine induction (Gazzaley and Benson, 2002). These results suggest that the estrogen-induced spine increase is dependent on excitation through the NMDA receptor as well as activation of Protein Kinase A (PKA) and CREB phosphorylation. Other studies have examined the eVect of calcium calmodulin-dependent protein kinase type II (CAM kinase II) on estrogen-induced spine increases. The reason for this is that CAM kinase II is important in maintaining and stabilizing dendritic spines during development (Wu et al., 1996). Research has suggested that CAM kinase II is not required for the induction of spines but is required for the maintenance of spines. Intestinally, all of the pathways that estrogen uses to cause spine induction are dependent on calcium inﬂux into the spine through the NMDA receptor. During development, CAM kinase II has been shown to be required for stable synapses to be formed. The rise in CAM kinase II occurs simultaneously with a decrease in glutamic acid decarboxylase (GAD) levels in inhibitory interneurons, suggesting that both are required to allow for new formation as well as to stabilize preexisting synapses (Gazzaley and Benson, 2002). This process has been observed in the visual system in response to monocular deprivation, suggesting that CAM kinase

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II, PKA, CREB phosphorylation, and GAD are all involved in spine formation, but depending on the brain region may be induced by diVerent factors. In addition, similar ﬁndings of increased spine density have been seen in the medial prefrontal cortex following corticosteroid injections (Seib and Wellman, 2003).

IV. Long-Term Potentiation and Learning

The second example of long-term potentiation also occurs in the hippocampus. Long-term potentiation is probably one of the best documented examples of plasticity in the brain. Studies have shown that morphological changes take place following the induction of long-term potentiation. Several studies have shown increases in the number of spines and the number of presynaptic boutons (Toni et al., 1999). This was further shown by studies that blocked proteins important for growth blocks in long-term potentiation (Bozdagi et al., 2000). Studies inducing long-term potentiation through exposure to forskalin have shown longlasting increases in spine density and the number of synapses (Tominaga-Yoshino et al., 2002). In the same study, blocking of PKA suppressed the increase in spine density, suggesting a role for PKA in long-term potentiation similar to that seen in exposure to estrogen, and activation of PKA led to an increase in the number of spines on distal dendrites (Tominaga-Yoshino et al., 2002). These changes appear to occur in young and young adults as opposed to adults, which do not appear to show similar widespread morphological changes. These data suggest that plasticity in the brain can occur in multiple ways. Research has also suggested that like estrogen-induced spine development, long-term potentiation is regulated through the NMDA receptor and is calcium dependent (Vanderklish and Edelman, 2002). Research has shown that activation of the NMDA receptor not only leads to spine lengthening and sprouting, but retraction of existing spines as well. These data suggest therefore that other factors must be involved in sprouting of spines other than calcium and activation of the NMDA receptor. Vanderklish and Edelman (2002) have shown that activation of the group 1 metabolic glutamate receptor can also induce increases in spine length. However, blocking of the same receptor does not alone suppress the increase in spine length, suggesting that calcium mobilization is very important in inducing the increase in spine number and length (Vanderklish and Edelman, 2002). When the metabolic glutamate receptor and calcium mobilization were both blocked, a suppression of spine length was observed. These data also suggest that the control of calcium release may be what causes increases in spine length and number versus spine retraction. Research has shown that low levels of calcium mobilization favor an increase in spine number and length, whereas high levels of calcium

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mobilization cause a retraction in spine number (Vanderklish and Edelman, 2002). Activation of the NMDA receptor has been linked to the translation of proteins involved in the formation of cytoskeletal proteins and, therefore, a combination of activation of both NMDA and metabolic glutamate receptors may be required to direct the type of morphological alteration observed. There are examples of learning that correlate with a decrease in spines as opposed to an increase. These examples occur in early learning and are best described in songbirds following auditory and sexual imprinting. Songbirds show a decrease in spine density during the acquisition phase of learning their song (LieshoV and Bischof, 2003). Additionally, during sexual imprinting, a male zebra ﬁnch reared in isolation and then exposed to a female exhibits an irreversible spine reduction found in two nuclei; the medial and lateral neohyperstriatum. However, in a third nucleus, the archineostriatum caudale (ANC), a reversible increase in spine density is observed (LieshoV and Bischof, 2003). The reversibility of the change in spine density suggests that it occurs in response to a change in complexity of the bird’s environment. When a male zebra ﬁnch is exposed to a female and then removed from the presence of the female, the number of spines in the ANC decreases back to the preexisting levels (LieshoV and Bischof, 2003). Additionally, the number of spines in the ANC is lower in zebra ﬁnches reared in isolation as opposed to those raised in a bird sanctuary. Research in rats examining activity-induced dendritic development showed that blockage of sodium channels by tetrodotoxin caused no changes in dendritic length or number, but did show that there were more immature spines as compared to nontreated cells (Drakew et al., 1999). These data suggest that activity is important in controlling dendritic development but is not the sole factor inﬂuencing development. In the cortex, evidence has shown that dendrites receive not only excitatory synapses but inhibitory synapses and can receive dual excitatory and inhibitory synapses (Keller, 2002). Research examining spine number in the somatosensory cortex following whisker stimulation showed a dramatic increase in spine density (Keller, 2002). An increase in synapse density on spines with no change in dendrites was shown. Interestingly, there was an increase in the number of both inhibitory and excitatory synapses. The increase in excitatory synapse was modest as compared to the increase in inhibitory synapses (Keller, 2002). Additionally, 4 days after stimulation the total spine density and the density of excitatory synapses were back to normal, but the number of inhibitory synapse was still increased as well as the number of dually innervated spines (Keller, 2002). These data suggest that activity-induced morphological changes in the cortex occur not only at excitatory spinous synapses, but also aVect inhibitory synapses. These data also lend evidence to the theory that cortical dendritic spines are constantly changing in response to neural activity.

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V. Alterations in Neuropil Components

The aforementioned discussion focused on normal development and plasticity involving one main component of the neuropil, dendritic spines. The other components of neuropil are axons and dendrites. While these are important in information processing, dendritic spines are the structures that receive incoming glutametergic, dopaminergic, and GABAergic synapses. Therefore, alterations in spines may have a greater eVect on cellular communication. However, without dendrites there are no spines and without axons there is no incoming information, so in the following discussion we will examine all three components of the neuropil, how they are altered in various disorders, and what that may mean for cellular communication.

VI. Alzheimer’s Disease

Alzheimer’s is one of the most well-studied neuropathological disorders. Alzheimer’s is characterized by marked cell loss and is identiﬁed neuropathologically by the presence of plaques and tangles. Behaviorally, Alzheimer’s is characterized by severe cognitive impairment with changes in personality. Much research has suggested alterations in dendritic morphology. These studies have shown a signiﬁcant decrease in the number of dendritic spines (Ferrer and Gullotta, 1990). It is thought that these changes may contribute to the cognitive problems observed in Alzheimer’s patients. More recent research has begun to examine if there are alterations in the remaining spines. While a loss of dendrites is surely suggestive of aberrant cellular communication, alterations in morphology or cytoskeletal components of the existing spines may also lead to aberrant cellular communication as well. A study by Shim and Lubec (2002) examined the expression of drebrin. Drebrin is a protein found in spines and is colocalized with actin ﬁlaments (Asada et al., 1994). It is thought to be an actin regulatory element and to control the assembly and disassembly of actin ﬁlaments. Shim and Lubec (2002) found a signiﬁcant decrease in the expression of drebrin in the frontal and temporal cortices but not in the cerebellum. Other research has suggested that drebrin plays a role in neurite extension and spines containing drebrin were signiﬁcantly longer (Hayashi and Shirao, 1999). These data suggest a role for drebrin in pre- and postsynaptic communication through the regulation of neuritic processes. Other research using immunohistochemical techniques examined immunoreactivity in the hippocampus of Alzheimer’s and found a signiﬁcant reduction in drebrin immunoreactivity (Haigaya et al., 1996). A loss of drebrin may

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be suggestive of alterations in actin ﬁlaments. Research has also suggested that actin, through its regulatory-binding proteins, of which drebrin is one, is responsible for the localization of neurotransmitter receptors on the postsynaptic membrane (Shirao and Sekino, 2001). Drebrin therefore may also have a role in long-term potentiation and that a loss of drebrin may result in a disorganization of actin ﬁlaments and alterations in spine plasticity, which in turn would aVect long-term potentiation, as well as other cellular physiological events. Interestingly, many patients with Down’s syndrome develop Alzheimer-like changes very early in life. In the same study by Shim and Lubec (2002), the expression of drebrin in tissue from Down’s syndrome patients was examined. Research into neuropathological alterations in Down’s syndrome has shown a decrease in dendritic spines and therefore may also show alterations in the expression and distribution of drebrin (Wisniewski et al., 1986). As with Alzheimer’s, Shim and Lubec (2002) found a signiﬁcant decrease in drebrin in the frontal and temporal cortices but not in the cerebellum. Additionally, Weitzdoerfer et al. (2001) found a decrease in drebrin during development in Down’s syndrome brains. These data lend conﬁrmation to the growing body of data suggesting dendritic spine abnormalities as well as a loss of dendritic spines and atrophy of the dendritic arbor in infants and children with Downs’ syndrome. Down’s syndrome, unlike Alzheimer’s, is a disorder of development; however, both disorders show similar changes in dendritic spines, suggesting that spines may be quite vulnerable to changes in their environment and that a loss of or change in structure can lead to severe cognitive dysfunction.

VII. Human Immunodeficiency Virus Encephalitis

Human immunodeﬁciency virus (HIV) has been associated with multiple neocortical alterations. Research has shown subtle changes such as atrophy of the frontal and temporal lobes, spongiosis in layers I–III, and neuronal cell loss (Artigas et al., 1989). Other research has shown a more marked loss of soma volume in the frontoorbital cortex in brains from patients with acquired immunodeﬁciency syndrome (AIDS), which has been associated with dementia (Ketzler et al., 1990). Wiley et al. (1991) have shown a reduction in neocortical width with a decrease in large neurons in the frontal, parietal, and temporal cortex of patients with human immunodeﬁciency virus encephalitis (HIVE). Additionally, Wiley et al. (1991) showed a decrease in syanptophysin-like immunoreactivity in the frontal cortex, suggesting a loss of dendritic spines or dendrites. Masliah et al. (1992) showed no diVerence between controls and HIV tissue without encephalitis. However, HIVE material showed a marked reduction in apical dendritic

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spine numbers, which worsened as one moved further away from the soma. The apical dendrites tended to be swollen, diminished in length, and vacuolated (Masliah et al., 1992). They also showed that many of the remaining spines were foreshortened and deformed in their morphology, suggesting that the remaining spines and dendrites may have problems with information processing. The alterations in dendritic morphology may be in response to a loss of incoming information or to disconnected circuits, which could also lead to problems in cognition observed in patients with HIVE. However, it is still unclear whether the observed changes are due to direct infection of the neurons with HIV or to neurotoxic factors released by microglia that are virally infected. Some data suggest that microglia that are virally infected release some factors that can interact with the NMDA receptor and that these factors may alter the functioning of the cell by altering the functioning of the NMDA receptor (Giulian et al., 1990). Again the evidence is not conclusive and more research needs to be done to examine why the apical dendrites and spines are aVected in HIVE.

VIII. Huntington’s Chorea

Huntington’s disease (HD) is a dominantly inherited adult-onset neurodegenerative disorder. Neuropathologically it is characterized as a loss of medium-sized spiny striatal neurons and cortical pyramidal cells. Behaviorally the disease is characterized by choreiform movements, cognitive impairments, and other behavioral problems (Vonsattel et al., 1985). Research using transgenic mice expressing the full-length mutant huntingtin showed behavioral abnormalities as early as 2 months of age, but the neuronal loss was not seen until much later (Reddy et al., 1998). This coincides with human data suggesting that in some cases the disease can manifest itself with behavioral alterations and no evidence of neurodegeneration. Research by Guidetti et al. (2001) examined dendritic morphology in the striatum and sensorimotor cortex. They found a decrease in the number of primary dendritic spines, a thickening of the proximal portion of primary dendrites, and a decrease in the surface area of somata and dendrites. The results were observed in both the striatum and the cortex, but the alterations were more pronounced in the striatum than in the cortex. These changes in morphology were observed in the absence of cell loss in either area, suggesting that neurodegeneration does not need to occur in order to have the behavioral and cognitive problems associated with HD. These data, therefore, suggest that subtle changes in dendritic architecture can lead to rather dramatic behavioral and cognitive abnormalities.

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IX. Schizophrenia

Schizophrenia is one of the most disabling of neuropsychiatric disorders whose pathogenic mechanisms are complex and poorly understood. Schizophrenia is a multifaceted disease where patients exhibit a variety of symptoms ranging from psychosis to cognitive deﬁcits. A preponderance of evidence suggests that a combination of multigenic factors and early developmental insults could lead to a variety of brain abnormalities, which involve cortical and subcortical structures. Mounting evidence appears to implicate the prefrontal cortex (PFC) in schizophrenia (Beasley and Reynolds, 1997; Benes et al., 1986, 1991; Bertolino et al., 1999; Broadbelt et al., 2002; Buxhoeveden et al., 2000; Daviss and Lewis, 1995; Dean et al., 1999; Garey et al., 1998; Glantz and Lewis, 1997, 2000; Honer et al., 1997; Jones et al., 2002; Kalus et al., 2000; Lewis et al., 2001; PeroneBizzozero et al., 1996; Pierri et al., 2001; Reynolds and Beasley, 2001; Thompson et al., 1998; for reviews, see Harrison, 1999; Hirsch et al., 1997; Jones, 2001; Shapiro, 1993). The prefrontal cortex is an important region involved in higher cognitive function, working memory, mental imagery, willed action, and active memory (Frith and Dolan, 1996) and therefore may play a role in the cognitive deﬁcits observed in schizophrenia. Behavioral assessments of patients with schizophrenia show problems with spatial learning and verbal memory tasks, which are functions subserved by the PFC (Peters et al., 2000; Weickert et al., 2000). Developmental studies of animals following lesions of either the medial dorsal (MD) nucleus of the thalamus or the PFC have found alterations in a variety of behaviors similar to those exhibited by patients with schizophrenia. For example, Harrison and Mair (1996) showed that young rats with lesions in either the thalamus or the cortex exhibited deﬁcits in the delayed nonmatching to sample test. IsseroV et al. (1982) showed similar results with young monkeys performing spatial memory tasks following a lesion of the MD. Aggleton and Mishkin (1983) showed impairments of visual recognition following lesions of the medial dorsal nucleus. Finally, Stokes and Best (1990) showed deﬁcits in ‘‘reference’’ and working memory in rats following a lesion of the MD. Postmortem studies conﬁrm the involvement of the medial dorsal nucleus. One of the most consistent ﬁndings in schizophrenia is the loss of cells and the decrease in volume of the MD nucleus (Byne et al., 2001a,b; Pakkenberg, 1990, 1992; Popken et al., 2000; Young et al., 2000; Staal et al., 2000). This ﬁnding is important for several reasons. The ﬁrst is that the medial dorsal nucleus has reciprocal connections with much of the prefrontal cortex, which has been implicated in schizophrenia (Faull and Mehler, 1994; Goldman-Rakic and Porrino, 1985; Kuroda et al., 1995a,b). Second, research suggests that the maturation of prefrontal cortical cells during development is dependent on the activity from the medial dorsal nucleus (van Pelt et al., 1996). Third, myelination of the axons from the medial dorsal nucleus occurs during

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the onset of schizophrenia (Benes, 1989). These data, in conjunction with imaging and postmortem human studies (Harvey et al., 1992, 1993; Purohit et al., 1993), as well as animal lesion studies (Aggleton and Mishkin, 1983; Harrison and Mair, 1996; IsseroV et al., 1982; Stokes and Best, 1990), suggest alterations in information processing in the PFC. Morphological studies in the prefrontal cortex have begun to show consistent changes that could have an eVect on information processing through a loss of synaptic surface area. Prefrontal cortical studies of pyramidal cells have shown a decrease in MAP2, a protein found in dendrites and cell bodies (Decamilli et al., 1984; Fischer et al., 1987), and in layers III and V of areas 9 and 32 of the prefrontal cortex ( Jones et al., 2002). These data suggest a possible loss of dendritic material and/or a change in soma size. A second study showed a loss of primary and secondary basilar dendrites in area 32 of the prefrontal cortex (Broadbelt et al., 2002), which suggests that the decrease in MAP2 is due to a loss of dendritic material. Preliminary data from our laboratory suggest similar changes in area 9. A second protein that acts as a marker for dendrites is neurogranin (Li et al., 2000). Neurogranin is localized to cell bodies, dendrites, and spines (AlvarezBolado et al., 1996). Functionally, neurogranin acts as an upstream regulator of calcium by binding calmodulin (Ho Pak et al., 2000; Prichard et al., 1999). A change in the expression of neurogranin would, therefore, suggest possible changes in internal cell signaling. Behavioral assessments of neurogranin knockout mice showed impairment in spatial learning, as well as a decrease in basal levels of activated CaM kinase II (Ho Pak et al., 2000). Behavioral data from patients with schizophrenia, as well as data from animal models, suggest alterations in spatial learning (Aggleton and Mishkin, 1983; Harrison and Mair, 1996; Harvey et al., 1992, 1993, IsseroV et al., 1982; Purohit et al., 1993; Stokes and Best, 1990). These data implicate neurogranin as having a possible role in the altered functioning of pyramidal cells in the prefrontal cortex. Preliminary qualitative analysis from our laboratory suggests that neurogranin in subjects with schizophrenia is localized predominately to the cell bodies, not in the processes of pyramidal cells. Preliminary quantitative results suggest a 50% decrease in staining in layer V and a 70% decrease in staining in layer III of area 9. Additional studies examining morphology have shown a decrease in spine density (Garey et al., 1998; Glantz and Lewis, 2000). Finally, a study by Pierri et al. (2001) has shown a decrease in the soma size of pyramidal cells in layer III in the prefrontal cortex. Soma size correlates directly with the number of dendrites a cell produces. Therefore, a change in soma size might aVect the number of primary dendrites produced by the pyramidal cells. These data together suggest alterations in synaptic surface area on the pyramidal cells through a loss of dendrites and spines. A loss of synaptic surface area could alter the number and type of input received by the cell and, therefore, alter the outgoing information. While it is diYcult to deﬁne the underlying mechanisms that cause changes in pyramidal cell morphology, such changes may be

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indicative of a greater problem in signal transduction through a loss of synaptic surface area and input to the cells.

X. Possible Link between Calcium and Dendritic Alterations

Neurons adapt their neuritic ﬁeld to maintain a particular level of bioelectric activity (Baker and van Pelt, 1997; Kossel et al., 1997; van Ooyan et al., 1995; Wise et al., 1979). Changes in the perceived activity of a cell, therefore, will be reﬂected in the number and length of dendritic branches. Research has shown that once the level of bioelectric activity is set, changes in activity around the cell cannot alter the neurites further (van Ooyan et al., 1995). Electrical stimulation or depolarization can increase neurite outgrowth and these eVects are dependent on the inﬂux of extracellular calcium (Kater et al., 1988; Petit et al., 1988; Ramakers et al., 2001). Growth cone extension rates are dependent on an optimal level of intracellular calcium, and if calcium levels fall below this level, growth cones will become smaller and stop elongating or retract (Kater et al., 1988; Ramakers et al., 2001). Research has shown that lowering the level of intracellular calcium arrested axonal outgrowth and stopped the net addition of dendrites and dendritic branching. Blocking calcium inﬂux decreased dendritic branching as well (Ramakers et al., 2001). These researchers also showed that the generation and maintenance of dendrites are dependent on actin polymerization, which in turn is dependent on intracellular calcium levels. Research in schizophrenia has suggested a loss of dendrites and spines as well as a decrease in soma size. These data imply that alterations in calcium levels during development could lead to morphological changes. Calcium inﬂux is controlled by activation of the NMDA receptor. Binding of glutamate to the NMDA receptor causes an inﬂux of calcium, which triggers the release of calcium from internal stores and the release of calmodulin from neurogranin (Chakravarthy et al., 1999). Calcium, when bound to calmodulin, causes several diVerent events to occur within the cell. First the complex can bind to the NMDA R1 receptor, causing it to have a decreased open probability (Chakravarthy et al., 1999). PKC binds to a similar site on the NMDA R1 receptor, causing the opposite eVect (Chakravarthy et al., 1999). Neurogranin activity, therefore, could have an eVect on how, when, and how long the NMDA receptor channel opens through its regulation of calcium and calmodulin. Second, calcium–calmodulin can activate Cam kinase II (Chakravarthy et al., 1999). Cam kinase II is found both pre- and postsynaptically and is also activated through the binding of calcium to calmodulin (Chakravarthy et al., 1999). Developmentally, CaM kinase II has been shown to be important in controlling neuronal arborization both presynaptically and postsynaptically

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(Borodinsky et al., 2002; Mainen and Sejnowski, 1996; Zou and Cline, 1999). The size of the dendritic arbors is very important to cell function. Arbors that are too expansive or too small can alter the transfer of information and in turn alter the integration of information (Mainen and Sejnowski, 1996). Zou and Cline (1999) inhibited Cam kinase II activity and found that endogenous Cam kinase II activity is required to control the elaboration of pre- and postsynaptic terminals. A study by Borodinsky et al. (2002) showed similar results using diVerent methods. Borodinsky et al. (2002) found that activation of the L-type calcium channel induced neurite outgrowth through the activation of CaM kinase II and appeared to control neural outgrowth and complexity. A study has linked CaM kinase II to MAP2 and dendritic branching (Hely et al., 2001). CaM kinase II dephosphorylates MAP2 and during development this situation will promote elongation (not branching), whereas phosphorylated MAP2 will increase dendritic branching (Audersirk et al., 1997; Diez-Guerra and Avila, 1994; Hely et al., 2001). Changes, therefore, in Cam kinase II levels developmentally can have an adverse eVect on the phosphorylation state of MAP2 and alter dendritic branching and length. Taken together, these data suggest, as mentioned earlier, that dendritic integrity is dependent on calcium levels and that changes in calcium levels can cause aberrant dendritic development, as well as alterations in the maintenance of existing dendritic arbors. Therefore, it will become important to examine further the role of calcium in diseases showing alterations in neuropil elements.

XI. Conclusion

These are just some of the diseases that aVect dendritic architecture; others include metabolic disorders, Creutzfeldt–Jakob disease, and Picks disease to name a few. It is interesting that no matter whether the disorders are developmental in origin, degenerative, or acquired by exposure to a virus, they all involve cognitive impairments. The link between cognitive impairments and morphological changes seems to be a loss of or an alteration in the morphology of dendrites or spines or both dendrites and spines. This suggests that is does not take dramatic cell loss in order to have cognitive problems. Most of the described disorders do not show signiﬁcant cell loss from the beginning or at all during the duration of the disease, yet they do show dendritic morphological alterations. These changes could be secondary to alterations in neurotransmission, especially through the NMDA or dopamine receptors leading to altered calcium inﬂux. A proper balance of calcium within the cell appears to be very important in maintaining dendritic cytoarchitecture. More research needs to be done to better understand how the inﬂuence of calcium may play a

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role in many of the disorders that aVect neuropil elements and, more speciﬁcally, dendritic plasticity.

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SCHIZOPHRENIA AS A DISORDER OF NEUROPLASTICITY

Robert E. McCullumsmith, Sarah M. Clinton, and James H. Meador-Woodruff Department of Psychiatry and Mental Health Research Institute University of Michigan Medical School Ann Arbor, Michigan 48109

I. II. III. IV. V.

Introduction Clinical Features of Schizophrenia Synaptic Plasticity and Schizophrenia Substrates of Neuroplasticity Glutamatergic Dysfunction in Schizophrenia A. Phencyclidine as a Pharmacological Model of Schizophrenia B. Decreased Cerebrospinal Fluid Glutamate Concentrations in Schizophrenia C. In Vivo Evidence of Decreased Glutamatergic Markers in Schizophrenia D. Evidence of Decreased Excitatory Amino Acids in Postmortem Brain E. Alterations of Receptors and Associated Intracellular Molecules Relevant to Plasticity in Postmortem Brain F. Glutamate-Based Pharmacological Treatment in Schizophrenia VI. Conclusions References

Schizophrenia is a devastating mental illness aVecting millions worldwide with signiﬁcant ﬁnancial and emotional burdens for aZicted persons, their families, and society. Considering schizophrenia as a disorder of neuroplasticity permits integration of competing neurochemical and neurodevelopmental hypotheses. Recent advances have linked the pathophysiology of schizophrenia with abnormalities of the glutamate neurotransmitter system. Elements of glutamatergic neurotransmission implicated in schizophrenia, including glutamate receptors and receptor-associated molecules, have critical roles in long-term potentiation, a molecular correlate of neuroplasticity. We suggest that schizophrenia can be considered a disorder of plasticity, associated with molecular abnormalities of the glutamate synapse. I. Introduction

Recent advances in the understanding of the molecular substrates of neuroplasticity, combined with advances in elucidating mechanisms of pathophysiology in psychiatric illnesses, suggest that schizophrenia is a disorder of neuroplasticity at a cellular and molecular level. This review discusses the clinical features of INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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schizophrenia and describes this illness in terms of dysfunctional neuroplasticity. Molecular substrates common to physiological processes that underlie neuroplasticity and neurotransmitter models of schizophrenia are then discussed. Reports demonstrating alterations of molecules in the brain associated with plasticity in schizophrenia are then reviewed. We conceptualize schizophrenia as a disorder of neuroplasticity, associated, in particular, with molecular abnormalities of glutamatergic neurotransmission.

II. Clinical Features of Schizophrenia

Schizophrenia is a devastating illness that aZicts over 2 million people in the United States and over 50 million people worldwide (Buchanan and Carpenter, 2000). In many ways, this is the most serious of all psychiatric illnesses: more hospital beds (psychiatric and medical combined) are ﬁlled by persons with schizophrenia than any other medical condition (Buchanan and Carpenter, 2000). This disorder is characterized by a constellation of clinical ﬁndings, often divided into positive and negative symptoms (Buchanan and Carpenter, 2000). Positive symptoms include dramatic hallucinations, which are most often auditory. Patients report that they hear voices that are clearly located outside of their heads, most often engaged in a running commentary on their thoughts and behaviors. Other common positive symptoms include paranoid delusions and disorders of thought processes. Much more insidious and debilitating are the negative symptoms, which are associated with the diminution of normal social behaviors, and include withdrawal, decreased spontaneous communication, decreased eye contact, decreased or muted facial expression and vocal inﬂection, and diminished spontaneous movement (Buchanan and Carpenter, 2000). Few individuals suVering from schizophrenia have all of these symptoms, but the persistence of several characteristic symptoms, such as auditory hallucinations, must be present in order for someone to be diagnosed with this disorder (Buchanan and Carpenter, 2000). The onset of schizophrenia is typically postpubertal, frequently occurring when an individual is in the later years of high school or just beginning college (Alda et al., 1996). The so-called ‘‘ﬁrst psychotic break’’ or initial episode does not always have an identiﬁable trigger (Larsen et al., 1996a,b; Lieberman et al., 1996). Schizophrenia is not a static condition, and the vast majority of patients experience a progressive deterioration in their ability to function over their lifetimes (Buchanan and Carpenter, 2000). Frequent hospitalizations, medication side eVects, and the psychotic symptoms themselves hinder vocational and social development, preventing many persons with schizophrenia from developing or maintaining the skills to live independently, leading to signiﬁcant ﬁnancial and emotional burdens for families of aVected individuals.

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III. Synaptic Plasticity and Schizophrenia

The onset of the modern biological revolution in psychiatry in part started with the discovery of the phenothiazine drug chlorpromazine as a treatment for various psychiatric maladies (Healy, 2002; Swazey, 1974). Prior to the discovery of this drug and other antipsychotic medications, persons with psychotic illnesses such as schizophrenia were oVered a variety of treatments with questionable beneﬁt and were frequently institutionalized for long periods of time (Shorter, 1998). The superior eYcacy of chlorpromazine compared to previous treatment modalities revolutionized the clinical management of psychiatric illness by providing a pharmacological intervention that attenuated or extinguished the positive symptoms of schizophrenia (Healy, 2002; Swazey, 1974). Subsequent research demonstrated that these potent antipsychotic eVects were associated with dopamine D2 receptor antagonism, a deﬁning pharmacological feature of earlier antipsychotics (Hyttel et al., 1985; Wilson et al., 1998). These observations led to formulation of the dopamine hypothesis of schizophrenia, which suggests that excess dopaminergic activity contributes to psychotic psychopathology (Meltzer and Stahl, 1976; Sayed and Garrison, 1983). Over the years, reﬁnement of this neurochemical hypothesis has led to inclusion of other neurotransmitter systems. For example, ingestion of phencyclidine (PCP), a noncompetitive antagonist of the NMDA subtype of glutamate receptor, can precipitate a schizophreniform psychosis in nonpsychiatrically ill persons and exacerbate psychotic symptoms in persons with schizophrenia ( Javitt and Zukin, 1991; Tamminga, 1999). Based on these clinical eVects of PCP and similar drugs, altered glutamate neurotransmission has been identiﬁed as another candidate neurotransmitter to explain elements of the pathophysiology of schizophrenia (Coyle, 1996; Tamminga, 1999). Interestingly, recent work has demonstrated convergence of signaling by dopamine, glutamate, and other neurotransmitter systems at the cellular level, providing a rationale for integration of these apparently disparate neuorochemical models (Greengard et al., 1998). Despite these advances, the complexity of schizophrenia is not readily explained by a static neurochemical model. The onset of schizophrenia is typically in late adolescence or early adulthood (Alda et al., 1996). The onset of positive and negative symptoms in a previously normally functioning person, coupled with a lifetime of waxing and waning symptoms, accompanied by the possibility of a steady decline in social, occupational, and cognitive functioning, has led to longitudinal models that take into account genetic and environmental factors. In a thoughtful review by Marenco and Weinberger (2000), the authors summarize a prevailing paradigm, the neurodevelopmental hypothesis of schizophrenia, as ‘‘a subtle disease process aVecting critical circuits in the brain during early development and reaching full-blown consequences during adolescence or early

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adulthood.’’ Data supporting this hypothesis include studies suggesting that schizophrenia is associated with late winter births in urban environments, as well as a number of other prenatal, perinatal, and postnatal events (reviewed in Lewis and Levitt, 2002; Marenco and Weinberger, 2000). An alternative to the neurodevelopmental hypothesis, the neurodegenerative model, suggests a longitudinal, progressive course where various types of neurotoxic events lead to pathological changes in brain circuitry that manifest as signs and symptoms of schizophrenia (Deutsch et al., 2001). Excitotoxicity, dysregulated apoptosis, oxidative stress, and other pathological processes have been implicated in this model (Deutsch et al., 2001; Weinberger and McClure, 2002). Although conceptually compelling due to the age of onset and the chronic course of schizophrenia, a major shortcoming of any neurodegenerative hypothesis is the absence of consistently reported pathological lesions in the brain in schizophrenia (Weinberger and McClure, 2002). An alternative formulation of the pathophysiology of schizophrenia is in terms of neuroplasticity. Plasticity is the ability of a system to reversibly change or alter a response to a stimulus in an activity-dependent manner (Gordon, 1969; Malenka and Nicoll, 1999). Neuroplasticity refers to the ability of the brain to change and adapt in response to multimodal input, to make new circuits by strengthening or weakening speciﬁc synapses, by changing the number of dendritic spines, or through an alteration in the number of neurons or glia via cell division or apoptosis (Smythies, 2002). Such changes in neural substrates impact learning, memory, and other cognitive functions, permitting the brain to function in a plastic manner. In many ways, schizophrenia is an illness where aZicted individuals have great diYculty interacting with and adapting to the environment. For example, hallucinations are experienced as intense and dramatic sensory stimuli that have no basis in reality, whereas paranoid delusions may interfere with the ability to relate and interact normally with friends, family, or co-workers. In many cases, persons with schizophrenia are unable to function or interact socially, with common deﬁcit symptoms including poor eye contact, ﬂat aVect, and withdrawal. Thus, aZicted persons are both perceiving misinterpreted sensory stimuli and reacting dysfunctionally to appropriately received sensory information. Because neuroplasticity involves the ability of the brain to react in an activity-dependent manner to speciﬁc stimuli, schizophrenia may be thought of as a disorder of dysfunctional neuroplasticity. Considering schizophrenia as a disorder of neuroplasticity permits integration of neurodevelopmental, neurodegenerative, and neurochemical ﬁndings and observations. For example, dysregulation of activity-dependent changes in cognitive processes such as learning and memory could be a result of miswiring of brain circuitry during development, a result of neurotoxic events that may alter the synaptic pruning that occurs in the postadolescent period, or both, whereas

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either degenerative or developmental abnormalities may lead to detectable alterations in neurochemical substrates of long-term potentiation (LTP), an eVector of neuroplasticity (Malenka and Nicoll, 1999). While a full consideration of these competing hypotheses is beyond the scope of this review, this chapter discusses evidence for schizophrenia as a disorder of neuroplasticity in terms of substrates common to physiological activities that underlie neuroplasticity, such as long-term potentiation, and speciﬁc neurochemical alterations found in schizophrenia.

IV. Substrates of Neuroplasticity

Identiﬁcation of the biological mechanisms that underlie plasticity has been an area of intense interest for many years. Repetitive activation of excitatory synapses increases synaptic strength, a phenomena called long-term potentiation due to the persistence of the eVect for hours to days (Malenka and Nicoll, 1999). LTP and the related phenomena long-term depression (LTD) have been demonstrated in excitatory synapses in brain regions implicated in learning and memory, including the hippocampus, neocortex, and some subcortical structures (Malenka and Nicoll, 1999). There are several hallmark features of LTP: (1) LTP may be induced diVerentially in independent synapses of the same cell, as it is input speciﬁc; (2) induction of long-lasting LTP requires de novo synthesis of mRNA and protein; and (3) LTP may be induced at independent synapses adjacent to an active synapse if the activating stimuli are suYciently strong and temporally coordinated, a property called associativity (Malenka and Nicoll, 1999). LTP is a reversible, focal, input-speciﬁc increase in synaptic strength believed to be a molecular correlate of learning and memory. A full review of the diVerent types of LTP described in the literature is beyond the scope of this review; we focus on the widely studied form of LTP originally characterized in the CA1 region of the hippocampus (Malenka and Nicoll, 1999). Facilitation of LTP requires a localized increase in intracellular calcium in the dendritic spine. Calcium levels are typically increased via activation of the ionotropic NMDA subtype of glutamate receptor, which gates both calcium and sodium ions (Malenka and Nicoll, 1999). Pharmacological regulation of the NMDA receptor depends on the unique combination of glutamate, glycine/ d-serine, polyamine, phencyclidine/MK-801, pH, and zinc-binding sites on the assembled receptor (Hollmann and Heinemann, 1994; Lynch and Guttmann, 2001). In addition, magnesium ions block the receptor channel at physiological concentrations. This blockade is voltage dependent; partial depolarization of the cell membrane, usually via activation of the AMPA subtype of glutamate receptor, extrudes these magnesium ions. Therefore, both presynaptic glutamate release and AMPA receptor-mediated postsynaptic depolarization are typically required for NMDA receptor-mediated calcium inﬂux.

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The putative involvement of other molecules in LTP has been examined. G-protein-coupled metabotropic glutamate receptors and the kainate subtype of glutamate receptors likely modulate the initiation of LTP (Anwyl, 1999; Ben-Ari and Aniksztein, 1995; Bortolotto et al., 2003). Various signaling molecules have also been implicated. For example, autophosphorylation of calcium–calmodulindependent kinase II (CaMKII) is a requirement for LTP, whereas other kinases such as protein kinase C and protein kinase A likely contribute to LTP to a lesser extent (Carroll et al., 1998; Tzounopoulos et al., 1998). Recent investigations into the molecular mechanisms of LTP have demonstrated that the rapid cycling of AMPA receptors to and from the postsynaptic density is a critical event. Some synapses only possess NMDA receptors and are functionally silent prior to the colocalization of AMPA receptors at the postsynaptic density (Malenka and Nicoll, 1999). AMPA receptors may be recruited to the synapse by three distinct mechanisms: a constitutive pool, a regulated pool, and a golgiderived newly synthesized pool (Contractor and Heinemann, 2002). Cycling and integration of AMPA receptors to postsynaptic density by these mechanisms involve a number of recently characterized molecules that possess AMPA receptor subunitspeciﬁc protein-binding domains and are regulated via phosphorylation or palmitoylation (Contractor and Heinemann, 2002; McGee and Bredt, 2003) (Fig. 1). GRIP, PICK, stargazin, and ABP-L have all been implicated in AMPA receptor traYcking and thus likely have critical roles in LTP (Contractor and Heinemann, 2002; McGee and Bredt, 2003). Receptor-associated molecules have also been identiﬁed for the NMDA receptor, including NF-L, SAP102, yotiao, PSD95, and PSD93 (Contractor and Heinemann, 2002; McGee and Bredt, 2003) (Fig. 1). Some of these molecules speciﬁcally bind C-terminal consensus sequences called PDZ domains, named for three proteins with this motif: PSD95, Drosophila disc-large tumor suppressor gene (D1g-A) product, and ZO-1 a tight junction protein (Ehlers et al., 1998; Lin et al., 1998; Sheng and Pak, 2000). PDZ and related binding domains link neurotransmitter receptors with kinases, phospholipases, and other well-characterized signal transduction or receptor-traYcking pathways. The receptor-associated proteins for both NMDA and AMPA form signaling complexes within the postsynaptic density that mediate signaling-dependent modiﬁcations in the content, morphology, and function of the postsynaptic density (Contractor and Heinemann, 2002; McGee and Bredt, 2003). For example, the CaMKII-mediated phosphorylation of newly synthesized and assembled AMPA receptors promotes interaction with SAP97 and stargazin (Cai et al., 2002; Chen et al., 2000; Lisman and Zhabotinsky, 2001). The AMPA receptor–SAP97– stargazin complex may laterally translocate and colocalize in the postsynaptic density with the NMDA receptor (Lisman and Zhabotinsky, 2001). In summary, a number of molecules have been localized to postsynaptic neurons of glutamatergic synapses in brain regions where LTP has been observed. AMPA and NMDA receptors and their aYliated signaling, traYcking,

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Fig. 1. Binding sites and receptor subunits for NMDA and AMPA subtypes of the glutamate receptor and subunit-associated intracellular signaling proteins.

scaVolding, and anchoring proteins have a central role in the activation and regulation of LTP, a phenomena believed to be essential for learning, memory, and other cognitive functions that underlie neuroplasticity. V. Glutamatergic Dysfunction in Schizophrenia

Given that schizophrenia can be considered a disorder of neuroplasticity, and the central role of glutamatergic neurotransmission in molecular events that underlie plasticity, this section discusses evidence for glutamatergic abnormalities in schizophrenia. A. Phencyclidine as a Pharmacological Model of Schizophrenia Perhaps the most compelling evidence implicating glutamate dysfunction in schizophrenia is the fact that phencyclidine and related compounds such as ketamine, which are antagonists of the NMDA receptor, can induce both positive and negative symptoms of schizophrenia, including cognitive deﬁcits ( Javitt and

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Zukin, 1991; Tamminga, 1999). Moreover, these compounds can exacerbate both positive and negative symptoms in persons with schizophrenia (Lahti et al., 1995). Chronic administration of PCP-like compounds may provide a more valid model of schizophrenia than acute treatment because it induces persistent psychotic symptomatology and reduces frontal lobe blood ﬂow and glucose utilization, eVects remarkably similar to the ‘‘hypofrontality’’ described in schizophrenia (Hertzmann et al., 1990; Javitt and Zukin, 1991). Acute PCP administration leads to increased glutamate release in the prefrontal cortex of rodents (Moghaddam and Adams, 1998; Moghaddam et al., 1997). Few studies, however, have evaluated the eVects of chronic PCP exposure on glutamatergic systems. Electrophysiological data reveal a heightened depolarization of prefrontal cortical pyramidal neurons of rats treated chronically with PCP following a local application of NMDA (Arvanov and Wang, 1999; Yu et al., 2002). Chronic PCP treatment also alters the expression in brain of NMDA receptor subunits, as well as the subunit stoichiometry of the NMDA receptor (Yu et al., 2002). To date, no studies have directly examined extracellular glutamate levels in the prefrontal cortex following long-term PCP treatment.

B. Decreased Cerebrospinal Fluid Glutamate Concentrations in Schizophrenia In the early 1980s, decreased glutamate levels were reported in the cerebrospinal ﬂuid (CSF) of patients with schizophrenia, suggesting that glutamate deﬁciency may have a role in the pathophysiology of this illness (Kim et al., 1980). Subsequent studies have provided inconsistent results, including ﬁndings of decreased CSF glutamate concentrations and unchanged glutamate levels in serum, plasma, and CSF (Alfredsson and Wiesel, 1989; Bjerkenstedt et al., 1985; Deutsch et al., 1989; Gattaz et al., 1985; Korpi et al., 1987; Macciardi et al., 1990; Perry, 1982). Antipsychotic treatment and other factors may inﬂuence CSF glutamate levels, which may account for discrepancies among these earlier studies (Gattaz et al., 1985; GoV et al., 1996). In a more recent study, CSF levels of glutamate and other amino acids [aspartate, Nacetylaspartate (NAA), and N-acetylaspartylglutamate (NAAG)], clinical symptomatology, and brain structure were measured in antipsychotic-free schizophrenic patients in order to examine associations between glutamatergic markers and other illness-related factors. While signiﬁcant diVerences in amino acid levels were not detected in the group with schizophrenia, signiﬁcant negative correlations between CSF glutamate concentrations and degree of both structural atrophy and clinical symptomatology were found, indicating that greater brain atrophy and cognitive impairment in schizophrenia are associated with lower glutamate levels (Tsai et al., 1998a). Because CSF excitatory amino acid levels appear to be inﬂuenced by the clinical state and severity of brain atrophy, both of which may vary

SCHIZOPHRENIA AS A DISORDER OF NEUROPLASTICITY

27

substantially among patients with schizophrenia, these data may also help explain the inconsistency among previous CSF studies in schizophrenia. C. In Vivo Evidence of Decreased Glutamatergic Markers in Schizophrenia Magnetic resonance spectroscopy (MRS) has been used to study possible glutamatergic abnormalities in vivo in the brain in schizophrenia (Kegeles et al., 1998). Glutamate itself is currently diYcult to resolve using this method without quite powerful magnets, although a trend for decreased glutamate levels in the thalamus in schizophrenia has been reported in one study (Kegeles et al., 1998; Omori et al., 1997). One of the most consistent MRS ﬁndings in schizophrenia is a decreased level of the amino acid NAA in the prefrontal cortex, temporal cortex, thalamus, basal ganglia, and cerebellum (Table I). Glutamate and NAA are produced by the enzymatic breakdown of NAAG. While the exact role of NAA is not fully understood, it is considered a neuronal marker, and its relative abundance is generally thought to reﬂect neuronal density, function, and/or viability (Kegeles et al., 1998). Others have speculated that decreased NAA levels may be associated with decreased glutamate levels, as both are produced by the enzymatic breakdown of NAAG. These ﬁndings may be associated with diminished glutamate neurotransmission in certain brain regions in schizophrenia; however, the more commonly accepted interpretation is that decreased NAA levels reﬂect neuronal or glial dysfunction in the frontal cortex, temporal structures, and the thalamus in schizophrenia, which is consistent with postmortem and in vivo imaging studies describing abnormalities in these regions (Andreasen et al., 1997; Shenton et al., 2001). D. Evidence of Decreased Excitatory Amino Acids in Postmortem Brain Postmortem studies of glutamate and its related metabolites have found decreased levels of aspartate and glutamate in schizophrenia. In addition, activity of the enzyme N-acetyl--linked acidic dipeptidase (NAALADase), which cleaves NAAG into NAA and glutamate, was decreased, whereas increased levels of NAAG, a substrate for NAALADase, were found in the prefrontal cortex and hippocampus (Tsai et al., 1995). These ﬁndings are consistent with diminished activity at glutamate receptors in regions that have been implicated in the pathophysiology of schizophrenia, especially the prefrontal cortex (Coyle, 1996; GoV and Wine, 1997). Because LTP requires normally functioning NMDA receptors, reduced prefrontal glutamate release and/or NMDA receptor activity may aVect the activation of LTP in cortical regions, which in turn may be associated with deﬁcits in working memory and other cognitive tasks attributed to the neocortex.

28

McCULLUMSMITH et al.

TABLE I H MR Spectroscopy Evidence for Glutamatergic Abnormalities in Schizophrenia

1

Glutamate and related metabolitesa

Findings

Frontal cortex

NAA NAA Ratio NAA/Cr Ratio NAA/Cr; NAA/Ch Ratio NAA/Cr Ratio NAA/Cr NAA NAA Ratio NAA/Cr, NAA/Ch Glutamate Glutamine

# None # # # # None None None None "

Deicken et al. (1997a) Steel et al. (2001) Choe et al. (1994) Bertolino et al. (1998) Cecil et al. (1999) Callicott et al. (2000) Stanley et al. (1996) Buckley et al. (1994) Fukuzako et al. (1995) Bartha et al. (1997) Bartha et al. (1997)

Temporal cortex

NAA NAA NAA

# # #

NAA Ratio NAA/Cr Ratio NAA/Cr, NAA/Ch Ratio NAA/Cr

# # # #

Ratio NAA/Cr, NAA/Ch Ratio NAA/Cr Ratio NAA/Cr NAA NAA, glutamate, glutamine Glutamate, NAA Glutamine

# # # None None None "

Nasrallah et al. (1994) Maier et al. (1995) Deicken et al. (1997b, 1998) Maier et al. (2000) Renshaw et al. (1995) Fukuzako et al. (1995) Yurgelun-Todd et al. (1996) Bertolino et al. (1998) Cecil et al. (1999) Callicott et al. (2000) Buckley et al. (1994) Bartha et al. (1999) Theberge et al. (2002) Theberge et al. (2002)

Basal ganglia

Ratio NAA/Ch Ratio NAA/Cr Ratio NAA/Cr, NAA/Ch

# None None

Fujimoto et al. (1996) Sharma et al. (1992) Bertolino et al. (1998)

Thalamus

Glutamate and NAA Glutamate, NAA Glutamine NAA NAA NAA NAA NAA NAA

None None " # # # None None None

Omori et al. (1997) Theberge et al. (2002) Theberge et al. (2002) Deicken et al. (2000) Auer et al. (2001) Ende et al. (2001) Heimberg et al. (1998) Bertolino et al. (1998) Delamillieure et al. (2000, 2002)

Cerebellum

NAA

#

Deicken et al. (2001)

Brain region

a

NAA, N-acetylaspartate; Cr, creatine; Ch, choline.

Reference

SCHIZOPHRENIA AS A DISORDER OF NEUROPLASTICITY

29

E. Alterations of Receptors and Associated Intracellular Molecules Relevant to Plasticity in Postmortem Brain Studies evaluating NMDA and AMPA receptor subunit and binding site expression in the brain are complicated by the structure and subunit composition of these receptors. NMDA receptor subunits are encoded by seven genes: NR1, NR2A–NR2D, NR3A, and NR3B (Eriksson et al., 2002; Hollmann and Heinemann, 1994; Matsuda et al., 2002; Nishi et al., 2001) (Fig. 1). NR1 can be expressed as one of eight isoforms due to the alternative splicing of exons 5, 21, and 22 (Durand et al., 1993; Hollmann and Heinemann, 1994; Nakanishi, 1992). AMPA receptor subunits are derived from four diVerent genes, gluR1–gluR4 (Fig. 1). Subunits associated with the AMPA receptor also exist in multiple forms due to alternative splicing and editing of their respective transcripts (Hollmann and Heinemann, 1994). Accordingly, there is the potential for heterogeneity in both AMPA and NMDA receptors based on subunit composition, as well as transcriptional and posttranscriptional modiﬁcation of individual subunits. The expression of NMDA receptor subunit transcripts and binding sites has been evaluated in the brain in schizophrenia (Table II). Studies on the expression of ionotropic glutamate receptors in the brain in psychiatric illnesses have focused almost exclusively on schizophrenia and have concentrated on cortical and medial temporal lobe structures. Relatively few studies have focused on the expression of NMDA receptors in subcortical limbic structures. While there are discrepancies among some of these studies, alterations in NMDA receptor subunit and binding site expression are complex and region speciﬁc (Table II). There is evidence for shifts in subunit stoichiometry and increased binding to at least some of the NMDA-binding sites, primarily in cortical areas (Akbarian et al., 1996; Dracheva et al., 2001; Grimwood et al., 1999; Humphries et al., 1996; Kornhuber et al., 1989; Sokolov, 1998). The AMPA receptor has been studied extensively at multiple levels of gene expression in the brain in schizophrenia, as summarized in Table III. Data from these studies result in one of the more robust and reproducible sets of ﬁndings in this ﬁeld; many past studies have found that AMPA receptor expression is decreased in the hippocampus and related structures in schizophrenia, occurring at the levels of both transcript and subunit protein expression (Breese et al., 1995; Eastwood et al., 1995, 1997a,b; Harrison et al., 1991; Kerwin et al., 1990). With a few exceptions, AMPA receptor expression tends to be unchanged in other cortical and subcortical areas (Breese et al., 1995; Eastwood et al., 1995, 1997b; Freed et al., 1993; Sokolov, 1998). Taken together, these studies imply two important generalizations about the expression of AMPA and NMDA receptors in the brain in schizophrenia. First, most abnormalities have been reported in limbic cortical and hippocampal regions, with relative sparing of subcortical structures. Second, both NMDA and AMPA

TABLE II NMDA Receptor Expression in Schizophrenia

30

Level of gene expression

Techniquea

Receptor-binding sites

Autoradiography

MK-801

None

Homogenate binding

MK-801

"

MK-801

None

L-689, 560

"

L-689, 560 L-689, 560

None "

Temporal cortex

Grimwood et al. (1999)

L-689, 560 CGP 39653 Ifenprodil Ifenprodil L-689, 560

None None " None "

Motor cortex Temporal cortex, motor cortex Temporal cortex Motor cortex Temporal cortex

Nudmamud and Reynolds (2001)

L-689, 560 Ifenprodil MDL105, 519 MK-801, GCP39653 Ifenprodil MDL105, 519

None # # None None None

Prefrontal cortex Thalamus Thalamus Thalamus Caudate, putamen, accumbens

Homogenate binding Homogenate binding

Homogenate binding Autoradiography

Autoradiography

Probe(s)b

Finding

Brain region

Caudate, putamen, nucleus accumbens Putamen Frontal cortex, entophinal region, hippocampus, amygdala Caudate, putamen, nucleus accumbens

Reference

Noga et al. (1997) Kornhuber et al. (1989)

Aparicio-Legarza et al. (1998)

Ibrahim et al. (2000)

Meador-Woodruff et al. (2001)

Autoradiography Autoradiography Autoradiography

MK-801 GCP39653 MK-801 MK-801 MK-801 NMDA MK-801

None None # None " None None

CA3 CA1, CA2, dentate gyrus Anterior cingulate Hippocampus Prefrontal cortex

Zavitsanou et al. (2002) Gao et al. (2000) Noga et al. (2001)

ISH

NR2D

"

Prefrontal cortex

Akbarian et al. (1996)

NR1, NR2A-C NR1, NR2A-D NR1, NR2B,C NR2A,D NR1 NR1 NR1, NR2A-D NR1 NR2A NR2B NR1 w/exon 5 NR1 NR1 NR1 NR1, NR2A NR2B

None None # None # None None # None " " # #c Noned " None

Prefrontal cortex Cerebellum, parietotemporal cortex Thalamus Thalamus Dentate gyrus CA3 Accumbens, caudate, putamen Hippocampus

Autoradiography

Subunit mRNA expression

ISH ISH 31

ISH ISH

ISH Northern blot RT-PCR RT-PCR a

Frontal, occipital, temporal cortex Temporal cortex Frontal cortex Frontal cortex Prefrontal, occipital cortex Prefrontal, occipital cortex

ISH, in situ hybridization; RT-PCR, reversed transcribed-polymerase chain reaction. All binding studies utilized 3H unless otherwise noted. c Neuroleptic-free schizophrenics vs. controls. d Neuroleptic-treated schizophrenics vs. control. b

Dean et al. (1999)

Ibrahim et al. (2000) Law and Deakin (2001) Meador-Woodruff et al. (2001) Gao et al. (2000)

Le Corre et al. (2000) Humphries et al. (1996) Sokolov (1998) Dracheva et al. (2001)

TABLE III AMPA Receptor Expression in Schizophrenia Level of gene expression Receptor-binding sites 32 Subunit protein expression

Techniquea

Probe(s)b

Finding

Brain region

Reference

Autoradiography

CNQX

"

Caudate

Noga et al. (1997)

Autoradiography Autoradiography Autoradiography Autoradiography Homogenate binding Autoradiography Autoradiography Autoradiography

CNQX CNQX CNQX AMPA CNQX AMPA AMPA AMPA AMPA

None # " None # None None # "

Putamen, nucleus accumbens Caudate, nucleus accumbens Prefrontal cortex Caudate, putamen, nucleus accumbens CA4, CA3 Frontal cortex, putamen, nucleus accumbens Thalamus CA2 Anterior cingulate

Noga and Wang (2002) Noga et al. (2001) Healy et al. (1998) Kerwin et al. (1990) Freed et al. (1993) Ibrahim et al. (2000) Gao et al. (2000) Zavitsanou et al. (2002)

Immunohistochemistry

GluR1

#

Parahippocampal gyrus

Eastwood et al. (1997b)

GluR2/3

None # None

GluR1 GluR2, GluR3

None None

CA1, CA3, CA4, subiculum, CA4 Dentate gyrus, CA1, CA3, subiculum, parahippocampal gyrus Hippocampus Cingulate cortex

Western blot

Breese et al. (1995)

Subunit mRNA expression

ISH

GluR1c

CA1, parahippocampal gyrus Dentate gyrus, CA3, CA4, subiculum CA1 Caudate, putamen, nucleus accumbens CA3 Dentate gyrus, CA1, CA4, subiculum Thalamus Thalamus Frontal cortex Frontal cortex Hippocampus Hippocampus Hippocampus Prefrontal cortex

ISH ISH

GluR1–4 GluR-K1

ISH

GluR1, 3 GluR2, 4 GluR1 GluR1 GluR2 flip GluR2 flop flip/flop ratio GluR2

None # # #

RT-PCR RT-PCR

Microarray

33

Dentate gyrus, CA3, CA4, subiculum

None # None None # None # None #d Nonee

GluR2c

a

#

ISH, in situ hybridization; RT-PCR, reversed transcribed-polymerase chain reaction. All binding studies utilized 3H unless otherwise noted. c Flip plus flop. d Neuroleptic free schizophrenics vs. controls. e Neuroleptic treated schizophrenics vs. control. b

Eastwood et al. (1995)

Healy et al. (1998) Harrison et al. (1991) Ibrahim et al. (2000) Sokolov (1998) Eastwood et al. (1997a)

Vawter et al. (2002)

34

McCULLUMSMITH et al.

receptors have been reported to be abnormal, with changes in binding sites as well as subunit changes suggestive of altered stoichiometry of subunit composition. The expression of NMDA and AMPA receptor-aYliated molecules in schizophrenia has been evaluated. Our laboratory found signiﬁcant increases in the expression of transcripts encoding the NMDA-associated proteins PSD95 and SAP102, but not PSD93, in the thalamus in schizophrenia (Table IV) (Clinton et al., 2003). Other groups have found changes in the expression of PSD95 mRNA in the occipital cortex and prefrontal cortex, whereas the expression of the PSD95 protein was unchanged in the hippocampus (Dracheva et al., 2001; Ohnuma et al., 2000; Toyooka et al., 2002) (Table IV). PSD95 and SAP102 are PDZ-containing proteins that facilitate the interface of NMDA receptors and intracellular signaling processes (Contractor and Heinemann, 2002; Kornau et al., 1995; McGee and Bredt, 2003; Muller et al., 1996; Niethammer et al., 1996). Interestingly, PSD95-deﬁcient mice exhibited enhanced LTP at diVerent frequencies of stimulation and impaired spatial learning, supporting a role for PSD95 in the coupling of NMDA receptors to LTP and neuroplasticity (Migaud et al., 1998). Changes in the expression of PSD95 and related molecules suggest that the functional link between NMDA receptors and intracellular signaling processes is abnormal in schizophrenia. We also found that NF-L mRNA expression was elevated in the thalamus in schizophrenia (Clinton et al., 2003). Although a portion of NF-L in thalamic cells may associate with the NR1 subunit to participate in NMDA function, the majority of NF-L associates with the other neuroﬁlament subunits to maintain the neuronal cytoskeleton. The impact of changes in the expression of scaVolding proteins such as NF-L on LTP and plasticity remains to be determined. Changes in AMPA receptor-aYliated molecules have also been reported. NSF mRNA was decreased in the prefrontal cortex in persons with schizophrenia in one microarray study, whereas NSF protein and mRNA levels were unchanged in another utilizing Western blots and reversed transcribed-polymerase chain reaction (Imai et al., 2001; Mirnics et al., 2000). A diVerent study found decreased levels of the GluR1 subunit-binding protein SAP97 in the prefrontal cortex in schizophrenia (Toyooka et al., 2002). NSF and SAP97 are components of the intracellular machinery that mobilizes AMPA receptors to the plasma membrane from constitutive and newly synthesized pools of receptors, respectively (Contractor and Heinemann, 2002; Kim and Lisman, 2001; McGee and Bredt, 2003; Sans et al., 2001). Localization of AMPA receptors to the PSD in juxtaposition with NMDA receptors is a critical element for the induction of LTP, whereas removal of the AMPA receptor is believed central for long-term depression (Malenka and Nicoll, 1999). Thus, changes in the expression of AMPA receptor-associated molecules may signiﬁcantly impact LTP-dependent processes such as learning and memory, supporting the hypothesis of dysfunctional neuroplasticity in schizophrenia.

TABLE IV Expression of Intracellular Glutamate Receptor-Associated Molecules in Schizophrenia Affiliated receptor

Techniquea

NMDA receptor

ISH ISH RT-PCR

35

Western

AMPA receptor

a

Microarray RT-PCR Western Western

Probe(s)

Finding

Brain region

PSD-95, SAP102, NF-L PSD-93 PSD-95 PSD-95 PSD-95 PSD-95 SAP102 PSD-95, PSD-93 SAP102, PSD-95, PSD-93 NSF NSF NSF SAP97 GRIP1 SAP97, GRIP1 SAP97, GRIP1

" None # None " None # None None # None None # None None None

Thalamus Thalamus Prefrontal cortex Hippocampus Occipital cortex Prefrontal cortex Hippocampus Hippocampus Prefrontal, occipital cortex Prefrontal cortex Prefrontal cortex Prefrontal cortex Prefrontal cortex Prefrontal cortex Hippocampus Occipital cortex

ISH, in situ hybridization; PSD, postsynaptic density.

Reference Clinton et al. (2003) Ohnuma et al. (2000) Dracheva et al. (2001) Toyooka et al. (2002)

Mirnics et al. (2000) Imai et al. (2001) Toyooka et al. (2002)

36

McCULLUMSMITH et al.

Few broad conclusions may be drawn about the expression of receptorassociated intracellular proteins in schizophrenia due to the relatively low number of published studies. However, there appear to be abnormalities of these molecules in regions where their aYliated receptors are also abnormal, such as the prefrontal cortex and the thalamus. In addition, changes in receptor-associated molecules are not necessarily in the same direction in schizophrenia as alterations in receptor expression. For example, while NR1 mRNA expression was decreased in the thalamus in schizophrenia, transcripts for the NR1-aYliated molecules PSD95, SAP102, and NF-L were increased. Such disparate results suggest diVerential regulation of the expression of NMDA receptor subunits and receptor-binding proteins. One of the most replicated ﬁndings in schizophrenia is a decrease in AMPA receptor expression in the hippocampus, a region of the brain where LTP has been studied the most extensively. The expression, colocalization with the NMDA receptor, and phosphorylation state of the AMPA receptor are all critical variables for LTP (Malenka and Nicoll, 1999). However, postmortem studies of AMPA receptor subunit and binding site expression in schizophrenia have been limited to examining the total pool of this receptor and its subunits. Decreases in AMPA receptor expression could be in the constitutive, regulated, or newly synthesized pools of these receptors. Region- and subunit-speciﬁc changes in NMDA receptor expression have also been reported in schizophrenia. NMDA receptor activation is an event central to LTP (Malenka and Nicoll, 1999). Thus, changes in NMDA receptor expression and stoichiometry may aVect LTP and neuroplasticity directly. Further, the characterization of NMDA and AMPA receptor-interacting proteins has provided novel substrates for neurochemical studies of schizophrenia. These novel molecules have a clear role for the facilitation of AMPA receptor cycling and the interface of AMPA and NMDA receptors with intracellular signaling pathways (Contractor and Heinemann, 2002; Malenka and Nicoll, 1999; McGee and Bredt, 2003). Initial ﬁndings in schizophrenia suggest that changes in these receptor-associated molecules may be more robust than detected changes in aYliated receptors. These converging lines of evidence support the concept of schizophrenia as a disorder of neuroplasticity, as LTP and related molecular correlates of learning and memory are likely altered in this illness.

F. Glutamate-Based Pharmacological Treatment in Schizophrenia The induction of schizophreniform symptoms by antagonists of the NMDA receptor has prompted eVorts to identify glutamate transmission-enhancing pharmacological treatments that may oVer therapeutic beneﬁt, particularly for the treatment of persistent negative and cognitive symptoms that do not respond to traditional antipsychotic treatment. Direct NMDA receptor agonists are not

SCHIZOPHRENIA AS A DISORDER OF NEUROPLASTICITY

37

feasible because of their propensity to become excitotoxic and poor penetration of the blood–brain barrier. A more promising target, however, is the glycine/dserine modulatory site of the NMDA receptor complex. Compounds that act at this site have been found to be NMDA receptor-promoting agents. Given in combination with traditional antipsychotic medications, these drugs produce signiﬁcant improvement in both negative symptoms and cognitive deﬁcits in schizophrenia (GoV and Wine, 1997) (Table V). Early clinical trials using low doses (5–15 g/day) of glycine had variable outcomes, largely due to the poor ability of glycine to cross the blood–brain barrier (Costa et al., 1990; Waziri, 1988). More recently, double-blind placebo-controlled crossover trials using high doses of glycine (30–60 g/day) added to antipsychotic treatments have been conducted. Those studies demonstrated selective improvement in negative symptoms and cognitive functioning (Heresco-Levy et al., 1996, 1999; Javitt et al., 1994; Leiderman et al., 1996). d-Serine, another full agonist at the glycine modulatory site, was given (30 mg/ kg/day) in addition to antipsychotics in a double-blind placebo-controlled trial. d-Serine treatment reduced both negative and positive symptoms and improved performance of the Wisconsin card sorting test. d-Serine crosses the blood–brain barrier more readily than glycine and has a higher aYnity for the glycine modulatory site, which may account for its greater eYcacy (Tsai et al., 1998b). d-Cycloserine, a rigid analog of d-alanine, acts as a partial agonist at the glycine modulatory site and has also been shown to improve negative symptoms in schizophrenia. Early trials with relatively high doses of d-cycloserine (250 mg/day) were ineVective in treating psychotic symptoms in schizophrenia (Cascella et al., 1994; Simeon et al., 1970). A later dose-ﬁnding trial (giving either placebo or 5, 15, 50, or 250 mg/day) showed that d-cycloserine signiﬁcantly improved negative symptoms and cognitive functioning only at a dose of 50 mg (GoV et al., 1995). Several subsequent studies have conﬁrmed that 50 mg/day of d-cycloserine given in combination with conventional antipsychotics improves negative symptoms by 10–23% (Evins et al., 2002; GoV et al., 1996, 1999; Heresco-Levy et al., 1998, 2002). These studies demonstrate that enhancing NMDA receptor function through activation of the glycine modulatory site signiﬁcantly impacts negative symptoms and improves cognitive functioning in schizophrenia. Another class of drugs that may be useful for the treatment of schizophrenia are AMPAkines, compounds that act as positive modulators of the AMPA receptor. AMPAkines have been shown to increase the peak and duration of glutamateinduced AMPA receptor-gated inward currents and to also enhance LTP in the hippocampal slice, as well as learning and memory performance (Larson et al., 1996). Because NMDA receptor activation requires partial membrane depolarization via AMPA receptors, agents that modulate AMPA receptor function positively may have therapeutic beneﬁts in schizophrenia (Tsai and Coyle, 2002). Preliminary results from placebo-controlled trials in a small number of patients

TABLE V Efficacy of Glutamate-Based Pharmacotherapies in Schizophrenia Glycine-site modulator Glycine

38

Dose/day

Positive symptoms

10 g 30–60 30–60 30–60 30–60 30 g 60 g

No No No No No No No

g g g g

effect effect effect effect effect effect effect

Negative symptoms

Cognitive symptoms

Reference

No effect Improved 36% improvement 20% improvement 30% improvement No effect No effect

No effect Not rated Improved Not rated 30% improvement Not rated No effect

Rosse et al. (1989) Javitt et al. (1994) Heresco-Levy et al. (1996) Leiderman et al. (1996) Heresco-Levy et al. (1999) Potkin et al. (1999) Evins et al. (2000)

d-Serine

30 mg/kg 30 mg/kg

Improved No effect

Improved No effect

Improved No effect

Tsai et al. (1998) Tsai et al. (1999)

d-Cycloserine

2g 250 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg

Worsened Worsened Not rated Not rated No effect Not rated No effect Not rated Not rated

No effect No effect 21% improvement 21% improvement Improved 23% improvement Worsened 10% improvement 15% improvement

No effect No effect Improved Not rated Not rated No effect Not rated No effect Not rated

Simeon et al. (1970) Cascella et al. (1994) Goff et al. (1995) Goff et al. (1996) Heresco-Levy et al. (1998) Goff et al. (1999b) Goff et al. (1999a) Evins et al. (2002) Heresco-Levy et al. (2002)

SCHIZOPHRENIA AS A DISORDER OF NEUROPLASTICITY

39

receiving clozapine and the AMPAkine CX516 suggest improved performance in tests of attention, memory, and distractibility (GoV and Coyle, 2001). Changes in glutamate levels measured in the CSF and the brain, glutamate metabolites measured by MRS, and glutamate receptor expression all suggest alterations of glutamatergic neurotransmission in schizophrenia. This is further supported by an induction of psychotic symptoms following the administration of NMDA receptor antagonists and an improvement in positive and negative symptoms with compounds that promote NMDA receptor function. Interestingly, glycine-site agonists have been demonstrated to enhance LTP in the hippocampus and visual cortex, suggesting another possible mechanism for the beneﬁcial eVects of glycine-site agonists in schizophrenia (Abe et al., 1990; Ito and Hicks, 2001; Ramakers et al., 1993; Watanabe et al., 1992).

VI. Conclusions

Schizophrenia is a devastating illness aVecting nearly 1% of the population worldwide. Despite the currently available treatment regimens, schizophrenia exacts a costly emotional and ﬁnancial toll on aVected individuals, their family members, and society at large. Numerous observations suggest that schizophrenia may be a disorder of neuroplasticity. Interestingly, many mechanisms of neuroplasticity involve molecules associated with glutamatergic neurotransmission; many of these glutamatergic molecules have also been found to be abnormal in schizophrenia. We suggest that schizophrenia can be considered a disorder of plasticity, associated with molecular abnormalities of the glutamate synapse.

Acknowledgments

This work was supported by a Pfizer Postdoctoral Fellowship (REM) and MH53327 ( JMW ).

References

Abe, K., Xie, F. J., Watanabe, Y., and Saito, H. (1990). Glycine facilitates induction of long-term potentiation of evoked potential in rat hippocampus. Neurosci. Lett. 117, 87–92. Akbarian, S., Sucher, N. J., Bradley, D., Tafazzoli, A., Trinh, D., Hetrick, W. P., Potkin, S. G., Sandman, C. A., Bunney, W. E., Jr., and Jones, E. G. (1996). Selective alterations in gene expression for NMDA receptor subunits in prefrontal cortex of schizophrenics. J. Neurosci. 16, 19–30.

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THE SYNAPTIC PATHOLOGY OF SCHIZOPHRENIA: IS ABERRANT NEURODEVELOPMENT AND PLASTICITY TO BLAME?

Sharon L. Eastwood Department of Psychiatry University of Oxford, Warneford Hospital Oxford, OX3 7JX, United Kingdom

I. Introduction II. Studies of Proteins Associated with Synaptic Plasticity in Schizophrenia A. Growth-Associated Protein-43 Expression in Schizophrenia B. Neural Cell Adhesion Molecule in the Brain in Schizophrenia III. Can the Synaptic Pathology of Schizophrenia Be Related to Changes in the Expression of Genes Involved in Development and Plasticity? A. Presynaptic Protein Expression Is Altered in Schizophrenia B. Cerebellar Reelin and Semaphorin 3A Expression in Schizophrenia C. Altered Reelin Expression Is Related to Cortical Synaptic Pathology D. How May Reelin and Semaphorin 3A Expression Contribute to Altered Synaptic Plasticity and Pathology in Schizophrenia? IV. Discussion and Future Directions References

Synaptic pathology is a feature of the brain in schizophrenia, denoted by alterations in the expression of synaptic proteins. In the absence of data indicative of neurodegenerative processes, the neuropathological features of schizophrenia suggest that the major pathogenic process in the disorder is one of aberrant development. Molecular evidence in support of a neurodevelopmental origin of schizophrenia has come from studies examining the expression of key developmental genes. However, as many of these genes are also involved in synaptic plasticity, their altered expression in schizophrenia also suggests that the disorder may be one of aberrant synaptic plasticity. The aim of this review is to explore whether aberrant development and synaptic plasticity may underlie the synaptic pathology of schizophrenia. It does this in two ways. First, studies in schizophrenia of the expression of two synaptic genes important in synaptic remodeling and plasticity are reviewed, changes in which may be indicative of aberrant synaptic plasticity in the disorder. Second, the possible relationship between the expression of genes involved in development and plasticity with that of presynaptic proteins is examined. Such a relationship, in combination with their altered expression in schizophrenia, may indicate whether developmental and plasticity-related processes may contribute to the synaptic pathology of the

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disorder. A brief discussion on the possible origins of the synaptic pathology of schizophrenia, and possible future studies, concludes the review.

I. Introduction

Many structural alterations occur in the brain in schizophrenia (see Harrison, 1999; Shapiro, 1993), and the prevailing hypothesis is that these are caused by an anomaly in brain development due to genetic and environmental factors (Lewis and Levitt, 2002; Marceno and Weinberger, 2000; Weinberger, 1987). Robust macroscopic changes of increased ventricular volume and decreased cerebral volume are accompanied by histological correlates, including reduced neuropil and decreased neuronal size (rather than neuronal loss). Together with other evidence, these neuropathological features of schizophrenia suggest that decreases in cortical volume may reﬂect changes in the synaptic, dendritic, and axonal components of the neuropil in the disorder (see Harrison, 1999), and it is feasible that such synaptic changes may underlie the aberrant functional connectivity widely purported to occur in the disorder (Andreason et al., 1996, 1999; Benes, 2000; Bullmore et al., 1998; Friston and Frith, 1995; McGuire and Frith, 1996). For these reasons, many researchers have focused on determining whether synaptic alterations are a feature of the brain in schizophrenia. To do this, presynaptic proteins have been widely utilized as proxy markers of synapses, and their altered expression is mostly interpreted as indicative of changes in synaptic density (or size), thereby denoting synaptic pathology (Eastwood et al., 1994; Masliah et al., 1990). Although data are sometimes inconsistent, for some brain regions, including the hippocampus, many studies have found molecular evidence in support of a synaptic pathology of schizophrenia (see review by Honer and Young). Demonstrating that a pathology exists is the ﬁrst step to gaining a better understanding of the processes that may be involved in schizophrenia, the next is to hypothesize and investigate what factors may be contributing to such a pathology. It is widely accepted that schizophrenia is a disorder of aberrant neurodevelopment, and as such, many groups are investigating whether the expression of key developmental genes is altered in schizophrenia. However, one of the fundamental issues associated with postmortem studies of gene expression is that they are only able to examine a snapshot of the events that may be taking place in the brain in schizophrenia. It is not possible to determine whether changes in the expression of genes of interest reﬂect incidents immediately prior to death or are representative of ongoing molecular changes that may have been present throughout the lifetime of the subject. This issue is discussed later, but regardless of the timing of events, the altered expression of key developmental genes in schizophrenia may indicate which processes may have contributed, or still be

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contributing, to the synaptic pathology of schizophrenia. One widely promoted possible process is synaptic plasticity, and it has been proposed that aberrant synaptic plasticity may be a persisting manifestation of the aberrant neurodevelopment thought to underlie schizophrenia. This review explores the question of whether aberrant plasticity may contribute to the pathogenesis of schizophrenia, and in particular, whether the synaptic pathology of schizophrenia can be related to changes in the expression of developmental genes also involved in synaptic plasticity. It begins with a review of studies of two synaptic proteins thought to be involved in events mediating synaptic plasticity, which also play a role in axon formation and guidance in development.

II. Studies of Proteins Associated with Synaptic Plasticity in Schizophrenia

The molecular mechanisms underlying synaptic remodeling and plasticity are not thoroughly understood and have mostly been studied in speciﬁc features of synaptic plasticity, learning and memory, such as long-term potentiation (LTP) and long-term depression (LTD). One review listed more than 100 possible candidates implicated in these forms of plasticity (Sanes and Lichtman, 1999), and despite the little we know, what is certain is that there is not one molecule solely responsible for determining the capacity of a neuron for synaptic plasticity. Hence, although changes in the expression of any of these genes in schizophrenia are supportive of ideas of aberrant synaptic plasticity in the disorder, the underlying caveat is that alone they may not be suYcient to cause it. Interestingly, of the candidates listed, many are genes important during brain development and include those known to play a role in axon guidance and synapse formation. Studies of two such genes in the brain in schizophrenia are reviewed here.

A. Growth-Associated Protein-43 Expression in Schizophrenia Growth-associated protein-43 (GAP-43; also known as F1, B-50, or neuromodulin) is a presynaptic phosphoprotein involved in neurodevelopment, plasticity, and injury response. It is a principal substrate for protein kinase C (PKC) and aVects several intracellular messenger systems through its interactions with calmodulin and GTP-binding proteins. This section gives a brief overview on the roles of GAP-43 in development and plasticity and the reasoning behind it being a protein of interest in studies of schizophrenia. For more comprehensive summaries on GAP-43 function, the reader is directed to Benowitz and Routtenberg (1997) and Oestreicher et al. (1997).

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GAP-43 is ﬁrst detected in postmitotic neurons and becomes segregated to the developing axon, thereby providing an early molecular marker of normal neuronal polarity (Goslin and Banker, 1990; Goslin et al., 1990). It remains concentrated in growth cones and axons, being most highly expressed during development, after which it is downregulated and a more restricted pattern of expression is observed in adult rat and human brain (de la Monte et al., 1989; Neve et al., 1988). Support for a key role of GAP-43 in axon growth and guidance during development comes from experimental manipulations of its expression. For example, downregulation of GAP-43 in culture results in a failure of axon outgrowth (Aigner and Caroni, 1993, 1995; Shea et al., 1991), whereas overexpression in a transgenic mouse model lead to aberrant connectivity, with axons overshooting their normal targets (Aigner et al., 1995). The continued expression of GAP-43 into adulthood is thought to reﬂect its role in LTP, neurotransmission, and synaptic plasticity. GAP-43 undergoes a persistent change in phosphorylation during LTP, which is proposed to lead to the interaction of GAP-43 with the cytoskeleton, thereby altering the motility of the nerve ending and allowing synaptic reorganization (Benowitz and Routtenberg, 1997). GAP-43 phosphorylation is also thought to be involved in the modulation of neurotransmitter release (Iannazzo, 2001), and other evidence suggests that GAP-43 is involved in the calcium-dependent fusion of synaptic vesicles to the presynaptic membrane (Haruta et al., 1997). Taken together, these ﬁndings suggest that the selected expression of GAP-43 by certain neuronal populations may be related to their greater capacity for synaptic plasticity, a supposition supported by evidence from experimental (Bendotti et al., 1994; Chao and McEwan, 1994; Levin and Dunn-Meynell, 1993; Lin et al., 1992; Liu et al., 1996; Masliah et al., 1991; Palacios et al., 1994) and neuropathological lesions (Parhad et al., 1992), where GAP-43 expression is observed to increase. Early observations of GAP-43 expression in adulthood in both human and rat brain suggested that it was restricted primarily to limbic and association cortical regions (Benowitz et al., 1989; de la Monte et al., 1989; Kruger et al., 1993; Neve et al., 1988; Ng et al., 1988). This patten of expression in brain regions often implicated in the symptomatology and pathophysiology of schizophrenia (Harrison, 1999; McGuire and Frith, 1996; Pearlson et al., 1996) led to suggestions that altered GAP-43 expression in the disorder may underlie the involvement of these brain regions in schizophrenia through its role in neurodevelopment and/or plasticity and neurotransmission. Investigations of GAP-43 expression in schizophrenia are summarized in Table I, and overall conclusions are discussed later. Although data demonstrate that GAP-43 expression is altered in schizophrenia, it is inconsistent, both in the direction of change and in the brain regions involved. Broadly speaking, studies of GAP-43 mRNA more often than not demonstrate decreased GAP-43 expression in schizophrenia, whereas protein studies appear to show the opposite pattern of change. On face value, this ﬁnding

TABLE I Studies of Growth-Associated Protein-43 in the Brain in Schizophrenia Brain regiona mRNA studies Eastwood and Harrison (1998)

Webster et al. (2001) Weickert et al. (2001) 51

Protein studies Perrone-Bizzozero et al. (1996)

Blennow et al. (1999) Honer et al. (1999) Eastwood and Harrison (2001) a

Methodb

Sample size (controls/cases)

Mean age (years) (controls/cases)

Summary of findings

HC

ISHH

11/11

63/57

Decreased (25–60%) in all subfields except DG and CA1

BA46 BA17 BA22 BA24 HC BA46 BA46

ISHH ISHH ISHH ISHH ISHH RPA ISHH

11/11 11/11 11/11 11/11 10/11 21/17 6/6

63/57 63/57 63/57 63/57 49/49 49/51 49/43

Unchanged Decreased 35% Unchanged Decreased 28% Decreased nonsignificantly (30–70%) Decreased 40% Decreased 25–29% in layers III, V, and Via

BA9 BA10 BA20 BA17 HC BA24 Frontal BA24

IB IB IB IB WB, ICC WB, ICC ELISA WB

4/5 6/6 4/6 6/10 20/17 20/17 10/13 15/15

60/52 41/44 51/43 53/43 75/80 75/80 45/48 47/45

Increased 120% Increased 50% Increased 100% Decreased nonsignificantly (12%) Increased 41% Increased 52% Unchanged Decreased nonsignificantly (40%)

HC, hippocampal formation; BA, Brodmann area. ELISA, enzyme-linked immunoadsorbent assay; IB, immunoblotting; ICC, immunocytochemistry; ISHH, in situ hybridization histochemistry; RPA, RNase protection assay; WB, Western blotting. b

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may suggest that the posttranscriptional regulation of GAP-43 expression is abnormal in schizophrenia, and indeed, it is known that GAP-43 expression is controlled by factors inﬂuencing mRNA stability rather than turnover (see Neve et al., 1999; Tsai et al., 1997). Hence, although GAP-43 mRNA may be decreased in schizophrenia, if the normal control of its stability is altered such that the mRNA half-life is extended in the disorder, then the net result may be an increase in GAP-43 translation. However, a more simple explanation may be that there are to date too few studies conducted on the same subjects examining both measures of GAP-43 expression in schizophrenia. Until such studies have been undertaken, interpretation of the discrepancy between mRNA and protein studies of GAP-43 is mostly a matter of conjecture. Some consistencies in data are apparent. GAP-43 mRNA is decreased in the hippocampus (Eastwood and Harrison, 1998), and the pattern and degree of loss were repeated in a diVerent brain series, albeit these ﬁndings were not statistically signiﬁcant (Webster et al., 2001). Disappointingly, a large Western blotting study of hippocampal GAP-43 reported a large increase in GAP-43 expression in schizophrenia (Blennow et al., 1999). Although controls and subjects with schizophrenia were reported to be matched for age and brain weight, Blennow and colleagues (1999) failed to specify whether the two comparison groups were also matched for postmortem interval; if they were not, it is possible that the change in GAP-43 detected in schizophrenia may have been driven by a shorter postmortem interval in subjects with the disorder. Reassuringly, a study examining GAP-43 expression in a well-matched series using immunocytochemistry has replicated the decreased expression of GAP-43 in the CA4 subﬁeld of the hippocampus (Chambers et al., 2003). Hence, at least in the hippocampus, decreased expression of GAP-43 appears to be a robust ﬁnding in schizophrenia. Finally, although data summarized in Table I demonstrate that GAP-43 expression is altered in schizophrenia, not all brain regions appear to be aVected, even in those studies where several diVerent regions were available from the same subjects (see Eastwood and Harrison, 1998; Perrone-Bizozero et al., 1996). Contrary to earlier suggestions that GAP-43 expression in adulthood was restricted to the limbic and association cortex, GAP-43 mRNA was detected robustly in the primary visual cortex (Eastwood and Harrison, 1998; Webster et al., 1997) and decreased in this brain region in schizophrenia (Eastwood and Harrison, 1998). In contrast, GAP-43 expression was unchanged in the frontal cortex (Eastwood and Harrison, 1998; Honer et al., 1999), a brain region often implicated in the pathophysiology of schizophrenia (Goldman-Rakic et al., 1997; Harrison, 1999). Hence, the anatomical distribution of changes in GAP43 expression in schizophrenia is neither uniform nor restricted to regions other ﬁndings have suggested to be perhaps most aVected in the disease. In summary, GAP-43 expression is altered in schizophrenia, although more studies are required to elucidate the nature and extent of its involvement in the

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disorder. Until such time, it is diYcult to interpret the pattern of pathology or to speculate upon what biological signiﬁcance altered GAP-43 expression may play in the pathophysiology of schizophrenia. However, given its roles in axon guidance, neurotransmission, and plasticity, data do support suggestions that aberrant synaptic plasticity may be contributing to the synaptic pathology of schizophrenia.

B. Neural Cell Adhesion Molecule in the Brain in Schizophrenia Neural cell adhesion molecule (NCAM) was initially characterized as a cell recognition molecule of the immunoglobulin superfamily involved in cell–cell adhesion during development (Edelman, 1984), with subsequent studies indicating that it is highly expressed in synaptic terminals (Persohn and Schachner, 1987; Persohn et al., 1989). NCAM exists in three major isoforms of 180, 140, and 120 kDa through alternative splicing of a single gene (Cunningham et al., 1987), with a fourth putative isoform (NCAM 105–115 kDa) being reported in human brain (Vawter et al., 1998a). A variable alternatively spliced exon (VASE) can also be inserted into any of the major isoforms and is thought to be associated with decreased neural plasticity (see Vawter et al., 1998b). NCAM undergoes posttranslational modiﬁcation during development by the addition of complex side chains composed of polysialic acid (PSA), a linear homopolymer of -2, 8-linked sialic acid. Polysialylated NCAM (PSA-NCAM) is expressed abundantly throughout the brain during development and into the early postnatal period (Seki and Arai, 1993a), after which a more restricted pattern of expression is reported in adulthood, with PSA-NCAM expression mostly conﬁned to regions, such as the hippocampus, capable of plastic reorganization (Arellano et al., 2002; Bonfanti et al., 1992; Seki and Arai, 1993b; Theodosis et al., 1991). The addition of PSA to NCAM during development has been suggested to result in a decrease in NCAM-mediated cell adhesion and to thereby promote developmental events such as cell migration and axon growth (see Bruses and Rutishauser, 2001; Fields and Itoh, 1996). Experimental manipulation of PSANCAM expression using a variety of methods has shown that it mostly plays a permissive role in axon guidance during development and is important in the regulation and promotion of axon growth and fasiculation (for reviews see Bruses and Rutishauser, 2001; Cremer et al., 2000). For example, studies of hippocampal formation in transgenic mice devoid of PSA-NCAM have demonstrated its importance in axonal growth and pathﬁnding and in the establishment of normal patterns of synaptic connectivity (Cremer et al., 2000). PSA-NCAM is also important for some modes of neuronal migration and has been shown to inhibit migration in which neurons move forward by using each other as a substrate

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(chain migration), such as that undertaken by olfactory bulb precursor cells from the subventricular zone (Ono et al., 1994). NCAM polysialylation is progressively downregulated with increasing age and is replaced by nonsialylated adult NCAM, which have increased adhesive properties, a feature most likely to be associated with a change in the role of NCAM from one promoting plasticity to that of synapse stabilization. For example, excessive NCAM expression has been demonstrated to inhibit the formation of new synapses (Martin and Kandel, 1996). However, there appears to be an optimum level of NCAM expression for synaptic plasticity, as blockade of NCAM inhibits LTP (Lu¨thi et al., 1994; Rønn et al., 1995), whereas LTP is impaired in hippocampal slice preparations from NCAM-deﬁcient mice (Cremer et al., 1998). Hence, the role of NCAM in adult synaptic plasticity is most likely to be a balance between weakening cell–cell interactions to permit structural changes to allow the formation of new synapses (in which PSA-NCAM may play a particular role; see Cremer et al., 2000; Rønn et al., 2000; Rutishauser and Landmesser, 1996) and the strengthening and consolidation of potentiated synaptic connections. In this respect it is of interest that patterns of neuronal activity regulate the expression and posttranslational modiﬁcation of NCAM. Stimulation of AMPA receptors activates the NCAM promoter in slice preparations (Holst et al., 1998), whereas polysialylation may be regulated by neuronal activity (see Rutishauser and Landmesser, 1996), and in turn, enzymatic removal of sialic acid from membrane preparations modulates AMPA receptor binding (HoVman et al., 1997). Hence, neuronal activity and structural remodeling through changes in NCAM expression may be intimately linked. Given the aforementional roles of NCAM in development and plasticityrelated ‘events in the adult brain (Bruses and Rutishauer, 2001; Kiss et al., 2001; Rønn et al., 2000), a role for NCAM in the pathogenesis of schizophrenia and other psychiatric disorders has been proposed (Vawter, 2000). Support for such a supposition has come from studies of NCAM knockout mice that exhibit neuropathological changes often reported in schizophrenia, including deﬁcits in neuronal migration (Tomasiewicz et al., 1993) and ventricular enlargement (Wood et al., 1998), one of the most robust neuropathological ﬁndings reported in schizophrenia (see Harrison, 1999). Investigations of NCAM in the brain in schizophrenia are summarized in Table II with overall conclusions discussed later. To date, no mRNA studies of NCAM expression in schizophrenia have been undertaken, and only a few protein studies of NCAM in the hippocampus and frontal cortex have been conducted. The one study of PSA-NCAM in schizophrenia reported a signiﬁcant decrease in the density of immunoreactive neurons in the CA4 subﬁeld of the hippocampus (Barbeau et al., 1995). Total NCAM immunoreactivity is increased in the cingulate cortex (Honer et al., 1997), but as this was an ELISA study, the identity of the isoform(s) contributing to this ﬁnding

TABLE II Studies of Neural Cell Adhesion Molecule (NCAM) in the Brain in Schizophrenia

Barbeau et al. (1995)

Brain regiona

Methodb

Anterior frontal

WB, ICC

HC

Sample size (controls/cases)

Mean age (years) (controls/cases)

9/10

73/58

9/10

73/58

c

HC BA24

WB ELISA

7/8 24/18

47/49

Vawter et al. (1998a)

HC

WB

13/16

52/52

BA9/46

WB

10/10

53/55

HC BA9/46 HC

WB WB WB

13/16 10/10 13/16

52/52 53/55 52/52

55

Breese et al. (1995) Honer et al. (1997)

Vawter et al. (1998b) Vawter et al. (1999)

a

Summary of findings NCAM 180, 140, 120 isoforms unchanged NCAM 180, 140, 120 isoforms unchanged. Decreased density (20–95%) of polysialylated (embryonic) NCAM-positive neurons in CA4 Unchanged Total NCAM immunoreactivity increased 22%. Increased ratio of NCAM to synaptophysin 105- to 115-kDa NCAM isoform increased 55%. Other isoforms unchanged 105- to 115-kDa NCAM isoform increased 46%. Other isoforms unchanged VASE NCAM unchanged VASE NCAM unchanged Secreted NCAM unchanged. Increased ratio of cytosolic 105- to 115-kDA NCAM to synaptophysin

HC, hippocampal formation; BA, Brodmann area. ELISA, enzyme-linked immunoadsorbent assay; ICC, immunocytochemistry; WB, Western blotting. c Not given in this study. b

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could not be determined. Vawter and colleagues (1998a) have examined this question using Western blotting and have detected an increase in the expression of a cytosolic 105- to 115-kDa isoform in schizophrenia, but not in any of the three major NCAM isoforms. This 105- to 115-kDa NCAM isoform was not identiﬁed in the only other Western blotting study conducted in schizophrenia (Barbeau et al., 1995), but may have been due to diVerences in sample preparation between the two groups. Vawter and colleagues proposed that their ﬁnding of the increased cytosolic 105- to 115-kDa NCAM isoform in schizophrenia may be related to changes in the production of the secreted form of NCAM in the disorder. However, Western blotting using a speciﬁc antibody to secreted NCAM failed to reveal any diVerence in the expression of this isoform in schizophrenia (Vawter et al., 1999). The scarcity of studies of NCAM expression in the brain in schizophrenia makes interpretation of the ﬁndings diYcult, and at ﬁrst glance, the decreased PSA-NCAM expression reported by Barbeau and colleagues may seem to be in conﬂict with the ﬁndings of increased nonpolysialyated NCAM (Honer et al., 1997; Vawter et al., 1998a). However, both ﬁndings may be taken in support of ideas of decreased synaptic plasticity in schizophrenia. Data from several studies suggest that PSA-NCAM is an important regulator of synaptic plasticity (see Cremer et al., 2000), and removal of PSA from NCAM in vitro results in an inhibition of LTP in hippocampal slice preparations (Becker et al., 1996; Muller et al., 1996). Therefore, decreased PSA-NCAM reported in the hippocampus in schizophrenia may be indicative of a diminished capacity for synaptic plasticity and remodeling in the disorder. Likewise, excessive adult NCAM expression has been shown to inhibit the formation of new synapses (Martin and Kandel, 1996), and increased NCAM in the cortex in schizophrenia has been suggested to be related to an impairment in the processes necessary for long-term memory formation (see Honer et al., 1997). In addition to changes in NCAM expression, two groups have reported an increase in the ratio of NCAM to synaptophysin in schizophrenia (Honer et al., 1997; Vawter et al., 1999). These ﬁndings indicate that alterations in NCAM expression are not simply paralleling changes in presynaptic protein expression, and the ratio of NCAM to synaptophysin has been suggested to provide an index of synaptic remodeling or plasticity ( Jo¨rgensen, 1995). The increased NCAM/synaptophysin ratio reported in schizophrenia has been interpreted as either representing a decrease of mature synapses (as suggested by studies of presynaptic protein expression) or an increase in immature synapses (see Vawter, 2000). Hence, taken together, the few positive reports of abnormalities in the expression of NCAM may be included as evidence in support of abnormalities in synaptic plasticity in schizophrenia. Given what is known about the role of NCAM in development and plasticity, it would be interesting to determine whether altered NCAM expression in schizophrenia could be related to other molecular or neurochemical changes reported

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in the disorder. For example, PSA-NCAM expression and insertion into the cell surface in culture are calcium dependent and modulated by neuronal activity (Kiss et al., 1994; Muller et al., 1996), ﬁndings that suggest that PSA-NCAM may be translocated to the synaptic membrane by exocytosis. It is therefore tempting to speculate that changes in the expression of presynaptic proteins involved in exocytosis that have been reported in schizophrenia (see review by Honer and Young) may be linked causally to decreased PSA-NCAM expression in the hippocampus in schizophrenia. Similarly, given that enhanced synaptic transmission modulated through AMPA receptor increases the activity of the NCAM promoter (Holst et al., 1998), it would be interesting to determine whether increased NCAM expression in the cortex in schizophrenia was in any way related to changes in AMPA receptors in schizophrenia. In this context, although there have not been very many studies of cortical AMPA receptors in schizophrenia, a few have detected increased AMPA binding in schizophrenia (Noga et al., 2001; Zavitsanou et al., 2002). Hence, although NCAM expression has not been widely examined in schizophrenia, data suggest that further studies may be warranted. The application of newly available technologies that allow the expression of many genes to be determined concurrently (i.e., microarrays, quantitative polymerase chain reaction, proteomics) will be valuable in determining whether changes in NCAM expression can be linked to the molecular neuropathology of schizophrenia.

III. Can the Synaptic Pathology of Schizophrenia Be Related to Changes in the Expression of Genes Involved in Development and Plasticity?

Modern neuropathological studies of schizophrenia suggest that synaptic pathology is a feature of schizophrenia, providing a plausible anatomical basis for the aberrant functional connectivity demonstrated in the disorder using brain imaging. This section summarizes some of our ﬁndings that have contributed to demonstrating the synaptic pathology of schizophrenia and follows with an outline of subsequent studies in which we have explored the possible relationship between presynaptic protein expression and genes involved in development and plasticity.

A. Presynaptic Protein Expression Is Altered in Schizophrenia Using a variety of techniques, we have consistently demonstrated that there are alterations in presynaptic protein expression in the hippocampal formation in schizophrenia. Utilizing in situ hybridization histochemistry (ISHH),

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immunoautoradiography (IAR), and Western blotting to examine synaptic proteins and their mRNAs, hippocampal synaptic pathology was indicated by the reduced expression of synaptophysin (Eastwood and Harrison, 1995, 1999; Eastwood et al., 1995), GAP-43 (Eastwood and Harrison, 1998), complexin I and II (Eastwood and Harrison, 2000; Harrison and Eastwood, 1998), and, more recently, the vesicular glutamate transporter VGLUT1 (unpublished observations). An extension of our study of synaptic protein expression to other brain regions demonstrated that synaptic changes are also a feature of the cerebellum (Eastwood et al., 2001) and, to a lesser extent, the neocortex (Eastwood and Harrison, 2001; Eastwood et al., 2000). These ﬁndings are in keeping with other studies of synaptic protein expression in schizophrenia and suggest, at least in the hippocampus (Browning et al., 1993; Fatemi et al., 2001a; Vawter et al., 2002; Webster et al., 2001; Young et al., 1998), that synaptic pathology is a robust ﬁnding. In the absence of data supportive of neurodegenerative processes (Harrison, 1999; Marenco and Weinberger, 2000), the default explanation is that synaptic pathology is developmental in origin. The focus of our recent research has been to search for evidence supportive of such a developmental origin of schizophrenia.

B. Cerebellar Reelin and Semaphorin 3A Expression in Schizophrenia One approach to examine whether aberrant development may underlie schizophrenia is to study the expression of genes known to play key roles in brain development (Beasley et al., 2001, 2002; Cotter et al., 1998; Fatemi et al., 2000, 2001b; Guidotti et al., 2000; Ilia et al., 2002; Miyaoko et al., 1999; Novak et al., 2002). Given our ﬁndings suggesting that synaptic pathology is a feature of schizophrenia (summarized earlier), we have studied two genes important in axon formation: branching and synaptogenesis. The ﬁrst, reelin, is a key molecule during corticogenesis and is important in guiding the migration and positioning of proliferating neurons, thereby inﬂuencing cortical lamination, columnarity, and synaptic connectivity (Borrell et al., 1999; Nishikawa et al., 2002; Ogawa et al., 1995; Rice and Curran, 2001). During development, reelin is secreted by Cajal-Retzius cells in the marginal zone (D’Arcangelo et al., 1995; Del Rio et al., 1997; Meyer and GoYnet, 1998; Meyer et al., 2000) and acts on neurons and maybe glia (Fo¨rster et al., 2002) via several receptor-mediated mechanisms. Hence, reelin is thought to provide mostly trophic or attractant cues (Rice and Curran, 2001), and expression of this gene is decreased in schizophrenia (Fatemi et al., 2000, 2001b; Guidotti et al., 2000). The second, semaphorin 3A (sema3A, also known as collapsin-1 or semaphorin D), is a secreted chemorepellant belonging to a family of intercellular signaling proteins involved in axonal guidance during neurodevelopment (Kolodkin, 1996). Sema3A has been shown in mammals to be a key chemorepellant for several axonal populations and an

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inhibitor of axonal branching, thereby determining the balance of synaptic inputs to particular cell types (Bagnard et al., 1998; Nakamura et al., 2000; Raper, 2000; Steup et al., 1999). Given these broadly opposing roles and data showing that presynaptic molecular markers of synapses are decreased in schizophrenia, we hypothesized that the expression of reelin and sema3A may be related to the synaptic pathology of schizophrenia. In particular, we predicted that there may be an inverse relationship between sema3A and local presynaptic marker genes, whereas decreased synaptic protein expression may be correlated with reduced reelin expression in schizophrenia. To explore these predictions, we examined sema3A and reelin in schizophrenia and related it to indices of synaptic pathology. The cerebellum was chosen as the ﬁrst brain region of interest for three reasons. First, because it is one of the few human brain regions in which both reelin (DeSilva et al., 1997; Impagnatiello et al., 1998) and sema3A (Giger et al., 1998) expression is known to persist into adulthood. Second, the cerebellum is of increasing interest as a node in a cortico-thalamo-cerebellar circuit proposed to be of central importance in schizophrenia (Andreasen, 1999; Andreasen et al., 1996). Third, the expression of presynaptic proteins (synaptophysin and complexin II mRNAs) was decreased in schizophrenia in this brain series (Eastwood et al., 2001). Consistent with other studies, reelin mRNA was decreased in schizophrenia, and as predicted, presynaptic protein expression correlated with that of reelin in the cerebellum (Eastwood et al., 2003). In contrast, sema3A was increased in the cerebellum in schizophrenia and inversely correlated with presynaptic protein and reelin expression (Eastwood et al., 2003). These data suggest, simplistically, that the equilibrium between repulsive and attractant processes on synaptic modeling and growth is altered in schizophrenia and may contribute to the synaptic pathology of schizophrenia. An analogous process may underlie the cognitive and behavioral impairments observed in adult rats after the neonatal administration of epidermal growth factor (Futamura et al., 2003). Reelin and sema3A are unlikely to be solely responsible for the putative disequilibrium in schizophrenia; indeed, another inhibitory protein, Nogo, is also elevated (Novak et al., 2002). However, reelin and sema3A are useful representatives of the genes involved in the balance between inhibitory and attractant processes, and further studies are underway to determine whether similar alterations in their expression may be contributing to synaptic changes in other brain regions in schizophrenia.

C. Altered Reelin Expression Is Related to Cortical Synaptic Pathology Although decreased reelin expression is a robust ﬁnding in schizophrenia (see review by Costa, Grayson, and Guidotti), most studies have examined reelin mRNA using RT-PCR, but as these were homogenate-based studies, they were

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not able to determine which neurons were contributing to the decrement in reelin mRNA. For this reason we have coupled ISHH using an 35S-labeled oligonucleotide probe speciﬁc to reelin mRNA with emulsion autoradiography. This technique enables the identiﬁcation of neurons expressing reelin and also allows the quantiﬁcation of reelin mRNA over individual cells. Utilizing this methodology, we have begun to examine reelin expression in those brain regions exhibiting synaptic pathology (demonstrated by decreased presynaptic protein expression) to determine whether the relationship between reelin expression and synaptic pathology observed in the cerebellum was restricted to this brain region or represented a more widespread phenomenon associated with synaptic changes in other parts of the brain. Unfortunately, the limited expression of sema3A in the adult brain (Giger et al., 1998) did not allow similar studies to be undertaken for this protein. To date, we have examined reelin mRNA in the superior temporal gyrus (Eastwood and Harrison, 2003) and hippocampal formation (unpublished observations.) In both regions, reelin was expressed by most layer I neurons (including neurons adjacent to the hippocampal ﬁssure) and some interneurons across the cortical ribbon (in agreement with immunocytochemical studies). In addition, reelin was expressed robustly by many interstitial white matter neurons, and decreased reelin expression by these neurons was associated with an increase in their density in the superﬁcial white matter of the superior temporal gyrus (Eastwood and Harrison, 2003). Reelin mRNA was also quantiﬁed in layer I of the superior temporal gyrus, and a reduction in the number of layer I neurons expressing reelin and a decrease in cellular reelin mRNA by these neurons were detected in schizophrenia (Fig. 1). Of particular interest in relation to our ﬁnding of synaptic changes in this brain region in schizophrenia (Eastwood et al., 2001) were signiﬁcant positive correlations between synaptophysin mRNA and reelin expression, both in terms of the density of reelin expressing neurons in layer I (Fig. 2) and in the cellular quantiﬁcation of its mRNA (not shown). Hence, as observed in the cerebellum, decreased reelin expression in schizophrenia (Figs. 3A and B) is associated with reduced synaptophysin mRNA (Figs. 3C and D) in the superior temporal gyrus. Similar relationships between reelin and presynaptic protein expression were detected in hippocampal formation, although the smaller number of subjects included in both studies resulted in these correlations only trending toward signiﬁcance (not shown). Further studies are in progress, but data obtained to date suggest that changes in the expression of reelin and synaptic proteins in schizophrenia are related and that the altered expression of a key development gene may contribute to the synaptic pathology of the disorder.

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Fig. 1. Reelin expression in the superior temporal gyrus in schizophrenia. The expression of reelin is decreased in layer I of the superior temporal gyrus in schizophrenia. Quantification of reelin expression, both in terms of the number of neurons expressing this transcript (top) and in the amount of reelin mRNA measured over individual neurons (bottom), revealed a decrease in reelin mRNA in schizophrenia, significantly so for the latter ( *p ¼ 0.036).

D. How May Reelin and Semaphorin 3A Expression Contribute to Altered Synaptic Plasticity and Pathology in Schizophrenia? In combination, the altered balance between sema3A and reelin expression in the cerebellum in schizophrenia suggests that repulsive processes may have the greatest inﬂuence. Hence, by inhibiting normal processes involved in axonal growth and synaptogenesis, changes in the expression of these two genes may

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Fig. 2. Relationship between reelin and synaptophysin mRNA in the superior temporal gyrus. Reelin expression is correlated significantly and positively with synaptophysin mRNA in the superior temporal gyrus in both controls and subjects with schizophrenia, with the latter demonstrating lower levels of expression of both transcripts.

contribute to the synaptic pathology of schizophrenia, whenever they originated. The persistent expression of sema3A and reelin in adulthood and their altered expression detected postmortem in schizophrenia, however, suggest that they may also be involved with ongoing events, including synaptic plasticity; this issue is discussed further later. Most studies of sema3A have concentrated on its roles during development. However, later involvement of the semaphorins, including sema3A, in synaptic plasticity has been described, which may represent a continuation of their earlier functions during development (Fournier and Strittmatter, 2001; Gavazzi, 2001; Pasterkamp and Verhaagen, 2001). Studies of the role(s) of sema3A in the adult brain have concentrated on lesion models of nerve regeneration and have revealed changes in sema3A expression during axon regeneration, often reciprocally with that of GAP-43 (see Pasterkamp et al., 1998). These ﬁndings suggest that sema3A is involved in synaptic plasticity, and it would be interesting to determine what eVect downregulation of sema3A expression may have on other models of synaptic plasticity, such as LTP and LTD. To date, no such studies have been conducted in sema3A-deﬁcient mice. Evidence in support of a role for reelin in synaptic plasticity has come from a study showing that reelin and its receptors cooperate to enhance synaptic plasticity (Weeber et al., 2002). Two apolipoprotein E (apoE) receptors, the very low density lipoprotein (VLDL) receptor and apoE receptor 2 (apoER2), are also receptors for reelin and both appear to be necessary for the reelin-dependent enhancement of synaptic transmission. In experiments using hippocampal slice preparations from knockout and wild-type mice, reelin signiﬁcantly augmented

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Fig. 3. Reelin and synaptophysin expression in the superior temporal gyrus. Cellular reelin expression in human superior temporal gyrus viewed under dark field in a representative control (A) and schizophrenic (B) subject. Decreased reelin expression is associated with reduced synaptophysin mRNA, as demonstrated by the greater autoradiographic signal observed in a representative control subject (C) as compared to a subject with schizophrenia (D). Bar: 30 mm.

LTP in slices from wild-type mice, but not in those from either VLDLR- or apoER2-deﬁcient mice (Weeber et al., 2002). In addition, although baseline synaptic transmission was normal in apoER2-deﬁcient mice, they exhibited a profound decrease in LTP. Weeber and colleagues (2002) have suggested a hypothetical model for the action of reelin on LTP and proposed that enhancement of LTP results through the activation of tyrosine kinases, which may alter the activity of NMDA receptors. In addition, through interaction of the ApoER2 receptor with scaVolding proteins, which interact functionally with the microtubluleassociated molecular motor kinesin, reelin signaling may also regulate axonal transport. Hence, decreased reelin expression in schizophrenia could conceivably be linked to a dysfunction in the transport of presynaptic proteins to the synapse,

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which could contribute to altered synaptic plasticity and pathology of the disorder. Although speculative, this association between reelin expression and axonal transport suggests a possible mechanism that may underlie the positive correlations detected between reelin and synaptic protein expression (Fig. 2).

IV. Discussion and Future Directions

The title of this review questioned whether the synaptic pathology of schizophrenia may be blamed on aberrant development and plasticity. Although a deﬁnitive answer cannot be given, a growing body of evidence suggests that processes common to development and plasticity may be related to the synaptic pathology of schizophrenia. Indeed, it is becoming more apparent that developmentally important genes are frequently involved later in neuroplasticity and that aberrant synaptic plasticity may be the adult manifestation of persistent changes in the expression of key developmental genes. However, it is impossible to determine from postmortem studies of schizophrenia when these processes may ﬁrst have occurred, and although signiﬁcant correlations between the expression of synaptic proteins and that of key developmental genes suggest a relationship between development and plasticity with the synaptic pathology of schizophrenia, they do not establish causality. To do this, in vitro and animal models are needed to augment future human postmortem studies. In light of the relationship detected between reelin and synaptic protein expression, the heterozygous reeler (rl +/) mouse (HRM) may provide one such animal model to explore the aforementioned issues. Of interest in itself to schizophrenia research, HRM expresses approximately half the reelin mRNA of the wild-type mouse, a discrepancy comparable to that reported in schizophrenia, and exhibits morphological, neurochemical, and behavioral (sensorimotor gating) alterations similar to those observed in the disorder (Ballmaier et al., 2002; Costa et al., 2002; Tueting et al., 1999). As such, the HRM may provide an appropriate model with which to determine the downstream consequences of decreased reelin expression on synaptic protein expression and plasticity. Furthermore, by examining the HRM at diVerent ages and comparing its presynaptic protein expression to that of wild-type mice, it may be possible to determine if presynaptic changes (as seen in schizophrenia) occur in the HRM and whether any such changes are present from birth. In conclusion, further human postmortem studies (especially those that utilize methodologies allowing the examination of several genes of interest at one time), together with appropriate animal and in vitro models, will help establish whether schizophrenia is a disorder of synaptic plasticity. Although at this stage it cannot

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be said conclusively that aberrant development and plasticity underlie the synaptic pathology of schizophrenia, available data suggest that the likely answer is yes.

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Rice, D. S., and Curran, T. (2001). Role of the reelin signaling pathway in central nervous system development. Annu. Rev. Neurosci. 24, 1005–1039. Rønn, L. C., Berezin, V., and Bock, E. (2000). The neural cell adhesion molecule in synaptic plasticity and ageing. Int. J. Dev. Neurosci. 18, 193–199. Rønn, L. C., Bock, E., Linnemann, D., and Jahnsen, H. (1995). NCAM-antibodies modulate induction of long-term potentiation in rat hippocampal CA1. Brain Res. 677, 145–151. Rutishauser, U., and Landmesser, L. (1996). Polysialic acid in the vertebrate nervous system: A promoter of plasticity in cell-cell interactions. Trends Neurosci. 19, 422–427. Sanes, J. R., and Lichtman, J. W. (1999). Can molecules explain long-term potentiation? Nature Neurosci. 2, 597–604. Seki, T., and Arai, Y. (1993a). Distribution and possible roles of the highly polysialylated neural cell adhesion molecule (NCAM-H) in the developing and adult central nervous system. Neurosci. Res. 17, 265–290. Seki, T., and Arai, Y. (1993b). Highly polysialylated neural cell adhesion molecule (NCAM-H) is expressed by newly generated granule cells in the dentate gyrus of the adult rat. J. Neurosci. 13, 2351–2358. Shapiro, R. M. (1993). Regional neuropathology in schizophrenia: Where are we? Where are we going? Schizophr. Res. 10, 187–239. Shea, T. B., Perrone-Bizzozero, N. I., Beermann, M. L., and Benowitz, L. I. (1991). Phospholipidmediated delivery of anti-GAP-43 antibodies into neuroblastoma cells prevents neuritogenesis. J. Neurosci. 11, 1685–1690. Steup, A., Ninnemann, O., Savaskan, N. E., Nitsch, R., Puschel, A. W., and Skutella, T. (1999). Semaphorin D acts as a repulsive factor for entorhinal and hippocampal neurons. Eur. J. Neurosci. 11, 729–734. Theodosis, D. T., Rougon, G., and Poulain, D. A. (1991). Retention of embryonic features by an adult neuronal system capable of plasticity: Polysialylated neural cell adhesion molecule in the hypothalamo-neurohypophysial system. Proc. Natl. Acad. Sci. USA 88, 5494–5498. Tomasiewicz, H., Ono, K., Yee, D., Thompson, C., Goridis, C., Rutishauser, U., and Magnuson, T. (1993). Genetic deletion of a neural cell adhesion molecule variant (N-CAM-180) produces distinct defects in the central nervous system. Neuron 11, 1163–1174. Tsai, K. C., Cansino, V. V., Kohn, D. T., Neve, R. L., and Perrone-Bizzozero, N. I. (1997). Posttranscriptional regulation of the GAP-43 gene by specific sequences in the 30 untranslated region of the mRNA. J. Neurosci. 17, 1950–1958. Tueting, P., Costa, E., Dwivedi, Y., Guidotti, A., Impagnatiello, F., Manev, R., and Pesold, C. (1999). The phenotypic characteristics of heterozygous reeler mouse. Neuroreport 10, 1329–1334. Vawter, M. P. (2000). Dysregulation of the neural cell adhesion molecule and neuropsychiatric disorders. Eur. J. Pharmacol. 405, 385–395. Vawter, M. P., Cannon-Spoor, H. E., Hemperly, J. J., Hyde, T. M., VanderPutten, D. M., Kleinman, J. E., and Freed, W. J. (1998a). Abnormal expression of cell recognition molecules in schizophrenia. Exp. Neurol. 149, 424–432. Vawter, M. P., Hemperly, J. J., Hyde, T. M., Bachus, S. E., VanderPutten, D. M., Howard, A. L., Cannon-Spoor, H. E., McCoy, M. T., Webster, M. J., Kleinman, J. E., and Freed, W. J. (1998b). VASE-containing N-CAM isoforms are increased in the hippocampus in bipolar disorder but not schizophrenia. Exp. Neurol. 154, 1–11. Vawter, M. P., Howard, A. L., Hyde, T. M., Kleinman, J. E., and Freed, W. J. (1999). Alterations of hippocampal secreted N-CAM in bipolar disorder and synaptophysin in schizophrenia. Mol. Psychiat. 4, 467–475. Vawter, M. P., Thatcher, L., Usen, N., Hyde, T. M., Kleinman, J. E., and Freed, W. J. (2002). Reduction of synapsin in the hippocampus of patients with bipolar disorder and schizophrenia. Mol. Psychiat. 7, 571–578.

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NEUROCHEMICAL BASIS FOR AN EPIGENETIC VISION OF SYNAPTIC ORGANIZATION

E. Costa, D. R. Grayson, M. Veldic, and A. Guidotti Psychiatric Institute Department of Psychiatry, College of Medicine University of Illinois at Chicago Chicago, IL 60612

I. II. III. IV.

V.

VI. VII. VIII.

Introduction Conceptual Background for the Definition of Phenotypes and Genotypes Epigenetics and Evolution Biochemical Processes Included in Epigenetic Phenomena A. DNA Methylation B. DNA Cytosine Methylransferases C. CpG Islands D. The Influence of Methylation on Transcription Epigenetics and Synaptic Plasticity A. Postsynaptic Density (PSD) Organization at Excitatory Synapses B. Inhibitory Synapses C. Dynamic Aspects of PSD Organization D. Transmitter Receptor Stability in the Membrane Epigenetics Today The Epigenetic Concept in Psychiatry Conclusions References

I. Introduction

The term ‘‘epigenetic’’ was ﬁrst proposed by Conrad H. Waddington (1905–1975) in 1934 before genes became linked to DNA (Waddington, 1934). With this term, Waddington probably intended to stress that a change in genetic action is causally linked to changes in one or more pathways operative in development. In 1956, when research on the nature of the hereditary material led to the discovery of the important role of DNA in inheritance, the dynamic conception of genetic action was associated with the popular idea that the role of DNA fully coincided with the concepts of gene function (Waddington, 1956). In the same year, the term epigenetic was not only resurrected, but during this interval (1934–1956) additional interpretations had arisen, which, in the background of molecular and developmental biology, added further complexity to the term INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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‘‘epigenetic.’’ Although both developmental and molecular genetic studies explicitly mention the Waddington tradition in their reference to the term epigenetic, the signiﬁcance of this term underwent dramatic changes over the years that almost ignored the need to include a number of parallel deﬁnitions of this term. In its widest sense, epigenetic had shifted the original focus on the genetic action in embryology to encompass organismic organization allowing links between inheritance and epigenetic phenomena. However, the most popular deﬁnition of ‘‘epigenetics’’ is much more restricted as it refers to the study of alterations in the expression of speciﬁc DNA sequences that cannot be explained by classical DNA mutations (Waddington, 1966). Nevertheless, this apparently simple deﬁnition places epigenetics, as the term literally indicates, beyond genetics, signifying a major turn away from its connection with the central dogma of molecular biology. Currently, epigenetics not only oVers spectacular new insights on gene regulation and heredity, but it also profoundly challenges the way we think about genetics, evolution, and development, especially now that epigenetic phenomena are seen to occur in the entire realm of living creatures.

II. Conceptual Background for the Definition of Phenotypes and Genotypes

Although causation and plasticity refer to diametrically opposite aspects of phenotypic changeability, what they have in common is that they express an array of phenotypic variations uncoupled from genetic variation. Recognizing this and accounting for it is central to a genetic approach to psychiatric disorders. Hence, the distinction between epigenetics and developmental genetics includes a diVerence in focus, with epigenetics stressing complex developmental networks expressed with redundancy and compensatory mechanisms. In contrast, developmental genetics is more concerned with the hierarchies of actions that lead to gene eVects acting on the phenotype. Today, the situation is diVerent because all developmental biologists tend to think and discuss heritability in terms of complex gene networks stressing the signiﬁcance of interactions. Hence, the epigenetics perspective has to a large extent replaced that of classical developmental genetics. Nevertheless, it would be inappropriate to think that epigenetics is synonymous with developmental biology. Developmental biology is a much broader discipline, embracing all aspects of embryology, regeneration, growth, and aging. Although genes are basic to all of these, it is possible to study many important aspects of development without worrying about genes. Epigenetics, in the sense that Waddington (1968) proposed and used this term, is already a part of developmental biology but still remains a speciﬁc way of looking at this discipline without being synonymous. Although developmental and molecular genetic studies mention the Waddington tradition, a new and additional interpretation has

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arisen in their reference to the term epigenetics. This term was originally used to indicate genetic-related modiﬁcations in embryology. Today, this limitation has been removed because epigenetics now includes links with an evaluation of why epigenetic phenomena appear to be heritable. III. Epigenetics and Evolution

Evolution was central to the thinking of the proposers of the epigenetic concept. They were interested in the origins of the switches between alternative phenotypes and in the evolutionary routes bringing about either an increase or a decrease in plasticity and canalization. Hence, epigenetics was a discipline that was to give a platform to both evolutionary theory and embryology. The experiments in which Waddington (1966) genetically assimilated various induced characters in Drosophila are an appropriate example of the increase in latitude obtained by applying an epigenetic approach to evolution. A related research program that is popular today includes the study of the evolution of reaction norms and ranges that express phenotypic plasticity in diVerent environmental conditions. Epigenetics therefore relates to several diVerent branches of biology; it stands at the intersection of developmental biology and genetics underlying everything that is evolutionary biology ( Jablonka and Lamp, 2002). Hall (1992) was among the ﬁrst to notice that epigenetics was beginning to take on a new ﬂavor when epigenetic control became the sum of genetic and nongenetic factors acting on cells and selectively controlling gene expression. This produces an increase in phenotype complexity during development, but the scope of epigenetics was further narrowed as the last decade of the last millenium progressed. Mechanisms of epigenetic control now include inheritance of spectra of gene activities in each specialized cell. In addition to the DNA code, there is the superimposition of an additional layer of information composed of the heredity material. In many cases, this heredity is very stable and is described as epigenetic inheritance. IV. Biochemical Processes Included in Epigenetic Phenomena

A. DNA Methylation DNA methylation is essential for mammalian development (Li et al., 1992) but despite 25 years of investigation on this precise topic, we still do not know exactly why. Nevertheless, chromosomal epigenetic changes—the so-called

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epimutations—very likely are possible because the nucleotide sequence is not the only form of genetic information that a given cell receives. The ﬁrst epimutations were reported by the Nobelist Barbara McClintock (1984), when she noted that in maize, transposons underwent cycles of inactivity during methylation. While originally discovered in maize, transposons are mobile genetic elements found in virtually all genomes. The most abundant mobile elements in mammals are nonviral retrotransposons, which make up the main classes of moderately repeated DNA sequences found in mammalian genomes (Lodish et al., 2000). Chromosomal proteins and DNA methylation can be inherited with important phenotypic consequences (WolVe and Matzke, 1999). In mammals, coding sequences are methylated but also some noncoding (CpG island) sequences are methylated as well (Yoder et al., 1997). Of course, the human genome contains far less exonic DNA (<2%) than transposons (>45%), which therefore contribute more to the overall level of cytosine methylation (International Human Genome Sequencing Consortium, 2001). Methylation is one of the mechanisms for keeping transposons in a quiescent state. Naively, when transposons lose methylation they become transcriptionally active and can jump to new locations, but when these genes are hypermethylated they remain silenced.

B. DNA Cytosine Methyltransferases We know that in mammals there are three active DNA cytosine methyltransferases (DNMT1, 3a, and 3b) (Okano et al., 1998). Also, we have understood how the methylation signal in the 5 position of a cytosine ring alters the appearance of the major DNA groove to which DNA-binding proteins bind. These epigenetic methyl markers can be copied after DNA synthesis and can bring about changes in chromatin structure. In mammals, methylation of CpG-rich promoters prevents transcriptional initiation and can silence genes (e.g., the inactive X chromosome). It is currently believed that normal chromosome structure may be changed by methylation, which may impact cell function in characteristic pathological processes, for instance, the onset of special forms of cancer may be facilitated by methylation (Okano, 1998). It is also true that Drosophila melangoster, which does not methylate its genome, expresses a very high rate of spontaneous DNA mutation (from 50 to 85%) presumably elicited by the actions of transposable elements (Riddihough and Pennisi, 2001). In Arabidopsis, the DDM1 gene (decrease in DNA methylation 1) causes the reactivation of silenced gene sequences. DDM1 encodes a protein similar to the chromatin remodeling factor SW12/SNF2; some evidence suggests an intimate link between DNA methylation and chromatin structure (Gendrel et al., 2002), thus if DNA methylation is really a part of a host control system, it seems likely that chromatin must also be implicated. It has been suggested that in animals with

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a placenta, genomic imprinting involves a chromosomal dosage compensation, including host defense mechanisms directed against parasitic DNA (Riddihough and Pennisi, 2001). Other evidence suggests that normal chromosome structure may be aVected by methylation and that human diseases, including cancer (and possibly schizophrenia), express and are probably impacted by abnormal patterns of methylation (Esteller and Herman, 2002; Robertson, 2001). C. CpG Islands CpG nucleotides, which are sites for the expression of most of the genomic methylation, are underrepresented in DNA. Most often in promoters and ﬁrst exons, clusters of CpGs—termed CpG islands—are found in association with genes and their promoters. The exact deﬁnition of a CpG island is evolving. It was ﬁrst deﬁned by Gardiner-Garden and Frommer (1987) as a region greater than 200 bp with high GC content and an observed/expected ratio for the CpG occurrence >0.6. The salient property of a CpG island is that it is not methylated in the germline, thus ensuring its continued existence in the face of the strong mutagenic pressure of 5-methylcytosine deamination (Kishikawa et al., 2002). CpG islands can function as strong promoters and probably as replication origins (Delgado et al., 1998). Investigations on CpG island methylation show that as opposed to other regions where the majority of methylation is found, these islands are undermethylated. Probably, DNA methylases cannot have access to CpG islands that are often ﬂanked by Sp1 sites occupied by Sp1 transcription factors. However, if these sites represent a barrier to CpG island methylation, these barriers can be circumvented with the help of speciﬁc cues. Evidence for a targeting model comes from studies showing an association of DNMT1 and DNMT3a enzymes with ancillary proteins, including Rb, E1F1, histone deacetylases, and the transcription repressor RP58 (Fuks et al., 2001). D. The Influence of Methylation on Transcription Although it is frequently stated that ‘‘methylation blocks gene expression,’’ this statement is an oversimpliﬁcation of the facts. Methylation changes DNA– protein interactions, which may lead to changes in chromatin structure that can either increase or decrease transcription rates. Methylation of promoter CpG islands causes MBD protein binding with transcriptional repressors in complexes including HDACs and subsequent inhibition of transcription initiation ( Jones et al., 1998). One could marshal further evidence to deﬁne the role of MBDs on transcription, but current views hold that the role of the MBDs is still incompletely deﬁned. In addition, it appears clear that methylation does not stop transcript elongation in mammals. Methylation of silencer or insulator elements

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can interfere with the binding of cognate DNA-binding proteins and abolishes their suppressing action on gene expression (Li, 2002). The validity of many experiments testing relations between methylation and gene expression has been compromised because the precise sites of DNA methylation were not deﬁned. Methylation of cytosine is a major contributor to germline and somatic mutations that cause cancer. Immunodeﬁciency, centromeric instability, and mental retardation (Rett syndrome) are caused by changes in the methylation and methyl CpG binding protein machinery. Facial anomaly syndromes (FAS) express mutations in the DNMT3b gene, which lead to undermethylation of satellite DNA and cause speciﬁc chromosome decondensation (Okano et al., 1998). Rett syndrome is a neurodevelopmental disorder that results in mental retardation due to mutations in the methyl CpG-binding protein MeCP2 (Amir et al., 1999). Rett syndrome patients appear normal for the ﬁrst 6–18 months of life and then degenerate rapidly, suVering from severe dementia and autistic behavior (Chen et al., 2001). At the same time, it also seems plausible to believe that Rett syndrome patients may be unable to interpret the metabolic signiﬁcance of methylation signals. Data show that MeCP2 can assist in transcriptional silencing by binding to methylated CpG sites and by associating with chromatin-remodeling complexes ( Jones et al., 1998; Nan et al., 1998). Despite the increased attention received by MeCP2, the targets of its action remain unclear (Amir and Zoghbi, 2000). The study of these diseases suggests that methylation is not only needed to complete embryonic development, but is also required for developmental maturation after birth. The study of DNA methylation in mammals has been stimulated by the identiﬁcation of key enzymes that methylate DNA and their interactions with DNA and DNA-binding proteins, as well as by the link between methylation and changes in chromatin structure. A major role of methylation is the silencing of noncoding DNA in the genome. The DNA methylation ﬁeld is currently in a state of high activity as the links among stable epigenetic states, chromatin structure, and heterochomatization begin to become clear. Understanding how epigenetic states are generated and maintained is an area of research with high priority. This would then allow the development of appropriate strategies for the pharmacological manipulation of these states so as to alter changes in the expression of associated sets of genes.

V. Epigenetics and Synaptic Plasticity

Reelin and GAD67 gene expression is consistently downregulated in all postmortem brain regions of patients diagnosed with schizophrenia thus far examined (Guidotti et al., 2000; Impagnatiello et al., 1998). Reelin downregulation

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was conﬁrmed in the hippocampus of schizophrenia patients (Fatemi et al., 2001), and the GAD67 deﬁcit has been independently replicated (Akbarian et al., 1995; Volk et al., 2002). Other work has shown that the reelin promoter is regulated through the action of speciﬁc transcription factors and the interplay between transcription factor binding and promoter methylation (Chen et al., 2002). Reelin and other genes that are downregulated in the brains of patients diagnosed with schizophrenia may share a common epigenetic regulatory mechanism that is either hyperactive or operating in a biologically inappropriate manner. It seems plausible that promoter hypermethylation in the coordinate expression of reelin, and other genes expressed in GABAergic neurons (such as GAD67, GAD65, and Dnmt1), may underlie the symptomatology of schizophrenia. This is consistent with the increase in biochemical components associated with the expression of DNA methyltransferase 1 in GABAergic neurons of schizophrenia patients ( Veldic et al., 2004). A possible dysfunction of the GABAergic system in schizophrenia (for reviews, see Benes and Berretta, 2001; Costa et al., 2002, 2003) and the role of a reelin deﬁcit in schizophrenia etiopathology are major research avenues to be explored. Epidemiological and genetic studies on twins, assumed to be genetically concordant but discordant for schizophrenia, led to the inference that schizophrenia may be an epigenetic disorder ( Costa et al., 2003; McDonald et al., 2003; Petronis et al., 2001). Because the pathophysiology of schizophrenia is characterized by a dysfunction in cortical GABAergic circuitry (Costa et al., 2003; Guidotti et al., 2000), the hypothesis that an epigenetically acquired chromatin remodeling alteration, which is speciﬁcally localized to cortical GABAergic neurons, may play a central role in inducing some of the salient morphological features of cortical dendritic spine hypoplasticity requires additional attention. The consequent disruptions of pyramidal columnary assembly and synchronous ﬁring may induce important aspects of schizophrenia pathophysiology, including reelin expression downregulation. The release of GABA from GABAergic neurons is central to the regulation of depolarization-induced glutamatergic synaptic transmission. Moreover, reelin, secreted from GABAergic neurons, plays a role in spine mRNA translation and dendritic spine maturation (Dong et al., 2003). With this in mind, it becomes clear that a GABAergic neuron defect would result in altered glutamatergic signaling, which would subsequently be accompanied by alterations in dopaminergic and serotonergic synaptic transmission. As each of these transmitter receptor systems have been implicated in the pathogenesis of schizophrenia, it becomes relevant to discuss salient aspects of excitatory and inhibitory neurotransmitter synaptic organization. Many of the topics discussed in this section have been reviewed by Choquet and Triller (2003).

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A. Postsynaptic Density (PSD) Organization at Excitatory Synapses AMPA- and NMDA-selective glutamate ionotropic receptors are pentamers of subunit families that are assembled and expressed on postsynaptic neuronal membranes. These receptor complexes must be clustered spatially so as to provide suVicient ampliﬁcation of the presynaptic signal to locally depolarize the neuron. If multiple signals arriving at the neuron summate both spatially and temporally, the neuron becomes depolarized with subsequent neurotransmitter release. An important premise necessary to regulate this local depolarization is that receptor clustering in postsynaptic membranes occurs and that appropriate mechanisms are in place to ensure that receptor diVusion in the membrane is minimal. Studies of excitatory PSDs indicate that the structures previously viewed to be electron dense by electron microscopic analyses include a vast collection of protein complexes, which subserve this function at so-called asymmetric/excitatory synapses (Craven and Budt, 1988; Kennedy, 2000). That is, numerous anchoring proteins associated with ionotropic glutamate receptors form a vast molecular complex interacting with the receptors constituting a subsynaptic scaVold between these receptors and the neuronal cytoskeleton (reviewed in Sheng and Sala, 2001). In addition to the anchoring proteins themselves, various signaling proteins involved in signal transduction are located within this subsynaptic space (Garner et al., 2000). The glutamate receptors are concentrated at the center of the PSD and decrease as the distance from the center is increased. This is also true for type I metabotropic glutamate receptors, which are expressed as a ring surrounding the PSD (Baude et al., 1993). Mass spectrometry analyses of rat forebrain PSD fractions coupled with information associated with protein sequence databases have led to the detection and subsequent identiﬁcation of multiple proteins in this subcellular fraction (Walikonis et al., 2000). The implication of this study is that glutamate receptors interact either directly or indirectly with these intracellular proteins that reside within the PSD domain. Those proteins that interact directly with the postsynaptic receptors are the so-called scaVolding proteins. For instance, PSD-95 proteins (for example SAP90) interact directly with the NMDA receptor signaling complex (Kornau et al., 1997). PSD-95 represents an emerging family of four related proteins, called membrane-associated GuK (MAGuK) proteins, each of which contain multiple binding motifs and which are tightly colocalized with NMDA receptors at synapses. The glutamate receptor-interacting protein (GRIP) and proteins that interact with C kinase (PICK) interact preferentially with the C-terminal PDZbinding motifs of AMPA receptors (reviewed by Sheng and Lee, 2003). Homer interacts with metabotropic glutamate receptors (mGluRs) 1 and 5, which both signal through activation of phospholipase C and mobilization of intracellular calcium (Kreienkamp, 2002). Homer can function in part by forming homodimers. These primary scaVolding proteins are, in turn, associated with secondary

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scaVolding proteins. The guanylate kinase-associated protein (GKAP) interacts with PSD95 and Shank. Shank is a large protein with multiple protein-binding domains, which also forms homomultimers (Kennedy, 2000) and interacts with mGluRs through its interaction with Homer. GKAP can couple with PSD95– NMDAR to form complexes. A recent report indicates that through the interactions of primary and secondary scaVolding proteins, functionally distinct glutamate receptors may interact and communicate at the same synapse (Wenthold et al., 2003). For example, stargazin interacts directly with the PDZ domains of the MAGUK proteins (associated with NMDA receptors) and mediates synaptic targeting of AMPA receptors. Through a variety of interacting proteins and domains, NMDA, AMPA, and mGluRs respond to the neurotransmitter glutamate at the same synapse. In addition, some literature suggests that Homer, Shank, and PSD95–GKAP–Shank may be expressed characteristically at diVerent synaptic compartments (Usui et al., 2003). In addition to primary and secondary scaVolding proteins, many signaling proteins are connected within the PSD. A-kinase-associated/anchoring proteins (AKAPs) are also expressed in PSDs and facilitate the interaction of GluRs with the regulatory subunit of PKA (Michel and Scott, 2002). Ca2þ /calmodulindependent protein kinase II (CaMKII) interacts directly or indirectly with glutamate receptors and is the most abundant signaling protein in the PSD fraction. CaMKII is a target for calcium entering through transmitter-activated NMDA receptors and is required for synaptic plasticity in pyramidal neurons (Stevens et al., 1994). While it seems clear that the PSD contains a complex network of interacting proteins involved in synaptic transmission, issues related to changes in synaptic plasticity and long-term potentiation appear to involve dynamic movements of AMPA receptors into and out of the PSD mediated in part through NMDA receptor signaling (Sheng and Lee, 2003). Some adhesion proteins (neurexin and neuroligin) also contribute to the shaping of synapse formation and to transmitter receptor recruitment at PSDs. ScheiVele et al. (2000) provided a potential role for a neuroligin–neurexin complex in facilitating the formation of functional presynaptic elements in vitro. Neuroligins are transmembrane adhesion molecules that bind -neurexins (Ichtchenko et al., 1995). The interaction between neurexins and neuroligins appears to activate the formation of pools of presynaptic vesicle clusters (Cantallops and Cline, 2000). Neuroligins link the presynaptic compartment to the postsynaptic component through interactions with the PSD-95 and NMDA receptors. Similarly, cadherins can recruit kainate receptors (Coussen et al., 2002) and can regulate synapse shape and function and also the shape and volume of dendritic spines (Togashi et al., 2002). Homophilic cadherin interactions link kainate receptors with presynaptic terminals. In addition to cadherins, homophilic complexes form between synaptic cell adhesion molecule (SynCAM) and proteins called Sidekicks (Yamagata et al., 2002). The interaction

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of receptor–scaVold complexes with cytoskeleton proteins regulates receptor traVicking from given synapses. A model of the molecular organization of the PSD has been reviewed (Kennedy, 2000). This model shows the postsynaptic density in which AMPA and NMDA receptors are linked through interactions with the underlying scaVold, which is arranged in layers parallel to the membrane. In addition, mGluR receptors link to the IP3 and ionotropic receptors through interactions with the Homer and Shank proteins. Collectively, the emerging picture, while complex, provides a mechanism through which multiple glutamate receptors are able to integrate the presynaptic signal into a cohesive postsynaptic response that occurs at multiple levels.

B. Inhibitory Synapses Similar to the proteins that are expressed at excitatory PSDs, inhibitory receptors have evolved mechanisms to ensure receptor clustering in the synaptic domain. PSDs at inhibitory synapses are considerably less complex than those present at excitatory synapses. This has been known for some time from electron microscopic (EM) studies showing the inhibitory synapse to be symmetric whereas the electron dense excitatory PSD is asymmetric. Gephyrin is a 93kDa protein that copuriﬁes with the glycine receptor and was found to colocalize at glycinergic synapses (Triller et al., 1985). Evidence in support of this was provided by antisense knockdown experiments showing that gephyrin depletion was associated with a loss of receptor clustering (Feng et al., 1998; Kirsch et al., 1993). Similarly, gephyrin acts as a core protein of the scaVold and interacts with the cytoskeleton, binding to polymerized tubulin (Kirsch et al., 1995). The disruption of microtubules and microﬁlaments was shown to impair glycine receptor clustering (Kirsch and Betz, 1995). Gephyrin is also involved in the synaptic localization of some GABA-Rs. The mechanism appears to involve a direct interaction between gephyrin and the -2 subunit of the receptor (Essrich et al., 1998; Kneussel et al., 1999). In addition to gephyrin, GABA-A R-associated protein (GABARAP) plays a role in the intracellular traVicking of GABA-A Rs. GABARAP, like gephyrin, is a tubulin-binding protein and can function to assemble microtubules in the process of receptor clustering (Coyle et al., 2002). Collectively, scaVolding proteins function as essential components for receptor organization in the PSD. Additional speciﬁc mechanisms for receptor stabilization have not yet been identiﬁed. In fact, knock-out (KO) of scaVolding molecule expression leaves inhibitory and excitatory synapse function largely unaVected. This implies that a certain amount of compensation, ﬂexibility, and redundancy likely exists that we do not, as yet, fully appreciate. It seems clear that the primary and secondary scaVolding proteins present in PSDs very likely act as diVusion barriers within postsynaptic membranes. Hence, the dynamics of

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receptor protein mobility in PSDs may be guided by some of the aforementioned proteins and protein complexes, but the functional signiﬁcance of the receptor mobility in the PSD is just beginning to be appreciated.

C. Dynamic Aspects of PSD Organization As discussed earlier, the structure of the PSD is highly organized, with multiple proteins coupling the dynamics of the neurotransmitter response. It is also necessary to superimpose upon this organization the concept that receptor number and type may change in response to the quality, frequency, and duration of the presynaptic signal. Processes that have been described for many years, including LTP and LTD, underscore the dynamics of the synapse in terms of its ability to change in response to the presynaptic signal. The number of receptors expressed in a given PSD changes, but only in response to the proliferation of new presynaptic elements or to changes in the frequency and duration of their use. Receptor exocytosis, endocytosis, and membrane diVusion are highly linked processes, but in terms of neuronal function, the number of synapses is relatively stable, even though plasticity and renovation (turnover) rate can change (Trachtenberg et al., 2002). At excitatory synapses, dynamic movements of AMPA receptors into and out of the PSD likely underlie the expression of NMDA receptor-mediated changes in synaptic plasticity. The traVicking of AMPA receptors in response to complex variegation patterns in the context of scaVolding protein function has been reviewed (Sheng and Lee, 2003). Evidence shows that movement on the surface from nonsynaptic to synaptic sites is a critical aspect of AMPA receptor plasticity. However, the mechanisms responsible for regulating these processes are still being elucidated. Receptor synthesis/turnover cycle is regulated not only by neuronal activity, but probably also by the turnover of the scaVolding proteins as well. At which stage of the receptor recycling process scaVolding proteins play a role is still a matter of debate (Lee et al., 2002). Along these same lines, the rapid redistribution of PSD95 (Okabe et al., 1999) and the related protein PSD-Zip45 (Homer 1C) (Okabe et al., 2001) has been observed in hippocampal neurons. The dynamic behavior of these GFP-tagged proteins was analyzed using time-lapse confocal microscopy. These authors found that neuronal activity such as NMDA receptor-dependent calcium inﬂux or voltage-dependent calcium channel activation resulted in either receptor disassembly or clustering, respectively, thus underscoring a role for receptor stimulation in scaVolding protein turnover. At inhibitory synapses, both Gly Rs (Rasmussen et al., 2002) and GABA-A Rs (Barnes, 2001) are turned over rapidly. However, it has not been established whether the cycling of receptors in PSDs involves some of the aforementioned proteins that are expressed in PSDs. For

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instance, as discussed earlier, stargazin links AMPA-Rs to PSD95 (Chen et al., 2001) and is essential for the delivery of functional AMPA-Rs to PSD. Disrupting the stargazin–PSD95 interaction using a dominant-negative mutant leads to a reduced density of synaptic AMPA-R expression and an increased density of extrasynaptic AMPA-Rs (Schnell et al., 2002). Conversely, overexpression of synaptic PSD-95 results in an increase in synaptic AMPA-Rs.

D. Transmitter Receptor Stability in the Membrane A central question relevant to receptor clustering at PSDs is how to reach an understanding of the molecular mechanisms operative in the process that maintains the receptor in a given synaptic domain. What are the processes that negate or support receptor diVusion in membranes? Multiple mechanisms are likely operative, some of which have already been discussed. Linking the proteins to the subsynaptic scaVold through protein–protein interactions would provide an anchoring eVect. A second consideration is that a mechanism must be operative for maintaining the PSD proximal to the presynaptic component to ensure optimal activation kinetics. Interactions between the proteins in the PSD and the presynaptic membrane would immobilize the PSD to the presynaptic structure. Finally, the locale within the neuronal membrane domain proximal to the PSD must be ﬁnite and suViciently close to the reserve receptor pool. It seems probable that the size of a receptor cluster on the PSD would depend on receptor subunit aVinity for scaVolding molecules and on interactions with the scaVolding molecules available in the membrane. If they were bound to one another with the same aVinity as they bind to other proteins, then there would be a driving force for local accumulation at the plasma membrane. To facilitate receptor and scaVolding association, a high binding aVinity would prevent the fortuitous interaction of these proteins with other cytoskeletal proteins. An emerging concept is that receptor traVicking in and out of the PSD domain facing the presynaptic axon terminal is under the control of the presynaptic neuron ﬁring rate. It is possible that basal neuronal activity in synaptically connected neurons can also control AMPA-R movement (BorgdorV and Choquet, 2002). Elevations in postsynaptic intraneuronal Ca2þ due to activitydependent mechanisms are known to be important in the regulation of transmitter eVicacy. Perhaps the increase of Ca2þ triggers receptor immobilization and local accumulation in the neuronal surface. While the molecular mechanisms that underlie Ca2þ -dependent AMPA-R turnover are not clear, it may well be that Ca2þ facilitates and maintains receptor binding to the scaVolding proteins. Hence, the future challenge will be to understand the biophysical basis that maintains receptor traVicking considering the aVinities of receptor binding to

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receptor-stabilizing molecules at individual synapses in relation to neuronal activity. We have gained considerable insight into the molecular partners that integrate synaptic signaling among diVerent excitatory receptors and associated mechanisms that couple excitatory and inhibitory receptors to the PSD. We now seek a better understanding of how these processes are regulated in the context of synaptic plasticity. Moreover, it becomes relevant to consider the ways in which inhibitory receptor signaling modulates neuronal circuitry and the role that epigenetics plays in regulating these events.

VI. Epigenetics Today

By the end of the 20th century, epigenetics had grown to became a widely recognized subdiscipline of biology, but for many people, epigenetics had become almost synonymous with ‘‘epigenetic inheritance.’’ For example, a deﬁnition given in 2001 (Riddihough and Pennisi, 2001) was ‘‘The study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence.’’ Even as a deﬁnition of epigenetic inheritance rather than epigenetics, this wording presents problems because it excludes the regularly occurring developmental changes that alter gene function through the reorganization of DNA. The diViculty is that although one can usefully distinguish between DNA and non-DNA inheritance, no simple criteria exist for distinguishing between genetic and epigenetic phenomena. In general, genetics today deals with the transmission and processing of information in DNA, whereas epigenetics deals with its interpretation and integration with information from other sources. Epigenetics is therefore concerned with the systems of interactions that lead to predictable and usually functional phenotypic outcomes; it includes processes of spontaneous self-organization that depend on the physical and chemical properties of the internal and external environments as well as being involved in gene-dependent mechanisms. Because of the lack of consensus about what the term epigenetics means, Lederberg (2001) suggested that the term should be abandoned. He maintains that epigenetics in the Waddington sense and epigenetics in the modern sense have little in common, so retaining this word simply leads to confusion. It would be better, he suggests, to talk about nucleic, epinucleic, and extranucleic information rather than epigenetic information. It is true that epigenetics today is very diVerent from Waddington’s epigenetics, but the same can be said for many other terms in biology, including both ‘‘gene’’ and ‘‘genetics.’’ This has not been seen as a good reason for abandoning those terms. We feel that using Lederberg’s ‘‘nucleic,’’ ‘‘epinucleic,’’ and ‘‘extranucleic’’ information would not be helpful because information cannot be

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neatly parceled out in this way. One valuable aspect of the term epigenetics is that it has always been associated with the interactions of genes, their products, and the internal and external environment rather than with the individual facets of developmental regulation. Because of this, there is a continuity between epigenetics in Waddington’s sense and epigenetics today; both focus on alternative developmental pathways, on the development of networks underlying stability and ﬂexibility, and on the inﬂuence of environmental conditions of what happens in cells and organisms. It is only when epigenetics is equated solely with the inheritance of non-DNA variations that its original meaning is obscured.

VII. The Epigenetic Concept in Psychiatry

In psychiatry, epigenetics deﬁnes inheritance of a particular liability to a disease state and speciﬁcally indicates that such liability cannot be explained by Mendelian inheritance laws. For instance, it is diVicult to explain with these inheritance laws why identical twins are about 50% concordant for schizophrenia although their genome structure is virtually identical (McDonald et al., 2003). When Waddington ﬁrst proposed the epigenetic concept, the role of the gene in development was completely mysterious. In 1966, Conrad Waddington wrote ‘‘Some years ago, I introduced the word ‘epigenetics’ derived from the Aristotelian word ‘epigenesis,’ which had more or less passed into disuse, to indicate a suitable branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being.’’ Waddington had coined a very clever term. It related back to the Aristotelian theory of epigenesis, which stresses that developmental changes are gradual and qualitative but also links to current and future studies of heredity. ‘‘Epi’’ means ‘‘upon’’ or ‘‘over’’ and the ‘‘genetics’’ part of the word epigenetics implies that genes are involved, so the term reﬂected the need to study events ‘‘over’’ or ‘‘beyond’’ the gene. Waddington’s pictorial models (see Waddington, 1957) of epigenetics leave little doubt that he conceived development in terms of what today we would call diVerential gene regulation. He illustrated his way of thinking with drawings in which the developmental system was depicted as a landscape in which bifurcating and deepening valleys run down from a plateau. In this landscape, he depicted a fertilized egg located in an area in which it can choose diVerent paths. Clearly, Waddington’s epigenetics should not be confused with developmental genetics. Both are concerned with the same process but there are diVerences in perspective. In other words, epigenetics underlies a situation in which genetic variation does not lead to phenotypic variation and phenotypic diVerences are not associated with genetic diVerences. Plasticity is the other side of the coin. In fact, genetically identical cells or organisms can diVer in structure and function. For example,

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kidney, liver, and skin cells diVer phenotypically, these cells all inherit a diVerent phenotype, but the variation that is expressed and distinguishes each group of cells is epigenetic and not genetic. Similarly, morphological diVerences between worker bees and a queen bee are epigenetic, not genetic, because whether a larva becomes a worker or a queen is dependent on the way it is fed and not on its genotype.

VIII. Conclusions

Examination of recent publications including the term epigenetics in their titles shows that the scope of the subject is far less narrow than some current definitions suggest. It includes studies of the cellular regulatory networks that confer phenotypic stability related to developmentally regulated changes in DNA (such as those seen in the immune system), cell memory mechanisms (involving heritable changes in chromatin and DNA methylation), and self-propagating protein conformational changes inﬂuencing cellular structures. Cellular inheritance is an important aspect of some of these studies and there is growing interest in the transgenerational inheritance of some epigenetic variations. One of the most productive areas of research in the past decade has been the study of the controlled responses of cell genomes to severe environmental insults that involve DNA methylation, RNA-mediated gene silencing, and enzyme-mediated DNA rearrangements and repair ( Jenuwein, 2002). Much of this work stems from McClintock’s work (1984) and ideas on stress responses in plants, but it is very clearly within the epigenetic concepts in Waddington’s sense, particularly when, as commonly happens, it is discussed within an evolutionary framework. We believe that we have clariﬁed that morbidity related to epigenetic mechanisms is in a state of ﬂux that must be understood before becoming a potential target of pharmacological intervention in psychiatry. This is particularly important in view of current proposals of preemptive treatment of individuals at risk. In fact, it is very diVicult to assess morbidity vis-a`-vis an epigenetic process.

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MUSCARINIC RECEPTORS IN SCHIZOPHRENIA: IS THERE A ROLE FOR SYNAPTIC PLASTICITY?

Thomas J. Raedler Department of Psychiatry University Hospital Hamburg–Eppendorf 20246 Hamburg, Germany

I. II. III. IV.

Introduction Muscarinic Receptors Schizophrenia Synaptic Plasticity References

I. Introduction

Muscarinic cholinergic neurotransmission as a part of cholinergic neurotransmission constitutes an important factor of diVerent cognitive processes, including memory and learning. Five diVerent muscarinic receptor subtypes can be found in the brain in regionally varying distribution. While the exact function of these muscarinic receptor subtypes is not known, diVerent muscarinic receptor subtypes can have diVerent functional eVects. The focus of psychiatric research has been on neurotransmitter systems other than the muscarinic system. However, there has been increasing evidence that the muscarinic cholinergic system is part of the pathophysiological changes in diVerent psychiatric disorders. The role of the muscarinic receptor system in schizophrenia is reviewed. Changes in the muscarinic cholinergic system in schizophrenia should not be seen in isolation but as part of an overall disease process, particularly as the muscarinic receptor system is connected with other neurotransmitter systems at diVerent levels. Regarding schizophrenia, the muscarinic cholinergic system interacts with the dopamine system, which is still seen as the crucial receptor system in this devastating disorder. However, realization of the role of the muscarinic cholinergic system in schizophrenia has helped shape the pathophysiological concepts of this disorder and might lead to new pharmacological approaches. The muscarinic cholinergic system plays an important role in synaptic plasticity and is involved in diVerent mechanisms, including long-term potentiation (LTP) and long-term depression (LTD). Consistent with the complex nature of INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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the muscarinic cholinergic system, the eVects of muscarinic neurotransmission on synaptic plasticity diVer for diVerent brain regions, as well as with the muscarinic receptor subtype involved. Synaptic plasticity is a key feature of the molecular processes underlying learning and memory, and the muscarinic system is involved in some of these mechanisms. Little is known about the eVects of the muscarinic system on synaptic plasticity in psychiatric disorders. Given the central role of learning and memory in diVerent psychiatric disorders, further studies of the role of the muscarinic receptor system in synaptic plasticity seem warranted.

II. Muscarinic Receptors

Since the beginning of the last century, acetylcholine (ACh) has been recognized as a neurotransmitter. Acetylcholine is synthesized in neurons from acetyl-CoA and choline in a reaction catalyzed by the enzyme choline acetyltransferase. Glucose and citrate serve as the source for acetyl-CoA, while choline is transported by blood into the brain. Following activation of the neuron, acetylcholine is released into the synaptic cleft, where it binds to pre- and postsynaptic receptors. Acetylcholine is inactivated in the synaptic cleft by hydrolysis by the enzyme cholinesterase. Acetylcholinesterase is the most important of the cholinesterases and is speciﬁc for acetylcholine. Butyrylcholinesterase is less speciﬁc for acetylcholine. Following hydrolysis, choline is transported back into the presynaptic neuron through an active transport system and is recycled into the synthesis of acetylcholine. DiVerent substances aVect the inactivation of acetylcholine in the synaptic cleft and thus increase the concentration of acetylcholine. Organophosphates are irreversible inhibitors of cholinesterase, whereas physostigmine is a reversible inhibitor. Inhibitors of the acetylcholinesterase have been developed as pharmaceuticals and constitute the basis of the current pharmacological treatment of dementia. Cholinergic neurotransmission uses acetylcholine as a neurotransmitter in the central nervous system (CNS) as well as in the peripheral nervous system. In the latter system, acetylcholine has been identiﬁed as the transmitter of autonomic ganglia and the neuromuscular junction. Cholinergic neurons, including both interneurons and projection neurons, can be found throughout the entire central nervous system. Cholinergic interneurons are mainly located in the striatum and nucleus accumbens. Most cholinergic projection neurons are located in the basal forebrain and the brain stem. Based on their anatomical location and pattern of innervation, the following two cholinergic cell groups can be diVerentiated (Mesulam et al., 1983):

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1. Basal forebrain cholinergic neurons are located in the medial septum, the diagonal band of Broca, and the nucleus basalis of Maynert and innervate primarily the cerebral cortex and hippocampus. 2. Brain stem cholinergic neurons can be found in laterodorsal and pedunculopontine tegmental nuclei and project primarily to the midbrain and brain stem. Cholinergic neurons are crucial in a variety of central nervous functions, including motor function, cognitive processing, memory, arousal, attention, sleep, nociception, motivation, reward, mood, and psychosis. In addition to its activity in the CNS, acetylcholine also plays a major role in diVerent peripheral functions, such as heart rate, blood ﬂow, gastrointestinal tract motility, sweat production, and smooth muscle activity. In understanding the function of acetylcholine in the brain, a special emphasis has been put on the importance of acetylcholine for memory and learning (Deutsch, 1971; Drachman et al., 1974). In particular, cholinergic septohippocampal pathways play a crucial role in learning and memory (Bartus et al., 1982). Since the beginning of the last century, two distinct subclasses of the cholinergic system (the muscarinic and nicotinic system) have been known (Dale, 1914). DiVerent toxins with agonist and antagonist activity on the cholinergic system have helped identify the muscarinic and the nicotinic system. Muscarine, an alkaloid derived from a poisonous mushroom, serves as an agonist to the muscarinic system, whereas atropine, an alkaloid from deadly nightshade, is a powerful antagonist of the muscarinic system. The nicotinic system was deﬁned by the well-known agonist nicotine as well as the antagonist d-tubocurarine, an alkaloid used as an arrow poison. Muscarinic and nicotinic cholinergic receptors diVer regarding their function as well as their receptor structure. Nicotinic receptors consist of a variety of 17 diVerent subunits that can combine in various combinations (Picciotto et al., 2000). Nicotinic receptors are composed of 5 subunits surrounding a ligand-gated ion channel. The binding of acetylcholine to the nicotinic receptor leads to an activation of the ion channel, resulting in an inﬂow of sodium ions (Conti-Tronconi et al., 1982; Popot et al., 1976). As is typical for ligand-gated ion channel receptors, the activation of nicotinic receptors results in a rapid neural response. In contrast to ligand-gated ion channel receptors, another class of recetors, the so-called G-protein-coupled receptors, are coupled to GTP-binding proteins. These receptors can either activate or inhibit the adenylate cyclase, thus having an eVect on second messenger cyclic AMP (cAMP). Muscarinic receptors are coupled to GTP-binding proteins (Housley, 1992; Spiegel et al., 1992), resulting in a slower but potentially more sustained response to their activation. Muscarinic receptors belong to the superfamily of G-protein-coupled receptors (Kubo et al., 1986; Van Zwieten, 1991). Muscarinic receptors consist of seven

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transmembrane-spanning domains and are composed of 460–590 amino acids (Wess, 1993). The link between muscarinic receptors and the G-protein is thought to involve the third intracellular domain of the muscarinic receptor. Muscarinic receptors can be found on cholinergic and noncholinergic cells, both as auto- and heteroreceptors (Raiteri et al., 1984; Raiteri et al., 1990; Vizi et al., 1989; Wamsley et al., 1984). Five diVerent subtypes of muscarinic receptors (M1–M5) have been distinguished pharmacologically (Watling et al., 1995) and correspond to ﬁve diVerent genes (m1–m5) that have been cloned (Bonner et al., 1987, 1988; Buckley et al., 1989; Hulme et al., 1990). Pharmacological identiﬁcation of the diVerent subtypes of muscarinic receptors was hampered for a long time by the lack of subtype-speciﬁc agents. The molecularly and pharmacologically deﬁned subtypes have been found to correspond. Following the international convention (Caulﬁeld and Birdsall, 1998), the upper case nomenclature for the subtypes of the muscarinic receptor is used throughout this review. All ﬁve subtypes of the muscarinic receptor are found in the human brain, albeit in regionally varying concentrations (Levey et al., 1991). For example, basal ganglia express predominantly M1 and M4 receptors, as does the cortex, although in diVerent proportions, whereas the thalamus and brain stem have predominantly M2 receptors (Flynn and Mash, 1993; Li et al., 1991; Vilaro et al., 1991; Wall et al., 1991a,b; Yasuda et al., 1993). Overall, the M1, M3, and M4 subtypes are found abundantly in the brain (Buckley et al., 1988), where the M5 subtype is the least abundant (Liao et al., 1989; Vilaro et al., 1990). Muscarinic receptors have been further separated into two groups based on their eVects on the G-protein. M1, M3, and M5 muscarinic receptors have a stimulating eVect on G-protein, resulting in an activation of phospholipase C (PLC) and MAP kinase. M1, M3, and M5 receptors increase the intracellular concentration of Ca2þ and cAMP. M2 and M4 muscarinic receptors, however, inhibit adenylyl cyclase. Thus the diVerent subtypes of the muscarinic receptors exert opposite eVects on this second messenger system. Although all muscarinic receptor subtypes occur postsynaptically, the M2 receptor is also found on presynaptic neurons and serves as an autoreceptor (Baghdoyan et al., 1998; Mrzljak et al., 1993; Stoll et al., 2003). In the striatum, the M4 receptor constitutes the muscarinic autoreceptor (Zhang et al., 2002a). Knockout animals have helped clarify the role of the diVerent subtypes of the muscarinic receptor (e.g. Gomeza et al., 1999a,b; Hamilton et al., 1997; Shapiro et al., 1999). These knockout animals, predominantly mice, were altered genetically and do not express speciﬁc genes. Knockout animals lacking speciﬁc genes can be compared to wild-type animals, which express these genes. Thus the function of speciﬁc genes can be studied in vivo. The comparison of knockout and wild-type animals has helped identify the function of the diVerent subtypes of the muscarinic receptor. Wild-type animals develop seizures when muscarinic agonists are given in high doses. Animals that

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do not express muscarinic M1 receptors do not exhibit seizures upon administration of muscarinic agonists (Hamilton et al., 1997). Animals lacking the M2 receptor show pronounced deﬁcits in their movement and temperature control (Gomeza et al., 1999a). Furthermore, M2-deﬁcient animals do not show carbachol-induced bradycardia (Gomeza et al., 1999b). M4 receptor knockout mice have enhanced motor activity at baseline as well as a strongly increased locomotor response to dopamine D1 receptor agonists (Gomeza et al., 1999b). Muscarinic receptor knockout mice have also been used to diVerentiate the modulatory eVects of diVerent muscarinic receptor subtypes on calcium channels (Shapiro et al., 1999). Identiﬁcation of the exact function of the cholinergic system is complicated by the fact that both nicotinic and muscarinic cholinergic neurotransmission contribute to this function. In addition, both systems do not function in isolation, as cholinergic neurons interact closely with diVerent other neurotransmitter systems, including dopamine (Blaha et al., 1996; Lokwan et al., 1999; Zhang et al., 2002b). Interaction between the muscarinic system and other systems, particularly with the GABAergic system, is crucial for learning and memory (Wu et al., 2000). The muscarinic cholinergic system is known to be involved in diVerent neurological and psychiatric illnesses. A role of the cholinergic system has been identiﬁed in myasthenia gravis and related autoimmune disorders, such as the Lambert–Eaton mysthenic syndrome. Huntington’s disease involves the degeneration of cholinergic interneurons in the striatum. In all these disorders, as well as in Parkinson’s disease, pharmacologic treatment strategies that focus on the cholinergic system have proven eVective. Exciting and increasing amounts of data have been released on the role of the muscarinic cholinergic system in psychiatric disorders, particularly Alzheimer disease and schizophrenia. This review focuses on the role of muscarinic receptors in schizophrenia, as well as in synaptic plasticity.

III. Schizophrenia

Schizophrenia is a severe psychiatric illness that aVects about 1% of the population worldwide and exerts a huge toll on patients and their families. Schizophrenia remains a clinical diagnosis based on a typical symptom constellation and time course. So far, laboratory and technical examinations have not proven helpful in diagnosing schizophrenia per se, but are useful tools to rule out other diseases that can present with a similar clinical picture. The clinical symptoms of schizophrenia consist of a characteristic combination of hallucinations, delusions, disorganized thinking, disorganized behavior, and negative symptoms,

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such as apathy, anhedonia, and social withdrawal. In addition to these clinical symptoms, schizophrenia is frequently complicated by cognitive deﬁcits, mainly in the areas of attention, memory, executive functioning, and intelligence (Goldberg et al., 1993; Weickert et al., 2000). The negative and cognitive symptoms associated with schizophrenia can have a marked impact on the functional outcome of patients suVering from schizophrenia (Green, 1996). Over the past decades, research in schizophrenia has focused mainly on the neurotransmitter dopamine. Dopamine has been implicated in the pathophysiology and treatment of schizophrenia, and the dopamine hypothesis of schizophrenia has been a popular formulation to account for psychotic symptoms (for a review, see Carlsson, 1988). However, as the dopamine hypothesis of schizophrenia fails to explain all aspects of schizophrenia, other neurotransmitters, including glutamate, serotonin, and acetylcholine, have also been implicated. DiVerent pharmacological, endocrinological, and neuropathological observations suggest that the muscarinic system may be altered in schizophrenia. Treatment of schizophrenia subjects with antimuscarinic drugs used to be common practice, as anticholinergics help alleviate motor side eVects caused by older antipsychotics. In unmedicated schizophrenics, treatment with anticholinergics resulted in a worsening of psychosis, whereas negative symptoms of schizophrenia improved under anticholinergic treatment (Tandon et al., 1991, 1992). The worsening of psychosis under treatment with anticholinergic is consistent with reports of increased dopamine release after the application of anticholinergic agents (Dewey et al., 1993). At the same time, schizophrenic subjects frequently report an activating eVect of higher doses of anticholinergics, occasionally resulting in an abuse of anticholinergic medication (Zemishlany et al., 1996). Schizophrenic patients showed a shortening of rapid eye movement latencies after pretreatment with a muscarinic agonist as well as after a placebo (Riemann et al., 1994). In a cholinergic challenge test using the cholinesterase inhibitor pyridostigmine, the growth hormone response was increased in unmedicated schizophrenic subjects (O’Keane et al., 1994). These sleep and endocrine studies were interpreted as an indication that cholinergic tone is increased in schizophrenia. Tandon and Greden (1989) proposed that the cholinergic muscarinic system is altered in schizophrenia. Yeomans (1995) speculated later that schizophrenia may be caused by an overactivation of cholinergic neurons in the pedunculopontine and the laterodorsal tegmental nucleus, resulting in an activation of dopaminergic neurons. Further evidence for an involvement of the muscarinic cholinergic system in schizophrenia comes from neuropathological studies. Studies of postmortem tissue have found consistent reductions in muscarinic receptor density in schizophrenia. The ﬁrst study of brain tissue found reductions in muscarinic receptor binding in the frontal cortex of schizophrenic patients compared to normal

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controls (Bennett et al., 1979). However, this study focused on serotonin and dopamine receptors, and the muscarinic system was used only as a reference system. Unfortunately, this ﬁnding of a reduction of muscarinic receptors in schizophrenia was not followed up until many years later. More recent neuropathological studies from a group from Australia revealed signiﬁcant reductions of diVerent subtypes of the muscarinic receptors in diVerent brain regions of schizophrenic patients. Muscarinic M1 receptors, as well as M2 and M4, were found to be reduced in the caudate and putamen of schizophrenics (Crook et al., 1999; Dean et al., 1996). At the same time, levels of M1 mRNA were not decreased compared to healthy controls in the caudate and putamen of schizophrenic subjects (Dean et al. 2000). Looking at the hippocampus, muscarinic M1 and M4 receptor binding was reduced signiﬁcantly in diVerent subregions of the hippocampus, as well as in the dentate gyrpus, subiculum, and parahippocampal gyrus (Crook et al., 2000). This group extended their studies into the frontal cortex. Muscarinic M1 and M4 receptor binding was decreased in diVerent parts of the prefrontal cortex (Crook et al., 2001; Dean et al., 2002). By means of a diVerent technique, a group from the United Kingdom conﬁrmed decreased M1 receptor density in schizophrenia (Mancama et al., 2003). Using123I IQNB SPECT as an imaging tool to study muscarinic receptors in vivo, Raedler et al. (2003a) reported a decrease of muscarinic receptor availability in diVerent brain areas in unmedicated schizophrenic subjects. Using the same technique, we were able to show that olanzapine (Raedler et al., 2000) and clozapine (Raedler et al., 2003a), two atypical antipsychotics, also bind to the muscarinic receptor in vivo. The muscarinic system in schizophrenia has been evaluated as a potential novel pharmacological target for the treatment of psychosis. (5R, 6R)6-(3-propylthio1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane (PTAC) is a muscarinic receptor ligand with partial agonist eVects at muscarinic M2 and M4 receptors and antagonist eVects at M1, M3, and M5 receptors. PTAC selectively inhibits dopamine cell ﬁring, as well as the number of spontaneously active dopamine cells. As this substance proved to have functional dopamine receptor antagonistic properties in animals, it may be investigated further as a novel approach for the treatment of schizophrenia (Bymaster et al., 1998). Treatment with other muscarinic agonists, including xanomeline, resulted in behavioral responses similar to those seen after treatment with traditional antipsychotics in animal models that may be applicable to humans (Shannon et al., 1999). As a substantial proportion of schizophrenic patients do not respond adequately to treatment with currently available medications or suVer from severe side eVects, muscarinic agents may at some point represent a new therapeutic approach for the treatment of schizophrenia and other psychotic disorders.

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IV. Synaptic Plasticity

Cholinergic neurons undergo extensive changes during development (Hohmann et al., 1998). The cholinergic system is relatively late to mature (Nadler et al., 1974) and is subject to large-scale remodeling during postnatal development (Gould et al., 1989). The human brain does not develop in a static fashion but is constantly changing throughout life. In particular, synaptic connections undergo frequent changes that aVect how neurons interact with each other. Synaptic contacts that are in active use are strengthened. Synaptic contacts that are not used regularly are ‘‘pruned’’ (removed). Synaptic plasticity has been used as a phrase to describe these ongoing and essential changes in synaptic connections. Memory and learning depend critically on synaptic plasticity, i.e., the capacity of synapses to change their structure and function to satisfy particular functional or adaptive requirements. Synaptic plasticity is particularly important for the process of storage of long-term memory. Muscarinic cholinergic stimulation promotes the generation of LTP in the hippocampus (Huerta and Lisman, 1993), suggesting that the muscarinic cholinergic system plays a special role in the consolidation of long-term memory. The response of a postsynaptic neuron to stimulation can be altered in the short term as well as over a prolonged period of time. In LTP, the response of the postsynaptic response to stimulation is increased, whereas LTD results in a decrease of the postsynaptic response to stimulation. While the neurotransmitter glutamate plays a major role in both long-term potentiation and long-term depression, other neurotransmitters are also involved in these processes. DiVerent mechanisms are involved in LTP and LTD as part of synaptic plasticity. In terms of increasing synaptic strength, the molecular basis consists of increased dendritic branching, increased axonal collaterals, and the modiﬁcation of existing synapses, as well as the generation of new synapses. In terms of decreasing synaptic strength, synaptic pruning describes the process of removing unused synaptic contacts. On a molecular level, long-term potentiation and synaptic plasticity are mediated by changes in the expression of activity-dependent genes (Bailey et al., 1996). Regarding the muscarinic system, Albrecht et al. (2000) looked at the eVects of muscarinic receptors on the expression of diVerent genes through stimulation with the cholinergic agonist carbachol. Cholinergic stimulation of the muscarinic M1 and M3 receptor subtypes induced the immediate early gene CYR61. This gene induction was blocked by the muscarinic antagonist atropine. Stimulation of the M2 and M4 muscarinic receptor subtypes had little eVect on immediate early genes. In another study from the same group, the stimulation of muscarinic acetylcholine receptors resulted in the activation of diVerent genes, including transcription factors, signalling factors, and the acetylcholine esterase gene (von der Kammer et al., 2001).

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The muscarinic system with its critical role in memory and learning has also been evaluated for its potential role in synaptic plasticity. Several studies have looked at the eVects of muscarinic receptors on LTP and LTD as important parts of synaptic plasticity. Activation of muscarinic M1 receptors plays a crucial role for corticostriatal long-term potentiation (Calabresi et al., 1999). Using diVerent muscarinic and nicotinic agents, Gu and Singer (1993) showed that muscarinic but not nicotinic agents facilitated neuronal plasticity in the visual cortex. Pharmacological stimulation of muscarinic receptors has been associated with the induction of LTP in diVerent brain regions, including the hippocampus (Blitzer et al., 1990), dentate gyrus (Burgard and Sarvey, 1990), visual cortex (Brocher et al., 1992), and piriform gyrus (Patil et al., 1998). Stimulation of muscarinic receptors also results in the lasting potentiation of synaptic transmission in the hippocampus (Ito et al., 1998). Using diVerent cholinergic probes, Auerbach and Segal (1994, 1996) found muscarinic receptors to be involved in long-term depression and long-term potentiation in the hippocampus. Segal and Auerbach (1997) described a long-lasting increase in hippocampal reactivity to aVerent stimulation after the activation of postsynaptic M2 receptors, which they termed muscarinic long-term potentiation. A speciﬁc role of activation of muscarinic M2 receptors in long-term potentiation in the hippocampus was also found by other groups (Kojima et al., 1998). A facilitation of long-term potentiation after the administration of cholinergic agonists was similarly shown in the neocortex, whereas cholinergic antagonists blocked the induction of long-term potentiation (Boyd et al., 2000). Pharmacological blockade of muscarinic receptors prevented long-term potentiation in several other studies (Hess et al., 1999; Hirotsu et al., 1989; Katsuki et al., 1992; Kobayashi et al., 1997; Maalouf et al., 1998). While the aforementioned studies found that muscarinic stimulation led to long-term potentiation, other studies also found long-term depression after muscarinic stimulation. Massey et al. (2001) studied the role of acetylcholine in long-term potentiation and long-term depression in the perirhinal cortex of rats. Activation of cholinergic receptors by carbachol, a muscarinic agonist, produced long-term depression of synaptic transmission. The long-lasting depression depended on the activation of muscarinic M1 receptors, as the application of pirenzepine, a muscarinic M1 antagonist, prevented long-lasting depression. Similar eVects of carbachol-induced long-term depression were found in the visual cortex (Kirkwood et al., 1999). Taken together, these studies suggest that stimulation of the muscarinic system appears to have diVerent eVects on synaptic plasticity in diVerent parts of the brain. Apart from pharmacological studies, other techniques have also been used to assess the eVect of muscarinic neurotransmission on synaptic plasticity. Sawaki et al. (2002) used transcranial magnetic stimulation (TMS) to study the eVects of the muscarinic receptor antagonist scopolamine on excitability and use-dependent plasticity in the human motor system. The magnitude of

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training-induced changes in TMS-evoked movements was signiﬁcantly smaller after pretreatment with scopolamine than after placebo. As scopolamine decreased the magnitude of use-dependent plasticity, the authors concluded that muscarinic stimulation may have a facilitating eVect on synaptic plasticity in the motor system. In another study in humans, scopolamine similarly blocked the expected decrease in the visual threshold associated with light deprivation in the visual cortex (Boroojerdi et al., 2001). In an in vivo study in humans using functional magnetic resonance imaging (MRI), healthy controls were exposed to two diVerent tones while lying in the MRI scanner. One of these two tones was paired to a mildly aversive electric shock. Prior to the scan, subjects were pretreated with either the anticholinergic agent scopolamine or a placebo. In contrast with the placebo, the administration of scopolamine prevented the conditioning-speciﬁc blood oxygen level dependent (BOLD) response as a measure of hemodynamic changes. These results were interpreted to mean that muscarinic cholinergic neurotransmission modulates experience-dependent plasticity in the auditory cortex (Thiel et al., 2002). Further evidence for the role of muscarinic neurotransmission in synaptic plasticity comes from electrophysiological studies. Theta rhythm occurs during times of learning and facilitates the induction of synaptic plasticity, as synapses are in a state of increased plasticity during theta rhythm. Cholinergic agonists are able to induce theta rhythm and thus inﬂuence synaptic plasticity (Huerta and Lisman, 1993, 1996). A similar induction of synaptic plasticity through cholinergic stimulation also occurs in the hippocampus, a region of great importance for memory consolidation (Huerta and Lisman, 1995). Theta rhythm can be altered by the muscarinic antagonist atropine (Buzsaki et al., 1986). At the same time, the burst mode of theta rhythm can be restored with nicotinic antagonists (Cobb et al., 1999). Long-term potentiation in response to theta burst stimulation in the hippocampus was reduced in M1 receptor-deﬁcient mice (Anagnostaras et al., 2003). Findings on synaptic plasticity in schizophrenia have shown conﬂicting results. McGlashan and HoVman (2000) suggested a model of impaired synaptic connectivity arising from early developmental factors in schizophrenia. Alterations in synaptic structure have been found in diVerent brain regions, including basal ganglia (Kung et al., 1998; Uranova et al., 1996). A gene expression study looking at a large amount of genes in the entorhinal cortex found a decrease of synaptic proteins in schizophrenia (Hemby et al., 2002). DiVerent synaptic proteins have been found altered in schizophrenia. NCAM levels are increased in schizophrenia (Vawter et al., 2001). Synaptophysin, a presynaptic protein, was found decreased signiﬁcantly in brain tissue from schizophrenic patients (Honer et al., 1999; Landen et al., 1999; Webster et al., 2001). Synaptophysin tissue levels were reduced in the prefrontal cortex in schizophrenia, whereas mRNA levels were unchanged (Karson et al., 1999). In another study, the levels

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of mRNA for diVerent synaptic proteins were increased in schizophrenic patients (Sokolov et al., 2000). Other studies, however, failed to ﬁnd signiﬁcant changes in synaptic proteins in schizophrenia (Eastwood and Harrison, 2001). Antipsychotics as the main medication used in the treatment of schizophrenia have an eVect on synaptic plasticity (Konradi and Heckers, 2001) that can diVer for diVerent antipsychotics (Gemperle et al., 2003). Studies on the eVects of muscarinic receptors on synaptic plasticity in schizophrenia have not yet been performed. Taken together, these results suggest that activation of the muscarinic receptor system is one of the factors involved in synaptic plasticity. Muscarinic receptors play an important role in learning and memory, two cognitive functions that are crucial in diVerent neuropsychiatric disorders. At the same time, synaptic plasticity is one of the major mechanisms underlying learning and memory. While diVerent studies suggest an alteration of synaptic plasticity in schizophrenia, relatively little is known on the eVects of the muscarinic system on synaptic plasticity in this disorder. Given the central role of learning and memory in psychiatric disorders, further studies of the role of the muscarinic receptor system in synaptic plasticity seem warranted.

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SEROTONIN AND BRAIN DEVELOPMENT

Monsheel S. K. Sodhi and Elaine Sanders-Bush Departments of Pharmacology and Psychiatry Vanderbilt University Nashville, Tennessee 37232

I. Introduction II. The Discovery of Serotonin and Classification of Serotonin Receptors A. Distribution and Projections of the Serotonergic System III. The Role of Serotonin in Developmental Plasticity A. Serotonergic Projections during Brain Development B. Growth Factors Influencing the Development of Serotonergic Neurons C. The Role of Serotonin as a Growth Factor D. Serotonin Receptors and Developmental Plasticity IV. Manipulation of the Serotonergic System Alters Synaptic Plasticity A. Tryptophan and Serotonin Depletion Studies B. Experimental Models of Synaptic Plasticity V. Does Dysfunction of Serotonergic Signaling Result in Impaired Brain Development? A. The Role of Serotonin in Learning and Memory B. Autism and Serotonin C. The Role of Serotonin in Stress and Anxiety D. Serotonergic Influences on Synaptic Plasticity in AVective Disorders E. Altered Synaptic Plasticity in Schizophrenia F. Down’s Syndrome, Mental Retardation, and Serotonin VI. Conclusions References

The role of the serotonergic system in the neuroplastic events that create, repair, and degenerate the brain has been explored. Synaptic plasticity occurs throughout life and is critical during brain development. Evidence from biochemical, pharmacological, and clinical studies demonstrates the huge importance of an intact serotonergic system for normal central nervous system (CNS) function. Serotonin acts as a growth factor during embryogenesis, and serotonin receptor activity forms a crucial part of the cascade of events leading to changes in brain structure. The serotonergic system interacts with brain-derived neurotrophic factor (BDNF), S100, and other chemical messengers, in addition to its cross talk with the GABAergic, glutamatergic, and dopaminergic neurotransmitter systems. Disruption of these processes may contribute to CNS disorders that have been associated with impaired development. Furthermore, many psychiatric drugs alter serotonergic activity and have been shown to create changes in brain structure with long-term treatment. However, the mechanisms for their INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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therapeutic eYcacy are still unclear. Treatments for psychiatric illness are usually chronic and alleviate psychiatric symptoms, rather than cure these diseases. Therefore, greater exploration of the serotonin system during brain development and growth could lead to real progress in the discovery of treatments for mental disorders.

I. Introduction

Serotonin (5-hydroxytryptamine, 5-HT), the ‘‘happy hormone,’’ has a phylogenetically ancient role in neural transmission (Turlejski, 1996). Because the serotonergic system has a widespread distribution in the CNS, it inﬂuences almost every sphere of mammalian physiology, from cardiovascular regulation (Miyata et al., 2000; Nebigil et al., 2000; Thorin et al., 1990), respiration, the gastrointestinal system (Kato et al., 1999), pain sensitivity, and thermoregulation to more centrally controlled functions. The latter include the maintenance of circadian rhythm, appetite, aggression, sensorimotor activity, sexual behavior, mood, cognition, learning, and memory. Hence drugs with serotonergic activity are used to treat the aVective disorders schizophrenia (AbiDargham et al., 1996; Breier, 1995; Kapur and Remington, 1996; Meltzer, 2002; Ohuoha et al., 1993; Sodhi and Murray, 1997), anxiety (Gross et al., 2002), stress, eating disorders (Bray, 2000; GuyGrand, 1995; Halford, 2001; Heal et al., 1998; Heisler et al., 1998b; Hesselink and Sambunaris, 1995; Jallon and Picard, 2001; Koponen et al., 2002; Luque and Rey, 1999; McNeely and Goa, 1998; Prasad, 1998; Weissman, 2001), and deliberate self-harm (Holden, 1995). In addition, personality dysfunctions such as addictive behaviors, aggression, psychopathic and sociopathic behavior, attention-deﬁcit hyperactivity, and autism are also associated with altered serotonergic transmission. Indeed, new serotonin receptor ligands are being explored as possible treatments for Alzheimer’s disease, as they appear to improve memory (Sumiyoshi et al., 2001), obesity (Bray, 2000; Rothman and Baumann, 2002; Stunkard and Allison, 2003; Wechsler, 1998), and epilepsy (Chadwick et al., 1977; Chugani and Chugani, 2003; Dailey et al., 1992; Deahl and Trimble, 1991; Fromm et al., 1977; Heisler et al., 1998b; Lunardi et al., 1995; Monaco et al., 1995; Savic et al., 2001; Statnick et al., 1996; Yan et al., 1994). Although increasing knowledge of serotonergic function is propelling many advances in the therapeutics of psychiatric and behavioral disorders, drugs in clinical use often treat the disease symptoms instead of relieving or preventing the causes. Moreover, treatment regimes are often lengthy or lifelong, sometimes with severe side eVects. As yet the causes of psychiatric disease are unknown, therefore the role of serotonin in the etiology or progression of these disorders

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requires exploration in order to facilitate improvements in medication and prognoses. There is increasing support for the hypothesis that impaired development and synaptic plasticity contribute to the etiologies of many central nervous system (CNS) diseases. Plasticity is deﬁned as functionally relevant structural adaptations performed by the CNS following genetic or environmental challenges. Neuronal plasticity is essential for the survival of an individual in a constantly changing environment. It is a dynamic process based on the ability of neuronal systems, brain nuclei synapses, single nerve cells, and receptors to adapt to challenges. Plasticity reveals itself in a number of ways, which range from altered gene expression or changes in neurotransmitter release to changes in behavior or phenotype. Synaptic plasticity is constant throughout life and is especially important during development. Connections between neurons of the CNS are capable of being dismantled and reconstructed in response to changes in the physiological environment, therefore stress, malnutrition, sleep, hormones, and drugs can all produce changes in brain structure. Accumulating research suggests that serotonin plays an important role in synaptic plasticity and brain development. In this review we attempt to explore this evidence and its implications for impaired brain development and psychiatric illness.

II. The Discovery of Serotonin and Classification of Serotonin Receptors

The chemical 5-hydroxytryptamine (5-HT) was ﬁrst isolated in serum and, because of its powerful vasoconstrictive eVects, was dubbed ‘‘serotonin’’ (Rapport, 1948). Serotonin was later detected in the brain (Twarog and Page, 1953). In 1957, Gaddum and Picarelli reported the existence of multiple serotonin receptor subtypes, which they called 5-HT-M and 5-HT-D, after their antagonists, morphine and dibenzyline, respectively. Peroutka and Snyder (1979) reclassiﬁed these receptors based on radioligand-binding studies in brain homogenates. The 5-HT1 receptor was labeled by [3H]5-HT, whereas the 5-HT2 receptor (corresponding to 5-HT-D) was sensitive to the dopamine receptor ligand [3H]spiperone. By 1986, the M receptors were renamed 5-HT3 receptors (Bradley, 1986), were found to be the only ionotropic subtype of the 5-HT receptor, and were detected to be at low density in limbic and striatal areas (Abi-Dargham et al., 1993). [3H]Lysergic acid diethylamide (LSD), a psychotomimetic compound with a structure similar to serotonin, was found to have high aYnity for serotonin receptors. Subsequently, heterogeneity was revealed in the 5-HT1 receptor class; 5-HT1A receptors could be distinguished from 5-HT1B receptors (equivalent to the human 5-HT1D receptor) by the high aYnity of the former for spiperone (Pedigo et al., 1981). The use of receptor autoradiographic

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techniques demonstrated the existence of a third 5-HT1 receptor in the porcine choroid plexus, the 5-HT1C subtype (later renamed 5-HT2C), through its high aYnity for [3H]mesulergine and [3H]5-HT (Pazos et al., 1984). The application of molecular cloning techniques in the late 1980s revolutionized protein discovery and to date, 14 distinct subtypes of mammalian serotonin receptors have been cloned. Both molecular structure and pharmacological properties determine their classiﬁcation, and under a revised system, the serotonin receptors are now allocated to seven distinct families (Hoyer et al., 1994). The characteristics and distributions of the diVerent subtypes of serotonin receptors are summarized in Table I. A detailed review of their pharmacology has been compiled by Barnes and Sharp (1999).

A. Distribution and Projections of the Serotonergic System Serotonergic neurons are part of one of the most widely distributed neuronal systems in the mammalian brain; this neuronal network is also one of the earliest to develop in the embryo. Serotonin-containing neurons projecting to the forebrain originate in four brain stem nuclei, the principal of these being median and dorsal raphe nuclei. The dorsal raphe nucleus projects thin serotonin ﬁbers, which are more abundant in the cortex, whereas the median raphe nucleus provides thick serotonin ﬁbers with large varicosities that are relatively sparse and more abundant in the hippocampus (Kosofsky and Molliver, 1987). The hippocampus receives ﬁbers following a dorsomedial course through the cingulate cortex (reviewed by Rubenstein, 1998). During development of the cortex, the cortical regions have diVerent proﬁles for the maturation of serotonin axon terminals (D’Amato et al., 1987; DeFelipe et al., 1991). The thick and thin serotonin ﬁber systems innervate the cortex in diVerent time frames (Vu and Tork, 1992). Because the ﬁbers also change after injury and according to the target cell innervated, classiﬁcation according to the thickness of the ﬁbers is not consistent (Azmitia, 1999). Serotonin neurons innervate almost all areas of the brain ( Jacobs and Azmitia, 1992), and their projections and targets are summarized in Fig. 1.

III. The Role of Serotonin in Developmental Plasticity

A central tenet of neuroscience is that synaptic plasticity underlies behavioral plasticity and that information is coded by alterations in synaptic strength and connectivity in networks of neurons (Kandel and Spencer, 1968; Martin et al., 2000). Prior to its vital role as a neurotransmitter in adult brain, serotonin acts as a regulator of brain development. The latter is inextricably linked to the

TABLE I The Serotonin Receptorsa

Receptor The 5-HT1 family 5-HT1A

Structure (genetic locus)

7 TMD, intronless (5q11.2-q13)

Second messenger

G-protein-coupled # adenylate cyclase

Principal distribution

Hippocampus, lateral septum, cingulate and entorrhinal cortices, dorsal and median raphe nuclei

115 5-HT1B 7 TMD, intronless (previously (6q13) 5-HT1D) 5-HT1D 7 TMD, intronless (previously (1p34.3-36.3) 5-HT1D´)

5-HT1E

7 TMD, intronless (6q14-15)

Basal ganglia, especially substantia nigra, globus pallidus, ventral pallidum, and entopeduncular nucleus G-protein-coupled # Basal ganglia, especially adenylate cyclase substantia nigra, globus pallidus, caudate putamen, periaquaductal grey, hippocampus, cortex, olfactory cortex, dorsal raphe nucleus, locus coeruleus, and spinal cord Cortex including entorrhinal G-protein-coupled # adenylate cortex, basal ganglia, claustrum, hippocampus cyclase (subiculum), amygdale, hypothalamus G-protein-coupled # adenylate cyclase

Effect of receptor activation

# 5-HT release " ACh release in septohippocampal neurons " NE release in hypothalamus, and hippocampus, frontal cortex VTA # Glutamate release " Prolactin release, " growth hormone, " ACTH release # 5-HT release # ACh release

Clinical effects of receptor activation

Anxiolytic and antidepressant, e.g., buspirone, pindolol, 5-HT syndrome, hypothermia, hyperphagia, altered sexual behavior

Hypophagia, hypothermia, myoclonic jerks, antiaggressive properties, penile erection

" GABA release? # 5-HT release # ACh release?

(Continued )

TABLE I (Continued ) Receptor

5-HT1F

The 5-HT2 family 5-HT2A

Structure (genetic locus)

7 TMD, intronless (3q1)

Second messenger

G-protein-coupled # adenylate cyclase

Principal distribution

Cortex, especially cingulate and entorrhinal cortices, hippocampus (CA1-CA3 layers), basal ganglia, claustrum, caudate nucleus, dorsal raphe nucleus

Effect of receptor activation

Clinical effects of receptor activation

Relief of migraine?

116

G-protein-coupled Olfactory bulb, hippocampus, Antipsychotic (antagonist), Excitation of 5-HT neurons " phospholipase C cortex (neocortex, claustrum, Inhibition of NE neurons anxiolytic (antagonist), relief pyriform and entorhinal cortices), in locus coeruleus of sleep and eating disorders, caudate nucleus, and nucleus relief of migraine, # NE release in hippocampus accumbens hallucinogenic, hyperthermia " BDNF expression? " cortisol, " ACTH, " renin, " prolactin Mediation of mitogenic effects Anxiolysis (antagonist)? G-protein-coupled Cerebellum, lateral septum, 5-HT2B 7 TMD, " phospholipase C dorsal hippocampus and of 5-HT during neural 2 introns within medial amygdala, stomach development? ORF (2q36.3-37.1) fundus, heart Excitation of 5-HT neurons Antipsychotic (antagonist), 5-HT2C 7 TMD, 3 introns G-protein-coupled Choroid plexusolfactory nucleus, # NE and DA release in anxiolytic (antagonist), within ORF " phospholipase C pyriform, cingulate and (previously mesocortical/mesolimbic NE and antidepressant, relief of retrospinal cortices, nucleus (Xq24) 5-HT1C) migraine and sleep DA projections " cortisol, accumbens, hippocampus, " ACTH, " prolactin disorders, hypolocomotion, amygdala, subiculum, hypophagia, penile entorhinal cortex, caudate erection, anticonvulsant nucleus, substantia nigra pars compacta, striatum, thalamus, hypothalamus, frontal cortex 7 TMD, 2 introns within ORF (13q14-21)

5-HT3

5-HT4

Ligand-gated cation channel, several introns (11q23.1-23.2)

7 TMD, >5 introns (5q31-33)

" intracellular Na+:K+ ratio

Na+:K+ ratio G-protein-coupled " adenylate cyclase

Hippocampus, amygdale, superficial layers of cerebral cortex, limbic and basal ganglia structures, dorsal vagal complex in brain stem, gastrointestinal tract

Fast synaptic transmission in brain " 5-HT release # ACh release " GABA release " CCK release " DA release

Vomiting, relief of migraine (antagonist), anxiolysis (antagonist), " cognition (antagonist), # locomotion (antagonist), # reward (antagonist), # LTP, analgesia (antagonist)?

Hippocampus, basal ganglia and substantia nigra, atrium, and gastrointestinal tract

" 5-HT release

" cognition, anxiolysis and anxiogenesis (antagonist)

" ACh release " DA release 5-HT5A

5-HT5B

117

7 TMD, 1 intron (7q36.1) 7 TMD, 1 intron (2q11-13)

5-HT6

7 TMD, 2 introns (1p36-35)

5-HT7

7 TMD, 2 introns (10q21-24)

G-protein-coupled Cortex, hippocampus, thalamus, # adenylate cyclase olfactory bulb, hypothalamus, cerebellum, spinal cord G-protein-coupled Hippocampus, medial and lateral # adenylate cyclase habenula, dorsal raphe nucleus, olfactory bulb, entorrhinal and pyriform cortices G-protein-coupled Caudate nucleus, olfactory tubercles, " adenylate cyclase nucleus accumbens, hippocampus, cerebral cortex, striatum, stomach, adrenal glands G-protein-coupled Thalamus, hypothalamus, " adenylate cyclase hippocampus, cerebral cortex, amygdala

" locomotor activity

Antipsychotic (antagonist), antidepressant (antagonist), anxiolysis (antagonist) Antipsychotic (antagonist), antidepressant (antagonist), anticonvulsant (antagonist)

a Reviewed by Barnes and Sharpe (1999) and Crossland (2000). TMD, transmembrane domain; ORF, open reading frame; VTA, ventral tegmental area; NE, norepinepherine/noradrenaline; ACh, acetylcholine; GABA, -aminobutyric acid; CCK, cholescystokinin; DA, dopamine.

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Fig. 1. Serotonergic projections in the human brain, arising from the raphe nuclei. The serotonergic projections innervate sympathetic preganglionic neurons, the sensory glomeruli in olfactory bulb, the intermediate lobe of pituitary, the epithelial cells of choroid plexus, the lateral ventricles, the motor neurons of brainstem, the spinal cord, visual cortex and all regions of cerebral cortex. Other transmitter systems make specialized contacts with serotonergic targets: the dopaminergic neurons in substantia nigra, the noradrenergic neurons in locus coeruleus, pacemaker neurons of the suprachiasmatic nucleus, specialized calbindin GABA interneurons in hippocampus, and pyramidal cortical neurons. Cell types in close proximity to the serotonin fibers include glia, endothelial cells, eppendymal cells in addition to the pineal gland and subcommissural organ.

processes of long-term potentiation (LTP, described in Section V,A) and synaptic plasticity. The immature brain is thought to overdevelop, producing excess connections and cells. The redundant inputs are pruned according to their activity levels by apoptotic mechanisms guided by existing chemical systems. Therefore the ‘‘use it or lose it’’ principle states that unused connections are removed. Because serotonin is likely to be present earlier in development than other monoamine transmitter systems and because the turnover rate of serotonin is higher in the immature mammalian brain than at any other period (Hamon and Bourgoin, 1979), serotonin probably plays a key role in this developmental process (reviewed by Whitaker-Azmitia, 2001).

A. Serotonergic Projections during Brain Development The highest levels of serotonergic activity are detected early in development (Lidov and Molliver, 1982). Serotonergic neurons form a superior and inferior group of immature cells, which have distinct maturational and migration patterns

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(Lidov and Molliver, 1982; Wallace and Lauder, 1983). In both groups, serotonergic neurons form diVerent subsets of cells, and the raphe nuclei from which serotonergic ﬁbers project in the brain all have diVerent origins (Azmitia and Gannon, 1986). In the human, serotonergic neurons can be detected when the embryo is just 5 weeks old (Sundstrom et al., 1993), with rapid growth and multiplication until the 10th week of gestation (Kontur et al., 1993; Levallois et al., 1997; Shen et al., 1989). After 15 weeks, clustering of the serotonin cell bodies in the raphe nuclei is observed. The synaptic density of biogenic amine systems in the human cerebral cortex doubles from birth to 1 year old, when it reaches a peak value and decreases thereafter to adult levels by the age of 10 (Huttenlocher and Dabholkar, 1997). Similarly, levels of serotonin increase during the ﬁrst 2 years after birth and then decline to adult levels after the age of 5 (Hedner et al., 1986; Toth and Fekete, 1986). An equivalent time course for the development of the serotonergic system has been observed in the chicken (Kojima et al., 1988) and rodents (Rubenstein, 1998) indicating that these changes are conserved in evolution. The early arrival of the serotonergic system before other monoamines indicates that it may be required to guide the development of other neurotransmitter systems (Benes et al., 2000; Whitaker-Azmitia et al., 1996). The role of serotonin as a developmental signal is discussed in Section III,C. In the rat, axonal projections from the rostral raphe nuclei ascend to the midbrain and forebrain, whereas those of the caudal nuclei descend to the spinal cord (Wallace and Lauder, 1983). The descending ﬁbers enter the spinal cord by embryonic day 14 (E14) and innervate the preganglionic sympathetic neurons and somatic motor neurons. Here they start to form synapses at E17, with innervation of dorsal horn neurons occurring later. The rostral projections are visible soon after serotonin can be detected in the brain stem. The unbranched ﬁbers grow in the marginal zone as a fascicle in the median forebrain bundle, and by E15 they reach the diencephalon, where they branch out. By E17, medial ﬁbers from the medial forebrain bundle project to the frontal pole of the telencephalon, while lateral ﬁbers project to the hypothalamus and arrive at the rostral end of the brain, some crossing the midline in the supraoptic commissure. There is a simultaneous entry by serotonin ﬁbers into the telencephalon, the majority passing through the diagonal bands of Broca, the septal areas, and then projecting into the cerebral cortex. A small number of serotonin ﬁbers enter through the ganglionic eminences (Rubenstein, 1998). Interestingly, after birth there are nonraphe thalamocortical ﬁbers containing serotonin in the sensory neocortex. Because there is no serotonin synthesis within these neurons, serotonin must be transported to neighboring neurons from adjacent synapses (Lebrand et al., 1996), suggesting that all cells found to contain serotonin may not necessarily manufacture it. This may indicate a mechanism by which serotonin can act as a developmental signal in nonserotonergic cells (discussed in Section III,C).

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B. Growth Factors Influencing the Development of Serotonergic Neurons Several growth factors that inﬂuence serotonergic development are also important in synaptic plasticity events. These include the astroglial growth factor, S100, levels of which are increased by serotonin, indicating that serotonergic nerves can stimulate their own growth. Inhibition occurs via serotonergic receptors at the nerve terminals. Serotonin not only regulates the growth and development of its targets, but it is also autoregulatory (Whitaker-Azmitia, 2001). S100 is described in greater detail in the review by Rothermundt, Ponath, and Arolt. The serotonin system regulates its own diVerentiation by sequential activation of the 5-HT1A receptor, brain-derived neurotrophic factor and its receptor trkB (see the review by Guillin and colleagues), CREB, CREM, and ATF-1, among many of the signaling molecules under serotonergic inﬂuence (Herdegen and Leah, 1998). In addition, Petl, an ETS domain transcription factor, is closely associated with developing raphe serotonergic neurons. Other neurotrophins and growth factors control the diVerentiation of serotonergic neurons, including bone morphogenetic protein, and ciliary neurotrophic factor. Furthermore, consensus binding motifs for Petl have been detected in the 50 -untranslated regions of the human and mouse genes for the 5-HT1A receptor, serotonin transporter, aromatic l-amino acid decarboxylase (dopa decarboxylase), and tryptophan hydroxylase. These play important roles in the biosynthesis and degradation of serotonin from dietary tryptophan, as illustrated in Fig. 2. It follows that Petl may also be critical for the regulation of serotonin levels (Hendricks et al., 1999), which have profound eVects on brain development.

C. The Role of Serotonin as a Growth Factor The dietary precursor for serotonin, tryptophan, is found across the phylogenetic spectrum from lower plants to higher mammals. It is therefore an evolutionarily ancient chemical. Its chemical structure is shown in Fig. 2. Because tryptophan possesses an indole ring, it is capable of absorbing light and is vitally important for energy production during plant photosynthesis. Tryptophan can also be converted to auxin, which is a plant tropic factor, guiding plant growth and cell diVerentiation (reviewed by Azmitia, 2001). In animals, tryptophan is also the precursor for melatonin, a light-sensitive amine vital for the control of circadian rhythm. It follows that if auxin is a plant growth factor, then derivatives of tryptophan in animals could have similar functions. Serotonin alters the morphology of many mammalian cell types, including skeletal muscle (O’Steen et al., 1967), platelets (Leven et al., 1983), and ﬁbroblasts (Boswell et al., 1992). Once serotonergic terminals have developed in a target

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Fig. 2. A diagram summarizing the proposed signal transduction pathways of 5-HT2 receptor subtypes. These receptors belong to the superfamily of G-protein-coupled receptors and are specifically linked to the Gq protein, which activates phopholipase C (PLC) when stimulated by the receptor-agonist complex. This initiates the phosphoinositol second messenger cascade, producing inositol triphosphate (IP3) and diacylglycerol (DAG), which stimulate the release of calcium from intracellular stores and the activation of protein kinase C (PKC). Receptors with similar activity include the cholinergic muscarinic, metabotropic glutamate, and 2-adrenergic receptors. The 5-HT2C and M1 receptors may play a major role in the regulation of synaptic plasticity, since both receptors increase the mobilization of calcium ions (Ca2þ) from intracellular stores via PLC activation, which then activates Ca2þ/calmodulin, which opens L-type Ca2þ channels, leading to plastic events. (See Color Insert.)

region, serotonin release may inﬂuence neurogenesis (Lauder and Krebs, 1976, 1978), neuronal removal through apoptotic mechanisms, dendritic reﬁnement, cell migration, and synaptic plasticity (Chubakov et al., 1986, 1993; Lauder, 1990; Lauder et al., 1981). When these events are combined, they could produce the highly sophisticated organization of the hippocampus or the somatosensory maps called ‘‘barrel ﬁelds’’ (discussed in Section III,E,2). At a later developmental stage, serotonin directs dendritic growth. Serotonin activity alters the overall length of dendrites, the formation of dendritic spines, and branches into the hippocampus and cortex (Faber and Haring, 1999; Mazer et al., 1997; Okado et al., 1993; Wilson et al., 1998; Yan et al., 1997a). As the animal matures,

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Fig. 3. L-tryptophan is a dietary precursor of brain serotonin. Serotonin synthesis occurs as summarized above in the postganglionic serotonergic neurons.

increased serotonin levels inhibit normal spine formation, perhaps by a 5-HT1A receptor-induced mechanism indicated in Fig. 3 (discussed in Section III,D,1). Depletion of serotonin levels during the developmental years may cause a loss of synapses, which can be corrected by adulthood. However, in the aged animal this may lead to an increase in the number of synapses, a ‘‘reactive synaptogenesis,’’ or perhaps a retention of synapses normally lost (Whitaker-Azmitia, 2001). Therefore, early developmental disturbance has the potential to alter brain structure and repair much later in life.

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Studies of neurotoxin-induced serotonergic lesions of dorsal and medial raphe nuclei have detected changes in cell growth in basal ganglia regions and hypothalamic nuclei of adult rats. Newly generated granule cells can be identiﬁed by immunostaining for bromo-20 -deoxyuridine (BrdU) and the polysialylated form of neural cell adhesion molecule (PSA-NCAM). Decreases in PSA-NCAM staining have been observed after the inhibition of serotonin synthesis induced by parachlorophenylalanine (PCPA) administration, suggesting that serotonin may reduce adhesion by acting on PSA-NCAM expression in its environment and thus facilitate plasticity in adult brain. A normalization of PSA-NCAM staining two months after neurotoxin lesions has been associated with a partial restoration in serotonin ﬁber density in the nucleus accumbens and the supraoptic nucleus, indicating that PSA-NCAM may facilitate the sprouting of serotonin ﬁbers. As no changes in striatal PSA-NCAM staining have been observed after selective lesions of the dopaminergic pathway, serotonin appears to have a selective and critical role in adult brain plasticity (Brezun and Daszuta, 1999). Prenatal depletion of serotonin delays the onset of neurogenesis in serotonergic target regions. In the fetus evidence suggests that serotonin promotes the diVerentiation of cortical and hippocampal neurons. In the adult brain, studies indicate that serotonin may play a role in neuronal plasticity by maintaining the synaptic connections in the cortex and hippocampus (Azmitia et al., 1995; Chen et al., 1994; Mazer et al., 1997). Neuronal precursor cells persist in adulthood in two discrete regions, the subventricular zone and the hippocampal subgranular zone, as demonstrated in primates (Gould et al., 1998). Both inhibition of serotonin synthesis and selective lesions of serotonergic neurons are associated with decreases in the number of newly generated cells in the dentate gyrus, as well as in the subventricular zone (Brezun and Daszuta, 1999). Serotonin depletion studies are discussed in more detail in Section IV,A. In the periphery, serotonin has been shown to potentiate the mitogenic eVects of platelet-derived growth factor BB (Eddahibi et al., 1999). The mechanism for these eVects on morphology and cell proliferation could involve the phosphorylation of guanidine triphosphatase-activating protein, which is required for signal in smooth muscle cell mitogenesis induced by serotonin (Lee et al., 1997). Serotonergic activity also targets the cytoskeleton by inducing actin polymerization in addition to the regulation and maintenance of microtubules and microﬁlaments. The mechanism by which serotonin inﬂuences this growth is thought to involve the expression of S100, a neurotrophic factor derived from astroglial cells. S100 is released from glial cells after the activation of glial 5-HT1A receptors. S100 activity increases synaptic stability and promotes neuronal development (reviewed in the chapter by Rothermundt and colleagues). Interestingly, expression of the microtubule-associated protein tau is decreased in undiVerentiated neuroblastoma cells by high levels of serotonin, whereas low serotonin levels increased tau expression ( John et al., 1991). Alterations in tau phosphorylation

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induced by serotonin have been linked with the neuropathology of Alzheimer’s disease and other forms of neurodegeneration (Gudelsky and Yamamoto, 2003; Doraiswamy, 2003; Kovacs et al., 2003; Satoh et al., 1992; Yang and Schmitt, 2001).

D. Serotonin Receptors and Developmental Plasticity Several studies have demonstrated ﬂuctuations in the expression of serotonin receptor subtypes during brain development. In early developmental stages of the neocortex, the transient overexpression of these receptor subtypes has been demonstrated by autoradiography (Bar-Peled et al., 1991a; Dyck and Cynader, 1993a; Leslie et al., 1992; Lidow and Rakic, 1992). As outlined previously in Section III,C, accumulating data suggest that serotonin acts as a developmental signal for brain stem serotonin neurons and their target cells through the activation of serotonin receptors. Serotonin may act presynaptically by generating cAMP, which modulates hyperpolarization-activated cation channels (Ih channels) in axons. Compounds increasing intracellular cyclic adenosine monophosphate (cAMP) mimic and potentiate serotonin action (Dixon and Atwood, 1989; Enyeart, 1981), whereas adenyl cyclase inhibition reduces the serotonergic enhancement of synapse transmission (Dixon and Atwood, 1989). This modulaton increases synaptic strength and could be a mechanism by which serotonin regulates synaptic plasticity (Beaumont and Zucker, 2000). Table I summarizes the distribution and activity of the serotonin receptors, whereas Fig. 4 outlines the adaptive eVects of environmental challenges mediated by serotonin. The involvement of speciﬁc serotonin receptors in synaptic plasticity events is outlined next. 1. The Influence of the 5-HT1A Receptor on Synaptic Plasticity Evidence suggests that the 5-HT1A receptor is present early in development and is involved in the regulation of serotonergic system development (Bar-Peled et al., 1991a; Hillion et al., 1993). Experiments on pregnant rats have demonstrated that in utero exposure to PCPA, which depletes serotonin in rat brain (Lauder et al., 1985; Sanders-Bush et al., 1972a,b, 1974), reduces the postnatal expression of 5-HT1A receptors, suggesting that the system is autoregulatory (Lauder et al., 2000). 5-HT1A receptors are likely to be functional before birth because they can be up- or downregulated in fetal brain (Lauder et al., 2000; Whitaker-Azmitia et al., 1990). In human fetal brain, 5-HT1A mRNA expression peaks between 16 and 22 weeks (Bar-Peled et al., 1991a). Investigations in several brain regions demonstrate that the highest level of 5-HT1A mRNA expression occurs during the development of that region and declines after maturation has occurred. This phenomenon has been reported in the brain stem (Hillion et al.,

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1993), cerebellum (Daval et al., 1987), and visual cortex (Dyck and Cynader, 1993a). The 5-HT1A receptor is expressed at high density in the dentate gyrus. Evidence indicates that this receptor is involved in the development of dentate granule cells, and it is probable that it also plays a role in maintaining synaptic integrity in the adult. The 5-HT1A receptor has also been detected on glial cells, and its activation reduces the immunoreactivity of S100 in the dentate area of adult rats, indicating that loss of synapses is due to less neurotrophic activity. In a study performed by Wilson et al. (1998), decreased serotonin in the hippocampus produced a reduction of synaptic density in the dentate molecular layer, which was attributed to lower levels of S100 reductions by astrocytic 5-HT1A receptors (Wilson et al., 1998). In addition to eVects in the hippocampus, amygdala, and cortex, the serotonergic raphe neurons are thought to provide important modulatory eVects on motor output systems. Developmental increases in serotonergic innervation of the rat hypoglossal nucleus, which coincide with decreased 5-HT1A receptor expression by hypoglossal motor neurons (HMs), have been detected. After-spike hyperpolarization inhibition by serotonin on neonatal HMs is lost in juvenile HMs, probably due to 5-HT1A receptor activity. Therefore, 5-HT1A receptor regulation of neonatal HM function is lost in the adult (Bayliss et al., 1997). HMs contribute to innervation of the tongue muscles and to the maintenance of upper airway patency during respiration, which is important in the prevention of obstructive apneas during REM sleep (Remmers, 1990). 2. The Role of 5-HT2 Receptors in the Modulation of Plasticity In addition to the strong evidence supporting a developmental role for 5-HT1A receptors, similar data have been unearthed in investigations of 5-HT2 receptor subtypes. As with 5-HT1A receptor, the activity of 5-HT2 receptors is increased at critical stages of brain development. When 5-HT2R-mediated inositol phosphate levels have been measured, they are approximately 10-fold higher in developing brain compared with mature adult brain (Claustre et al., 1988). Another similarity with 5-HT1A receptor is that pre- or postnatal environmental stressors acting via the glutamatergic system during development can alter the number and function of 5-HT2 receptors permanently (Aghajanian and Marek, 1999; Meaney et al., 1994; Peters, 1988). Furthermore, pharmacological studies by Niitsu and colleagues (1995) suggest that 5-HT2A receptor activity regulates synaptogenesis in the embryo. In the mouse, serotonin modulates embryogenesis in a dose-dependent manner by the activation of 5-HT2 receptors (Choi et al., 1997, 1998; Moiseiwitsch and Lauder, 1995, 1997; Moiseiwitsch et al., 1998; Shuey et al., 1992, 1993; Yavarone et al., 1993). Lauder and colleagues (2000) investigated expression patterns of 5-HT2 receptors during mouse embryogenesis. DiVerential

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and overlapping spatiotemporal patterns of 5-HT2A, 5-HT2B, and 5-HT2C receptor immunoreactivity were observed during active phases of morphogenesis in a variety of embryonic tissues, including neuroepithelia of brain and spinal cord, notochord, somites, cranial neural crest, craniofacial mesenchyme and epithelia, heart myocardium and endocardial cushions, tooth germs, whisker follicles, cartilage, and striated muscle. Exposure of mouse embryos at the head fold stage to selective 5-HT2 receptor antagonists revealed potent developmental eVects. The most pronounced eVect was observed after the administration of ritanserin, which has high aYnity for all the 5-HT2 receptors, especially the 5-HT2B receptor subtype, and produced 100% malformed embryos. The 5-HT2A/2C receptor antagonist mianserin was 10-fold less potent, whereas ketanserin, which primarily targets 5-HT2A receptor, did not cause a signiﬁcant number of malformed embroys at any dose tested (Lambert and Lauder, 1999; Lauder, 2000). These data support previous evidence that serotonin acts as an important morphoregulatory signal during embryogenesis. As indicated in Table I and Fig. 4, 5-HT2A receptor and 5-HT2C receptor couple to the phosphoinositide (PI) hydrolysis signal transduction pathway. In 1995, Ike and colleagues demonstrated that during development a switch occurs in the functional 5-HT receptor in the rat hippocampus, from 5-HT2A to 5-HT2C, between the ﬁrst and third weeks of life. 5-HT2A receptor antagonists blocked serotonin-induced PI hydrolysis in the hippocampus of 7-day-old rats

Fig. 4. Schematic diagram illustrating the eVects of stress and synaptic plasticity on behavior. This scheme focuses on the role of serotonin. Excitatory amino acids, glucocorticoids, and brainderived neurotrophic factor (BDNF) are the topics of other articles in this issue. (See Color Insert.)

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but not in 21-day-old rats. In contrast, nonselective 5-HT2A/2C receptor antagonists blocked serotonin-mediated PI hydrolysis in both 7- and 21-day-old rats. These data support the idea that the serotonin-induced PI hydrolysis signalling in the hippocampus of 7-day-old rats is mediated predominantly by 5-HT2A receptor, but in 21-day-old rats the PI hydrolysis signal is mediated mainly by 5-HT2C receptor. Because there was no concurrent change in the gene expression, developmental changes in receptor density were ruled out as a mechanism for the observed diVerences, but instead the explanation probably lies in altered receptor activation. Therefore, 5-HT2A receptor predominates in neonatal hippocampus, whereas 5-HT2C receptor prevails as development progresses (Ike et al., 1995). 5-HT2A receptor promotes neuronal ﬁring by potentiating the activity of AMPA receptors and also by increasing intracellular calcium levels (Fig. 4), which increase the metabolic activity of the cell. There is enhanced glucogenesis, promoting cell proliferation. During development, 5-HT2A receptor has been localized to the neural folds, where intense cell growth occurs. Increased intracellular calcium concentration due to 5-HT2A receptor activation also stimulates protein kinase C (PKC) and increases the expression of several transcription factors. These include the immediate early gene, c-fos, and it is also coupled to the Jak/STAT pathway. The latter regulates the expression of myogenic genes and the glucose transporter GLUT3 in cultured skeletal myoblasts (Broydell et al., 1997). 5-HT2A receptors and 5-HT2C receptors also mediate the mitogenic eVects for serotonin when expressed at high density in ﬁbroblasts ( Julius et al., 1989, 1990). Therefore, the activation of 5-HT2 receptors inﬂuences apoptosis and cell growth and perhaps apoptotic mechanisms. 5-HT2A receptor has also been shown to inﬂuence postnatal development in the prefrontal cortex (PFC). This region of the brain comprises a large portion of the frontal lobe and undergoes progressive growth in mammals, reaching its greatest development in humans (Rakic and Goldman-Rakic, 1982). Functional studies have demonstrated that PFC plays a key role in cognitive functions (Fuster, 1991; Goldman-Rakic, 1987, 1995) and that PFC damage causes deﬁcits in memory (Kolb, 1984) and in the organization of future events (Fuster, 1985). Dysfunction of the PFC is thought to inﬂuence many CNS disorders, particularly in schizophrenia (discussed in Section V,E), as deﬁciencies in cognition and working memory are prominent symptoms in the disease (Goldman-Rakic, 1994; Weinberger and Berman, 1996). A critical developmental period for the neocortex in rodents occurs during the ﬁrst 2 postnatal weeks, which is also a period of intense synaptogenesis, as synaptic density increases ﬁvefold between P10 and P15 when it is almost at the level of adult brain (Micheva and Beaulieu, 1996). Neuronal activity plays a critical role during this time, and disruption during development produces enduring changes in cortical circuitry. The strong excitatory eVect produced by serotonin during this period is modulated mainly by 5-HT2A receptors and perhaps 5-HT7 receptors (Zhang, 2003).

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Cytoarchitecture is also altered in the presence of 5-HT2A receptor antagonists by modulating expression of the activity-regulated, cytoskeleton-associated protein (ARC). ARC is an eVector immediate early gene localized mainly in neuronal dendrites. Elevation of brain serotonin has been shown to increase the abundance of ARC mRNA abundance in the cingulate, orbital, frontal, and parietal cortices, as well as in the striatum, but conversely, produces a reduction in the CA1 region of the hippocampus. Pretreatment with a 5-HT2A receptor antagonist signiﬁcantly attenuates this eVect in the cortex. However, this attenuation is only partial and is absent in the CA1 region (Pei et al., 2000). Furthermore, administration of a single dose of lysergic acid diethylamide (LSD), which is a hallucinogen and 5-HT2 receptor agonist, has been shown to increase ARC expression in the cortex (Nichols and Sanders-Bush, 2002). The 5-HT2 receptor agonist DOI also stimulate a dose-dependent increase in ARC mRNA abundance in cortical areas, but has much weaker eVects on ARC mRNA in the striatum and no signiﬁcant eVects in the CA1, CA3, and the dentate gyrus (DG) of the hippocampus. This DOI eVect was completely blocked by ketanserin, indicating that it is mediated by 5-HT2A receptor (Pei et al., 2000). Astroglial cell growth is also modulated by 5-HT2 receptor activity. Several glial and glioma cell lines have been shown to express these receptors (Brismar, 1995; Ding et al., 1993; Hirst et al., 1998; Merzak et al., 1996; Wu et al., 1999). The serotonin-induced cell turnover in primary cultures of astroglia from the cortex, striatum, hippocampus, and brain stem is blocked by 5-HT2A receptor antagonists. Moreover, glioma cell migration and invasion can be modulated by serotonin (Merzak et al., 1996). Therefore, the role of serotonin as a growth factor in neurogenesis and synaptic plasticity is mediated in part by the activity of 5-HT2 receptors, and evidence that these processes may be impaired in psychiatric disorders is discussed in Sections V,D and V,E.

IV. Manipulation of the Serotonergic System Alters Synaptic Plasticity

The role of the serotonergic system has been tested in a variety of ways. The creation of lesions within serotonergic circuits alters transmission, as does reducing the production of the neurotransmitter and its precursors, by dietary tryptophan depletion. Conversely, inhibition of sensory or motor activity and investigations into the subsequent alterations in brain structure have provided insight into the serotonergic mechanisms involved in synaptic plasticity. This section discusses data from studies involving the manipulation of serotonergic activity.

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A. Tryptophan and Serotonin Depletion Studies Studies of serotonin depletion in rats using PCPA, a tryptophan hydroxylase inhibitor (Koe and Weissman, 1966), found that there was an increase in inducible nitric oxide synthase in the corpus callosum 2–5 weeks after treatment. In addition, neuronal nitric oxide synthase was increased in the striatum and hippocampus, demonstrating a close interaction between nitrergic and serotonergic systems during development (Ramos et al., 2002). PCPA depletion studies have also shown that the density of CA1 spines in the hippocampus and the dorsal raphe´ nucleus are maintained by serotonergic projections to these regions (Alves et al., 2002). The importance of serotonin in synaptic plasticity during development and in the maintenance of synapses in the adult has also been demonstrated in similar studies of chicken spinal cord (Okado et al., 1993). The role of serotonin in synaptic plasticity is underscored by the strong relationship between serotonin, S100, growth-associated protein-43 (GAP-43), and nitric oxide signaling. The latter increases cyclic guanidine monophosphate (cGMP) through guanyl cyclase activation (Southam and Garthwaite, 1993). Nitric oxide activity is thought to play a critical role in synaptic plasticity and mammalian brain development (Dinerman et al., 1994; Kandel and O’Dell, 1992; Roskams et al., 1994). Long-term depletion of serotonin was modeled in adult rats by the administration of PCPA for 1 week and was shown to increase binding at (S)-[3H]-amino-3hydroxy-5-methylisoxazol-4-proprionic acid receptors (AMPAr) in the cerebral cortex. In contrast, N-methyl-d-aspartate receptor (NMDAr) binding remained constant (Shutoh et al., 2000). Moreover, the maturation of motor neurons has been investigated in rats injected with PCPA in the lumbar region after birth. Postural dysfunctions were observed, including reduced ﬂexion of the knee and ankle and reduced extension of the hip, probably due to arrested development of these neurons (Pﬂieger et al., 2002). In similar experiments, adult rats have been shown to produce an elevated glial expression of S100, lowered serotonin levels, reduced densities of serotonin transporter (SERT), neuroﬁlament-200 and -68 ﬁbers (NF200 and NF-68, respectively), and altered cytoskeletal morphology. Even when serotonin levels normalize after treatment, cytoskeletal changes are still present in the striatum, whereas NF-200, NF-68, and SERT levels increased gradually (Ramos et al., 2000). These studies using PCPA demonstrate that serotonin appears to modulate synaptic plasticity by regulating AMPAr density and S100 release. An alternative method of depleting serotonergic transmission is through the control of tryptophan intake, which has been performed in rodents and in clinical studies (Section V,D). Tryptophan-restricted diets in rats produce increased glutamic acid decarboxylase (GAD) activity in the hippocampus and cerebral cortex between age 14 and 60 days. In contrast, -aminobutyric acid (GABA) activity decreases in these regions postnatally, but then increases in rats between 30 and 60 days of age. Reduced GABAergic inhibition by depletion in GABAergic

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cells at this stage of development would result in enhanced serotonergic transmission and glutamic acid decarboxylase (GAD) activity (Del Angel-Meza et al., 2002). In the pyramidal neurons of the prefrontal cortex, long-term plastic changes are observed in response to tryptophan depletion during development. At 40 days of age, rats have less profuse dendritic aborization and dendritic processes are enlarged, with an increased number of dendritic spines at 60 days (Gonzalez-Burgos et al., 1996). Additionally, reduced serotonin release is observed in prenatally malnourished anesthetized rats compared with well-nourished rats after electrical stimulation of the median raphe´ nucleus. However, the basal release of serotonin is higher before electrical stimulation in the malnourished group, perhaps due to the reduced density of serotonergic neurons leading to a decreased control of serotonin release. Electrical stimulation of the median raphe´ nucleus in the malnourished group would therefore enhance the inhibition of serotonin release through the negative feedback mechanisms controlled by 5-HT1A and 5-HT1D autoreceptors (Mokler et al., 1999). These changes in neurotransmitter function have important implications for cognitive processes, such as learning and memory (Section V,A), as tryptophan depletion has been shown to impair spatial learning, which is attributed to hippocampal function. In contrast, short-term memory is improved due to serotonergic activity in the prefrontal cortex (Gonzalez-Burgos et al., 1996). These ﬁndings are consistent with reports that agonists for 5-HT receptors and GABA receptors impair memory retention, whereas antagonists improve it (Farr et al., 2000). The clinical consequences of depletions in tryptophan intake or reduced serotonin levels in the brain are outlined in Section V,D. B. Experimental Models of Synaptic Plasticity 1. The Visual Cortex The mammalian visual cortex is comparatively straightforward to manipulate and therefore has been used as a model of postnatal synaptic plasticity. If one eye and not the other is deprived of vision, then permanent impairment to the vision of the deprived eye results, with the reassignment of cortical territory to the other eye. In kittens, this process begins at postnatal day 21 (P21), peaks after 4 to 6 weeks, and decreases gradually in the subsequent months (Cynader et al., 1980; Hubel and Wiesel, 1970). During this critical developmental period there are transient, regional, laminar, and columnal changes in the distribution of serotonergic aVerents and receptors; increases in 5-HT1A receptor and 5-HT2C receptor densities have been noted, which peak at P30 and P40, respectively, followed by a gradual reduction to adult levels (Dyck and Cynader, 1993b). Interestingly, 5-HT2C receptor transient expression is concentrated in columns within the striate cortex during this period. If the eye was removed shortly after birth,

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the 5-HT2C receptor columns were absent, and if the 5-HT2C receptor antagonist mesulergine was administered, visual cortex plasticity was lowered during this critical period (Wang et al., 1997). The importance of this receptor was underlined when electrophysiological studies of brain slices demonstrated that 5-HT activation of 5-HT2C receptor facilitated LTP or LTD in P60–P80 kittens (Kojic et al., 1997, 2000). It has been demonstrated further that 5-HT2C receptor activity provides a major contribution to LTP in layer 4 (Kojic et al., 2001). These receptors could play a signiﬁcant role in the regulation of synaptic plasticity, as they increase the mobilization of calcium ions (Ca2+) from intracellular stores via phospholipase C activation (Conn and Sanders-Bush, 1984, 1986a,b, 1987; Conn et al., 1986; Sanders-Bush and Conn, 1986), which is illustrated in Fig. 2. Evidence demonstrates that synaptic plasticity is induced after intracellular Ca2+ concentration rises, perhaps via the activation of NMDA receptors or voltagedependent Ca2+ channels (Perrier et al., 2002). 2. The Somatosensory Cortex Synaptogenesis, dendritic remodeling, and neurogenesis may all contribute to cortical columnarization. The primary somatosensory cortex (S1) of the rat has been studied extensively because of the segmented ‘‘barrel ﬁeld’’-checkered patterns on layer IV of S1 when immunostained for serotonin receptors. Each barrel is produced by axons extending to the individual whiskers of the rat, making this an ideal model to test the impact of serotonergic manipulations during brain development and synaptic plasticity. A transient production of serotonergic neurons occurs in the embryo, which may regulate the development of this area. Furthermore, several serotonin receptor subtypes, including 5-HT1B and 5-HT2A, transiently appear in a vibrissae-related pattern in S1 during early postnatal development (Chiaia et al., 1994; D’Amato et al., 1987; Lebrand et al., 1996; Rhoades et al., 1990). Raising serotonin levels in rats by the administration of speciﬁc monoamine oxidase A (MAOA) inhibitors leads to an increase in the size of the barrels, with apparent fusing of the adjacent barrels (Kesterson et al., 2002; Vitalis et al., 1998). In addition, MAOA gene knockout mice are devoid of the barrel pattern, which reappears when serotonin synthesis is inhibited (Cases et al., 1996). It has also been postulated that the elevation of cortical serotonin promotes GAP-43 expression before P8 in layer IV of S1. Increasing cortical serotonin leads to accelerated development of GAP-43 axons targeting the septal regions (Kesterson et al., 2002). Conversely, if serotonergic input is reduced, altered barrel patterns are observed and delayed maturation occurs (Bennett-Clarke et al., 1994; Blue et al., 1991; Osterheld-Haas et al., 1994; Persico et al., 2000). Moreover, serotonin transporter gene knockout mice display signiﬁcantly thinner barrels in the posteromedial barrel subﬁeld layer IV and an absence of barrel patterns in other subﬁelds of S1 (Persico et al., 2001).

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V. Does Dysfunction of Serotonergic Signaling Result in Impaired Brain Development?

We have already described the vastness of the serotonergic system within the brain and its widespread inﬂuence in almost every sphere of mammalian physiology (Section II). Furthermore, we have outlined the role of serotonin in the development of many neurotransmitter pathways and several regions of the brain (Section III). It therefore follows that a malfunctioning serotonergic system could be a contributory factor, if not a primary cause, of some developmental disorders. Synapses are probably the cellular substrate for learning and memory, therefore disruption of the serotonergic processes in brain development would lead to cognitive impairment. There are several behavioral and psychiatric disorders with cognitive dysfunction in which deviations from normal serotonergic activity have been discovered, such as schizophrenia. Addictive substances such as cocaine (Whitaker-Azmitia, 1998), alcohol (Kim et al., 1997; Sari et al., 2001; Tajuddin and Druse, 1993; Zhou et al., 2001), and nicotine (Muneoka et al., 1997) alter serotonin levels. These and similar substances are strongly contraindicated in pregnant women, as many have been shown to impair brain development after prenatal exposure. The serotonergic hypotheses of many psychiatric disorders are underpinned by pharmacological evidence. Many diseases are treated with serotonergic drugs, including those disorders thought to have developmental origins, such as schizophrenia, the aVective disorders, anxiety, and disorders of learning, such as autism and mental retardation. Behavioral problems, such as eating disorders, addiction, and stress-related disorders are also thought to be inﬂuenced by impaired serotonergic acitvity. Drugs are often administered for several weeks before a clinical response is observed, hence it is possible that structural changes are needed to alleviate psychiatric symptoms. The relationship of these disorders with serotonin and synaptic plasticity is discussed in the following sections. A. The Role of Serotonin in Learning and Memory Long-term potentiation (LTP) is thought to be the cellular mechanism through which memories are stored in the hippocampus (Teyler and Discenna, 1984). LTP is characterized by a long-term elevation in synaptic eYcacy after a short, high-frequency electrical stimulation of aVerent ﬁbers. Changes in synaptic density and the structure of neurons have been associated with LTP, i.e., LTP induces synaptic plasticity, which has a central role in nearly all models of learning and memory (reviewed by Silva, 2003). The phenomenon was ﬁrst reported in the perforant path–dentate gyrus synapse (Bliss and Gardner-Medwin, 1973) and has subsequently been detected in many other hippocampal pathways. The formation of new memories is thought to require the hippocampus and

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adjacent medial temporal lobe (including the archicortical hippocampus, entorrhinal, perirhinal, and parahippocampal cortices), but the ﬁnal storage of memories is probably in a widely distributed neocortical network. Lesion studies have suggested that there is a wide distribution of neocortical memory traces encoded in the strength of synaptic connections among neurons across large areas of the neocortex (Fries et al., 2003). Although glutamate and GABA receptors play a more major role in learning and memory (Zilles et al., 2000), serotonergic eVects have also been detected (Beaumont and Zucker, 2000; Klancnik et al., 1991; Mazer et al., 1997; Sarihi et al., 2000, 2003), and the regions implicated in memory storage are richly innervated by the serotonergic system (as described in Section II,A, Fig. 1, and Table I). Serotonin depletion inhibits LTP in the dentate gyrus, indicating a serotonergic modulation of LTP in this region. Also, stimulation of the median raphe´ nucleus has been shown to induce LTP in the dentate gyrus (Klancnik et al., 1991). Furthermore, reversible inactivation of serotonergic projections from the median raphe´ nucleus has been observed to enhance the maintenance of LTP in the dentate gyrus so that consolidation and retrieval but not acquisition of learned behavior occurs (Sarihi et al., 1999). This serotonergic lesion also enhances working memory tested by the Morris water maze (Sarihi et al., 2000). Therefore the median raphe´ nucleus has an inhibitory role in memory consolidation and retrieval in classic conditioning experiments and spatial memory (Sarihi et al., 2003). Developing serotonergic neurons are thought to regulate growth factors for dopamine neurons, a potential mechanism for serotonin and dopamine interactions in schizophrenia (Whitaker-Azmitia et al., 1995). Serotonin is required during synaptogenesis: if serotonin is depleted during the critical period for synaptogenesis in developing rats, the result is a decreased density of dendritic and synaptic markers in the brains of the adult animals. These rats show deﬁcits in learning and memory (Matsukawa et al., 1997; Mazer et al., 1997). Serotonin also plays a role in maintenance of the adult brain, as depletion of serotonin results in a loss of synapses (Azmitia, 1999; Okado et al., 2001). Behavioral experiments on 5-HT1A receptor gene knockout mice have demonstrated a deﬁcit in hippocampal-dependent spatial learning and memory tasks, such as the hidden platform version of the Morris water maze, but not in nonspatial tasks, such as the visible platform version of the Morris water maze. Absence of paired-pulse inhibition in the CA1 region of the hippocampus has also been reported, resulting in an abnormality in short-term neuroplasticity, although LTP was not impaired (Sarnyai et al., 2000). Similar dissociations between shortterm and long-term plasticity have been observed in -calmodulin kinase II (-CaMKII) knockout mice (Frankland et al., 2001; Glazewski et al., 2000; Waxham et al., 1996) and ataxin-1 knockout mice (Matilla et al., 1998), and it has been suggested that short-term plasticity enables storage of information about the timing of events (Buonomano and Merzenich, 1995). In the basolateral amygdala,

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-adrenergic receptors (AR) are colocalized with 5-HT1A receptors on excitatory nerve endings (Cheng et al., 1998; Huang et al., 1996), and when ARs are activated, they induce long-term enhancement of synaptic transmission in these neurons (Huang et al., 1996). Application of serotonin lowers this synaptic transmission. This eVect was mimicked by a 5-HT1A receptor agonist and blocked by a selective 5-HT1A receptor antagonist. Hence, LTP in the basal amygdala seems to be modulated by 5-HT1A receptor cross talk with ARs (Wang et al., 1999). 5-HT1A receptors also interact with glutamatergic receptors in prefrontal cortical (PFC) neurons. Postsynaptic PFC glutamatergic transmission is altered by serotonin via 5-HT1A receptor activation. This leads to a reduction in AMPA-evoked currents in PFC pyramidal neurons through a CaMKII-mediated mechanism, operated by the activation of protein phosphatase 1 and inhibition of protein kinase A. The 5-HT1A receptor/CaMKII mechanism may regulate the synaptic plasticity of PFC neurons (Cai et al., 2002). These events are crucial for learning and memory and for several forms of synaptic plasticity (Mayford and Kandel, 1999).

B. Autism and Serotonin Autism is a behavioral disorder of unknown etiology that is four times more common in boys than in girls. Symptoms include repetitive movements, lack of imaginative play, ritualistic behavior, and poor communication, leading to reduced social interaction. Bonding emotionally is limited and there is greater sensitivity to tactile (Ayres and Tickle, 1980) and auditory stimulation (Kientz and Dunn, 1997). The deﬁcit in communication only becomes apparent at age 2, when diagnoses are usually conﬁrmed. It was ﬁrst recognized that serotonin may be connected with the etiology of autism, when elevated platelet serotonin levels were reported in autistic patients (Boullin et al., 1970, 1971; Ritvo et al., 1970). Since then, neurobiological, pharmacological, and genetic data have added to the hypothesis that dysfunction of the serotonergic system plays a role in the development of autism (Betancur et al., 2002; Cook and Leventhal, 1996; Marazziti et al., 2000). The hypothesis is strengthened by increasing evidence for serotonergic involvement in brain development and the especially rich serotonergic innervation of limbic areas critical for emotional expression and social behavior. Depletion of the serotonin precursor tryptophan (discussed in Section IV, A) has been reported to cause a signiﬁcant deterioration in autistic patients (Cook and Leventhal, 1996; McDougle et al., 1993), and epidemiological studies detected a high frequency of prenatal exposure to cocaine (Davis et al., 1992) or alcohol (Nanson, 1992), which are both known to alter serotonergic activity. A positron emission tomography (PET) study of male autistic patients detected decreased serotonin synthesis in the cortex and thalamus, although there was an

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increase in the dentate nucleus (Chugani et al., 1997), which could lead to developmental abnormalities. In fact, postmortem studies have found reduced branching of dendrites in CA1 and CA3 hippocampal regions (Raymond et al., 1996), along with reduced hippocampal volumes, suggesting that the cells failed to mature and connections with the cortex may not have fully developed (Aylward et al., 1999). It is possible that the high levels of serotonin detected in autism would create a loss of serotonergic nerve terminals during brain development. This may explain why eVicacious treatments for autism enhance serotonergic transmission (Buitelaar and Willemsen-Swinkels, 2000; McDougle et al., 2000; Posey and McDougle, 2000). Drugs targeting the serotonin transporter (SERT), including the selective serotonin reuptake inhibitors (SSRIs) ﬂuoxetine, venlafaxine, ﬂuvoxamine, and clomipramine, are now widely used to treat autism (Fatemi et al., 1998; Hollander et al., 2000a; Namerow et al., 2003). SSRIs appear to alter many autistic symptoms, including social relatedness. Risperidone, an atypical antipsychotic with strong aYnity for many serotonin receptors, particularly 5-HT2A receptor, is also used frequently to treat autism. Neuroendocrine and behavioral challenge paradigms have found abnormal responses to the increased serotonin levels after the administration of fenﬂuramine or the serotonin precursor 5-hydroxytryptophan or the direct 5-HT1B/D receptor agonist sumatriptan. It has been suggested that 5-HT1D receptor supersensitivity may be responsible for the repetitive behaviors exhibited by autistic children (Hollander et al., 2000b). Evidence suggests that autism is a disorder of impaired brain development. The limited pharmacological data available provide circumstantial evidence that impaired serotoninergic activity may contribute to the etiology of this disorder. It is possible that the development of brain regions involved in communication and emotion are arrested by prenatal processes altering synaptic plasticity via a serotonergic mechanism that is triggered by either genetic or environmental factors.

C. The Role of Serotonin in Stress and Anxiety Serotonin is a chemical mediator of inﬂammation. Its secretion and physiological actions mediate stress and pain, aVecting both immune and nervous system functions through the hypothalamic–pituitary–adrenal (HPA) axis. Serotonin receptor dysfunction is well characterized in mental disturbances such as depression and anxiety. Considerable evidence supports the idea that the early postnatal period is a critical time for the establishment of lifelong anxiety-related behavior, which is a component of many psychiatric disorders. Moreover, clinical studies demonstrate that early life stressors, such as divorce or bereavement, increase susceptibility to anxiety and mood disorders in adulthood (Breier, 1989; Bulik et al., 2001; Kendler et al., 1992, 1993, 1996, 2000, 2002a,b; Kessler et al., 1997).

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The mechanism linking stress with abnormal plastic events during brain development is thought to involve 5-HT1A receptor activity (discussed in Section III,D). Chronic stress has been shown to produce speciﬁc downregulation of 5-HT1A receptors in the hippocampus ( Lopez et al., 1999; Pare and Tejani-Butt, 1996). This hippocampal deﬁcit may partially explain the cognitive dysfunction observed in patients with aVective disorders. Mice lacking the 5-HT1A receptor gene display increased anxiety in behavioral models such as the open ﬁeld test or elevated plus maze (Heisler et al., 1998a; Parks et al., 1998; Sarnyai et al., 2000; Sibille et al., 2000), and the anxious phenotype is associated with changes in GABAAR neurotransmission (Olivier et al., 2001). This behavior can be reversed by the selective reinstatement of 5-HT1A expression in the hippocampus and cortex during early postnatal development, but not by reinstatement in the adult or in raphe nuclei at any age (Gross et al., 2002). These data indicate that interrupted postnatal 5-HT1A receptor developmental processes contribute to anxiety behavior in adulthood. Active and passive stress responses have been compared in short (SAL) and long attack latency (LAL) mice, which are genetically selected mouse lines responding aggressively to an opponent in the intermale resident–intruder experiment. LAL mice have a characteristic chronic elevation in corticosterone levels and a decreased level of 5-HT1A receptors in the dentate gyrus, hippocampal CA1 region, lateral septum, and frontal cortex, but not in the dorsal raphe nucleus (Korte et al., 1996; van Riel et al., 2002). These ﬁndings are consistent with the reduced 5-HT1AR expression in mice exposed to chronic stress and the resultant elevation in glucocorticoid levels (Fernandes et al., 1997; Flugge, 1995; Lopez et al., 1998; McKittrick et al., 1995; Meijer et al., 1997a,b; Watanabe et al., 1993; Wissink et al., 2000). In addition to corticosteroids, sex steroids have been shown to alter behavior and synaptic plasticity. Stress-induced reductions in 5-HT1A receptors in the hippocampus can be renormalized by the administration of androgens (Flugge et al., 1997, 1998). Furthermore, hormone levels are known to alter mood, and there are strong links between estrogens and aVective disorders. Fluctuations in these hormones can cause mood swings, anxiety, or postnatal depression. The aVective symptoms, irritability, and anxiety associated with low estradiol levels in postmenopausal women are alleviated by estrogen replacement ( JoVe and Cohen, 1998; Soares et al., 2001). Furthermore, high levels of estrogens have been shown to increase dendrite growth and synaptic plasticity in the rat hippocampus (Foy et al., 1999; Good et al., 1999; Leranth et al., 2000). As with 5-HT1A receptor activity, estrogens reduce neuronal excitability in the basolateral amygdala (Edwards et al., 1999). Female mice lacking the estrogen receptor (ER) were shown to be more anxious and to have a lower threshold for the induction of synaptic plasticity in the basolateral amygdala, which coincided with increased 5-HT1A receptor expression in the medial amygdala (Krezel et al., 2001). Thus,

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the serotonin system may inﬂuence the steroid hormone control of synaptic plasticity. As described in Section III,D,1, 5-HT1A receptor activity inhibits synaptic plasticity in the amygdala, which is considered to be the sensorimotor interface for conditioned fear. Classic fear conditioning, a paradigm for the study of aversive learning and memory, is thought to depend on the integrity of the amygdala (Fendt and Fanselow, 1999; Rogan and LeDoux, 1996), although there is debate regarding the role of the hippocampus in this response (Maren et al., 1997; Radulovic et al., 1999). Studies either depleting or increasing serotonin have provided evidence for the role of serotonin in fear conditioning (Archer et al., 1982, 1984; Hashimoto et al., 1996; Inoue et al., 1996). A speciﬁc role for 5-HT1A receptor activity in this response has been indicated by studies of 5-HT1A receptor antagonists in combination with serotonin reuptake inhibition (Hashimoto et al., 1997). The 5-HT1A receptor agonist 8-OH-DPAT caused a deﬁcit in contextual fear due to postsynaptic 5-HT1A receptor activation in rats. Both responses were blocked by the 5-HT1A receptor antagonist WAY100635. Therefore, postsynaptic 5-HT1A receptor activation has been shown to interfere with learning processes in the acquisition of fear (Stiedl et al., 2000). In addition, PET studies of patients with major depression show decreased 5-HT1A receptor binding in the temporal lobe and in the limbic system (Drevets et al., 1999; Sargent et al., 2000), and 5-HT1A receptor agonists have anxiolytic properties in clinical and animal models (Feighner and Boyer, 1989; Menard and Treit, 1999). Although the 5-HT1A receptor plays an important role in anxiogenic processes, the 5-HT2B/2C receptor agonist m-chlorophenylpiperazine (mCPP) also induces anxiety. mCPP has been shown to reduce novelty-seeking behavior. In a two-box light/dark choice situation, mCPP has been found to decrease the time spent by mice in the lit box and the number of transitions between the light and dark boxes. This behavioral test has been validated for the assessment of novel compounds with anxiolytic or anxiogenic properties. Therefore, activation of 5-HT2C receptor enhances anxiety responses toward novel and aversive places (Griebel et al., 1991; Meert et al., 1997). Immobilization of animals is another stress-inducing behavioral paradigm. Immobilization decreases the expression of BDNF mRNA in the rat hippocampus, and this eVect could contribute to the atrophy of hippocampal neurons. Pretreatment with a selective 5-HT2A receptor antagonist signiﬁcantly blocks the inﬂuence of stress on the expression of BDNF mRNA. In contrast, pretreatment with either a selective 5-HT2C or a 5-HT1A receptor antagonist did not inﬂuence the stress-induced decrease in levels of BDNF mRNA levels (Vaidya et al., 1999). Furthermore, the inhibitory eVects of stress on the activity of periventricular hypophysial dopaminergic (PHDA) neurons have been shown to be mediated by serotonergic neurons, acting via 5HT2Rs, and this activity leads to an increase in the secretion of -melanocytestimulating hormone (-MSH) (Goudreau et al., 1993). Therefore, behavioral

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pharmacological evidence indicates an important role for the serotonergic system, together with steroid hormones, in the etiology for anxiety disorders.

D. Serotonergic Influences on Synaptic Plasticity in Affective Disorders The catecholamine hypothesis of the aVective disorders was proposed in 1965 based on the observation that the antihypertensive agent reserpine was found to lower the mood of patients (Schildkraut, 1965). This drug depletes catecholamines, therefore it was suggested that depression was caused by a dysfunction in the catecholamine system. Coppen (1967) modiﬁed this theory after the eVects of monoamine oxidase inhibitors (MAOI) on serotonin were observed by suggesting that depression was caused by reduced serotonin in the synapse (Fig. 2) (Coppen et al., 1967). However, the delay in patient response to antidepressants after administration (which is also seen in antipsychotic therapy) could not be correlated with the rapid eVects of antidepressant drugs on serotonin levels, and their eYcacy is thought to be related to more long-term changes caused by plastic events under the control of the monoamine transmitter systems (Charney et al., 1981). The monoamines, noradrenaline, serotonin, and dopamine, and their metabolites, 3-methoxy-4-hydroxyphenylglycol (MOPEG), 5-HIAA, and homovanillic acid (HVA), respectively, have been studied extensively in the blood, cerebrospinal ﬂuid (CSF), and postmortem brain of depressed patients. There is a general consensus after many clinical studies that MOPEG in the urine and CSF of depressed patients is reduced by about 25% in comparison with controls and that this shows a cyclic change when manic and depressed states are experienced by bipolar patients ( Johnstone, 1982). In 1976, Asberg and colleagues measured 5-HIAA in the CSF as an index of brain serotonin turnover (Fig. 2) and found a bimodal distribution in depressed patients. It was also observed that the severity of depression increased with decreased 5-HIAA levels in a subgroup of patients who were more prone to violent suicide attempts. Several studies have also measured decreases in HVA levels in depressed patients, but once methodological details were streamlined, no diVerences in CSF HVA, the dopamine metabolite, were observed between patients and controls. By contrast, more than 20 studies have detected a consistent association between high 5-HIAA levels and suicidal behavior, schizophrenia, personality disorder, and impulse control but not bipolar aVective disorder. A low concentration of 5-HIAA in patients is associated with an increased short-term risk for suicide in patients (reviewed by Asberg, 1997). It has been suggested that abnormalities in receptor sensitivity are present in depressed patients (Charney et al., 1981) and that they fail to make adaptive responses to stress or adverse stimuli due to dysfunctional neural plasticity. Therefore the mechanism of antidepressant action and perhaps the etiology of aVective illnesses may involve the induction of speciﬁc plastic changes by serotonergic activation.

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Alterations in levels of serotonin during the postnatal period produce longlasting changes in brain plasticity, including disruption of the maps of the sensory and visual cortices (Section IV,B), decreased density of dendritic spines (Yan et al., 1997b), and changes in the dentate gyrus (Section IV). One hypothesis has suggested that depression develops as a consequence of the poor availability of dietary tryptophan to the brain, which would reduce the availability of serotonin (Fig. 3). A study of plasma tryptophan in aggressive males found that high tryptophan levels correlated with anxiety (Cleare and Bond, 1995). Other investigations have associated tryptophan with feelings of well-being (Charney et al., 1982; Smith et al., 1997). Eighty to 90% acute depletion of dietary tryptophan has been associated with a lowering of mood in normal females but not in males in one study (Ellenbogen et al., 1996), and depletion of tryptophan has been shown to induce relapse of depression in more than 50% of patients during remission of the disease. This relapse was reversed when tryptophan levels were restored; free plasma tryptophan levels were found to be correlated inversely with depression scores during acute tryptophan depletion (Delgado et al., 1990; Smith et al., 1997). Changes in synaptic plasticity after tryptophan depletion are discussed in Section IV,A, and it is possible that these changes are involved in the etiology and treatment of the aVective disorders. Estrogens and aVective disorders are strongly linked, as described in the previous section, since ﬂuctuations in these hormones may cause mood swings, anxiety, or postnatal depression. High levels of estrogens have been shown to increase dendrite growth and synaptic plasticity in the rat hippocampus (Foy et al., 1999; Good et al., 1999; Leranth et al., 2000). Furthermore, as with 5-HT1A receptor stimulation, estrogens reduce neuronal excitability in the basolateral amygdala (Edwards et al., 1999). Investigations into the mechanisms by which endogenous factors stimulate neurogenesis have determined that serotonin and estradiol act through a common pathway to increase cell proliferation in the adult dentate gyrus. Neurogenesis has also been studied in animal models of depression. Ovariectomy is a method of depleting estrogens, creating depressive behavior in rodents. Combining ovariectomy with inhibition of serotonin synthesis using PCPA treatment produced approximately the same decreases in the number of BrdU and PSA-NCAM-immunolabeled cells (indicating a reduction in newly generated cells) in the subgranular layer as ovariectomy alone. Administration of 5-hydroxytryptophan (5-HTP), a precursor of serotonin (Fig. 3), has been shown to restore cell proliferation decreased primarily by ovariectomy, whereas estradiol does not reverse this change. Estrogen may regulate structural plasticity by stimulating PSA-NCAM expression independently of neurogenesis, as shown by the increases in the number of PSA-NCAM-labeled cells in pregnant rats. These data indicate that positive regulation of cell proliferation and neuroplasticity by serotonin and estrogen may contribute to the reduced hippocampal connectivity observed in depressed patients (Banasr et al., 2001).

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The aVective symptoms of irritability and anxiety, which are associated with low estradiol levels in postmenopausal women, are alleviated by estrogen replacement ( JoVe and Cohen, 1998; Soares and Cohen, 2001). Female mice lacking the estrogen receptor (ER) were shown to be more anxious and to have a lower threshold for the induction of synaptic plasticity in the basolateral amygdala, which coincided with increased 5-HT1A receptor expression in the medial amygdala (Krezel et al., 2001). Together, these studies demonstrate the link between the serotoninergic system and the steroid hormone control of synaptic plasticity in the aVective disorders. 1. Serotonin Receptors and Depression The regulatory actions of serotonin are mediated by serotonin receptors, therefore the distinct temporal expression patterns of serotonin receptors may reﬂect their changing roles during development. Serotonin is thought to regulate the production of neurotrophic factors in the CSF. 5-HT2C receptors are present at high density and probably play a role in CSF production. 5-HT2c receptors may mediate these regulatory functions of serotonin (Esterle and Sanders-Bush, 1992). Psychiatric disorders comprise an array of overlapping symptoms, and patients diagnosed with a variety of CNS disorders often suVer with sleep disturbances. Staner and colleagues (1992) demonstrated that 5-HT2 receptor antagonists produced increased slow wave sleep in healthy individuals, whereas depressed patients exhibited a smaller increase in slow wave sleep at the same dose of drug (Staner et al., 1992). 5-HT2 receptor antagonists have antidepressant potential (Eison, 1990; Marek et al., 1989), which may be related to an interaction with 5-HT1A receptors (Eison, 1990; Yocca et al., 1990). Alterations in 5-HT2A receptor expression and activity have been demonstrated in patients with a range of CNS disorders, and the changes could play a role in the pathogenesis and treatment of psychiatric disease (Burnet et al., 1996, 1999; Eastwood et al., 2001; Harrison and Geddes, 1996; Harrison and Burnet, 1997). An increase in postsynaptic 5-HT2A receptor binding in postmortem brains of patients may reveal a pathophysiological mechanism in aVective disorders (McKeith et al., 1987; Yates et al., 1990). The deﬁciency of serotonergic activity in depressed patients is thought to increase their vulnerability to the disease. Studies have shown low levels of serotonin in the brains of depressed patients who have committed suicide (Coppen and Doogan, 1988) and that there are altered densities of 5-HT2A receptors in the frontal cortex of suicide victims (Mann et al., 1986a,b,c; Stanley et al., 1986a,b), in depressed patients (McKeith et al., 1987), and in individuals at risk for suicidal behavior (Arango et al., 1997; Audenaert et al., 2001; Pandey et al., 1995, 1997; Stockmeier et al., 1997). Drug treatments for the aVective disorders, for example, antidepressants such as lithium, mianserin, and ﬂuoxetine, are thought to act through the serotonergic system and usually raise serotonin levels. Therefore, it has been proposed that

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there is a decrease in serotonergic activity in the brains of depressed patients, leading to an upregulation of 5-HT2A receptors. Evidence supporting this hypothesis includes the ﬁnding that there is increased 5-HT2A receptor-mediated phosphoinositide turnover in the platelets of patients with aVective disorders (Mikuni et al., 1992). Moreover, downregulation of brain 5-HT2 receptors has been demonstrated 2–4 weeks after the administration of many antidepressants (Peroutka and Snyder, 1980), such as lithium (Treiser and Kellar, 1980; Wajda et al., 1986), monoamine oxidase inhibitors (Kellar et al., 1981), some selective serotonin reuptake inhibitors (SSRIs) (Eison et al., 1991; Nelson et al., 1989; Stolz et al., 1983), 5-HT1A receptor partial agonists (Lafaille et al., 1991; Yocca et al., 1991), and clozapine (Hietala et al., 1992). Conversely, electroconvulsive therapy upregulates 5-HT2A receptor-binding sites (Burnet et al., 1996; Kellar et al., 1981). Alterations in 5-HT2A receptor binding also appear to be related to the severity of depressive symptoms, as clinically improved patients have similar densities of 5-HT2A receptors to controls (Yates et al., 1990). This evidence has been conﬁrmed by studies in platelets when patients were treated successfully with antidepressants (Biegon et al., 1990). Raising the level of serotonin in the synapse by the administration of antidepressants produces a cascade of events mediated by 5-HT2, 5-HT1A, 5-HT4, and 5-HT7 receptor subtypes. 5-HT2 receptor stimulation raises intracellular Ca2+ levels and activates CaM kinases, which lead to CREB phosphorylation at ser133. This increases expression of the BDNF gene, which promotes neuronal plasticity and survival. Similarly, 5-HT1A receptor and 5-HT7 receptor stimulation increases Raf, MEK, Erk 1, Erk 2, and Rsk 2 (critical for cell survival), which leads to phosphorylation of the proapoptotic protein BAD and inactivates it. Rsk also activates CREB phosphorylation, conferring cell survival by increasing expression of the antiapoptotic protein Bcl2 (reviewed by D’Sa, 2002; Duman, 2000). Ritanserin, a 5-HT2A receptor antagonist, has been reported to have anxiolytic or antidepressant properties in several studies (Bakish et al., 1993; Bersani et al., 1991; Ceulemans et al., 1985). Nefazodone blocks both noradrenaline and serotonin reuptake in addition to 5-HT2 receptors and has demonstrated antidepressant eYcacy in several controlled trials and was well tolerated by patients (Feighner et al., 1998). Another 5-HT2 receptor antagonist, mirtazapine, has been launched as a treatment for depression. Mirtazapine also acts as an antagonist at 2 and 5-HT3 receptors and this combined activity increases noradrenergic and serotonergic transmission, contributing to its therapeutic eYcacy (de Boer, 1996; de Boer et al., 1996; Dinan, 1996). The atypical antipsychotic drug clozapine has also demonstrated eYcacy in the treatment of depressive symptoms of bipolar aVective disorder, but may exacerbate mania (Frye et al., 1998). Although 5-HT2A receptors have been investigated extensively in studies of mood disorders, there is also evidence implicating altered 5-HT2C receptor function in these illnesses. Molecular genetic studies show that a single nucleotide

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polymorphism in the 5-HT2C receptor gene is associated with bipolar aVective disorder (Lerer et al., 2001) and that the locus of the 5-HT2C receptor gene, Xq24, is linked to this disease (Craddock and Jones, 1999, 2001). SSRIs have been shown to have high aYnity for 5-HT2C receptor, and antipsychotic drugs with high 5-HT2C receptor aYnity, such as clozapine, are also indicated for the treatment of bipolar disorder (Calabrese et al., 1991, 1996; Kimmel et al., 1994). Increased 5-HT2C receptor responsiveness accompanies the isolation-rearing model of anxiety/depression and may contribute to the enhanced response to stress and the increased neophobia observed. In isolation-reared rats, rapid downregulation of supersensitive 5-HT2C receptors may occur in the hippocampus following a serotonergic agonist challenge (Fone et al., 1996). 2. The Serotonin Transporter and Antidepressants A major site of action for many antidepressants is the serotonin transporter (SERT). A growing body of evidence indicates that variability in SERT gene expression inﬂuences temperamental traits, which could be determined by genetic factors, and may lead to psychopathology. Anxiety, depression, and aggressive behavior are all thought to be alleviated by altered SERT activity, but the mechanism of therapeutic eYcacy of SSRIs is still unclear and animal models provide a valuable tool for investigation. Animal models of depression have been varied, including procedures such as olfactory bulbectomy, maternal deprivation, and clomipramine administration. Dendritic spines could represent an anatomical marker for the enduring changes accompanying depression. Dendritic spines are the postsynaptic sites of most excitatory synapses, and their density increases during the ﬁrst 2 postnatal months in rat hippocampus. Synaptic development is altered signiﬁcantly in the hippocampus if there is variation in the levels of serotonin and norepinepherine during this time period. Norrholm and Ouimet (2000) have examined dendritic spine density in the CA1 region of the hippocampus and dentate gyrus of juvenile rats after acute and chronic exposure to antidepressant drugs. Acute antidepressant treatment has been reported to increase dendritic length and spine density, whereas chronic treatment with ﬂuoxetine, a selective serotonin reuptake inhibitor, arrests spine development into young adulthood. Further investigations have revealed that olfactory bulbectomy reduces spine density in CA1, CA3, and dentate gyrus compared to sham-operated controls. Chronic treatment with a nonspeciﬁc tricyclic antidepressant reverses the bulbectomy-induced reduction in dendritic spine density in CA1, CA3, and dentate gyrus. However, the atypical antidepressant mianserin, with 5-HT2A/2C receptor antagonist properties, only reversed this reduction in dentate gyrus. By contrast, other models of depression do not demonstrate this eVect. Hence enduring changes in hippocampal dendritic spine density could contribute to a neural mechanism speciﬁc to this model of depression (Norrholm and Quimet, 2001).

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It has been reported that the projections arising from the hippocampal structures to the hippocampo-medial prefrontal cortex (mPFC) are involved in the execution of higher cognitive functions in rats. The eVects of single and repeated antidepressant treatment on the synaptic eYcacy and synaptic plasticity in the rat medical (mPFC) pathway have been examined using the drug ﬂuvoxamine, a selective serotonin reuptake inhibitor similar to ﬂuoxetine. Fluvoxamine has been reported to enhance synaptic eYcacy in the hippocampo-mPFC pathway in a dose-dependent manner, and repeated treatments enhance synaptic plasticity, so that the establishment of long-term potentiation in the hippocampo-mPFC pathway improves signiﬁcantly. These ﬁndings may indicate the mechanism by which SSRIs produce their therapeutic eVects in depressive disorders (Ohashi et al., 2002). Investigations in mice lacking the SERT gene have demonstrated adaptive changes in 5-HT2A receptor function. Autoradiographic labeling of these receptors by the selective antagonist [3H]MDL 100,907 and saturation experiments with cortical membranes have revealed a new localization of 5-HT2A receptors in the external ﬁeld of striatum and regional variations in adaptive changes in the density of 5-HT2A receptors in SERT (-/-) mutants: a reduction of 30–40% in the claustrum, cerebral cortex, and lateral striatum when compared to wild-type mice (Rioux et al., 1999). Furthermore, immunohistochemistry of the cerebral cortex of SERT knockout mice has revealed a nearly complete absence of serotonin and of barrels, both at P7 and adulthood. VMAT2 knockout mice, which completely lack an activity-dependent vesicular release of monoamines, including serotonin, also display an absence of serotonin in the cortex but have almost normal barrel ﬁelds, albeit with some reduced postnatal growth. These data support the idea that transient SERT expression is required for barrel pattern formation, whereas activity-dependent vesicular serotonin release is not essential for this process (Persico et al., 2001). Although the signiﬁcance of these observations has yet to be fully explained, the importance of serotonin receptors and SERT in the etiology and treatment of the aVective disorders has been established. The development of depressive illness is reviewed more comprehensively by Lesch (2000, 2001). E. Altered Synaptic Plasticity in Schizophrenia 1. Is Schizophrenia Caused by Impaired Brain Development? The neurodevelopmental and neurodegeneration hypotheses of schizophrenia have been opposing etiological theories for many decades. The major glitch in the neurodegenerative hypothesis has been the failure to detect gliosis in postmortem schizophrenic brain (reviewed by Harrison, 1995). The alternative, neurodevelopmental hypothesis, proposes dysfunctional brain development and growth during embryogenesis and childhood before the emergence of

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schizophrenia during or after adolescence (Weinberger, 1995). Although the neurodevelopmental theory predominates, concurrent neurodegenerative processes that do not involve sustained gliosis have not been excluded (Woods, 1998). The neurodevelopmental hypothesis is supported by ﬁndings of delayed developmental milestones in preschizophrenic children, including learning and behavioral abnormalities, indicating abnormal brain function prior to the diagnosis of schizophrenia (Cannon et al., 1997; Chua et al., 1996; Hanson et al., 1976; Jones et al., 1994; Lewis and Levitt, 2002; Marenco et al., 2000; van Os et al., 1995). Murray and colleagues (1992) suggested that the age of disease onset could be used to categorize three forms of schizophrenia: congenital, adult onset, and late onset (Murray et al., 1992). The former can be traced to brain abnormalities during pre- or perinatal stages of development, with gradual increases in behavioral disturbances until the disease can be diagnosed in adolescence or early adulthood. The congenital form is thought to be more common in males (Pilowsky et al., 1993; Stober et al., 1998), whereas the late-onset disorder is more prevalent in females (Hafner, 2003; Palmer et al., 2001). Several neurodevelopmental mechanisms have been proposed. In addition to theories of brain lesions that occur before infancy and lie dormant until adolescence (Weinberger, 1987), it is also possible that a lesion occurs during adolescence before the onset of disease symptoms. Alternatively, computer simulations have proposed a model of schizophrenia in which reduced synaptic connectivity occurs after disturbed neural development during both perinatal and adolescent periods (McGlashan and HoVman, 2000). Alterations in brain structure have been observed in postmortem studies and neuroimaging of schizophrenia subjects. These changes have not been conclusive, as there is considerable overlap with the normal range measured in controls. In addition, there are several problems with these studies, including small sample sizes and patient drug history, because antipsychotic drugs have been shown to produce structural changes in the brain (Crow et al., 1986; Meredith et al., 2000). Even so, a consensus has been reached for some neurobiological changes that have been conﬁrmed by several independent studies summarized later. Initially, anatomical research of schizophrenic brain identiﬁed enlargements of the lateral and third ventricles in conjunction with a decrease in cortical volume. The latter was particularly prominent in the temporal lobe and medial temporal lobe structures, especially the hippocampal formation and amygdala (Lawrie and Abukmeil, 1998; Wright et al., 2000). Subcortical structures appear to be reduced in size, including some thalamic nuclei (Pakkenberg and Gundersen, 1989; Popken et al., 2000; Young et al., 2000) and the striatum (Keshavan et al., 1998). Reduced normal brain asymmetry is another consistent ﬁnding with increased structural variability in the left hemisphere of schizophrenia brains (reviewed by Harrison, 1999b). Considering the early anatomical evidence, Feinberg (1982) proposed that the central pathogenetic process in schizophrenia included altered synaptic pruning

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during adolescence (Feinberg, 1982). A smaller brain volume in schizophrenia could be explained by increased cell death, which would account for the decreased volume of the thalamus (Young et al., 2000), but not the changes in the cortex and hippocampus. These structures do not display cell loss (Pakkenberg, 1993) but instead there are smaller neurons and more densely packed cortical cells (Selemon et al., 1995, 1998; Weinberger, 1999). Altered synaptic plasticity has been inferred by these data, as neuronal cell body size correlates with the extent of dendritic arborization (Selemon and Goldman-Rakic, 1999). Moreover, fewer dendritic spines have been observed on cortical and hippocampal pyramidal neurons in schizophrenia subjects (Garey et al., 1998; Glantz et al., 2000; Rosoklija et al., 2000), and a decrease in the number of synapses in the prefrontal cortex and hippocampus in schizophrenia is suggested by the lower abundance of synaptic proteins in these areas (reviewed in the chapter by Eastwood). Altered synaptic plasticity would probably release a cascade of events altering the expression of genes controlling synaptic function, especially during critical developmental periods. Although the jury is still out on the underlying mechanisms surrounding these developmental disturbances, because the serotoninergic system plays such an important role both in neurodevelopment and in the pharmacotherapy of schizophrenia, it is possible that an aberrant serotonin-dependent developmental process contributes to the etiology of the disease. 2. The Serotonergic Hypothesis of Schizophrenia The idea that serotonergic activity contributes to the etiology of schizophrenia evolved from the observation that lysergic acid diethylamide, a drug structurally similar to serotonin, was hallucinogenic (Gaddum and Picarelli 1954; Jacobs et al., 1979; Pieri et al., 1978). Initially there were doubts about the relevance of these ﬁndings, because the psychosis induced by LSD involved primarily perceptual disturbances, i.e., there was a prevalence of visual rather than auditory hallucinations, an absence of thought disorder, and the preservation of aVect and insight, which is only found in a small proportion of patients with schizophrenic psychosis (Szara, 1967). However, these diVerences were minimized when LSD psychosis was compared with the early stages of schizophrenia rather than the chronic illness (Freedman and Chapman, 1973). The LSD psychosis has been found to be a close model for the reality distortion syndrome in schizophrenia but not for the negative symptoms prevalent in a subset of patients (Slade, 1976). Evidence suggests that LSD produces its psychotomimetic eVects through the stimulation of 5-HT2 receptors (Sanders-Bush et al., 1988; Sanders-Bush and Breeding, 1991), and because atypical antipsychotic drugs have high aYnities for these receptors, there is circumstantial pharmacological evidence for the role of serotonin receptors in the development of schizophrenia. The behaviors observed after the administration of serotonin receptor ligands and the distribution of serotonin receptors in brain regions involved in cognition and mood also support this

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hypothesis and are summarized in Table I. Also the importance of serotonin in development and neural plasticity has been established in previous sections of this review. Further evidence connecting serotonin with the development of schizophrenia includes the fact that serotonin levels can be altered during viral infections in development (Pletnikov et al., 2000; Popenenkova et al., 1977; Yamashita et al., 1989), malnutrition (Blatt et al., 1994; Kaye et al., 1991; Manjarrez et al., 1996), social isolation (Whitaker-Azmitia et al., 2000), hypoxia (Kim et al., 1994), and stress (Lapiz et al., 2001; Read et al., 2001; Robinson and Becker, 1986) (discussed in Section V,C). Most of these are epidemiological risk factors for schizophrenia later in life (reviewed by Lewis and Levitt, 2002) and are associated with altered brain development in the embryo and early childhood (Miller and Azmitia, 1999). a. Serotonin Receptors and Pharmacological Studies of Schizophrenia. The core symptoms distinguishing schizophrenia from other mental disorders according to DSM IV diagnostic criteria are delusions and hallucinations, which contribute to the loss of insight, which is characteristic of the disease. It is recognized that hallucinations occur after activation of the 5-HT2 receptors because, LSD acts potently at 5-HT2 receptor sites, as do the substituted phenethylamine hallucinogens such as mescaline (Aghajanian, 1994). Moreover, the aYnity of these compounds for 5-HT2 receptor sites has been found to be closely related to their potency as hallucinogens in humans (Glennon et al., 1984; Titeler et al., 1988). Human studies of the psilocybin indicate that 5-HT2 receptors are involved in hallucinogenesis (Vollenweider and Geyer, 2001; Vollenweider et al., 1998). Evidence from antipsychotic treatment studies also supports the serotonergic hypothesis. Although the discovery of antidopaminergic antipsychotic drugs meant that this hypothesis was eclipsed temporarily by the dopamine theory of schizophrenia, interest in serotonin reemerged with the discovery of atypical antipsychotic treatments. The archetype atypical drug clozapine is an antipsychotic with high eYcacy in patients exhibiting a poor response to conventional antidopaminergic antipsychotics. Clozapine has been found to have a higher aYnity for serotonin receptors than for dopamine receptors (Meltzer et al., 1989a,b,c). It is still undecided whether these pharmacological indications are symptomatic or causal in schizophrenia and therefore biochemical and postmortem studies have been conducted to address this question. The proposed dysfunction in synaptic plasticity in schizophrenia could unleash a cascade of changes in the expression of genes controlling synaptic function, especially during critical developmental periods. Because serotonin plays a vital role during development (discussed in Sections III and IV), investigations of gene expression in schizophrenia may provide etiological clues. In fact, postmortem studies have found alterations in the abundance of several 5-HTRs. Binding sites for 5-HT1A receptors are increased in the prefrontal cortex, cingulate cortex, and temporal cortex in schizophrenia (Burnet et al., 1996; Gurevich et al., 1997;

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Hashimoto et al., 1991, 1993; Simpson et al., 1996; Sumiyoshi et al., 1996), with no changes in the encoding mRNA (Burnet et al., 1996). Reductions in the abundance of 5-HT2A receptor mRNA have been reported in the prefrontal, cingulate, temporal, and occipital cortices (Burnet et al., 1996; Hernandez et al., 2000), and although there have been some failures to replicate these data, the majority of studies have demonstrated a decrease in 5-HT2A-binding sites in the prefrontal cortex in schizophrenia (reviewed by Harrison and Burnet, 1997; Harrison and Geddes, 1996). Postmortem studies have also demonstrated that the abundance of 5-HT2A receptor is reduced in schizophrenia in brain regions shown to have important neuropathological alterations speciﬁc to the disease (reviewed by Harrison, 1999a). Molecular genetic studies have shown associations between single nucleotide polymorphisms (SNPs) in the 5-HT2A receptor gene and schizophrenia (Inayama et al., 1996; Sodhi et al., 1999a; Spurlock et al., 1998; Williams et al., 1996, 1997) and antipsychotic drugs are being increasingly developed to target this receptor (reviewed by Sodhi and Murray, 1997). Because some behavioral responses elicited by acute doses of LSD resemble symptoms of schizophrenia, characterization of gene expression proﬁles after LSD may provide clues about the etiology of the disease. A small group of genes within the rat prefrontal cortex were found to have altered expression: ania3, ARC, c-fos, I- , krox-20 (erg2), neuron-derived orphan receptor 1(Nor1), and serum glucocorticoid kinase. These ﬁndings lend weight to the developmental hypothesis of schizophrenia because many of these proteins alter synaptic plasticity (Nichols and Sanders-Bush, 2002) and the prefrontal cortex is a region associated with neurochemical and structural alteractions in schizophrenia (Goldman-Rakic, 1994; Weinberger and Berman, 1996), these ﬁndings provide support for the developmental hypothesis of schizophrenia (Section V,E,1). The response of these genes to LSD administration was reported to be dynamic with diVering rates of expression changes. ARC was the most highly expressed gene and was also the gene with the greatest increase in expression after LSD treatment (Nichols et al., 2003). The majority of expression increases were due to activation of 5-HT2A receptor. Furthermore, Pei and colleagues (2000) observed that 5-HT2A receptor antagonists produced cytoarchitectural changes by modulating the expression of ARC. In another study, LSD administration produced a ﬁve- to eightfold increase in fos-like immunoreactivity in the medial prefrontal cortex, anterior cingulate cortex, and central nucleus of amygdala. LSD activation of the medial prefrontal cortex and anterior cingulate cortex was found to be mediated by 5-HT2A receptor, whereas 5-HT2A receptor activation in the amygdala was just a component of the response (Gresch et al., 2002). Although attention has focused on the 5-HT2C receptor subtype, the 5-HT2C receptor subtype may also be relevant, as lysergic acid diethylamide is a highaYnity agonist at 5-HT2CR (Burris et al., 1991). Furthermore, several genetic studies have detected associations between 5-HT2C receptor cys23ser polymorphism and long-term hospitalization in schizophrenia (Segman et al., 1997),

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and between both 5-HT2A receptor and 5-HT2C receptor genes and hallucinations in dementia (Holmes et al., 1998). Variation in both these genes has also been associated with a therapeutic response to clozapine (Arranz et al., 1995, 1996, 1998; Masellis et al., 1995, 1998; Sodhi et al., 1995, 1999b). The importance of 5-HT2C receptor in CNS disorders is also emphasized by the potentially fatal neurological deﬁcits observed in 5-HT2C receptor knockout mice (Tecott et al., 1995). In addition, interesting changes have been detected in 5-HT2C receptor RNA editing, a posttranscriptional process by which 5-HT2C receptor mRNA undergoes editing to produce several receptor variants, some with pharmacological diVerences (Burns et al., 1997). Elevated expression of 5-HT2C receptor mRNA unedited and partially edited isoforms has been detected in the dorsolateral prefrontal cortex of schizophrenia subjects treated with antidopaminergic drugs (Sodhi et al., 2001). Because the unedited 5-HT2C receptor exhibits greater G-protein coupling and constitutive activity (Herrick-Davis et al., 1999; Niswender et al., 1999), reduced RNA editing of the receptor may increase 5-HT2C receptor activity in the dorsolateral prefrontal cortex in schizophrenia. Although the association between 5-HT2c RNA editing and schizophrenia has not been replicated (Dracheva et al., 2003), 5-HT2c RNA editing has been associated with suicide in several studies (Gurevich et al., 2002; Iwamoto and kato, 2003; Niswender et al., 2001). Improved methodology and increased sample numbers will facilitate investigations into this exciting phenomenon. Examination of other serotonin receptors has revealed reductions in hippocampal levels of both 5-HT6 receptor mRNA and 5-HT7 receptor mRNA in the prefrontal cortex in schizophrenia (East et al., 2002a, b). Serotonin also binds to the serotonin transporter, which has also been examined in schizophrenia research because it is well established that ligands for SERT alter mood and SERT antagonists are widely used to treat depressive disorders (Section V, D). Some studies have reported a decreased density of transporter-binding sites in the schizophrenic frontal cortex (Joyce et al., 1993; Laruelle et al., 1993; Ohuoha et al., 1993), although others have reported no change (Dean et al., 1995, 1996; Gurevich and Joyce, 1997). In contrast, SERT mRNA levels appear to be increased in this brain region. However, the drug histories of the psychiatric case and control groups diVer in postmortem studies and could confound these data (Hernandez et al., 2000). Furthermore, reduced SERT-binding site densities have been observed in the cingulate cortex in schizophrenia, but are increased in striatum ( Joyce et al., 1993). In the hippocampus, no alterations in the density of transporter-binding sites have been found, but a lower aYnity of the transporter for [3H]paroxetine in schizophrenia has been reported (Dean et al., 1995). Therefore, changes in serotonin receptor and SERT gene expression, altered serotonin levels, and behavioral studies provide support for the hypothesis that a dysfunctional serotonergic system alters synaptic plasticity suViciently to cause developmental changes leading to schizophrenia.

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F. Down’s Syndrome, Mental Retardation, and Serotonin In contrast with disorders such as schizophrenia and autism, Down’s syndrome is associated with reduced levels of serotonin, detected ﬁrst in blood (Boullin and O’Brien, 1971; Tu and Zellweger, 1965), then in cerebrospinal ﬂuid (Scott et al., 1983), and ﬁnally in studies of postmortem brain (Mann et al., 1985). Neurotransmitter deﬁcits in Down’s Syndrome are comparable to those found in Alzheimer’s disease (Godridge et al., 1987). In Down’s syndrome, the expected peak of 5-HT1A receptor density during development is greater than in control postmortem brain, but this decreases below controls after birth (Bar-Peled et al., 1991b). This may explain the ﬁndings of initial dendritic overdevelopment, followed by hypertrophy (Becker et al., 1986; Takashima et al., 1994). It has been proposed that similar to autism, the serotonergic system fails to mature and form appropriate connections in the brains of Down’s syndrome children (Okado et al., 2001). In Down’s syndrome adults there is a region-speciﬁc increase in serotonin. These increases occur in the frontal and occipital cortices (Gulesserian et al., 2000), while there are reductions in the thalamus, caudate, cerebellum, and temporal cortex (Seidl et al., 1999). Pharmacological evidence supports a serotonergic role in the development of the symptoms of Down’s syndrome, as serotonergic drugs are especially useful in the treatment of the aggressive symptoms, self-harm, cognitive impairment, and depression exhibited by patients (Gedye, 1990, 1991). Interestingly, the gene for S100 is located on chromosome 21, which is in trisomy in Down’s syndrome (Ueda et al., 1994a,b). Overexpression of the S100 protein has been detected in postmortem brain and lymphocytes of Down’s syndrome patients, prenatally and in adults (Kato et al., 1990). S100 produced by astrocytes is used as a trophic factor by serotonergic neurons, and a positive correlation occurs among serotonergic neuron density, spine density of pyramidal cell dendrites, 5HT1AR-binding sites, and S100 expression (Ueda et al., 1995, 1996). The level of S100 also correlates with the degree of mental impairment in the patients (Kato et al., 1990). Moreover, a transgenic mouse developed to overexpress S100 possesses neuropathology and symptoms resembling Down’s syndrome (reviewed by Whitaker-Azmitia, 2001). Therefore, as with the other psychiatric disorders discussed, a component of the developmental pathology observed in Down’s syndrome can be attributed to dysfunctions of synaptic plasticity modulated by the serotonergic system.

VI. Conclusions

This review has considered the role of serotonin and serotonergic receptors in the neuroplastic events that create, repair, and degenerate the brain. Research spanning more than ﬁve decades has shown that serotonergic projections in the

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brain have a widespread distribution and that these projections interact with other neurotransmitter systems, thereby inﬂuencing many, if not all, physiological functions. Evidence from biochemical, pharmacological, and clinical studies demonstrates the huge importance of an intact serotonergic system to support brain development and neurogenesis in the maintenance of normal CNS function. Serotonin acts as a growth factor and inﬂuences other growth factors during development. The high level of serotonin function during these crucial stages of brain development and the pharmacological evidence for impaired serotonergic function in several disparate brain disorders underpins the importance of understanding this highly complex system. Increased insight may facilitate real progress in drug development, with the ultimate goal of preventing and even curing these debilitating illnesses. References

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PRESYNAPTIC PROTEINS AND SCHIZOPHRENIA

William G. Honer and Clint E. Young Center for Complex Disorders Department of Psychiatry University of British Columbia

I. Introduction II. Presynaptic Proteins A. Synaptophysin B. SNAP-25 C. Syntaxin D. VAMP E. N-Ethylmaleimide-Sensitive Factor ( NSF ) F. Complexins G. Synapsins H. Rab3a I. Synaptotagmin III. Are All Presynaptic Proteins AVected Equally within a Single Brain Region? IV. Are DiVerent Brain Regions AVected Equally for a Given Presynaptic Protein? V. What Are the Relationships between mRNA and Protein Findings When Both Are Measured in the Same Study? VI. Microarray Studies VII. Summary References

I. Introduction

There are two general models of synaptic dysfunction in schizophrenia. The ﬁrst relates synaptic dysfunction to a neurodevelopmental model of the illness (Feinberg, 1982–1983, 1990). A disturbance in the process of brain development is proposed to interfere with ‘‘synaptic pruning’’ or the modiﬁcation of synaptic number during childhood and early adolescence. In this model, more than the expected number of synapses are lost, and the neuropil compartment in schizophrenia is reduced as a consequence (Selemon and Goldman-Rakic, 1999). The second model proposes a disturbance in the more dynamic process of synapse modiﬁcation and plasticity (Haracz, 1984; Stevens, 1994). This process could result in loss of synapses in some brain regions. Equally importantly, the process of reinnervation and plasticity could result in altered synaptic connectivity without net synaptic loss (or with a net gain) in other brain regions. Progress toward understanding synaptic dysfunction in schizophrenia will require investigation of INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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postmortem samples and animal models, as direct in vivo studies of synapses in humans are quite limited with present imaging techniques. This review focuses on proteins found to be enriched in presynaptic terminals and examination of the evidence for alterations in these proteins and their coding mRNA in postmortem tissue obtained from individuals with a history of schizophrenia. Recent reviews have considered the usefulness of postmortem studies of synaptic connectivity in the context of the results of lesion studies in animals, the eVects of electrophysiological and behavioral manipulations, and the possible implications of antipsychotic drug treatment (Honer, 2002). The techniques applied to study presynaptic proteins were described and evaluated (Honer et al., 2000). This review ﬁrst provides a brief description of the presynaptic proteins to be considered. The results of studies in schizophrenia are updated with the ﬁndings of recent papers not covered in previous reviews. The main purpose is to consider three questions concerning presynaptic proteins and their coding mRNAs: (1) are all presynaptic proteins aVected equally within a single brain region, (2) are diVerent brain regions aVected equally and for a given presynaptic protein, (3) what are the relationships between mRNA and protein ﬁndings when both are measured in the same study? Papers were selected for inclusion based on the availability of a. data from one protein or mRNA and multiple regions studied in the same case series; b. data from multiple proteins or mRNAs studied in one region in the same samples; c. data from both protein and mRNA in the same samples; d. or, where a comparison paper exists, using the same series of samples allowing (a) or (b) to be accomplished. II. Presynaptic Proteins

Several families of proteins are enriched in presynaptic terminals and are involved in the process of chemical neurotransmission. A select subset of these proteins has been studied in postmortem tissue from individuals who suVered from schizophrenia.

A. Synaptophysin This is an integral protein of the synaptic vesicle membrane and is found ubiquitously in presynaptic terminals ( Jahn et al., 1985; Navone et al., 1986; Wiedenmann and Franke, 1985). Although the precise function is still subject

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to debate, this protein interacts with other presynaptic proteins, including VAMP (see later). The gene coding for synaptophysin is located on Xp11.23-p11.22. B. SNAP-25 This presynaptic membrane protein, along with syntaxin and VAMP, forms the SNARE complex, a macromolecular assembly vital for membrane fusion during chemical neurotransmission (Oyler et al., 1989; So¨llner et al., 1993a,b). SNAP-25 is relatively enriched in some presynaptic terminals compared with others (Duc and Catsicas, 1995; Geddes et al., 1990; Oyler et al., 1989). Glutamate, serotonin, and dopamine release are aVected by alterations in SNAP-25 (Raber et al., 1997). The SNAP-25 gene is located on chromosome 20p12-p11.2. C. Syntaxin This family of proteins is found in the presynaptic membrane (Bennett et al., 1992) and appears to be enriched in speciﬁc terminal types, particularly asymmetric-type terminals (Sesack and Snyder, 1995) (see also Fig. 1). Syntaxin binds to SNAP-25, voltage-gated calcium channels, and a range of other proteins that modulate syntaxin function (Mochida, 2000). The gene coding for syntaxin 1A is located on chromosome 7q11.23. D. VAMP This protein is also called synaptobrevin and is the vesicular protein member of the SNARE complex (Trimble et al., 1988). There are several isoforms of the protein, with diVerent distributions in brain regions (Trimble et al., 1990). The VAMP-1 and VAMP-2 genes are located on chromosomes 12p and 17p13.1, respectively. E. N-Ethylmaleimide-Sensitive Factor (NSF) This molecule is located in the cytoplasm of the presynaptic terminal and acts with -SNAP in the termination phase of the interaction between the SNARE proteins (So¨llner et al., 1993a,b). The gene coding for NSF is located on chromosome 17q21. F. Complexins The small complexin I and complexin II proteins interact with syntaxin and are thought to modulate the SNARE complex activity (Ishizuka et al., 1995; McMahon et al., 1995; Takahashi et al., 1995). Complexin I is enriched

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Fig. 1. Variability in the distribution of presynaptic proteins within a brain region. A doubleimmunostaining technique was used to investigate the distribution of presynaptic proteins within the human hippocampus. The technique involved immunostaining with one antibody, reacting with the soluble chromogen AEC, obtaining an image, followed by stripping the tissue section of reaction product, and then reimmunostaining with a second antibody. To validate the technique, the immunostaining pattern of an antibody reactive with myelin basic protein (antibody SMI-94, green, white matter areas) and an antibody reactive with the presynaptic protein synaptophysin (antibody SP15, red, gray matter areas) is shown (A). No overlap was detected. (B) Green color represents immunostaining for syntaxin (antibody SP6) and red synaptophysin (antibody SP15). As expected, there was considerable overlap, but areas with relative enrichment of one presynaptic protein compared with the other were also observed. (See Color Insert.)

PRESYNAPTIC PROTEINS AND SCHIZOPHRENIA

179

in inhibitory, GABAergic terminals, and complexin II is enriched in excitatory, glutamatergic terminals (Takahashi et al., 1995; Yamada et al., 1999). The genes coding for complexin I and complexin II are located on chromosomes 4p16.3 and 5q35.3, respectively.

G. Synapsins This family of phosphorylated presynaptic proteins interacts with the surface of the synaptic vesicle and appears to be involved with various forms of neural plasticity (Greengard et al., 1993). The gene coding for synapsin I is located on Xp11.23, synapsin II on chromosome 3p25, and synapsin III on chromosome 22q12.3.

H. Rab3a This GTP-binding protein family is located in the presynaptic cytoplasm and is associated with the vesicle membrane (Su¨dhof, 1995). Rab proteins appear to alter the dynamics of synaptic vesicle fusion with the presynaptic membrane (Rothman and So¨llner, 1997). The gene coding for Rab3a is located on chromosome 2q21.3.

I. Synaptotagmin This protein is thought to be the calcium sensor that acts to trigger exocytosis ( Jahn et al., 2003). Synaptotagmins are associated with both the synaptic vesicle membrane and the presynaptic terminal membrane. The gene coding for synaptotagmin I is located on chromosome 12cen-q21.

III. Are All Presynaptic Proteins Affected Equally within a Single Brain Region?

Eighteen studies contributed to this section, with measurements of two or more proteins or complementary mRNA in the same brain region. The ﬁndings are summarized in Table I. To determine if all proteins were aVected to the same extent in a single region, we deﬁned discordance as statistically signiﬁcant illnessrelated eVects on one protein but not on others or as diVerences in magnitude of 50% or more where multiple proteins exhibited illness-related eVects. According to this approach, concordant results were observed across proteins in only 4

TABLE I Studies of Multiple Presynaptic Proteins in the Same Brain Region in Cases of Schizophrenia (sch) Compared with Controls (con) mRNA or protein: Antibody

Assaya

Cases

Regionb

Finding

Combined protein and mRNA studies Harrison and Eastwood (1998)

180 Karson et al. (1999)

Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin Complexin

I: SP33 II: LP27 I mRNA II mRNA I: SP33 II: LP27 I mRNA II mRNA I: SP33 II: LP27 I: SP33 II: LP27 I mRNA II mRNA I: SP33 II: LP27 I mRNA II mRNA I: SP33 II: LP27

Synaptophysin: ICN SNAP-25: SMI-81 Synaptophysin: ICN

IAR

11 con 11 sch

ISH

DG ML CA4

IAR ISH

CA3

IAR IAR

CA1

ISH

Subiculum

IAR ISH

PHG

IAR IB homogenate Northern blot

12 con 14 sch 12 con

BA10 (frontal) BA10 (frontal)

c

ND ND ND Decreased ND ND ND ND ND Decreased ND ND ND Decreased ND ND ND Decreased ND Decreased

69%

12%

59%

84% 14%

Decreased 25% Decreased 50% ND

SNAP-25: SMI-81 Imai et al. (2001)

Eastwood et al. (2001)

14 sch

Synapsin II NSF Synapsin II NSF

mRNA RT-PCR

Synaptophysin

ISH

Complexin I Complexin II Synaptophysin: SY38 (DAKO) Complexin I: SP33 Complexin II: LP27

IB

8 6 10 8

con sch con sch

16 con

ND BA46 (frontal) BA46 (frontal) Cerebellum

16 sch IAR

ND ND ND ND Decreased 31% GCL, ND Purkinje ND Decreased 36% GCL ND ND Decreased 15–20% GCL, ND ML

Protein studies 181

Browning et al. (1993)

Synapsin I Synapsin IIb Synaptophysin

IB homogenate

7 con 7 sch

Honer et al. (1997)

Synaptophysin: SP4

ELISA: homogenate

24 con

SNAP-25: SP12 Syntaxin: SP6 Gabriel et al. (1997)

Synaptophysin: EP10 Synaptophysin: SP4 SNAP-25: SP14 Syntaxin: SP6 Synaptophysin: EP10/SP4

Hippocampus

Decreased 43% ND ND

Cingulate

ND

18 sch ELISA: homogenate

ND Increased 55%

15 con

BA24

Increased 33%

17 sch

(Cingulate)

12 con

BA20

Increased 40% Increased 34% Increased 47% ND

(Continued )

TABLE I (Continued ) mRNA or protein: Antibody

Assaya

SNAP-25: SP14 Syntaxin: SP6 Young et al. (1998)

Synaptophysin: EP10

ELISA: homogenate

SNAP-25: SP12 Synaptophysin: EP10 SNAP-25: SP12 Synaptophysin: EP10 SNAP-25: SP12 182

Davidsson et al. (1999)

Eastwood and Harrison (2001)

Synaptophysin: clone Rab3a: clone 42.2 Synaptophysin: clone Rab3a: clone 42.2 Synaptophysin: clone Rab3a: clone 42.2 Synaptophysin: clone Rab3a: clone 42.2 Synaptophysin: clone Rab3a: clone 42.2 Synaptophysin: clone Rab3a: clone 42.2

ICC

7.2

IB

7.2

IB

7.2

IB

7.2

IB

7.2

IB

7.2

IB

Cases

Regionb

Finding

19 sch

(Temporal), BA7 (Frontal), BA8 (Parietal)

ND ND

13 con

Hippocampus

ND

13 12 12 12 12

sch con sch con sch

10 13 12 18 10 15 6 5 9 13 7 12

con sch con sch con sch con sch con sch con sch

Synaptophysin: SY38 (DAKO)

IB synaptosomal

15 con

Complexin I: SP33 Complexin II: LP27

Fraction

15 sch

Hippocampus DG GCL Hippocampus BA32/33 (Cingulate) BA39/40 (Parietal) BA45/46 (Frontal) BA21/22 (Temporal) Cerebellum Cingulate

ND ND Decreased 5–20% Increased 56% Decreased 42% Decreased Decreased Decreased Decreased ND Decreased ND Decreased ND ND ND ND ND ND ND

31% 30% 33% 33% 22% 22%

Honer et al. (1999, 2002)

Syntaxin: SP6 SNAP-25: SP12

ELISA

VAMP: SP10 Synaptophysin: EP10 Vawter et al. (2002b)

11 con 7 sch

Anterior frontal

ND Decreased 28% sch nonsuicide ND Decreased 32% sch nonsuicide

Hippocampus

ND

(Nonsuicide) 6 sch (suicide)

183

Synapsin la: G304, G143 polyclonal Synapsin IIa: G304, G143 polyclonal Synapsin IIIa: G304, G143 polyclonal Synaptophysin: SVP38

IB membrane/cytosol fractions

13 con 16 sch

Sawada et al. (2002)

Complexin I: SP33 Complexin II: LP27

ELISA

Mukaetova-Ladinska et al. (2002)

SNAP-25: SP12 Syntaxin: SP8 Synaptophysin: EP10

ELISA Synaptosomal

Halim et al. (2003)

SNAP-25: Vector Syntaxin: SP8 VAMP: SP10 Synaptophysin: SP15

IB homogenate

23 con 18 sch

Synapsin 1A Synapsin 1B Synaptophysin

mRNA RT-PCR

4 6 6 9 13

Decreased 32% Decreased 32% ND 11 con 13 sch 8 con 8 sch

Anterior frontal

Decreased 33% ND

Cerebellum

Decreased 25% ND ND

Prefrontal

ND ND Decreased 22% ND

BA21 (temporal)

ND ND ND

mRNA studies Tcherepanov and Sokolov (1997)

con < 75 years con > 75 con > 75 sch < 75 sch > 75

(Continued )

TABLE I (Continued ) mRNA or protein: Antibody

Assaya

Cases

Synapsin 1A

Regionb BA22 (temporal)

Increased 100% in sch < 75 ND ND

BA22

Increased young ND Increased young Increased Increased

Synapsin 1B Synaptophysin Sokolov et al. (2000)

184 Eastwood and Harrison (2000)

Synaptotagmin I

mRNA RT-PCR

6 con < 79

(temporal)

Rab3a Synaptobrevin I

3 con > 79

Syntaxin 1A SNAP-25

7 sch < 79 7 sch > 79

Complexin I Complexin II Complexin I Complexin II

ISH

15 con 15 sch

Finding

CA4 CA3, subiculum, PHG

63%

60% 39% 54%

Decreased 50% Decreased 40% ND ND

a IAR, immunoautoradiography; ISH, in situ hybridization; IB, immunoblot; ICC, immunocytochemistry; RT-PCR, real-time polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay. b DG, dentate gyrus; ML, molecular layer; CA, Ammon’s horn; PHG, parahippocampal gyrus; GCL, granule cell layer. c No diVerence.

PRESYNAPTIC PROTEINS AND SCHIZOPHRENIA

185

studies. In each of the other 14 studies, individual presynaptic proteins or the coding mRNAs were aVected diVerentially in schizophrenia compared with control samples. Synaptophysin is a ubiquitous presynaptic protein and is likely the best marker of overall synaptic terminal density. There were 13 studies where synaptophysin mRNA or protein was measured in conjunction with another presynaptic protein (Table I). In 11 of these studies (Table II) there was discordance between schizophrenia-related eVects on synaptophysin and schizophrenia-related eVects on other presynaptic proteins.

IV. Are Different Brain Regions Affected Equally for a Given Presynaptic Protein?

Seventeen studies were located with multiple brain regions assessed or multiple subregions in the case of the hippocampus (Table III). Virtually all indicated that the eVects of schizophrenia on presynaptic proteins or coding mRNAs were diVerent in at least one brain region compared with other regions. Further evidence for divergent eVects on diVerent brain regions is found by considering regions with at least 4 diVerent reports (of diVerent proteins or multiple reports concerning the same protein). Within the hippocampus, mRNA coding for presynaptic proteins appears aVected most frequently by schizophrenia in CA4 (5/7 reports) and aVected least frequently in CA1 (1/5) or the subiculum (2/6). For protein measurements, the granule cell layer of the dentate gyrus was reported to be aVected in 4/4 studies. In contrast, CA4 was aVected in only 2/9 studies and CA1 in only 1/6. For the neocortex, proteins were aVected in cingulate (BA24 or BA32/33) in 6/7 studies; the prefrontal cortex (BA9) was next most likely to be aVected (3/5). The primary visual cortex was less likely to demonstrate presynaptic protein abnormalities (1/5).

V. What Are the Relationships between mRNA and Protein Findings When Both Are Measured in the Same Study?

Only six studies examined mRNA and protein in the same samples (Table IV ). Four of these are from the Harrison and Eastwood group and generally use in situ hybridization for mRNA and immunoautoradiography for protein. In several of the individual regions studied, diVerences both in mRNA and in protein between schizophrenia and controls were observed. More frequently, however, diVerences in mRNA were observed in the absence of diVerences in protein. In another report comparing Northern blotting and immunoblotting,

TABLE II Studies Demonstrating Discordance between Findings for Vesicular Membrane Protein Synaptophysin and Other Presynaptic Proteins in Schizophrenia (sch)a

Region Browning et al. (1993) Tcherepanov and Sokolov (1997) Honer et al. (1997) Young et al. (1998) Davidsson et al. (1999) 186

Karson et al. (1999)

Mukaetova-Ladinska et al. (2002) Halim et al. (2003)

Other protein: Antibody

Difference in schizophrenia

Hippocampus BA22 (temporal)

NDb ND

Synapsin I Synapsin 1A (mRNA)

Decreased 43% Increased 100%

Cingulate Hippocampus GCL BA39/40 (parietal) BA10 (frontal) Anterior frontal

ND ND Increased 56% ND ND Decreased 25% Decreased 32%

Hippocampus

mRNA decreased 31% Protein ND ND

Increased 55% Decreased 5–20% Decreased 42% Decreased 22% Decreased 31% Decreased 50% ND ND ND Decreased 15–20% Decreased 32%

Decreased 22%

Cerebellum

ND

Syntaxin: SP6 SNAP-25: SP12 SNAP-25: SP12 Rab3a: clone 42.2 Rab3a: clone 42.2 SNAP-25: SMI-81 Syntaxin: SP6 VAMP: SP10 Complexin I mRNA Complexin II Synapsin IIa: G304, G143 polyclonal Synapsin IIIa: G304, G143 polyclonal SNAP-25: SP12

Prefrontal

ND

VAMP

Honer et al. (1999, 2002) Eastwood et al. (2001) Vawter et al. (2002b)

Synaptophysin

Decreased 32% Decreased 25%

a Discordance between synaptophysin and other synaptic protein results was defined as greater than 50% diVerences, significant versus nonsignificant diVerences, or significant diVerences in diVerent directions. b No diVerence.

TABLE III Studies with One Presynaptic Protein Assayed in Multiple Brain Regions with Approximate Mean Differences mRNA or protein: Antibody

Assay

Regionsa

Cases

Finding

Combined protein and mRNA studies Eastwood et al. (1995)

mRNA Synaptophysin (M776 DAKO)

ISH ICC

13 con 7 sch

DG CA4 CA3 CA1 Subiculum

187

PHG Harrison and Eastwood (1998)

Complexin I

ISH

Complexin I: SP33

IAR

Complexin II

ISH

11 con 11 sch

CA4 CA3 Subiculum PHG DG CA4 CA3 CA1 Subiculum PHG DG CA4 CA3 CA1 Subiculum PHG

mRNA ND, protein ND mRNA decreased 38%, protein ND mRNA decreased 35%, protein ND mRNA ND, protein ND mRNA decreased 34%, protein ND mRNA decreased 27%, protein ND ND ND ND ND ND ND ND ND ND ND Decreased Decreased ND Decreased Decreased Decreased

57% 69% 48% 59% 84% (Continued )

TABLE III (Continued ) mRNA or protein: Antibody

Eastwood et al. (2000)

Assay

Complexin II: LP27

IAR

Synaptophysin mRNA Synaptophysin: SY38 DAKO

ISH

Regionsa

Cases

11 con 11 sch

IAR

Finding

DG CA4 CA3 CA1 Subiculum PHG

ND ND Decreased 12% ND ND Decreased 14%

BA9/46 (frontal) BA24 (cingulate) BA22 (temporal)

mRNA ND, IAR ND mRNA ND, IAR ND mRNA decreased 26%, IAR ND mRNA decreased 44%, IAR decreased 14% mRNA ND, IAR ND, WB ND mRNA ND, IAR ND mRNA ND, IAR ND

188

BA17 (occipital) Synaptophysin mRNA

ISH

8 con

BA9/46 (frontal)

Synaptophysin: SY38 DAKO

IAR WB synaptosomal

8 sch

BA22 (temporal) BA17 (occipital)

Synaptophysin (M776 DAKO)

IAR

13 con

ML DG

10–11 sch

CA4 CA3 CA1 Subiculum

Protein studies Eastwood and Harrison (1995)

PHG

Left ND, right decreased 20% ND ND ND Left ND, right decreased 23% Left ND, right decreased 20%

Perrone-Bizzozero et al. (1996)

Blennow et al. (1996)

Synaptophysin (polyclonal DAKO)

IB synaptosomal membranes

4–6 con

BA9 (frontal)

Decreased 23%

5–10 sch

BA10 (frontal) BA17 (occipital) BA20 (temporal)

Decreased 44% ND Decreased 26%

39 19 9 7

Thalamus BA9 (frontal) Hippocampus

Left decreased 50%, right ND ND Decreased 18%

IB

con sch con sch

Synaptotagmin I (clone 41.1)

IB delipidized homogenates

Glantz and Lewis (1997)

Synaptophysin (Sigma and Boehringer)

ICC

10 con 10 sch

BA9 (frontal) BA46 (frontal) BA17 (occipital)

Decreased 15% Decreased 15% ND

Gabriel et al. (1997)

Synaptophysin: EP10

ELISA: homogenate

12–15 con 8–19 sch

BA24 (cingulate)

Increased 33%

BA20 (temporal), BA7 (frontal), BA8 (parietal) BA24 (cingulate) BA20 (temporal), BA7 (frontal), BA8 (parietal) BA24 (cingulate) BA20 (temporal), BA7 (frontal), BA8 (parietal) BA24 (cingulate) BA20 (temporal), BA7 (frontal), BA8 (parietal)

ND

189

Rab3a (clone 42.2)

Synaptophysin: SP4

SNAP-25: SP14

Syntaxin: SP6

Young et al. (1998)

SNAP-25: SP12

ICC

12 con

Hippocampus

Increased 40% ND Increased 34% ND Increased 47% ND

Decreased 5–20%

(Continued )

TABLE III (Continued ) mRNA or protein: Antibody

Assay

Cases

Regionsa

Finding

t-

12 sch

Thompson et al. (1998)

ICC

12 con 12 sch

SNAP-25: SMI-81

IB: synaptosomal membranes

10 9 7 4 6 10 5 5

con sch con sch con sch con sch

10 13 12 18 10 15 6 5 9 13 7 12

con sch con sch con sch con sch con sch con sch

190

Synaptophysin: EP10

Davidsson et al. (1999)

Synaptophysin (clone 7.2)

IB

Dentate granule cell layer Subiculum Presubiculum Hippocampus Dentate granule cell layer Subiculum Presubiculum

Decreased 42% Decreased 7% ND ND Increased 56% ND ND

BA17 (occipital)

ND

BA20 (temporal)

Decreased 33%

BA9 (frontal)

Increased 32%

BA10 (frontal)

Decreased 56%

Hippocampus

Decreased 31%

BA32/33 (cingulate)

Decreased 33%

BA39/40 (parietal)

ND

BA45/46 (frontal)

ND

BA21/22 (temporal)

ND

Cerebellum

ND

Rab3a (clone 42.2)

IB

21 15 10 13 12 18 10 15 6 5 9 13 7 12

con sch con sch con sch con sch con sch con sch con sch

Thalamus

Decreased 55%

Hippocampus

Decreased 30%

BA32/33 (cingulate)

Decreased 33%

BA39/40 (parietal)

Decreased 22%

BA45/46 (frontal)

Decreased 31%

BA21/22 (temporal)

ND

Cerebellum

ND

SNAP-25: SMI-81

ICC

15 con 15 sch

Hippocampus Dentate granule cell layer

ND Decreased 51%

Nowakowski et al. (2002)

Synapsin I: clone Cl 46.1

ICC

16 con

OML

Decreased 46%

17 sch

IML GCL CA4 CA4-MF CA1-SO

Decreased Decreased Decreased Decreased ND

BA21 (temporal) BA22 (temporal)

ND Increased 100% in sch < 75

BA21 (temporal) BA22 (temporal)

ND ND

191

Fatemi et al. (2001)

56% 40% 31% 26%

mRNA studies Tcherepanov and Sokolov (1997)

Synapsin 1A

Synapsin 1B

mRNA RT-PCR

4 con < 75 6 con > 75 9 sch < 75 13 sch > 75

(Continued )

TABLE III (Continued ) mRNA or protein: Antibody

Assay

Cases

Synaptophysin

Regionsa

Finding

BA21 (temporal) BA22 (temporal)

ND ND

Synaptophysin mRNA

ISH

11 con 6 sch

DG CA4 CA3 CA1 Subiculum PHG

ND ND Decreased 50% ND ND Decreased 56%

Eastwood and Harrison (2000)

Complexin I

ISH

15 con 15 sch

CA4 CA3 Subiculum PHG DG CA4 CA3 CA1 Subiculum PHG

Decreased 50% ND ND ND Decreased 13% Decreased 40% ND ND ND ND

ISH

10 con 11 sch

DG CA4 CA3 CA1 Subiculum ERC

ND Decreased 62% Decreased 43% ND ND ND

192

Eastwood and Harrison (1999)

Complexin II

Webster et al. (2001)

Synaptophysin mRNA

a ERC, entorpheral cortex; DG, dentate gyrus; OML, outer molecular layer; IML, inner molecular layer; GCL, granule cell layer; CA, Ammon’s horn; MF, mossy fiber; SO, stratum oriens.

TABLE IV Studies Evaluating Protein and mRNA Together Region

Protein (antibody)

Cases

Change

Hippocampus Parahippocampal gyrus

Synaptophysin mRNA ISH ICC (M776 DAKO)

13 con 7 sch

mRNA decreased 27–38% CA3, CA4, subiculum, parahippocampal gyrus mRNA ND CA1 Protein all areas ND

Harrison and Eastwood (1998)

CA4 CA3 Subiculum PHG DG CA4 CA3 CA1 Subiculum PHG

Complexin 1 mRNA ISH, IAR (SP33)

11 con 11 sch

mRNA mRNA mRNA mRNA mRNA mRNA mRNA mRNA mRNA mRNA

ND, protein ND ND, protein ND ND, protein ND ND, protein ND decreased 57%, protein ND decreased 69%, protein ND ND, protein decreased 12% decreased 48%, protein ND decreased 59%, protein ND decreased 84%, protein decreased 14%

Karson et al. (1999)

BA10 (frontal)

Synaptophysin mRNA Northern blot IB homogenate SNAP-25 mRNA Northern blot IB homogenate

12 con 14 sch

mRNA Protein mRNA Protein

ND decreased 25% ND decreased 50%

Eastwood et al. (2000)

BA9/46 (frontal) BA24 (cingulate) BA22 (temporal) BA17 (occipital)

ISH IAR

11 con 11 sch

mRNA ND, IAR ND mRNA ND, IAR ND mRNA decreased 26%, IAR ND mRNA decreased 44%, IAR decreased 14%

193

Eastwood et al. (1995)

Complexin II mRNA ISH, IAR (LP27)

(Continued )

TABLE IV (Continued ) Region

194

Eastwood et al. (2001)

Imai et al. (2001)

Protein (antibody)

Cases

Change

BA9/46 (frontal) BA22 (temporal) BA17 (occipital)

ISH IAR IB synaptosomal

8 con 8 sch

mRNA ND, IAR ND, WB ND mRNA ND, IAR ND mRNA ND, IAR ND

Cerebellum

Synaptophysin mRNA ISH, IAR SY38 (DAKO) Complexin I mRNA ISH, IAR (SP33) Complexin II mRNA ISH, IAR (LP27)

16 con 16 sch

mRNA decreased 31%, protein ND

Synapsin II mRNA, IB NSF mRNA, IB

8–10 con 6–8 sch

BA46 (frontal)

mRNA ND, protein ND mRNA decreased 36%, protein decreased 15–20% mRNA ND, protein ND mRNA ND, protein ND

PRESYNAPTIC PROTEINS AND SCHIZOPHRENIA

195

he latter technique demonstrated diVerences in protein in schizophrenia, whereas mRNA was unchanged (Karson et al., 1999). More studies are required to determine the relationship between changes in mRNA coding for presynaptic proteins in schizophrenia and the amounts of the proteins themselves.

VI. Microarray Studies

Results of several microarray studies also contribute to the questions posed earlier. The ﬁrst study considered gene expression patterns in prefrontal cortex. Overall, genes involved with presynaptic function were reported to have lower expression in schizophrenia (Mirnics et al., 2000). These included NSF and synapsin II, as well as synaptojanin 1 and synaptotagmin V. Of note, diVerent patterns of relative expression of these genes were observed when comparing across cases, and other genes coding for presynaptic proteins also exhibited more subtle changes in expression in schizophrenia. Limited support for the involvement of presynaptic protein genes was provided by one independent microarray study of the prefrontal cortex (Vawter et al., 2002a), but not by another (Hakak et al., 2001). Entorhinal cortex layer II neurons (also known as pre- cells) are the origin of the perforant pathway projection to the molecular layer of the dentate gyrus and Ammon’s horn of the hippocampus. Several studies suggest that these neurons are positioned abnormally in schizophrenia, indicating a disturbance of brain development (reviewed in Falkai et al., 2000). Furthermore, the terminal ﬁeld of the projection from these cells appears to have lower presynaptic proteins, including synaptophysin (Eastwood and Harrison, 1995), synapsin I (Nowakowski et al., 2002), and SNAP-25 (Young et al., 1998). In an examination of gene expression in pre- cells with a microarray strategy, a decreased expression of SNAP-25, synaptotagmins I and IV, and synaptophysin was detected, along with an increased expression of syntaxin (Hemby et al., 2002). This study supports ﬁndings reviewed previously, indicating that in the same population of neurons, divergent expression patterns for presynaptic proteins occurred in schizophrenia.

VII. Summary

Overall, the studies reviewed here generally indicate that diVerences exist in presynaptic proteins and their mRNAs between schizophrenia and control samples. However, the individual proteins aVected and the speciﬁc regions altered are not always consistent between studies. The ubiquitous presynaptic

196

HONER AND YOUNG

protein synaptophysin is not always aVected, even in the presence of alterations of other presynaptic proteins. Synaptic pathology in schizophrenia does not appear to be diVuse. Between-protein and between-region eVects on presynaptic proteins in schizophrenia are observed. There may be some eVects of abnormal brain development on synaptic connectivity in schizophrenia. However, the abnormalities in presynaptic proteins may also represent an ongoing dynamic equilibrium among synapse formation, remodeling, and elimination, which may diVer in schizophrenia compared with health.

References

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MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING

Svetlana V. Kyosseva Department of Biochemistry and Molecular Biology University of Arkansas for Medical Sciences Little Rock, Arkansas 72205

I. Introduction II. Mitogen-Activated Protein (MAP) Kinase Cascades A. Extracellular Signal-Regulated Kinase B. c-Jun N-Terminal Kinase C. p38 MAP Kinase D. MAP Kinase Phosphatases E. Transcription Factor Targets III. Role of MAP Kinases in the Central Nervous System IV. MAP Kinases in Schizophrenia V. MAP Kinases in the Phencyclidine Rat Model of Schizophrenia VI. MAP Kinases and Psychiatric Disorders VII. Conclusions and Future Directions References

The mechanism by which cells respond to extracellular stimuli involves a series of signal transduction events across the cell membrane and through the cytoplasm to the nucleus. Mitogen-activated protein (MAP) kinases are important mediators of signal transduction and play a key role in the regulation of many cellular processes, such as cell growth and proliferation, diVerentiation, and apoptosis. In mammalian cells, three major groups of MAP kinases have been identiﬁed: extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase ( JNK), and p38 MAP kinase. It is well documented that ERK is typically stimulated by growth-related signals, whereas the JNK and p38 MAP kinase cascades are activated by various stress stimuli. Studies have indicated that MAP kinases are expressed abundantly in the central nervous system (CNS) and that ERK is involved in long-lasting neuronal plasticity, including long-term potentiation and memory consolidation. While the role of ERK in neuronal plasticity and behavioral adaptation is beginning to emerge, the role of MAP kinase signal transduction cascades in major psychiatric disorders, including schizophrenia, is not well understood. This review outlines the intermediates of this signaling cascade and downstream transcription factor targets and recent evidence implicating MAP kinases to important biological functions in the CNS. Evidence from human postmortem studies, as well as from the phencyclidine model of schizophrenia, that

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diVerent MAP kinase cascades may be involved in the pathogenesis of schizophrenia, and potentially in other psychiatric disorders, is presented. Knowledge of MAP kinase signaling will aid greatly in our ability to understand causal changes in disease process and may lead to new therapeutic approaches in controlling or treating schizophrenia.

I. Introduction

How signals from the surface of neurons get relayed to the nucleus and other points within the cells (signal transduction) is a dimension of neurotransmission in schizophrenia that is recently beginning to receive attention. The brain is a signal-processing organ responsible for mental health. Its function is to receive, integrate, and respond to stimuli, as well as to adapt to environmental changes. Therefore, poorly generated brain signals, or their misinterpretation, lead to dysfunction in thinking, memory, and behavior. A diverse array of extracellular stimuli, such as neurotransmitters, growth factors, neurotrophins, and hormones, can trigger activation of the mitogen-activated protein (MAP) kinase cascades in neurons (Fukunaga and Miyamoto, 1998). On activation, MAP kinases are translocated to the nucleus and cause subsequent activation of immediate early genes through the regulation of transcription and therefore can produce a cascade of responses that may lead to functional and morphological alterations of neurons (Herdegen and Leah, 1998; Sheng and Greenberg, 1990). MAP kinase cascades are one of the most intensively studied signal transcduction pathways shown to control changes in gene expression, cytoskeletal organization, and cell division (Hill and Treisman, 1995; SchaeVer and Weber, 1999; Whitmarsh and Davis, 2000). It has become evident that MAP kinase signaling cascades are critical for the initiation of cellular mechanisms that embody, retain, and modify gene expression in neurons. Several reviews have emphasized the important role of MAP kinases in the regulation of neuronal function, especially the role of ERK in synaptic plasticity, learning, and memory (Adams and Sweatt, 2002; Fukunaga and Miyamoto, 1998; Grewal et al., 1999; Sweatt, 2001). Schizophrenia is a brain disorder that aVects approximately 1% of the population worldwide and is characterized by a disruption in cognition, including information and visual processing, executive functions, attention, memory, and emotion. Given the pivotal role of MAP kinases in the integration, ampliﬁcation, and regulation of signal transduction, it is not surprising that alterations in the expression and/or function of various intermediates of MAP kinase cascades are involved in the neuropathophysiological events occurring in the brain in schizophrenia.

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II. Mitogen-Activated Protein (MAP) Kinase Cascades

MAP kinases are a family of evolutionary well-conserved proteins that are expressed in all eukaryotic cells. In neurons, extracellular stimuli, such as nerve growth factor (Boulton et al., 1991; Miyasaka et al., 1990), neurotrophins (Cavanaugh et al., 2001; Huang and Reichardt, 2001; Kaplan and Miller, 2000), ion channels associated with N-methyl-d-aspartate (NMDA) receptors (Bading and Greenberg, 1991; Chandler et al., 2001; Fiore et al., 1993b; Kurino et al., 1995; Xia et al., 1996), muscarinic acetylcholine receptors (Rosenblum et al., 2000; Takagi et al., 2002), serotonin 5-HT (Errico et al., 2001) and dopamine receptors (Gerfen et al., 2002), and nitric oxide (NO) (Kanterewicz et al., 1998) can trigger the activation of extracellular signal-regulated kinases (ERKs), members of the MAP kinase family. Two other groups of MAP kinases, c-jun N-terminal kinase ( JNK) and p38 MAP kinase, are activated by various stress stimuli, including cytokines (Dunn et al., 2002), osmotic shock (Yamagishi et al., 2001), and NO (Cheng et al., 2001; Ghatan et al., 2000). It is important to note the selective activation of distinct MAP kinase cascades in response to diVerent extracellular stimuli leading to a wide variety of cellular functions. Activation of JNK and p38 has been shown to be necessary for the induction of apoptosis in neuronal cells, whereas ERK regulates cell growth and diVerentiation (Xia et al., 1995). However, there is increasing evidence for cross talk between distinct MAP kinase cascades (Zhang et al., 2001). A common feature of the three major MAP kinase groups is that typically they are organized in a three-kinase module (Fig. 1), consisting of a MAP kinase (MAPK), a MAP kinase activator or MAP kinase kinase (MAPKK), and a MEK activator or MAP kinase kinase kinase (MAPKKK) (SchaeVer and Weber, 1999; Widmann et al., 1999). MAP kinases are serine/ threonine kinases and are activated and phosphorylated on a Thr-X-Tyr motif. ERK has the dual phosphorylation motif Thr-Glu-Tyr, JNK has Thr-Pro-Tyr, and the Thr-Gly-Tyr motif is present in p38 MAP kinase (Davis, 1995). The overall sequence identity among ERK, JNK, and p38 MAP kinases is 40–45%.

A. Extracellular Signal-Regulated Kinase The ERK (p44 ERK1/p42 ERK2) cascade constitutes a major signaling conserved throughout evolution and is stimulated in mammalian cells via tyrosine kinase receptors (TKRs) and G-protein-coupled receptors (GPCRs) through both Ras-dependent and Ras-independent pathways (Lewis et al., 1998). Stimulation of TKRs leads to the activation of receptor tyrosine kinase domains, leading to recruitment of several adapter proteins, such as Shc, Grb2, and SOS (Son of sevenless), and transducing the signal to GTP-binding proteins of the Ras family

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Fig. 1. MAP kinase signaling cascades. In mammalian cells, the three major groups of MAP kinases, ERK, JNK, and p38, are activated by various extracellular stimuli, leading to the phosphorylation of specific transcription factors. See text for full details and discussion. (See Color Insert.)

(H-Ras, K-Ras, N-Ras, Rap1) (Fig. 1). Activated Ras-GTP can activate MAPKKK of the Raf family (A-Raf, B-Raf, c-Raf-1), which in turn phosphorylates and activates MAPKK, MEK1/2 and MAPK, ERK1/2. Many GPCRs can also activate the ERK1/2 cascade. When Gi-coupled receptors are involved, activation of phosphatidylinositol-3-kinase is required, in addition to Shc, Grb2, Sos, Ras, Raf, and MEK (Touhara et al., 1995). There are currently seven MAP kinases cloned deﬁned as ERK1–7. ERK1 and ERK2 were the ﬁrst identiﬁed members of ERK/MAP kinases and the most widely studied members of the MAP kinase cascade (Boulton et al., 1990). They are expressed ubiquitously and have 90% sequence identity (Boulton et al., 1991). However, some studies have indicated that the two ERK isoforms may be regulated diVerently (review

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by Adams and Sweatt, 2002). ERK1-deﬁcient mice are viable and fertile, whereas ERK2-deﬁcient mice are embryonic lethal (Pouyssegur et al., 2002). ERK1/2 activation is essential for cell growth by controlling nucleotide synthesis, gene expression, and protein synthesis (Whitmarsh and Davis, 2000). The other ERK isoforms are less well characterized and understood. It has been shown that ERK3 is activated by protein kinase C isoforms (Sauma and Friedman, 1996), ERK4 is activated in response to growth factors (Peng et al., 1996), and ERK5 is stimulated by reactive oxygen species (Abe et al., 1997). ERK6 is highly expressed in human skeletal muscle (Lechner et al., 1996) and is cloned ERK7 (Abe et al., 1999).

B. c-Jun N-Terminal Kinase A MAP kinase, which was activated in response to cellular stress stimuli, was isolated and cloned by two groups. The human homolog was named JNK (Derijard et al., 1994), and the rat homolog was called stress-activated protein kinase (SAPK) (Kyriakis et al., 1994). JNK has been shown to be activated through a variety of cell surface receptors, including TKRs, GPCRs, and cytokine receptors (CRs) (Fig. 1). In contrast to ERK, JNKs are activated by the Rho family of small GTPases, including Rho, Rac, and Cdc42. These proteins in turn activate MEKK1/3, MKK4/7, and JNK. In addition to MEKK1/3, apoptosis signalregulated kinase (ASK) 1, mixed lineage kinases (MLKs), and TGF -activated kinase (TAK) 1 regulate the JNK cascade. Activation of JNK has been implicated to play a role in apoptosis, oncogenic transformation, inﬂammation, development, and diVerentiation of the immune system, as well as in renal, hepatic, and cardiovascular function (Tibbles and Woodgett, 1999). There are currently three mammalian JNK isoforms: JNK1, JNK2, and JNK3. The JNK1 p54 and JNK2 p46 isoforms are expressed ubiquitously whereas JNK3 is expressed selectively in neurons (Davis, 2000). It has been shown that JNK knockout mice are viable, but that JNK is required for many aspects of normal cellular physiology, including apoptosis and immune responses (Weston and Davis, 2002).

C. p38 MAP Kinase p38 MAP kinase was ﬁrst identiﬁed in Saccharomyces cerevisiae as a protein kinase activated by hyperosmolarity, Hog1 (Han et al., 1994). Four isoforms of p38 MAP kinase (, , , and ), which derive from diVerent genes, have been identiﬁed (Goedert et al., 1997; Han et al., 1994; Jiang et al., 1996). DiVerent p38 isoforms are activated by cellular stress (UV light, osmotic and heat shock, lipopolysaccharides), cytokines (interleukin-1 and tumor necrosis factor-),

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GPCRs (Widmann et al., 1999), and NO (Cheng et al., 2001; Ghatan et al., 2000). Similar to JNK, activation of p38 MAP kinase through either stress or cell surface receptors involves members of the Rho family, which can activate and phosphorylate MEKK1/4, MLKs, TAK1, ASK1, and MAPKK MKK3/6. The activation of p38 MAP kinases is generally associated with apoptosis; however, their function is still poorly deﬁned and understood. It is suggested that p38 may be involved in myocardial cell growth and sarcomeric organization (Zechner et al., 1997) and plays a role in the immune response (Dong et al., 2002).

D. MAP Kinase Phosphatases MAP kinase phosphatases (MKPs) are dual-speciﬁcity phosphatases capable of dephosphorylating both threonine and tyrosine residues within the ‘‘activation loop’’ of MAP kinases. Evidence indicates that MKPs participate in the negative feedback control of MAP kinase activation. At least nine MKPs have been isolated and characterized in mammalian cells (Keyse, 2000). Studies have shown that certain MKPs display distinct substrate speciﬁcity for diVerent MAP kinases in vitro and in vivo (Slack et al., 2001; Zhao and Zhang, 2001). For example, MKP3 is highly speciﬁc more than 100-fold for ERK2 than p38 in vitro (Groom et al., 1996). Another interesting feature of MKPs is their distinct expression pattern and subcellular localization. MKP-1 and MKP-2 are localized within the nucleus, MKP-3 appears to be cytosolic, and MKP-4 is present in the cytosol as well as in the nucleus (Camps et al., 2000). What physiological role MKPs play in the overall control of MAP kinase signaling cascades is not fully understood. It has been shown that disruption of the MKP-1 gene does not aVect mouse development and MAP kinase activity in cell cultures from these animals (Dorfman et al., 1996).

E. Transcription Factor Targets A major role for MAP kinases is to transmit signals from the cell surface to the nucleus, where the transcription of speciﬁc genes is induced by the phosphorylation and activation of transcription factors (Whitmarsh and Davis, 1996). This leads to an altered transcription of genes that regulate growth, apoptosis, diVerentiation, or altered posttranscriptional events. One of the most widely studied nuclear targets of MAP kinase in mammals is the activator protein (AP-1) transcription factor (Whitmarsh and Davis, 1996). AP-1 is a homodimer and/or heterodimeric complex composed of members of Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) families. Dimerization can also occur with other transcription factor families, such as cAMP response element-binding

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protein (CREB) and activating transcription factor (ATF) (Herdegen and Leah, 1998). AP-1 is present in the promoter regions of certain neuronal genes that encode neuropeptides, neurotransmitter receptors, or tyrosine hydrohylase (Chaudhuri, 1997; Nestler et al., 1997). The regulation of c-fos is the best characterized of the AP-1 transcription factor genes (Pennypacker, 1998). The c-fos promoter contains several regulatory elements, including CRE, serum response element (SRE), and a Sis-inducible enhancer (SIE). The CRE is recognized by the CREB and ATF, whereas the SRE is recognized by a complex of serum response factor (SRF) and ternary complex factor (TCF) that includes Elk-1. The SIE site contains the signal transducer and activator of transcription (STAT) factor that is activated by cytokines and growth factors. The regulation of AP-1 activity is complex, and its function and role has been reviewed extensively elsewhere (Karin et al., 1997; Shaulian and Karin, 2002). Two other potential nuclear targets of MAP kinase signaling are the Ets family of transcription factors that have been implicated in lymphoid development and the myc/max transcription factor family that contributes to proliferation, diVerentiation, and apoptosis. As described earlier, MAP kinase cascades play a key role in integrating a variety of cell surface signals. They transmit signals through a complex array of intracellular proteins to downstream nuclear targets, which can amplify or modify the signal at any point in order to achieve coordinated and adaptive responses, including development, diVerentiation, apoptosis, or diVerent types of physiological and pathological events at the cellular level.

III. Role of MAP Kinases in the Central Nervous System

The three MAP kinase groups (ERK, JNK, and p38) are abundant in the central nervous system (CNS) and are activated during various physiological and pathological events (Fukunaga and Miyamoto, 1998). Both ERK1 and ERK2 are highly expressed in the postmitotic neurons, and ERK2 is localized in neuronal cell bodies and dendrites (Fiore et al., 1993a). Several groups have reported a selective activation of ERK2 by various stimuli (Bading and Greenberg, 1991; English and Sweatt, 1996, 1997; Fiore et al., 1993b). The diVerential activation of the two isoforms raises the possibility of a diVerential function of ERK in neurons, as studies have shown that ERK1 knockout mice cannot compensate for the loss of ERK2. JNKs are expressed heterogeneously in the adult rat brain and distributed diVerentially from ERKs (Carbonni et al., 1997; Carletti et al., 1995). p38 MAP kinase has been shown to be expressed in various tissues and cells (Kawasaki et al., 1997; Xia et al., 1995). In addition, Maruyama et al. (2000) demonstrated a cell type-speciﬁc expression of these kinases in the CNS with a strong immunoreactivity observed in myelin sheath-like structures, but not in

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axons. Accumulating evidence suggests physiological and pathological roles of MAP kinases in the CNS. The three MAP kinases are activated diVerentially in the rat brain following electroconvulsive shock, an eVective treatment for psychiatric disorders (Ahn et al., 2000; Baraban et al., 1993; Oh et al., 1999), and brain ischemia (Gu et al., 2001; Nozaki et al., 2001). Dash et al. (2002) showed that ERK is involved in cognitive and motor deﬁcits following experimental traumatic brain injury in rats. There is now considerable evidence suggesting a role of MAP kinase signaling cascades in the CNS and especially the involvement of ERKs in synaptic plasticity, learning, and memory. Because long-term neuronal plasticity and memory consolidation require de novo gene expression and protein synthesis, this made MAP kinase cascades attractive candidates in the regulation of synaptic plasticity and long-term memory (LTM). In the rat, activation of ERK was reported to be required for the expression of LTM induced by a fear conditioning (Atkins et al., 1998). Furthermore, results revealed that the MAP kinase cascade is involved in long-term spatial memory in the rat (Blum et al., 1999; Selcher et al., 1999). Moreover, Zhen et al. (2001) demonstrated a learning-dependent activation of ERKs and p38 MAP kinase in rabbits. Evidence also suggests that ERK and JNK are activated speciﬁcally and diVerentially in the insular cortex after exposure to a novel taste and that this activation is required for the consolidation of long-term taste memory (Berman et al., 1998). The ERK cascade is also involved in longterm potentiation (LTP) and long-term depression (LTD). A number of studies have provided evidence that the activation of ERKs is necessary both for the induction of LTP in the hippocampus (English and Sweatt, 1996; Rosenblum et al., 2002) and the insular cortex ( Jones et al., 1999) and for the LTD in Purkinjie cells (Kawasaki et al., 1999). Taken together, these results strongly suggest that MAP kinase signal transduction is an important component in long-term plasticity and learning and memory formation. It is apparent that MAP kinase cascades play a critical role in maintenance of CNS through the determination of survival or cell death depending on the cell type, as well as the type, duration, and intensity of the stimulus. JNK and p38 have been implicated in apoptosis in neuronal cells, whereas ERK regulates cell growth and diVerentiation (Xia et al., 1995). For example, the involvement of JNK and p38 in glutamate-induced apoptosis in cerebellar granule cells was observed (Kawasaki et al., 1997). However, studies have demonstrated that neuronal diVerentiation can also be mediated by p38 in PC12 cells (Iwasaki et al., 1999; Morooka and Nishida, 1998) and that ERK1/2 are involved in glutamateinduced apoptosis in cortical neurons ( Jiang et al., 2000). It is increasingly recognized that transcription factors play a critical role in the development and functioning of the normal nervous system, as well as in the adaptation of the nervous system to various stimuli, such as injury, growth factors, drug treatment, and physiological and pathological events (Duman et al., 1999;

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Herdegen and Leah, 1998). Perhaps CREB has been the best studied transcription factor in the CNS and has been shown to be implicated in synaptic plasticity and long-term memory formation in both invertebrates and vertebrates (Impey et al., 1998; Shaywitz and Greenberg, 1999; Silva et al., 1998). Evidence from diVerent species has indicated that transcription factor CREB regulates the synthesis of proteins necessary for long-term memory formation. In addition, CREB is required for the stability of new and reactivated fear memories (Kida et al., 2002). In vitro studies suggest that CREB is also a critical mediator in protecting neurons of cell death. For example, in mice lacking the CREB gene, sensory neurons exhibit excess apoptosis and degeneration, whereas excess apoptosis was not detected in the CNS (Lonze et al., 2002). ATF-2 is expressed at high levels in human and murine brain and is most abundant in the cerebrum, cerebellum, and forebrain. The other transcription targets of MAP kinases, such as Elk-1, Fos, and Jun family of transcription factors, are also highly expressed in neurons and play an important role in development and neuronal function (Herdegen and Leah, 1998).

IV. MAP Kinases in Schizophrenia

Although much has been discovered about the prominent role of MAP kinases in regulating gene expression in the CNS, little is known about their role and function in major psychiatric disorders, including schizophrenia. Schizophrenia is a complex neuropsychiatric disorder of uncertain etiology and pathophysiology. Evidence exists for both neurodevelopmental and neurodenegerative components in the pathophysiology of schizophrenia (Ashe et al., 2001). Neurochemical studies have shown that several neurotransmitters, including glutamate, dopamine, GABA, serotonine, and acetylcholine, may be implicated in the pathophysiology of this disorder (Benes, 2000). The expression of brain-derived neutrophic factor (BDNF) and the gene-encoding regulator of G-protein signaling 4 (RGS4) has also been demonstrated to be abnormal in schizophrenia (Mirnics et al., 2001; Takahashi et al., 2000). It has been suggested that the altered expression of RGS4 may have a profound eVect on GPCR-mediated signaling. Given the fact that the aforementioned neurotransmitters activate MAP kinases via TKRs and GPCRs, as well as the important role of MAP kinase cascades in regulation of neuronal function, there is increased interest in understanding their role in the pathogenesis of schizophrenia and other psychiatric disorders. As a ﬁrst step toward understanding the possible involvement of MAP kinase cascades in schizophrenia and whether these proteins could be targets of neuropathological events, we determined the expression of ERK1 and ERK2 in postmortem brain samples from schizophrenic patients and control subjects

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(Kyosseva et al., 1999). Total levels of ERK2, i.e., both active and inactive forms, were increased signiﬁcantly, at about 30% in the cerebellum and 40% in the thalamus (Fig. 2A). Trends of lower expression of ERK2 protein were also found in mesopontine tegmentum in schizophrenic patients. However, no signiﬁcant change was observed in the expression of ERK1 in postmortem cerebellum, thalamus, frontal pole (Broadman’s area, BA 10), or mesopontine tegmentum. As mentioned previously, several groups have found that glutamate receptor activation in neurons can lead to selective activation of ERK2 (Bading and Greenberg, 1991; English and Sweatt, 1996; Fiore et al., 1993b). The mechanism of this selective activation of ERKs is still unknown. A possible explanation of this phenomena is the speciﬁc activation of upstream activators of ERK, localization,

Fig. 2. Protein levels of ERK2 (A), JNK (B), and p38 (C) determined by Western blots in the cerebellum (CB), thalamus (TH), Broadman’s area 10 (BA), and mesopontine tegmentum (MT) of patients with schizophrenia (s) and control (c) subjects. Autoradiograms from 10 schizophrenic and 12 control subjects were scanned; results are presented in densitometry units. Values are means SD. Modified from Kyosseva et al. (1999), with permission from the Society of Biological Psychiatry. (See Color Insert.)

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and scaVolding (Adams and Sweatt, 2002). In contrast to ERK2, the protein expression of the two other members of the MAP kinase family, JNK and p38, were unchanged between schizophrenia and controls in all the brain areas studied (Figs. 2B and 2C). Interestingly, the expression of p38 MAP kinase was undetectable in the thalamus, which suggest that they are expressed diVerentially in human brain. Next, we sought to determine whether the observed increased levels of ERK were associated with changes in the levels of MKP-2, a dualspeciﬁcity threonine/tyrosine phosphatase that displays selectivity for dephosphorylation and inactivation of ERK. Indeed, the expression of MKP-2 was decreased signiﬁcantly in the cerebellum (Kyosseva et al., 1999) and thalamus (S. V. Kyosseva, unpublished observation) in patients with schizophrenia. Elevated levels of ERK could result from a decrease in the expression of phosphatases, an increase of upstream intermediates in the pathway, or both. Therefore, we examined the upstream activators in the ERK pathway and found that MEK and c-Raf are also increased in schizophrenia compared to controls. Taken together, these results suggest that speciﬁcally the entire ERK pathway is abnormal in the cerebellum and in part in the thalamus in schizophrenia. Additionally, we also described an increase in protein levels of several transcription factors, such as Elk-1, CREB, ATF-2, and c-Jun in postmortem cerebellum in schizophrenia (Kyosseva et al., 2000; Todorova et al., 2003), as well as an elevation of c-fos and c-jun mRNA in thalamus (S. V. Kyosseva, unpublished results). Furthermore, we demonstrated that the protein level of transcription factor STAT3 associated with inﬂammatory activation does not change in the cerebellum of patients with schizophrenia (Kyosseva et al., 2000). It is possible that alterations of the ERK pathway and downstream transcription factors observed in brain of patients with schizophrenia are due to chronic antipsychotic medications, as most of the schizophrenic patients had received antipsychotic drugs over many years during their illness. However, chronic administration of haloperidol and risperidon to rats did not result in alterations of ERK, MEK, CREB, ATF-2, and c-Jun (Kyosseva et al., 1999, 2000; Todorova et al., 2003). These ﬁndings suggest that alterations of ERK pathway are not likely due to antipsychotic drug treatment and thus may represent a neuropathological feature of schizophrenia, although interpretation of these data should be made with caution because it is coming from two diVerent species.

V. MAP Kinases in the Phencyclidine Rat Model of Schizophrenia

Several neurochemical hypotheses of schizophrenia, including dopamine and glutamate, have been developed based on biochemical, pharmacological, imaging, and behavioral studies. The dopamine hypothesis is based on the observation that amphetamine and other dopaminergic agents induced schizophrenia-like

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symptoms, whereas the NMDA hypothesis suggests that phencyclidine (PCP) and other NMDA antagonists also induced symptoms that closely resemble positive, negative, and cognitive symptoms of schizophrenia ( Javitt and Zukin, 1991). It has been suggested that PCP can be a useful drug-induced model of schizophrenia (Sams-Dodd, 1999; Thornberg and Saklad, 1996), although such a complex neuopsychiatric disease is diYcult to model completely and special care has to be taken when related work is carried out. We explored the PCP rodent model of schizophrenia and designed a study to determine whether repeated PCP administration to rats could produce similar changes in the ERK pathway to those observed in schizophrenia. PCP in doses of 2.5, 10, 18, and 25 mg/kg/day or saline was delivered via subcutaneous osmotic minipumps for 10 days (Kyosseva et al., 2001). The role of MAP kinase cascades leading to ERK, JNK, and p38 activation following chronic treatment of PCP in rat brain was determined using phospho-speciﬁc antibodies. As can be seen in Fig. 3 there is a dose-dependent activation in the phosphorylation of ERK1 and ERK2 in the cerebellum and no detectable activation of ERK1, ERK2, JNK1, JNK2, and p38 in the brain stem, frontal cortex, or hippocampus. The expression of activated JNK1, JNK2, and p38 was not changed signiﬁcantly in the cerebellum as well. A time-dependent activation of MEK by PCP infused for 3, 10, and 20 days was also observed in the cerebellum (Kyosseva et al., 2001). These data suggest a possible molecular mechanism for the common features between schizophrenia and PCP-induced psychosis, and it is believed that PCP infusion into rats can produce a promising model for investigating the role of MAP kinases in schizophrenia.

Fig. 3. Dose-dependent eVects of PCP on ERK, JNK, and p38 in the rat cerebellum (CB), brain stem (BS), frontal cortex (FC), and hippocampus (HC). Cytosol fractions from saline (lane 1) and 2.5 mg (lane 2), 10 mg (lane 3), 18 mg (lane 4), and 25 mg (lane 5) PCP-infused rats were subjected to Western blot for 10 days. Reproduced from Kyosseva et al. (2001). (See Color Insert.)

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In summary, the presented ﬁndings from human postmortem studies and the PCP rodent model of schizophrenia strongly suggest that schizophrenia may be due, at least in part, to abnormalities of the ERK pathway.

VI. MAP Kinases and Psychiatric Disorders

Major depressive disorder and bipolar aVective disorder are common and severe psychiatric disorders with still poorly understood etiology and pathophysiology. Although they are highly heritable disorders, nongenetic factors, including environmental, can play a role in the disease process. It has been shown that schizophrenia and bipolar disorder are similar in several epidemiological aspects, such as age of onset, lifetime risk, course of illness, and genetic susceptibility (Berrettini, 2000). Despite the research eVorts, the biological and pathological markers associated with speciﬁc psychiatric disorders remain poorly documented. The ﬁndings of abnormalities in the ERK pathway raise questions as to whether the observed changes are speciﬁc to the cerebellum of schizophrenic patients or whether these abnormalities can occur in the cerebellum of patients with a major psychiatric disorder. Using diVerent patient populations of schizophrenia, bipolar disorder, major depression, and a control group from the Stanley Foundation Neuropathology Consortium, we determined the protein expression of ERK, JNK, and p38 MAP kinase in the cerebellum (cerebella hemisphere). Unpublished results show that in contrast to cerebellar vermis, used in previous investigations, the ERK2 protein was decreased in the cerebella hemisphere in schizophrenic patients without any change in bipolar and depressive disorder. The lack of ERK2 abnormalities in the cerebellum of bipolar and depressed patients in contrast to that seen in schizophrenic patients suggests regional and disease-speciﬁc changes in ERK2 expression. Again, no alteration of ERK1 protein between control and patients group was observed. The trend of lower JNK1 and JNK2 levels was found only in patients with major depression. Surprisingly, the expression of p38 showed a signiﬁcant two-fold decrease in schizophrenia and depression. Because ASK-1 is a MAPKKK involved in stress-induced signaling through JNK and p38 pathways and plays a role in apoptosis, we also determined the expression of ASK-1. However, the protein levels of ASK-1 were not altered in any patients group. Based on current data, it appears that there is possible cross talk among the three MAP kinases, as the integration of signals may take place at many levels, which is upstream or within the cascades. Therefore, a comprehensive knowledge of the upstream activators of JNK and p38 is needed to provide insights about their involvement in schizophrenia and other major psychoses. These are very preliminary results and more analyses are needed to elucidate the molecular mechanism underlying the observed abnormalities.

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Nonetheless it can be hypothesized that MAP kinases are expressed and regulated diVerentially in a neuroanatomically diVerent pattern in schizophrenia and depression but not in bipolar disorder. Evidence for a possible role of MAP kinases in depression has also been provided by Dwivedi et al. (2001). They observed that the activity and expression of ERK1 and ERK2 were decreased signiﬁcantly in BA 8, 9, and 10 and the hippocampus of depressed suicide subjects without any change in the cerebellum. The aforementioned ﬁndings suggest involvement of the ERK pathway in cerebellar abnormalities speciﬁcally in schizophrenia, but not in other psychiatric disorders. Available evidence supports the idea that a disturbed neuronal circuitry linking the cerebellum via connections with both the dorsolateral prefrontal cortex and the thalamus may contribute signiﬁcantly to cognition-related symptoms in schizophrenia (Andreasen et al., 1998).

VII. Conclusions and Future Directions

In recent years, we have witnessed a mounting body of data suggesting that MAP kinase cascades regulate neuronal survival, diVerentiation, and plasticity, perhaps reﬂecting our growing interest in understanding the role of this signaling pathway in schizophrenia and other psychiatric disorders. The evidence reviewed in this chapter indicates that MAP kinase-signaling cascades are undoubtedly involved in neuropathological events that occur in the brain in schizophrenia. Moreover, there are compelling reasons to believe that MAP kinases are regulated diVerentially in diVerent brain regions in schizophrenia and major depression and may play a role in psychiatric disorders. Further studies are required to establish the precise molecular mechanisms responsible for such speciﬁc alterations of MAP kinases in order to determine where and when a particular signaling pathway is impaired. It is also hoped that this research may lead to the development of better molecular markers in investigating the pathophysiology of schizophrenia and other psychiatric disorders. The existence of speciﬁc MAP kinase inhibitors may set the stage for future therapeutic interventions aimed at correcting the abnormalities of MAP kinase signaling.

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POSTSYNAPTIC DENSITY SCAFFOLDING PROTEINS AT EXCITATORY SYNAPSE AND DISORDERS OF SYNAPTIC PLASTICITY: IMPLICATIONS FOR HUMAN BEHAVIOR PATHOLOGIES

Andrea de Bartolomeis and Germano Fiore Laboratory of Molecular Psychiatry Department of Neuroscience and Behavioral Sciences University Medical School of Naples, Federico II, Italy

I. Introduction II. Structural and Functional Organization of Postsynaptic Density (PSD) Proteins: An Overview A. ScaVolding Proteins B. MAGUK Proteins C. Protein Kinases and Protein Phosphatases D. Receptors E. PSD Proteins and the Concept of Microdomain F. PSD Proteins and Receptor DiVusion III. PSD-95/SAP90 A. Structure B. Functions C. PSD-95/SAP90 and Neuropsychiatric Disorders IV. Shank/ProSAP Proteins A. Structure B. Functions C. Shank3 and the 22q13.3 Deletion Syndrome V. SAP97 A. Structure B. Functions C. SAP97 and Schizophrenia VI. Homer Proteins A. Structure B. Functions C. Homer 1a Induction and Implication for Neuropsychiatric Disorders VII. Conclusive Remarks References

Excitatory synapses are characterized by an electron-dense thickening at the cytoplasmic surface of the postsynaptic membrane, called the postsynaptic density (PSD). The PSD is a ﬁbrous specialization of the submembrane cytoskeleton approximately 30–40 nm thick and about 100 nm wide. Hundreds of molecules have been identiﬁed in the PSD: ion-gated and G-protein-coupled receptors, association, adaptors, and scaVolding proteins, key enzymes involved INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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in phosphorylation–dephosphorylation mechanisms, and cytoskeletal proteins. Each of these proteins may have a pivotal function in setting the molecular scenario for the development of synaptic plasticity. ScaVolding proteins are major players in the organization of the postsynaptic signal transduction machinery, they regulate receptor traYcking and clustering, modulate axon pathﬁnding, and drive the correct targeting of neuronal proteins to their appropriate cytoplasmic compartment. Emerging ﬁndings suggest a relevant involvement of PSD scaVolding/adaptor proteins in behavior modulation in animal models of synaptic plasticity disorders and pharmacological isomorphisms.

I. Introduction

Synaptic plasticity can be viewed as the result of a highly regulated form of neuronal communication (Bear and Malenka, 1994; Sheng and Kim, 2002). Among the structures associated with the chemical synapse, postsynaptic density (PSD) has recently attracted special interest for the sophisticated mechanisms of molecule interaction and regulation of signal transduction that it shows (Kennedy, 1997, 2000; Scannevin and Huganir, 2000). Postsynaptic density at the excitatory synapse is a disc-shaped structure that can be recognized under electron microscopy as an organelle of about 50 nm thick, localized beneath the postsynaptic membrane of the type I synapse (axodentric synapse) prominently and facing the presynaptic active zone (Kennedy, 2000). The PSD can be puriﬁed from brain tissue by diVerential centrifugation, and several proteins have been identiﬁed by means of mass spectrometry (Yamauchi, 2002). Ionic and G-protein-coupled glutamate receptors, scaVolding proteins, adaptor and receptor interactor proteins, kinases and phosphatases, key enzymes, and cytoskeleton proteins have been described at the PSD (Garner et al., 2000; Kennedy, 1998; Sheng and Pak, 2000; Yamauchi, 2002; ZiV, 1997), constituting a dynamic protein lattice (Scannevin and Huganir, 2000) with a highly regulated molecular organization. PSD proteins are thought to play a major role in neurodevelopment (Foa et al., 2001), dendritic spine formation (Sala et al., 2001), receptor electrophysiological property regulation (Yamada et al., 1999a,b), receptor clustering (Ehlers et al., 1996; Kornau et al., 1997; Sheng, 1996; Sheng and Kim, 2002), and linking membrane receptors directly to second messenger signaling (Xiao et al., 1998). Moreover, ﬁndings have shown a putative role for PSD proteins in human behavior pathologies and potential implications in pharmacotherapy (de Bartolomeis et al., 2002; Nesslinger et al., 1994; Ohnuma et al., 2000; Prasad et al., 2000). All the major components of the PSD have been implicated directly or indirectly in synaptic plasticity regulation; it is beyond the scope of this review to describe the massive amount of ﬁndings on the role of PSD

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proteins in neuronal function (Sheng and Kim, 2002). This review focuses on PSD scaVolding proteins at the glutamatergic postsynaptic density that have been demonstrated to be involved speciﬁcally in disorders of synaptic plasticity, with special emphasis on those proteins for which data are available linking them with human behavior pathologies and, possibly, with their treatment. Nevertheless, animal models and pharmacologic isomorphisms are considered when pertinent to better understand the pathophysiology of synaptic plasticity disorders in humans.

II. Structural and Functional Organization of Postsynaptic Density (PSD) Proteins: An Overview

The PSD at the excitatory synapse is a hierarchically and highly dynamic organized structure where signal transduction is processed, modiﬁed, integrated, and propagated. Proteins of the PSD may be classiﬁed according to their function in scaVolding, association, adhesion, cytoskeletal elements, and as kinase and phosphatase enzymes (Yamauchi, 2002). Each PSD protein may have a pivotal function in setting the scenario for the development of synaptic plasticity, and emerging ﬁndings point to an implication of PSD proteins in behavior modulation in animal models, as well as in pharmacological isomorphisms.

A. Scaffolding Proteins Among PSD molecules believed to have a direct role in synaptic plasticity, scaVolding proteins deserve special attention for their multifunctional property of adaptor proteins (ZiV, 1997). ScaVolding proteins may inﬂuence the function and availability of receptors by clustering and connecting them physically with other pivotal PSD proteins as well as with intracytoplasmic molecules. Considering the high concentration of glutamate receptors of ionic type [amino-3-hydroxy-5methyl-4-isoxazole propionate (AMPA) and N-methyl-d-aspartate (NMDA) receptors] and metabotropic type (mGluR) at PSD of excitatory synapses, the correct alignment and the precise spatial distribution of these receptors, as well as the speciﬁc interaction with intracytoplasmic proteins responsible for integrating the signal transduction, are crucial requirements. Postsynaptic density 95/synapse-associated protein 90 (PSD-95/SAP90) (Cho et al., 1992; Kistner et al., 1993), Homer family (Brakeman et al., 1997; Xiao et al., 1998), and glutamate receptor interacting protein (GRIP) (Dong et al., 1997, 1999) are the major postsynaptic density proteins at glutamatergic synapse with a

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scaVolding function and interacting with the NMDA NR2 subunit, type I mGluR (mGluR1 and mGluR5) and AMPA receptors (AMPA-R), respectively. It is worth emphasizing that, based on the nomenclature of glutamate receptorassociated proteins, the scaVolding proteins may be also classiﬁed as glutamate receptor interactors, a group including several other molecules regulating receptor signal transduction but with no fully demonstrated scaVolding function, such as Yotiao (Lin et al., 1998), AMPA receptor-binding protein (ABP) (Srivastava et al., 1998), mammalian Lin seven (MALS) ( Jo et al., 1999), neural activity-regulated pentatraxin (NARP) (O’Brien et al., 1999), and protein interacting with C kinase (PICK 1) (Xia et al., 1999). Finally, protein members of the same family of PSD proteins may have diVerent functions; however, a clear dissection between scaVolding and anchoring or adaptor function may not be clear-cut. This is the case in the Homer family with Homer 1b included in the scaVolding group of proteins and Homer 1c demonstrated to have a scaVolding but also anchoring function (Ciruela et al., 2000).

B. MAGUK Proteins Based on gene structure and domain interaction speciﬁcity, some scaVolding proteins also belong to a large group of membrane-associated proteins called membrane-associated guanylate kinase (MAGUK) proteins (Niethammer et al., 1996). This group includes, among others, PSD 95/SAP90 (Cho et al., 1992), PSD 93/chapsyn-110 (Kim et al., 1996), SAP 97 (Muller et al., 1995), and SAP 102. In addition to the guanylate kinase domain, MAGUK proteins contain a SH3 domain and three PDZ domains (see Section III). The complexity of MAGUK protein structure may explain the multiple modulator functions of these groups of proteins demonstrated to be involved not only in receptor clustering, but also in regulation of the electrophysiological properties of the receptor (Yamada et al., 1999a), as well as in linking glutamate receptors directly to second messenger signaling systems (Brenman et al., 1996a).

C. Protein Kinases and Protein Phosphatases Several kinase proteins have been identiﬁed at the PSD: calcium/calmodulindependent protein kinase type II (CaMKII), protein kinases A and C, MAP kinase, the Src family tyrosine kinase, the trkB receptor tyrosine kinase, and the Erb receptor tyrosine kinase. It is important to note that the same PSD proteins may be a substrate of diVerent kinases. For example, dynamin may be phosphorylated by PKA, Src family tyrosine kinase, or MAP kinase.

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CaMKII is the most abundant kinase at PSD. Four diVerent isoforms (, , , and ) are known to aggregate to form a dodecameric enzyme. The functionally active form of the kinase in neurons is represented by the and subunits, with the subunit largely prevailing in the holoenzyme over the subunit. CaMKII may undergo autophosphorylation, followed by translocation to the PSD where the kinase is believed to enhance the synaptic strength by phosphorylating ion channels and receptors. ScaVolding, association, and interactor, as well as cytoskeleton PSD proteins, are substrates for the CaMKII. PSD 95/SAP90, SAP 97, guanylate kinase-associated protein (GKAP), rasGTPase-activating protein, and synaptic GTPase-activating protein (SynGAP) family proteins have several consensus sequences of the phosphorylation site RXXXS/T. Even the precise function of CaMKII-dependent phosphorylation of diVerent PSD proteins has yet to be unraveled. It is thought to represent a relevant mechanism in the maintenance of dendritic architecture and synaptic plasticity (Means, 2000). The following sequence of events has been suggested: the release of neurotransmitter at presynaptic terminal triggers the Ca2þ inﬂux through the NMDA and AMPA receptors, and calcium activates the postsynaptic CaMKII, which translocates from the cytosol to the postsynaptic density and phosphorylates (activating or inhibiting) several PSD proteins (Soderling, 2000). For example, the phosphorylation and inhibition of SynGAP may result in potentiation of the mitogen-activated protein kinase pathway (Chen et al., 1998; Kim et al., 1998).

D. Receptors NMDA, kainate, AMPA, and metabotropic aglutamate receptors have all been identiﬁed in the PSD glutamatergic synapse. With regard to the receptor– scaVolding proteins interaction, the NMDA receptor (NMDA-R) may be considered to be the core of the PSD based on its role, together with AMPA-Rs, in long-term potentiation (LTP), considered a highly regulated process of synaptic plasticity. LTP has been reported to be impaired by the disruption of normal PSD scaVolding protein function (Migaud et al., 1998). Several proteins at the PSD have been demonstrated to interact with NMDA-Rs; moreover, this receptor binds directly at least two PSD scaVolding and anchoring proteins, PSD-95/ SAP90 and Yotiao, through, respectively, the NR2 and NR1 subunits. Metabotropic glutamate receptors are divided into three groups. Type I mGluRs, including mGluR1 and mGluR5, are the major targets of the Homer family and have been localized speciﬁcally around the periphery of PSD (Migaud et al., 1998; Thomas, 2002). AMPA-Rs have been shown originally to be clustered at excitatory synapses by GRIP (Dong et al., 1997). However, other reports also suggest that AMPA-Rs

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may interact with GRIP 2 without apparent clustering (Srivastava et al., 1998). It is remarkable that GRIP may interact with B-ephrin receptor tyrosine kinases, which have a pivotal role in the induction of LTP in hippocampal mossy ﬁbers (Bruckner et al., 1999; Contractor et al., 2002).

E. PSD Proteins and the Concept of Microdomain An emerging concept strongly correlated to PSD organization as a multifunctional structure implicated in synaptic plasticity regulation is the junctional signaling microdomain (Delmas and Brown, 2002). Several lines of evidence suggest that the plasma membrane is specialized locally, both structurally and functionally, into distinct microdomains and subdomains. The suggested role of the microdomains is to ensure a highly speciﬁc and selective downstream signaling, avoiding interactions between molecules whose activation would introduce noise or contrasting eVects in the downstream propagation of the transduction signal. The PSD may have a signiﬁcant impact in the organization of microdomains, as key molecules of the PSD are involved in signal transduction and scaVolding/ adaptor proteins are the elements that physically link, in a cooperative manner, diVerent intracytoplasmic ‘‘transducers.’’ The functional importance of PSD proteins in signaling microdomains architecture can be appreciated especially considering the role of Homer proteins (both the constitutive and the inducible isoforms, see later) in the spatial and temporal Ca2þ-regulated modulation of signal transduction. Homer constitutive proteins may interact through the EVH1/WH1 (EVH1, Ena/Vasp homology 1; WH1, Wasp homology 1) domain with type I mGluRs (whose activation may modulate the cytosolic calcium concentration) and IP3Rs (inositol 1,4,5-triphospate receptors). Moreover, the ryanodin receptor type I (RyR1) binds a proline-rich sequence of Homer (Shiraishi et al., 1999; Sun et al., 1998; Xiao et al., 1998). In this way, Homer acts as a scaVolding element that links together major molecules involved in the regulation of cytosolic calcium (Serge et al., 2002). In summary, junctional signaling microdomains may represent a highly eVective strategy developed to ensure speciﬁcity, speed, and selectivity in the propagation of signal transduction.

F. PSD Proteins and Receptor Diffusion A relevant role of postsynaptic density proteins with scaVolding and association function has been recognized also in receptor diVusion that, together with endocytosis and exocytosis, is a crucial mechanism regulating the movement of receptors in the postsynaptic membrane during plasticity (Blanpied et al., 2002;

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Tovar and Westbrook, 2002). It has been only recently that the role of lateral diVusion in setting the number of receptors functionally available for neurotransmitter binding has been fully appreciated. Both the disappearance of the receptors from the cell surface and the dispersal of receptors from the synaptic to extrasynaptic membrane may be responsible for an apparent net loss of receptors from a postsynaptic site. Receptor diVusion can be viewed as a lateral movement of receptors in the plane of the membrane mainly between synaptic and extrasynaptic pools (Choquet and Triller, 2003). An example of the relevance of postsynaptic density proteins and their complex interactions in receptor diVusion comes from the stargazin–PSD-95/SAP90 interaction in synaptic AMPA-R number regulation. AMPA receptors are localized to synapses by direct binding of the ﬁrst two PDZ domain of PSD-95/SAP90 to the AMPA-R-associated protein, Stargazin. Increased levels of PSD-95 in hippocampal slice cultures recruit new AMPA-Rs to synapses, but this does not change the number of AMPA-Rs expressed to the membrane surface. Overexpression of Stargazin increases the level of extrasynaptic AMPA-Rs. Removing the PDZ-binding site of Stargazin, by deleting the last four amino acids at its C terminus, causes a reduction of its clustering and of AMPA excitatory postsynaptic currents (EPSC). Taken together, these ﬁndings point to a mechanism of AMPA-R transport between synaptic and extrasynaptic membrane, regulated by Stargazin binding to synaptic PSD-95/SAP90, trapping AMPA-Rs (Schnell et al., 2002). Fast movements and periods of receptor immobility have been described on the surface of neurites. The relative time spent in each state may be modulated by scaVolding protein expression. It is important to stress that when receptors are clustered by PSD proteins, they are not completely immobilized but may move within the cluster. In summary, many lines of evidence suggest an involvement of scaVolding and adaptor proteins in the regulation of receptor lateral diVusion.

III. PSD-95/SAP90

A. Structure PSD-95/SAP90 is an 80-kDa protein that migrates at 95 kDa on SDS–polyacrylamide gels and is a close homolog of the Drosophila protein Discs-large (Dlg) (Cho et al. 1992). PSD-95/SAP90 is a MAGUK protein that contains a catalytically inactive guanylate kinase-like (GK) domain in the carboxyl-terminal region, an Src homology 3 (SH3) domain in the central zone, and three PDZ (PDZ1, 2, 3) domains at the amino-terminal end. The GK domain binds GKAP directly, a

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Fig. 1. Domain organization and protein-binding partners of PSD-95/SAP90. (See Color Insert.)

major constituent of PSD able to interact with Shanks/ProSAPs (see later) and therefore is important for protein–protein interaction (Kim et al., 1997) (Fig. 1). The GK and SH3 domains probably make a module responsible for the oligomerization of MAGUK scaVolding proteins. For example, several diVerent proteins, such as protein 4.1 and calmodulin, binding to the hinge region of the SH3 domain could subvert the intramolecular assembly of the SH3 and support intermolecular assembly (Lue et al., 1994; Masuko et al., 1999; McGee et al., 2001). The PDZ (PSD-95/Dlg/ZO-1) domains are modular domains of about 90 amino acids that bind speciﬁc sequences at the C termini portion of target proteins. Speciﬁcally, PDZ1 and PDZ2 can bind Shaker-type Kþ channels (Kv1.4), NMDA-R NR2 subunits, and Stargazin, a protein connecting PSD-95/SAP90 with AMPA-Rs (Chen et al., 2000; Kim et al., 1995; Kornau et al., 1995; Schnell et al., 2002); PDZ3 binds neuroligins (neuronal cell adhesion molecules) and CRIPT (a cysteine-rich interactor of PDZ3), a microtubule-associated protein (Irie et al., 1997; Niethammer et al., 1998). Furthermore, PDZ2 is able to bind neuronal nitric oxic synthase (nNOS) through a PDZ–PDZ interaction, establishing a crucial functional connection (Brenman et al., 1996a). Finally, SynGAP, a Ras-GTPase-activating protein, interacts with all three PDZ domains of PSD-95/SAP90 (Kim et al., 1998). Long et al. (2003) proposed that PDZ1 and PDZ2 behave in tandem, PDZ12, promoting the dimerization of receptors and ion channels or stabilizing the receptor dimer. B. Functions 1. PSD-95/SAP90 and Assembly of PSD The ﬁrst steps of PSD-95/SAP90 clustering function are its multimerization ability through N-terminal domains and its anchorage property to the plasma membrane by palmitoylation of two N-terminal cysteines (Hsueh et al., 1997;

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Topinka and Bredt, 1998). Palmitoylation is crucial for molecule clustering and for the synaptic localization of PSD-95/SAP90 (Craven et al., 1999). This process in heterologous cells and primary hippocampal cultures may be prevented by conjugation with 2-bromopalmitate, resulting in dispersed synaptic clusters of PSD95/SAP90, end in a decreased number of synaptic AMPA-Rs (El-Husseini Ael et al., 2002). This ﬁnding supports the hypothesis that, through an interaction with Stargazin, AMPA-Rs localized into intracellular compartments are driven to the cell surface. These AMPA-Rs/Stargazin complexes are kept at the synapse by interaction with PSD-95/SAP90 (Schnell et al., 2002). PSD-95/SAP90 seems therefore a pivotal modulator of AMPA-R targeting. PSD-95/SAP90 also interacts with subunits of the kainate receptors. The KA2 subunit of the kainate receptor interacts with both SH3 and GK domains, whereas the GluR6 subunit binds speciﬁcally to the PDZ1 domain. This interaction leads to clustering and incomplete kainate receptor desensitization (Garcia et al., 1998). In addition, PSD-95/SAP90 cooperates with Kv1.4. When PSD-95/SAP90 is expressed heterologously together with Kv1.4, the expressed proteins become colocalized in plaque-like clusters whereas when the Kv1.4 channel and PSD-95/ SAP90 are expressed individually, the proteins are localized diVusely throughout the cellular membranes or the cytosol (Kim et al., 1995). PDZ2 seems to be a key domain for Kv1.4 clustering (Imamura et al., 2002). These ﬁndings are supported by evidence that the genetic disruption of D1g (see earlier discussion) causes great changes in the morphology and physiology of synapses and interferes with synaptic localization of Kv1.4. (Budnik, 1996; Tejedor et al., 1997). However, work by Rasband et al. (2002) denies the clustering ability of PSD-95/SAP90. Indeed, they have found, at juxtaparanodal regions adjacent to the node of Ranvier, that Kv1 clustering is normal in a mutant mouse lacking juxtaparanodal PSD-95/SAP90 (Rasband et al., 2002). Further studies are needed in order to clarify this issue. PSD-95/SAP90 synaptic interaction with NMDA-Rs is not yet completely deﬁned. In mutant mice lacking PSD-95/SAP90, the localization of NMDA-R is normal and, according to this ﬁnding, mice expressing the NR2 subunit in a C-terminally truncated form show no change in the synaptic localization of NMDA-R (Migaud et al., 1998; Sprengel et al., 1998). Nevertheless, the NR2A subunit in a C-terminally truncated form impairs synaptic but not extrasynaptic localization of NMDA-Rs; moreover, during synapse development, PSD-95/ SAP90 clusters before NMDA-R apparently form a scaVold to which the receptors later attach (Rao et al., 1998; Sanes and Lichtman, 1999; Steigerwald et al., 2000). 2. PSD-95/SAP90 and Synaptic Plasticity More than 100 molecules have been associated with synaptic plasticity, namely with LTP and long-term depression (LTD) (Sanes and Lichtman, 1999), nevertheless the role of PSD-95/SAP90 in the development of these events

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may be crucial. Migaud et al. (1998) demonstrated that mutant mice lacking PSD95/SAP90 have an enhancement of LTP and a learning impairment. Mutant mice showed a signiﬁcantly larger potentiation of synaptic transmission so that various stimulation frequencies (1, 5, 10, 20, 100 Hz) induce LTP. Furthermore, the analysis of spatial learning by means of the water maze test demonstrated that mutant mice have a marked inability to learn the position of the hidden platform. This enhanced LTP seems unrelated to the SynGAP–MAPK pathway (Grant and O’Dell, 2001; Komiyama et al., 2002). Moreover, PSD-95/SAP90 connects NMDA-Rs with nNOS, enzyme well known to be involved in LTP and LTD induction (Christopherson et al., 1999; Wu et al., 1997). This association may modulate nNOS function. Suppressing the expression of PSD-95/SAP90 attenuates nitric oxide neurotoxicity (Sattler et al., 1999). PSD-95/SAP90 also promotes nNOS phosphorylation at Ser847 induced by CaMKII, which reduces enzyme catalytic activity (Komeima et al., 2000; Watanabe et al., 2003). PSD-95/SAP90 functionally modulates NMDA-Rs and its overexpression increases AMPAR-mediated synaptic transmission (Beique and Andrade, 2003; Yamada et al., 2002). This last ﬁnding is related to a previous report demonstrating that PSD95/SAP90 overexpression increases synaptic AMPA-R levels, miniature excitatory postsynaptic current (mEPSCs) amplitude, and the size and number of dendridic spines (El-Husseini et al., 2000). According to these results, PSD-95/ SAP90 reduced expression seems responsible for hippocampal neuronal death through CaMKII transduction pathway activation (Gardoni et al., 2002).

C. PSD-95/SAP90 and Neuropsychiatric Disorders Evidence suggests that PSD-95/SAP90 may be involved in the pathophysiology of some neuropsychiatric disorders, such as schizophrenia, ischemia, and Huntington’s disease (HD). The glutamatergic system is involved in the pathophysiology of psychosis. Indeed, NMDA-R hypofunction has been proposed as a model of schizophrenia (Olney and Farber, 1995). NMDA noncompetitive receptor antagonists, such as ketamine and phencyclidine (PCP), induce a schizophrenia-like psychotic state in normal subjects that closely mimics both positive and negative symptoms of the disorder and that is suppressed by treatment with clozapine but not with haloperidol ( Javitt and Zukin, 1991; Krystal et al., 1994, 1999; Malhotra et al., 1997). Moreover, ketamine and PCP may exacerbate psychotic symptoms in schizophrenic patients (Lahti et al., 1995). Two studies investigated PSD-95/SAP90 mRNA expression in diVerent brain areas of schizophrenic subjects. In the ﬁrst one, in situ hybridization histochemistry (ISHH) showed a signiﬁcant PSD-95/SAP90 gene expression decrease in Broadmann’s area 9 of the prefrontal cortex but not in the hippocampus of

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schizophrenic patients compared to normal controls (Ohnuma et al., 2000). In the second one, Dracheva et al. (2001) compared PSD-95/SAP90 expression in patients with schizophrenia, patients with Alzheimer’s disease, and normal controls. The level of PSD-95/SAP90 mRNA expression in the occipital cortex (Broadmann’s area 17) of schizophrenic subjects was signiﬁcantly higher compared to normal subjects, where no signiﬁcant changes were observed in the dorsolateral prefrontal cortex (Broadmann’s area 46). PSD-95/SAP90 gene expression was unchanged in the brains of the Alzheimer’s disease group (Dracheva et al., 2001). In animal models, NMDA-R blockade induced by the administration of MK801, an uncompetitive NMDA-R antagonist, produces a signiﬁcant increase of PSD-95/SAP90 mRNA levels in parietal, entorhinal, temporal, and perirhinal rat corticies (Linden et al., 2001). Moreover, PSD-95/SAP90 gene expression is unmodiﬁed after the acute administration of antipsychotics drugs such as haloperidol and olanzapine in several cortical and subcortical regions, including the prefrontal cortex (cG30 area) caudate-putamen, and accumbens (de Bartolomeis et al., 2002). These ﬁndings, taken together, suggest an involvement of PSD-95/ SAP90 in schizophrenia, but other studies are needed to better understand the glutamatergic transmission role in the pathophysiology of psychosis. The ﬁrst evidence of a PSD-95/SAP90 implication in brain ischemia came from the studies of Hu et al. (1998) and Takagi et al. (2000). These authors demonstrated that an ischemic challenge produces a slight decrease of PSD-95/ SAP90 expression (Hu et al. 1998), a decreased association of PSD-95/SAP90 with NR2A and NR2B NMDA-R subunits, and a reduction in the size of protein complexes containing PSD-95/SAP90 (Takagi et al., 2000). A pivotal report by Aarts et al. (2002) demonstrated that perturbing in vivo the interaction between PSD-95/SAP90 and NMDA-Rs reduces the ischemic brain damage induced in rats by transient middle cerebral artery occlusion (MCAO) by the intraluminar suture method. The administration of peptides that bind PSD-95/SAP90 PDZ2 domains, dissociating the NMDA-R–PSD-95/SAP90 complex, decreases the total cerebral infarction volume and improves motor deﬁcits (Aarts et al., 2002). This ﬁnding may suggest new strategies for treating stroke. Huntington’s disease is a dominant inherited neurodegenerative disorder characterized by choreiform movement, psychiatric disturbances, and cognitive decline (Martin and Gusella, 1986). HD is caused by a polyglutamine expansion of huntingtin, the protein codiﬁed by the HD gene (The Huntington’s Disease Collaborative Research Group, 1993). Normal huntingtin is enriched in dendrites and nerve terminals and is associated with microtubule complexes and synaptic vescicles. It binds the SH3 domain of PSD-95/SAP90 through its N-terminal proline region, while polyglutamine expansion inhibits the interaction between huntingtin and PSD-95/SAP90. Furthermore, in HD patients the huntingtin association with PSD-95/SAP90 is about 80% lower compared to normal subjects

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(Sun et al., 2001). These data suggest that normal huntingtin sequesters PSD-95/ SAP90, avoiding neuronal toxicity, whereas polyglutamine-expanded huntingtin may sensitize and activate NMDA-Rs, causing neuronal toxicity. New ﬁndings support the view that PSD-95/SAP90 is also involved in neuropathic pain and in kainic acid-induced seizures (Garry et al., 2003; Wyneken et al., 2001).

IV. Shank/ProSAP Proteins

A. Structure Shank/ProSAP (SH3 domain and ankyrin repeat containing protein/ proline-rich synapse-associated protein) proteins are a family of three members: Shank1, Shank2, and Shank3. Shanks/ProSAPs are multidomain proteins containing, from N to C termini the following structures: an ANK (ankyrin) repeat region, a SH3 domain, a PZD domain, a proline-rich (PRO) region, a ppI (cortactin binding) domain, and a sterile motif (SAM) domain (Fig. 2). The domain organization of Shank/ProSAP proteins appears to be regulated by alternative splicing, Shank1 lacks the ppI domain, and ANK seems to be absent in Shank2 (Boeckers et al., 1999; Lim et al., 1999). In rat brain, Shank/ProSAP mRNAs are expressed diVerentially in several brain areas, namely Shank1 is abundant in the cortex, hippocampus, and amygdala; Shank2 and Shank3 show a similar distribution in the cortex and hippocampus, but in the cerebellum, Shank2 is found in Purkinje cells, whereas Shank3 is only expressed in the granule cell layer (Sheng and Kim, 2000). The ANK repeat region of Shank1 and Shank3 binds -fodrin, a multidomain protein containing 22 spectrine repeats, one SH3 domain, and two EF hand calcium-binding motifs (Carlin et al., 1983). -Fodrin is a cytoskeletal protein

Fig. 2. Domain organization and protein-binding partners of Shank/ProSAP proteins. (See Color Insert.)

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interacting with actine and calmodulin, expressed in the neuronal dendritic compartment mainly in the dendridic spines and in PSD (Glenney et al., 1983). The ANK repeat region also interacts with the C-terminal zone of Sharpin, a PSD component able to homomultimerize through the N-terminal extreme (Lim et al., 2001). Therefore, Sharpin may cross-link multiple Shank proteins. The SH3 domain interacting partners are to date unclear, indeed Sheng and Kim (2000) reported, in GST-pulldown experiments, that the SH3 domain binds to GRIP, but no more studies have been published to support this ﬁnding. The PDZ domain binds GKAP, somatostatin receptor type 2 (SSTR2), the calcium-independent latrotoxin receptor (IRL), and PIX, a guanine nucleotide exchange factor for Rac1 and Cdc42 small GTPases (Boeckers et al., 1999; Kreienkamp et al., 2000; Park et al., 2003; Zitzer et al., 1999a,b). The interaction with GKAP links, through PSD-95/SAP90, the NMDA-R macromolecular complex with Shanks/ProSAPs. The PRO region is more than 1000 residues long and is rich in proline and serine. Three proteins shown to bind to the PRO region are Homer, dynamin-2, and IRSp53 (insulin receptor tyrosine kinase substrate protein). Homer (see later) is a scaVolding molecule linking type I mGluRs with releasable Ca2þ intracellular pools (Tu et al., 1998, 1999). Dynamin-2 is a member of endocytic machinery, interacting with a short serine-rich sequence within the PRO region (Okamoto et al., 2001). IRSp53 is a signaling molecule that acts downstream of small G-proteins such as cdc42 and rac (Bockmann et al., 2002; Soltau et al., 2002). The ppI domain is a prolin-rich cluster within the PRO region that mediates the interaction with cortactin, an F-acting-binding protein enriched in cell matrix contact sites, in lamellipodia of cultured cells, and in growth cones of cultured neurons (Du et al., 1998; Wu and Parsons, 1993). Furthermore, cortactin translocates to the cell periphery by means of rac1 and reallocates to synapses after glutamate stimulation (Naisbitt et al., 1999; Weed et al., 1998). Therefore, cortactin may play a key role in the cytoskeletal organization produced by intracellular and extracellular stimuli. The SAM domain of Shanks/ProSAPs interacts with itself in vitro and may be involved in a tail-to-tail multimerization of Shank proteins (Naisbitt et al., 1999).

B. Functions Although the Shank/ProSAP structure is well characterized, to date only two reports can help us understand Shank/ProSAP functions. Sala et al. (2001) showed that Shank1 overexpression, in transfected primary hippocampal neurons, stimulates the maturation and growth of dendritic spines. Namely, Shank1 expression during the ﬁrst week, when endogenous Shank1 is expressed at low levels, accelerates the development of mushroom spines; moreover, during

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the second week, when endogenous Shank1 is increased, Shank1 overexpression promotes spine head enlargement. Furthermore, dominant-negative Shank1 mutants cause a reduction in spine density. Intriguingly, Shank1 postsynaptic overexpression seems to enhance a presynaptic function (Sala et al., 2001). Park et al. (2003) demonstrated that overexpression of Shank1B in cultured hippocampal neurons promotes recruitment of PIX and PAK (p21-activated kinase) in dendritic spines. PIX is a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases; PAK is a family of Rac/Cdc42-activated serine/threonine kinases that, through PIX, interacts with Rac1/Cdc42 (Bagrodia and Cerione, 1999; Manser et al., 1998). PIX activates Rac1 and Cdc42 small GTPases involved in neurite initiation, growth, guidance, branching, polarity, and synapse formation (Luo, 2000). These ﬁndings suggest that Shank may regulate spine dynamics through the synaptic accumulation of PIX IX and local activation of the Rac1-PAK signaling pathway. C. Shank3 and the 22q13.3 Deletion Syndrome The 22q13.3 deletion syndrome is a neurogenetic syndrome characterized by severe expressive language delay, mild mental retardation, and facial dysmorphisms (Nesslinger et al., 1994; Prasad et al., 2000). The ﬁrst indication of a Shank3 gene involvement in 22q13.3 deletion syndrome came from Wong et al. (1997). In a child with clinical signs of 22q13.3 deletion syndrome (severely delayed expressive language, mild mental retardation, hypotonia, dolichocephaly, epicanthic folds, bulbous nose, and lax joints), Bonaglia et al. (2001) showed a balanced translocation between chromosomes 12 and 22, causing a disruption of the Shank3 gene at exon 21. This result suggests a Shank3 association with this syndrome (Bonaglia et al., 2001). V. SAP97

A. Structure Synapse-associated protein 97 kDa (SAP97), together with PSD-95/SAP90, SAP102, and PSD-93/chapsyn-110, is a member of the MAGUK protein family (Muller et al. 1995). SAP97 is a multidomain protein, characterized from the C to N-terminal region by a GK domain, a SH3 domain, and three PDZ domains. Three further regions can be recognized: the HOOK/U5 region (protein sequences situated between the GK and the SH3 domain), the S97N region (protein sequences N-terminal to PDZ1), and the MRE (MAGUK recruitment) domain (Fig. 3) (Karnak et al., 2002; Lee et al., 2002; Wu et al., 2002). The

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Fig. 3. Domain organization and protein-binding partners of SAP97. The SAP97 interaction with the NR2A subunit of the NMDA receptor is not shown. (See Color Insert.)

S97N region and the MRE domain may coincide, but because this issue is not yet clear, they will be dealt with separately. The GK domain binds GKAP; interestingly, this association is inhibited by intramolecular interactions of the SH3 domain and HOOK/U5 region with the GK domain and is enhanced by the S97N region binding with the SH3 domain and HOOK/U5 region ( Wu et al., 2000). The GK and SH3 domain of SAP97 mediate binding to AKAP79/150 (A-kinase anchoring protein 79 kDa in humans and 150 kDa in rodents), a protein binding the regulatory subunits of protein kinase A (PKA) and targeting PKA to various subcellular compartments (Colledge et al., 2000; Colledge and Scott, 1999). The HOOK/U5 region interacts with calmodulin, the principal mediator of calcium-dependent signaling, with low to intermediate aYnity in a calcium-dependent manner (Paarmann et al., 2002). The PDZ1 domain binds the GluR6 subunits of kainate receptors weakly and has been proposed to interact with GluR1 subunits of AMPA-Rs (Mehta et al., 2001). The PDZ2 domain interacts with GluR1 subunits of AMPA-Rs (Leonard et al., 1998). This association is speciﬁc; indeed, GluR1 does not bind PDZ domains of other MAGUKs and is dependent on a GluR1 SSG sequence located outside the PDZ-binding motif (Cai et al., 2002). The PDZ2 domain is also able to bind the inward rectiﬁer potassium channels Kir2.2 and Kir3.2c (Hibino et al., 2000; Leonoudakis et al., 2001). The PDZ3 domain is in charge of binding with tumor necrosis factor converting enzyme (TACE), an enzyme responsible for ectodomain shedding of TNF- (Peiretti et al., 2003). Finally, Bassand et al. (1999) showed that the C-terminal tail of NR2A interacts strongly with portions of SAP97 encompassing the three PDZ domains, suggesting a SAP97 interaction with NMDA-R (Bassand et al., 1999). The MRE domain binds DLG3 (discs large 3), DLG2 (discs large 2), and calcium/calmodulin-dependent serine protein kinase (CASK), three members of MAGUK family proteins (Karnak et al., 2002). The S97N region binds myosin VI, a minus

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end-directed actin-dependent motor protein, able to move the particles/vesicles toward the minus or pointed end of actin ﬁlaments (Cho et al., 1992; Wells et al., 1999; Wu et al., 2002). Whereas other MAGUKs were found only in neuronal cells, SAP97 is also located in non neuronal tissues such as epithelial/endothelial, mesenchymal, and hematopoietic cells (Brenman et al., 1996b; Caruana and Bernstein, 2001; Cho et al., 1992). Furthermore, it has been discovered only recently that SAP97 is both presynaptic and postsynaptic and that it is enriched in PSD ( ValtschanoV et al., 2000). SAP97 is found in diVerent brain areas such as the cerebellum and forebrain, but is especially expressed in the pyramidal layers of the hippocampus, where SAP97 is located in apical dendrites through the stratum radiatum and stratum lucidum in the CA1 and CA3 areas, respectively (Bassand et al., 1999).

B. Functions SAP97 is involved in AMPA-R targeting. The interaction between SAP97 and GluR1 subunits occurs early in the secretory pathway, whereas the receptors are in the endoplasmic reticulum or cis-Golgi (ER-CG) (Sans et al., 2001). SAP97 is enriched in the PSD, but is also abundant in the cytoplasm and interacts with intracellular membranes. This peculiar intracellular localization is consistent with a role in the organization of membrane proteins and seems not related to GK and SH3 domain truncation (Klocker et al., 2002). Moreover, few synaptic AMPA-Rs bind SAP97. These ﬁndings may suggest that SAP97 carries GluR1 from ER-CG to the plasma membrane and, once there, dissociates from the receptor complex (Sans et al., 2001). Considering that SAP97 binds myosin VI (see earlier discussion), a suggestive hypothesis is that SAP97 serves as a molecular link between GluR1 and myosin VI during the translocation of AMPA-Rs to and from the postsynaptic membrane ( Wu et al., 2002). SAP97 is probably involved in hippocampal synaptic plasticity. The phosphorylation state of the GluR1 subunit is most likely associated with AMPA-R current intensity; indeed the phosphorylation of Ser845 on GluR1 enhances native and recombinant AMPA-R currents, whereas Ser845 is dephosphorylated during LTD (Banke et al., 2000; Lee et al., 2000). SAP97 binds AKAP79/150 (see earlier discussion), a multivalent anchoring protein associated with PKA and PP2B ( protein phosphatase 2B). PKA and PP2B are, respectively, in charge of GluR1 phosphorylation and dephosphorylation (Colledge et al., 2000; Tavalin et al., 2002). Taken together, these results demonstrate that SAP97 is a member of a complex involved in the modulation of GluR1 receptor currents. Moreover, SAP97 may regulate Kir3.2c function. Kir3.2c is a neuronal inwardly rectifying Kþ channel activated directly by G-proteins, is coupled to

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several inhibitory receptors, is involved in the generation of slow inhibitory postsynaptic potentials, and is localized at the postsynaptic membrane in dopaminergic neurons (Inanobe et al., 1999; Luscher et al., 1997; Yamada et al., 1998). Hibino et al. (2000) showed that SAP97 induces the sensitization of Kir3.2c to G-protein stimulation, and the GK domain seems to play a major role in this function (Hibino et al., 2000).

C. SAP97 and Schizophrenia Toyooka et al. (2002) showed an association of SAP97 with schizophrenia. The authors have investigated the expression of several PDZ proteins, including PSD95/SAP90, SAP97, PSD-93/chapsyn-110, GRIP1, and SAP102 in postmortem human brain tissue of schizophrenic patients by means of Western blot analysis. The regions analyzed were the dorsolateral prefrontal cortex (Broadmann’s area 46), the occipital cortex (Broadmann’s area 17), CA regions, and the dentate gyrus of the hippocampus. SAP97 levels were decreased signiﬁcantly in the dorsolateral prefrontal cortex of schizophrenics, where GluR1 protein expression was also reduced. This ﬁnding suggests that SAP97 reduction may contribute to glutamatergic dysfunction in the dorsolateral prefrontal cortex. Intriguingly, levels of other PDZ proteins (PSD-95/SAP90, chapsyn-110, GRIP1) were found unmodiﬁed in the prefrontal cortex of schizophrenic subjects versus the control group (Toyooka et al., 2002). In an animal model, uncompetitive NMDA-R antagonists modify SAP97 mRNA expression. Indeed, MK-801 and phencyclidine administration increases SAP97 levels in the entorhinal cortex, whereas in superﬁcial layers of the parietal cortex, treatment with MK-801 decreases SAP97 mRNA expression (Linden et al., 2001). These ﬁndings represent further clues that the glutamatergic system is involved in the pathophysiology of psychosis.

VI. Homer Proteins

A. Structure Homer is a family of proteins that includes the following members: Homer 1b/c, 2a/b, 3, and Homer 1a. Homer 1b/c, 2a/b, and 3, called coiled-coil (CC), are expressed constitutively, whereas Homer 1a is inducible (Xiao et al., 1998). Genes coding for Homers are located, in humans, on chromosome 5 (Homer1), chromosome 15 (Homer2), and chromosome 19 (Homer3) (Xiao et al., 1998). Both constitutive and inducible isoforms are characterized by an

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Fig. 4. Domain organization and protein-binding partners of Homer proteins. (See Color Insert.)

EVH1/WH1 domain at the amino-terminal region, while only CC isoforms have a coiled-coil domain at the carboxyl-terminal extreme (Fig. 4). The EVH1/WH1 domain is about 110 amino acids long and, binding to PPxxF (single-letter amino acid code used, where ‘‘x’’ is any amino acid) motifs, mediates association with type I mGluRs, IP3Rs, RyR1, Shank/ProSAP proteins and dynamin 3 (Dyn3) (Brakeman et al., 1997; Feng et al., 2002; Gray et al., 2003; Tu et al., 1998, 1999). Type I mGluRs include mGluR1 and mGluR5, which are coupled to phospholipase C and activate phosphoinositide hydrolysis to produce IP3 and diacylglycerol (Nakanishi, 1994). IP3Rs and RyR1 play a central role in releasable Caþ2 intracellular pool modulation. Dyn3 is a mechanoenzyme localized within the PSD involved in the maintenance of dendritic morphology by regulating the outgrowth of dendritic protrusions and the morphogenesis of dendritic spines (Gray et al., 2003). The CC domain, about 200 amino acids long, is required for self-multimerization and contains typical leucine zipper motifs mediating homotypic interactions (Tadokoro et al., 1999; Xiao et al., 1998). Homer 1a lacks the CC region and therefore is unable to multimerize. The open reading frame spreads over 10 exons. Exon 1 codes for the 50 untranslated region (UTR), exons 2–5 code for the N-terminal EVH1 domain, and exons 6–10 code for the C-terminal CC motif and the 30 UTR. A substantial number of modulation elements have been recognized in the Homer1 gene promoter region, including SP1, AP1, GATA, E-box, and CRE (Bottai et al., 2002). Homer 1a presents a unique and highly conserved C-terminal tail of 11 residues. This 11 residue tail is coded by a 33 nucleotide sequence positioned into the

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intron between exon 5 and exon 6, and an alternative splicing, most likely triggered by synaptic activity (see later), allows the ending of the Homer 1a transcripts within intron 5 (Bottai et al., 2002). This mechanism extends the exon 5 sequence and generates an mRNA construct constituted by EVH1 domain coding exons but not by exons coding for the CC region. A similar transcription mechanism is also present in Ania-3, a less investigated activity-induced splice variant of Homer 1 that shows great similarity with Homer 1a. Further, Homer 1a mRNA contains several AUUUA repeats at 30 UTR. The AUUUA repeats are responsible for destabilizing the interaction with the translational machinery at the ribosomal level. The functional consequence of this organization of gene structure is a fast decay of the mRNA transduction (Bottai et al., 2002). The AUUUA repeats are common to other IEGs, while they lack in the Homer 1b/c mRNA (Soloviev et al., 2000; Xiao et al., 1998). However, compared to other early genes, e.g., c-fos, the mRNA for Homer 1A lasts relatively longer after its induction by synaptic activity. The fast and dramatic increase of Homer1a mRNA after stimuli is not paralled by an increase of similar magnitude in the Homer1a protein level (Kato et al., 1998). A possible explanation is a rapid degradation pattern of the protein due to the presence of a PEST sequence in the Homer1 and perhaps Homer2 C-terminal regions (Soloviev, 2000). PESTs are amino acid sequences acting as molecular markers. Indeed, proteins displaying PEST sequences in their primary structure are degraded by an obiquitinemediated system. Moreover, PEST sequences have been found particularly in proteins with a high turnover. However, the fast degradation pattern is typical of Homer 1a but not Homer 1b, Homer 2, or Homer 3 proteins (Ageta et al., 2001), whereas the PEST sequence is shared by almost all Homer isoforms. Because Homer 1a mutants lacking the 11 residue C-terminal tail are not degraded rapidly, it seems likely that the Homer 1a unique 11 residue tail might promote its fast degradation pattern (Ageta et al., 2001). It is remarkable that Homer proteins, as well as SPA97, have been identiﬁed not only in the nervous system, but also in various peripheral tissues, including cardiac and skeletal muscles (Sandona et al., 2000). Saito et al. (2002) found a novel Homer 1 isoform (Homer 1d) from the cardiac cDNA library identical to Homer 1b except for a unique N-terminal region.

B. Functions TraYcking of type I mGluRs is probably the most extensively investigated function of Homer proteins. Type I mGluR surface expression has been studied in diVerent heterologous expression systems. In HeLa cells, Homer 1b was found to trap mGluR1a and mGluR5 in the endoplasmic reticulum, causing a reduction of receptors at the plasma membrane. Moreover, mGluR5 point mutations

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interrupting the association with Homer inhibit this eVect and Homer 1a expression does not modify mGluR5 surface expression (Roche et al., 1999). In HEK-293 cells, both Homer 1c and Homer 1a induce plasma membrane surface clustering of mGluR1a (Ciruela et al., 1999, 2000). In contrast, Coutinho et al. (2001) showed, also in HEK-293 cells, that neither Homer 1a nor Homer 1c had any marked eVect on mGluR1a distribution, whereas coexpression of mGluR5 with Homer 1c resulted in reduced receptor surface localization and receptor intracellular cluster formation (Coutinho et al., 2001). In COS-7 cells, Homer 1c causes mGluR1a clustering but has no eVect on membrane targeting of mGluR1a (Tadokoro et al., 1999). Ango et al. (2002) investigated the role of Homer 1a and Homer 1b in the traYcking of mGluR5 in primary cultures of mouse cerebellar granule cells. In this paradigm, Homer 1b produces intracellular clustering of mGluR5 at synaptic sites, whereas Homer 1a inhibits the intracellular retention of receptors, allowing cell surface localization of mGluR5 at synaptic sites. Furthermore, Homer 1a increases the latency and the amplitude of mGluR5-mediated Ca2þ responses (Ango et al., 2002). Homer proteins can also regulate mGluR localization in speciﬁc neuronal compartments. Indeed, in cultured cerebellar granule cells, in the absence of Homer 1, mGluR5 are localized in the soma, whereas Homer 1b distributes mGluR5 at the dendritic synaptic sites and Homer 1a causes mGluR5 translocation to both dendrites and axons (Ango et al., 2000). Moreover, in cultured cortical neurons, Homer 1c increases the transport of mGluR1a to the dendrites (Ciruela et al., 2000). In addition, Homer proteins are involved in axon pathﬁnding in vivo. Indeed, in Xenopus optic tectal neurons, time-lapse imaging shows that interfering with Homer 1b/c causes axon pathﬁnding errors (Foa et al., 2001). Homer proteins connect type I mGluRs with releasable Caþ2 intracellular pools through IP3Rs and RyR1. This interaction links type I mGluRs with their downstream eVectors. Homer 1a expression, in transfected Purkinje cells, causes an amplitude reduction and a latency increase of mGluR-evoked Caþ2 responses, as compared with Homer 1b transfection (Tu et al., 1998). Furthermore, it is remarkable that both Homer 1b and Homer 1a might bind RyR1 and similarly enhance their responses to physiological and pharmacological stimuli such as Ca2þ, depolarization, and caVeine (Feng et al., 2002). Homer proteins modulate the coupling of type I mGluRs to N-type calcium and M-type potassium channels. Indeed, Homer CC may occlude signaling from type I mGluRs to G-proteins regulating N-type calcium and M-type potassium channels, whereas Homer 1a may revert this eVect (Kammermeier et al., 2000). Homer proteins may also modulate type I mGluR activation. Indeed, a pivotal paper of Ango et al. (2001) demonstrated, in cultured cerebellar granule cells, that Homer 3 knock down and Homer 1a induction produce an agonistindependent mGluR1a activation. Taken together, these ﬁndings suggest a model in which Homer 1a behaves as ‘‘dominant negative,’’ promoting disassembly of

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the type I mGluR signaling complex, modulating glutamatergic synaptic activity directly. Homers interaction with Shank proteins is involved in the regulation of dendritic spine morphology, namely the ability of Shank1 to induce spine enlargement (see earlier discussion) seems related to calcium release from intracellular stores. This release may be due to the synaptic recruitment of IP3Rs by Homers (Sala et al. 2001).

C. Homer 1a Induction and Implication for Neuropsychiatric Disorders Homer 1a is an immediate early gene (IEG), and several stimuli may induce its transient transcription. In a pivotal paper, Brakeman et al. (1997) demonstrated that Homer 1a mRNA upregulation in the hippocampus after electroconvulsive seizure and synaptic stimulus in the cortex by visual experience and in striatum by cocaine administration produces LTP (Brakeman et al., 1997). These data led some researchers to explore Homer 1a expression in diVerent experimental paradigms. In E1 mice, an animal model for human epilepsy, within 1 h from a tonic– clonic seizure, Homer 1a mRNA increases in the granule cell layer of dentate gyrus (DG) and a weaker signal occurs in pyramidal cells of the hippocampus and cortex. No signiﬁcant modiﬁcation was observed 8 h after seizure, suggesting a Homer 1a transient increase due to the tonic–clonic seizure (Morioka et al., 2001). In the rat kindling model of temporal lobe epilepsy, Potschka et al. (2002) demonstrated, by means of massively parallel signature sequencing and quantitative RT-PCR, a Homer 1a induction in the hippocampus of kindled rats. Furthermore, kindling of transgenic mice overexpressing Homer 1a was retarded signiﬁcantly compared to wild-type mice. These results suggest that Homer 1a may have anticonvulsant and antiepileptogenic eVects (Potschka et al., 2002). In rats, phencyclidine administration (see earlier discussion) has been reported to acutely induce Homer 1a mRNA in the prefrontal cortex and primary auditory cortex, and 24 h posttreatment, Homer 1a mRNA reduction in the retrosplenial cortex and dentate gyrus (Cochran et al., 2002). We also observed a Homer 1a increase in subcortical regions induced by subanesthetic and subconvulsant doses of ketamine, a dissociative anesthetic related structurally to PCP and with a complex receptor proﬁle including a noncompetitive NMDAR blockade (A. de Bartolomeis et al., in preparation). Due to the role of NMDA hypofunction in several animal models of behavioral disorder, it may be intriguing to further explore the putative role of the Homer family in the pathophysiology of psychosis-like conditions. A Homer involvement in the mechanism of action of drugs used in the treatment of psychosis is suggested by Homer 1a induction after the acute administration of antipsychotics. Indeed, we have shown, by means of ISHH, that

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acute administration of antipsychotics with diVerent pharmacodynamic proﬁles modulates Homer 1a expression with a speciﬁc pattern. Namely, haloperidol induced an increase of Homer 1a mRNA in the caudate-putamen and in both core and shell of the nucleus accumbens, whereas olanzapine does so in the core of nucleus accumbens only (de Bartolomeis et al., 2002). Moreover, we have investigated Homer 1a gene expression after acute treatment with haloperidol or clozapine alone or with the coadministration of d-cycloserine, a glycine partial agonist acting at the glycine-binding site of NMDA-R. This paradigm reproduces in animals an augmentation strategy proposed for schizophrenia treatment (GoV et al., 1999a,b). In this pharmacologic isomorphism, we have observed that haloperidol produces a Homer 1a mRNA increase in the caudate-putamen and nucleus accumbens, whereas clozapine induces Homer 1a only in the accumbens. Intriguingly, Homer 1a induction after both haloperidol and clozapine was attenuated in the same brain areas after the adjunction of d-cycloserine (Polese et al., 2002). Taken together, these ﬁndings suggest that Homer 1a is induced diVerently by typical (haloperidol) or atypical (olanzapine, clozapine) antipsychotics and suggest a potential role for Homer as a molecular link between glutamatergic and dopaminergic transmission. Drug addiction is considered a disorder of synaptic plasticity taking place in speciﬁc brain areas known to be linked to reward, craving, and withdrawal. Evidence suggests Homer 1a involvement in opioid addiction. Indeed, Ania-3 mRNA, a splice variant of Homer 1 almost identical to Homer 1a, was found to be increased in the rat prefrontal cortex after chronic treatment with morphine. After administration of naloxone, an opioid antagonist precipitating withdrawal, Ania-3 mRNA levels were still elevated, providing a clue of long-lasting induction (Ammon et al., 2003). Even if the exploration of Homer expression and function in the central nervous system (CNS) by psychotropic compounds is only beginning, it is worth noting that chronic imipramine treatment does not modify Homer 1a protein levels in any brain region, underlining that only certain speciﬁc psychotropic drugs may modulate Homer 1a expression (Matrisciano et al., 2002). Memory and learning are key functions in behavioral adaptation and may represent a higher expression of a modulation of complex synaptic plasticity. Interaction with a novel environment may be considered a simple and relatively naturalistic example of memory and learning task. Using ﬂuorescence in situ hybridization, Vazdarjanova et al. (2002) examined Homer 1a mRNA expression in a paradigm consisting in exploration of a novel environment for 5 min. Rats were sacriﬁced at 0, 8, 16, 25, or 35 min after exploration and confocal images were acquired for qualitative or quantitative analysis. Qualitative analysis has shown Homer 1a expression at 25 min in hippocampal CA1 and CA3 regions, and in the parietal cortex, these results were conﬁrmed by quantitative analysis of the

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CA1 region demonstrating a signiﬁcant increase of Homer 1a in the 25- and 35-min groups ( Vazdarjanova et al., 2002). These ﬁndings may provide evidence for a role of Homer 1a in learning and memory processes. Synaptic plasticity is believed to be aVected by light and circadian rhythms. Light induces a phase shift of activity rhythms only during a subjective night. Early gene activation in brain regions driving or related to circadian rhythms has been reported to be a neuronal marker of CNS pathways implicated in response to environmental light changes. The ﬁrst studies on Homer activation showed that Homer mRNA expression is induced rapidly in rat suprachiasmatic nucleus by light during the subjective night, pointing to an involvement of this protein and perhaps of the glutamate metabotropic receptormediated signaling system in circadian rhythm-related synaptic changes (Park et al., 1997). More recently, this issue has been investigated further with the ﬁnding that Homer1a mRNA exhibits a diurnal variation in the rat suprachiasmatic nucleus in vivo with the highest level during the midsubjective day (Nielsen et al., 2002). These results, taken together, point to the potential contribution of Homer in synaptic changes that may be relevant for behavior and disorders of behavior linked to the abnormal regulation of circadian rhythms in mammals. Finally, an elegant piece of evidence that strongly links Homer gene regulation to the synaptic plasticity and adaptation has been reported by Bottai et al. (2002), who demonstrated a synaptic activity-induced conversion of the intronic to exonic sequence in Homer 1a expression. By means of ﬂuorescent in situ hybridization, these authors showed that in mouse at a resting state of neuronal activity the entire Homer gene is transcribed constitutively to yield Homer 1b/c. After maximal electroconvulsive shock, a neuronal increase in the rate of transcription was detected, with most transcripts ending within the central codon (Bottai et al., 2002). In reviewing the role of Homer in synaptic plasticity disorders and its putative involvement in behavior, a report on Homer gene manipulation in Drosophila that may shed light on Homer function in unexpected animal behaviors needs to be considered (Diagana et al., 2002). A single gene encoding a protein homologous to the mammalian Homers has been identiﬁed (Kato et al., 1998; Xiao et al., 1998) and shown to be expressed in the CNS of Drosophila where it is localized in the endoplasmic reticular and targeted to dendritic spines. A mutant ﬂy carrying a deletion in the Homer gene (Homer102), which removes the ﬁrst two exons and half of the third exon, was generated. No gross abnormalities in brain morphology were observed in mutant ﬂies compared to the wild type. However, mutation of Drosophila Homer disrupted the control of locomotor activity and was responsible for deﬁcits in courtship conditioning (Diagana et al., 2002). Caution in translational neuroscience is always necessary, especially with behavioral ﬁndings in lower species. However, the role of the Homer signaling

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pathway in mating behavior is certainly fascinating and it would be interesting to explore its potential involvement in similar behavioral patterns in mammalians.

VII. Conclusive Remarks

Converging evidence suggests a pivotal role of PSD proteins in neurodevelopment, axon pathﬁnding, receptor number regulation and clustering, and maintenance of dendritic architecture, as well as in synaptic plasticity complex expression such as LTP. It may be conceivable that disruption of the normal structure and function of scaVolding, adaptor, or kinase proteins at PSD may strongly impact synaptic plasticity. Animal models and pharmacologic isomorphisms, as well as genetic and postmortem studies, all indicate that PSD proteins may be regarded as potential key molecules in the pathophysiology of complex disorders of synaptic plasticity and are worth further studies for elucidating their role in human behavior pathologies.

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PROSTAGLANDIN-MEDIATED SIGNALING IN SCHIZOPHRENIA

S. Smesny Department of Psychiatry University of Jena Jena, Germany

I. II. III. IV.

Introduction Prostaglandin Hypothesis of Schizophrenia Membrane Phospholipid Hypothesis of Schizophrenia Niacin Tests in the Field of Schizophrenia A. Clinical Studies Using Oral Niacin Stimulation B. The Topical Niacin Skin Test in Schizophrenia C. Niacin Skin Test and Psychopathology V. Summary References

I. Introduction

Phospholipid compounds contain 30–60% of essential fatty acids, which in turn are essential substrates for the formation of various cell signaling systems. Some of these fatty acids, e.g., arachidonic acid (AA) or dihomogammalinolenic acid (DGLA), are converted to prostaglandins (PGs), a group of cyclized acidic arachidonic acid derivatives, which are important because of the variety of hormonelike physiological functions. Depending on the number of double bonds in their side chains, PGs were termed series 1, series 2, and series 3. Series 1 PGs are formed from DGLA, series 2 PGs are formed from AA, and series 3 PGs are formed from eicosapentaenoic acid (EPA). Only DGLA and AA are found in phospholipids; DGLA has one double bond in the side chain and AA has two double bonds (Horrobin, 1978). The release of AA from the sn2 position of phospholipids is generated by the activation of phospholipase A2 enzymes. The key enzymes that convert arachidonic acid to PGs are cyclooxygenase-1 and -2 (COX-1 and -2). PGs do not act as classic neurotransmitters. They are not stored in or secreted from synaptic vesicles and they are eVective in very low concentrations. Once synthesized, they diVuse rapidly to speciﬁc G-protein-linked membrane receptors and activate them. However, due to their short half-life, they can act only on the originating cell or on neighboring cells. Thus they are local mediators. PGs have their major function as inﬂammatory mediators in peripheral tissue, but they also have various eVects in the central nervous system, including profound eVects INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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on transmitter release and transmitter action. PGs can regulate processes such as long-term potentiation (Bazan and Serou, 1999; Chen et al., 2001; Wieraszko et al., 1993; Williams et al., 1998), nerve cell migration (Bix and Clark, 1998; Thomas and Huttunen, 1999), and cellular survival and death mechanisms (Das, 1999). Therefore, a number of investigators have examined the question of whether arachidonate metabolism and PGs are deregulated in schizophrenia.

II. Prostaglandin Hypothesis of Schizophrenia

In the 1970s, a concept was formed that centered the etiology of schizophrenia on PGs. Feldberg (1967) put forward the idea that schizophrenia may be due to an excess of PGs. He based his suggestion on the observation that PGE1 injections into animals produce catalepsy, which may be analogous to catatonia in humans. Further evidence came from studies on human platelets. Pandey (1977) investigated a small number of acutely psychotic schizophrenic patients. It was found that PGE1-stimulated cyclic adenosine monophosphate (cAMP) formation was enhanced in patients with acute exacerbation of their symptoms compared with chronic schizophrenic patients and healthy controls. In contrast, Horrobin (1977) supported the hypothesis that schizophrenia may be associated with a deﬁciency of PGE1. PGE1 is an important compound of the DGLA series (series 1) and was described to regulate the synthesis of PGs of the AA series (series 2) with a deﬁciency of PGE1 leading to excess formation of series 2 PGs (Horrobin, 1979). Also the PGE1 deﬁciency hypothesis was corroborated by investigations on human blood cells. All 20 unmedicated severe schizophrenic patients failed to increase the formation of PGE1 from DGLA in response to adenosine diphosphate (ADP). In contrast, 8 healthy, 10 depressed, and 8 manic individuals all increased PGE1 synthesis four- to ﬁvefold (Abdullah and Hamadah, 1975). Similarly, studies on peripheral blood cells of schizophrenia patients revealed a signiﬁcant decrease of PGE1-stimulated cAMP production in both acutely psychotic and chronic schizophrenia patients (Kafka et al., 1979; Kaiya et al., 1990; Ofuji et al., 1989; Rotrosen et al., 1978). Also, a large amount of indirect evidence (Horrobin, 1977, 1978; Horrobin et al., 1978) pointed to a PG deﬁciency in schizophrenia: the almost complete absence of rheumatoid arthritis in schizophrenic patients (Oken and Schulzer, 1999), which is associated with an overproduction of PGs, the altered thermosensitivity associated with schizophrenia (Hermesh et al., 2000); the resistance of schizophrenic patients to pain (Dworkin, 1994; Kudoh et al., 2000), which is mediated by PGs; and the anecdotally reported improvement of schizophrenia symptoms during febrile illnesses (brain PGE1 levels rise during fever) (Lipper and Werman, 1977). Additional support for the PG-based concept of schizophrenia

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came from an animal model in which amphetamine-induced stereotypic behavior in mice was reversed after intraventricular injections of PGs (Schwarz et al., 1982). Keeping in mind the limitations of this paradigm as a model of schizophrenia, the study nevertheless demonstrated the ability of PGs to inﬂuence a transmitter system assumed to be involved in the pathophysiology of schizophrenia. Although the inﬂuence of PGs on mental functions was anything but clariﬁed, the promising results between the late seventies and the early eighties led to several studies with PGs as therapeutic agents in schizophrenia. The antibiotic penicillin, which stimulates 1 series (not 2 series) PG formation (Horrobin, 1978), was able to prevent psychotic relapse in 8 of 10 severe chronic hospitalized schizophrenic patients (Chouinard et al., 1978). In contrast, administration of intravenous PGE1 to 7 unmedicated schizophrenic patients with decreased PGE1-mediated platelet aggregation led to improvement in only 2 of the cohort (Kaiya, 1987). However, this study was limited by a small sample size and unclear diagnostic criteria. Taken together, the evidence is very limited that treatment of schizophrenia patients with phospholipid-derived second messengers themselves, such as PGE1, is of any beneﬁt. More promising was the therapeutic use of PG precursors to schizophrenia patients. In a pilot trial, application of the PG precursor gamma linoleic acid (GLA) (an important component of evening primrose oil) in addition to neuroleptic medication led to a considerable improvement of schizophrenic symptoms in severe chronic hospitalized patients nonresponding to diVerent antipsychotic medication (Vaddadi, 1979).

III. Membrane Phospholipid Hypothesis of Schizophrenia

A new impulse for investigations on phospholipids and related second messenger systems in the ﬁeld of schizophrenia was given by the assumption that the function of literally all membrane-bound and membrane-associated proteins must in some way depend on the ﬁnal tertiary structure of neuronal membrane phospholipids. Therefore, metabolic steps higher up the arachidonate pathway moved into focus. First investigations again were performed on blood cells. Membrane breakdown products as lysophosphatidylcholine were increased signiﬁcantly in platelets of schizophrenic patients (Pangerl et al., 1991). Furthermore, the turnover of phosphatidylinositol (another important cerebral phospholipid) was found to be increased in schizophrenic patients (Demisch et al., 1992; Yao et al., 1992), whereas incorporation of arachidonic acid turned out to be decreased in patients compared to controls (Demisch et al., 1992; Yao and van Kammen, 1996). The introduction of new magnetic resonance-based techniques made it possible to also investigate cerebral phospholipid metabolism in vivo. In accordance with changes on blood cells, ﬁrst results using 31P magnetoresonance

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spectroscopy (31P-MRS) were suggestive of an upregulation of lipid breakdown processes in frontal (Keshavan et al., 1991; Pettegrew et al., 1991) and temporal (Calabrese et al., 1992; Fukuzako et al., 1999) brain regions of schizophrenic patients free of antipsychotic medication. Results pointed toward an increased phospholipid turnover, especially in the early phase of disorder (Stanley et al., 1994). Further evidence for the cooccurrence of deregulated phospholipid breakdown processes and schizophrenia came from investigations of phospholipase A2 (PLA2) enzyme activity. Gattaz and colleagues (1987, 1990, 1995) demonstrated ﬁrst an overactive PLA2 in plasma, serum, and platelets of patients with schizophrenia, a ﬁnding conﬁrmed by other groups (Lasch et al., 2003; Ross et al., 1997). First studies on postmortem brain tissue of schizophrenic patients revealed similar changes found in serum or peripheral blood cells. An investigation by Ross et al. (1999) examined the activity of two classes of PLA2, calcium stimulated and calcium independent, in autopsied temporal, prefrontal, and occipital cortices, putamen, hippocampus, and thalamus of 10 patients with schizophrenia as compared to controls. Calcium-independent PLA2 activity was increased 45% in the temporal cortex of patients but was not altered signiﬁcantly in other brain areas. In contrast, calciumstimulated PLA2 activity was decreased by 27–42% in the temporal and prefrontal cortices and putamen, with no signiﬁcant alterations in other brain regions. As decreased calcium-stimulated PLA2 activity has also been reported in the striatum of chronic cocaine users, it could be in part related to increased dopaminergic activity in schizophrenia. The increase of calcium-independent PLA2 activity was related to abnormal fatty acid metabolism (Ross et al., 1999). A considerable number of studies on peripheral blood cells and postmortem brain tissue have apparently shown similar metabolic changes. Therefore, investigators used methodically combined approaches to further explore systemic aspects of metabolic alterations observable in schizophrenia. A combined application of 31 P-MRS and a ﬂuorimetric PLA2 enzyme assay to a small sample of unmedicated schizophrenic patients demonstrated a positive correlation between increased cerebral phospholipid breakdown and increased PLA2 activity in blood serum (Smesny et al., 2000). Another 31P-MRS-based study in unmedicated ﬁrst-episode psychotic patients revealed moderate to strong correlations between brain phosphoester peaks and peripheral red cell membrane AA levels (Yao et al., 2000b). The gap between cerebral phospholipid alterations and reduced PGE1 formation in schizophrenic patients was closed by studies on polyunsaturated fatty acids (PUFA). As reviewed by Fenton et al. (2000), results of 15 studies indicate a depletion of PUFA in blood cells of patients with schizophrenia. Studies done on red cell membranes (Glen et al., 1994; Peet et al., 1995) suggested the existence of a group of schizophrenic patients with signiﬁcantly decreased concentrations of key fatty acids in which the decrease of PUFA was mainly attributed to AA and docosahexaenoic acid (DHA) depletion in membranes. Investigations on various animal models demonstrated similarities of PUFA distribution in

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peripheral blood cells and nervous tissue (Carlson et al., 1986; Connor et al., 1990, 1993; Lim and Suzuki, 2000). Accordingly, reduced PUFA levels have also been found in postmortem brains of schizophrenic patients (Horrobin et al., 1991; Yao et al., 2000) similar to the changes of PLA2 activity in blood cells and postmortem brain tissue. Results of increased cerebral phospholipid turnover, increased PLA2 activity, and membrane PUFA depletion supported the phospholipid hypothesis concept of schizophrenia and the notion that alterations of phospholipid metabolism are present throughout the body (Horrobin, 1996). The relevance of phospholipid alterations to psychiatric symptomatology and the pathophysiological mechanisms behind the disturbance were, however, far from being resolved. A 31P-MRS study by Shioiri and co-workers (1994) reported a signiﬁcant correlation between phospholipid alteration in the prefrontal brain and schizophrenic negative symptoms. Similarely, Glen et al. (1994) reported low levels of red cell membrane AA and DHA in a subgroup of schizophrenic patients with predominantly negative symptoms (apathy and social withdrawal). Patients with positive symptoms (e.g., delusions or hallucinations) showed normal fatty acid levels. Zubenko and Cohen (1986) reported disturbed membrane ﬂuidity of platelets only in schizophrenic patients with tardive dyskinesia (TD). In accordance with this ﬁnding, another group (Vaddadi et al., 1989) reported a signiﬁcant correlation between the depletion of red blood cell PUFAs and the occurrence of tardive dyskinesia. Given that the occurrence of negative symptoms indicates an insidiously deteriorating course of the disorder, and tardive dyskinesia an unfavorable hypersensitivity to neuroleptic treatment, phospholipid alterations may be taken as potential markers of poor prognosis and poor treatment response to antipsychotic medication. Phospholipase A2 is important for the breakdown of oxidative-damaged membrane lipids. Investigations of oxidative processes on membrane lipids demonstrated that polyunsaturated fatty acids are especially modiﬁed. PLA2 removes altered fatty acids from the membrane and enables detoxication by antioxidative defense systems (e.g., superoxide dismutase, glutathione peroxidase, catalase, and vitamin E). An impaired activity of superoxide dismutase (Mukerjee et al., 1996) and a concomitant increase in plasma lipid peroxides (Mahadik et al., 1998) have been reported in never-medicated ﬁrst-episode schizophrenic patients. Thereby, oxidative stress was associated with deterioration of school functioning from childhood to early adolescence and more pronounced negative symptomatology in the later course of the disorder. These ﬁndings indicate that a disturbance of phospholipid metabolism at the onset of psychosis may be caused by a compromised antioxidant defense. They are suggestive that oxidative injury might contribute to adverse developmental events in the pathogenic cascade of schizophrenia and to the development of a later deﬁcit syndrome.

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The assumption of dysfunctional phospholipid metabolism associated with schizophrenia is also supported by a number of genetic investigations, most of them at gene loci coding for PLA2 subtypes. Tay and co-workers (1995) identiﬁed a gene coding for cytosolic PLA2 at chromosome 1q25, a region that was later targeted by other groups. Hudson et al. (1996) investigated single chain nucleotide polymorphisms in the promotor region of this gene. They applied the so-called ‘‘haplotype relative risk’’ design (Terwilliger and Ott, 1992) to 44 triades of both parents and one oVspring each fulﬁlling DSM IV criteria for schizophrenia. That maternal and paternal allele that was not passed on to the oVspring served as the allele pair of a virtual control person. In 40% of schizophrenic subjects, over 50 polyadenosine repeats were found, demonstrating an association between the number of repeats and the manifestation of schizophrenia. This ﬁnding was replicated by Peet et al. (1998), who also found signiﬁcantly diVerent allelic frequencies in this PLA2 promoter region between patients with schizophrenia and normal controls. Genes of PLA2 and COX-2 were shown to be located at the same region (1q25) on chromosome 1 (Tay et al., 1995). Furthermore, the activity of cytosolic PLA2, COX, and PGD2 synthase has been reported to be regulated in a coordinated manner (Murakami et al., 1999). Another PLA2 gene locus of 69 kb has been described at chromosome 22q13.1, which includes at least 17 exons (Pontus et al., 1999). Interestingly, this genetic region [chromosome 22 (q11–13)] was also found by another group to be altered in schizophrenia (Deickert et al., 1997). In summary, available data suggest that abnormalities at diVerent PLA2 gene loci may also have causative importance for the deregulation of phospholipid metabolism, which is evident in schizophrenia. Integrating the results on cerebral phospholipids, PLA2, and PUFAs with earlier PG ﬁndings, Horrobin put forward the ‘‘membrane phospholipid hypothesis’’ of schizophrenia (Horrobin, 1996; Horrobin et al., 1998). In contrast to the PG-based concept, which attached special importance to a disturbed transmitter function of the PGs themselves, this extended concept included a systemic deregulation of phospholipid metabolism with cerebral membrane instability and a general disturbance of information ﬂow in a subgroup of schizophrenic patients. Within the scope of a neurodevelopmental concept, genetic abnormalities of key enzymes of phospholipid degradation and oxidative damage of neuronal membranes were hypothesized as main etiopathological mechanisms.

IV. Niacin Tests in the Field of Schizophrenia

As a consistent ﬁnding, most studies investigating phospholipid metabolism in schizophrenia showed that only a subgroup of patients, in general 30–50%, displayed some kind of alteration. Thus, although patients were selected in general

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according to DSM-IV or ICD-10 criteria for schizophrenia and thereby showed presumably a homogeneous psychopathology, only a certain number of them displayed metabolic alterations. Given that schizophrenia represents a syndrome with heterogeneous etiopathology (Garver et al., 2000) and that phospholipid alterations are of importance for the development or clinical manifestation of the disorder, then investigation of phospholipid biochemistry in schizophrenia oVers the possibility of deﬁning an intermediate phenotype (endophenotype). The search for biological criteria to deﬁne such endophenotypes has a long history. A number of markers (e.g., nail plexus visibility, eye tracking or evoked potentials: P50, P300) have been described in subgroups of schizophrenic patients according to DSM-IV or ICD-10 criteria, but none of these markers has reached signiﬁcant importance in clinical practice. The absence or diminution of a niacin (vitamin B3) ﬂush response (also called niacin sensitivity) in schizophrenia patients was proposed to indicate an endophenotype of schizophrenia with a deregulated arachidonate pathway (Ward, 2000). The hyperemic eVect of niacin was initially noticed in the ﬁeld of dermatology. Illig (1952) described marked interindividual diVerences of skin reaction in terms of intensity and pattern of skin response, with some people showing no skin reaction at all. Most of them were identiﬁed as suVering from neurodermatitis. Furthermore, niacin gained interest as a hypolipidemic agent with the potential to decrease mortality in patients with hyperlipidemia (Morrow et al., 1989). A diminished ﬂush response among individuals with schizophrenia was reported for the ﬁrst time by HoVer (1962). Horrobin (1980) contributed the clinical observation that the improvement of schizophrenia symptoms was associated with a restoration of normal ﬂushing. According to the PGE1 deﬁciency hypothesis, he initially suggested that this might be related to a normalization of defective conversion of PGE1 precursors (Horrobin, 1980; Horrobin et al., 1978). This assumption was further supported by the ﬁnding that niacin stimulation initiates an increase of PGE (the used assay was not able to diVerentiate between PGE1 and PGE2) (Eklund et al., 1979) and by the fact that niacin ﬂushing could be attenuated substantially by pretreatment with COX inhibitors, such as acethylsalicylic acid. However, research revealed other possible mechanisms behind the niacin skin phenomenon and thereby changed the interpretation of diminished niacin sensitivity in schizophrenia. In search for the ﬂush causing PG, Morrow et al. (1989) observed in three healthy subjects that the skin response after ingestion of 500 mg niacin was accompanied by a 800-, 430-, and 535-fold increase of the PGD2 metabolite 9- 11--PGF2 in plasma, which correlated with the intensity and duration of ﬂushing. Therefore, PGD2 (a vasodilator) was supposed to be the active mediator of the niacin-induced skin reaction. However, the fact that topical skin exposure to niacin also caused a signiﬁcant erythema and edema response left the question of the origin of PGD2. In a more recent study, Morrow’s group showed 58- to 122fold increased PGD2 levels and 25- to 33-fold increased levels of the PGD2

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metabolite 9--11--PGF2 in blood drawn from the antecupital vein draining the arm were the niacin was applied topically (Morrow et al., 1992). The release of PGD2 occurred dose dependently over a concentration range of 103 to 101 M. In blood drawn simultaneously from the contralateral arm, PG levels were not increased, indicating that PGD2 was released locally from the site of niacin stimulation. In addition, the observation that PGD2 levels after oral niacin stimulation in blood from superﬁcial veins draining the skin were 14 to 1200 times higher than the levels in arterial blood supplying the skin of the same arm led to the conclusion that the skin is the major site where PGD2 is released. The mechanism of action of PGD2 was further elucidated by animal studies. The identiﬁcation of niacin responsive cells in the skin goes back to results by Urade and colleagues (1989) in various rat tissues. Dermal macrophages were found to contain the highest concentration of PG synthase and were therefore assumed to have the greatest capacity for PGD2 production. The mechanism of ﬂush response has also been studied in vivo in the ears of guinea pigs (Andersson et al., 1977). The threshold doses of niacin (1–3 mg/kg) needed to raise the skin temperature of the ears and to increase the cAMP levels of this tissue were similar. In isolated slices from guinea pig ears, niacin increased the level of cAMP. Also the association of PGD2 formation with metabolic steps higher up the arachidonate pathway was demonstrated in animal studies. Investigations on colon tumors of rats (Rao et al., 1996) displayed connections among the membrane fatty acid proﬁle, PLA2 activity, and the formation of PGs by the action of COX-2. Studies on cultured mouse mast cells revealed a coordinated gene regulation of cPLA2, PG endoperoxide synthase (PGHS)-1, and hematopoetic PGD2 synthase, all enzymes necessary for the production of PGD2 from endogenously released arachidonic acid (Murakami et al., 1995). Thus, as it can be concluded so far, PGD2 released by skin macrophages may trigger an increase of intracellular cAMP production, which in turn causes a vasodilatation of superﬁcial skin microvessels—clinically observable as erythema and edema. As PGD2 is formed as a result of endogenously mobilized arachidonic acid from membrane phospholipids, niacin sensitivity is assumed to give indirect information about the availability of membrane-bound long-chain fatty acids and their bioactive derivatives (e.g., PGs). In terms of cell-to-cell signaling, niacin sensitivity indicates the capacity of peripheral immunocompetent cells (e.g., skin macrophages) to receive metabolic signals (e.g., via PGD2), to further process them (via cAMP production), and to mediate a physiological tissue response (in blood vessels).

A. Clinical Studies Using Oral Niacin Stimulation After the ﬁrst clinical observations by HoVer (1962) and Horrobin (1980) that were based on the assessment of ‘‘ﬂush obvious to the naked eye,’’ attempts were made to quantify niacin sensitivity within the scope of controlled clinical trials.

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Whereas Wilson and Douglass (1986) evaluated niacin sensitivity by measuring the blood ﬂow by a photoplethysmograph attached to the left ear lobe, Fiedler et al. (1986) used a malar thermal circulation index (MTCI) (Wilkin, 1982), which includes the approximation of peripheral skin temperature to body core temperature caused by an increase of skin perfusion after niacin stimulation. Both groups failed to elicit vasodilatation diVerences between medicated (Fiedler et al., 1986) and unmedicated (Wilson and Douglass, 1986) schizophrenic patients and controls, most likely attributable to comparatively low niacin doses and small patient sample sizes. More successful were the investigations by Rybakowski and Weterle (1991), who combined thermometric recordings (Wilkin, 1982) with visual assessments of skin color changes in 33 drug-free schizophrenic patients and controls. In all controls but only in 76% of schizophrenia patients was skin ﬂushing noted. Accordingly, the time of maximum temperature following niacin intake was delayed signiﬁcantly in nonﬂushing schizophrenic patients. A consecutive clinical trial (Hudson et al., 1997) on the basis of Wilkin’s MTCI method demonstrated that 42.9% of patients with schizophrenia had no change in temperature, whereas 94% of patients with bipolar disorder and 100% of normal controls did. As has been reported in PLA2 and PUFA studies, about 30–50% of schizophrenic patients seem to exhibit some kind of alteration of phospholipid metabolism. The fact that Hudson et al. (1997) identiﬁed a similar percentage of schizophrenic patients not responding to niacin stimulation raised the question of the association between niacin sensitivity and steps higher up the arachidonate pathway. In a double-blind multicenter study on medicated schizophrenic patients with primarily negative symptoms, Glen and colleagues (1996) took measures of red blood cell PUFAs and niacin ﬂush response. One-half of the schizophrenic patients failed to ﬂush and showed signiﬁcantly reduced levels of arachidonic and docosahexaenoic acids. Conversions from non-ﬂushing to ﬂushing during a 6-month supplementation period of the AA precursor GLA was predicted by an increase of AA levels in red blood cells irrespective of treatment. In a comparable study, Tavares et al. (2003) investigated the relationship between niacin response and PLA2 activity in 38 drug-free schizophrenic patients and 28 healthy controls. Twenty-two of these patients were reevaluated after 8 weeks of treatment with atypical neuroleptic drugs. Patients with diminished or absent niacin responses displayed the highest PLA2 activity as compared to those with normal niacin responses. Eight weeks of antipsychotic treatment led to a decrease of PLA2 activity, and 4 out of 13 patients with absent responses to niacin converted to niacin positive. The association between diminished niacin sensitivity, decreased AA availability (Glen et al., 1996), and increased PLA2 activity (Tavares et al., 2003) in schizophrenic patients suggests that niacin skin tests may be diagnostic value for the identiﬁcation of patients with disordered phospholipid metabolism. As niacin sensitivity normalized with fatty acid supplementation and

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neuroleptic treatment, niacin sensitivity may also be worth investigating as a marker of therapy response.

B. The Topical Niacin Skin Test in Schizophrenia Further research on niacin sensitivity was inﬂuenced by several methodical problems: the wide physiological variation of a normal skin response with about 10% of healthy people showing no response et all (Basketter and Wilhelm, 1996), the problem of integrating and quantifying all test information (pattern, intensity, and time course of erythema and edema), and the unsettling side eVects associated with the ﬂush reaction of the upper body after oral niacin application. Considerable progress in terms of test applicability has been reached by a new topical variant of skin stimulation introduced by Ward et al. (1998). Erythema was assessed after a topical application of four diVerent concentrations of niacin (0.1, 0.01, 0.001, and 0.0001 M ) in 5-min steps on the basis of an ordinate four-point rating scale (0, no response to 3, maximal cutaneous erythema) for the assessment of skin redness and a qualitative description (yes/no) of edema. In a preliminary study, Ward et al. (1998) demonstrated a zero or minimal niacin response in 83% of medicated schizophrenia patients and 23% of controls with the greatest degree of diVerentiation at 0.01 M niacin. Using the same test protocol, these results have also been conﬁrmed in unmedicated schizophrenia patients (Shah et al., 2000). A more elaborated seven-point rating scale has been introduced by Berger and McGorry (2001), which integrates erythema, edema, and time course of the skin response. In a population of ﬁrst-episode psychosis patients, about 40% had a strong impairment of skin reaction (Berger et al., 2002a). The quantiﬁcation of niacin sensitivity after topical niacin stimulation via optical reﬂection spectroscopy (ORS) (Smesny et al., 2001) and via blood ﬂow duplex sonography (Messamore et al., 2003) also revealed consistent results. In two consecutive ORSbased studies on diVerent populations of medicated schizophrenic patients, a signiﬁcantly diminished niacin sensitivity as compared to controls was demonstrated (Smesny et al., 2003a,b). In accordance with these results, Messamore et al. (2003) described a signiﬁcantly lower blood ﬂow response to higher niacin doses in schizophrenic patients measured by laser Doppler ﬂowmetry. A study combining Berger’s qualitative seven-point rating scale and ORS demonstrated that ORS is able to separate early psychosis patients from controls in a similar way as qualitative rating scales (Smesny et al., 2003a). These results suggest that both descriptive rating scales and objective assessment techniques are capable of distinguishing an endophenotype of patients with deregulated phospholipid metabolism.

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C. Niacin Skin Test and Psychopathology Most niacin skin test studies have demonstrated that a substantial proportion (between 25 and 50%) but not all schizophrenic patients have a dysfunctional phospholipid metabolism. This result seemed to be in line with the ﬁndings of PLA2 and PUFA studies. However, it was still unresolved whether the patient group deﬁned by diminished niacin sensitivity corresponds to the patient group with primarily negative symptoms described by Shioiri et al. (1994), Glen et al. (1994), and Mahadik et al. (1998). Accordingly, Puri et al. (2000) aimed to determine whether the ﬂushing status correlates with positive or negative schizophrenia symptoms. In 13 patients, a signiﬁcant positive correlation was found between the degree of ﬂushing and positive symptoms. Nonﬂushing patients were found to have signiﬁcantly higher negative symptom scores. Similar results have also been reported by other groups (Berger et al., 2002a). An optical reﬂection spectroscopic investigation was also able to demonstrate a negative correlation between skin erythema and negative symptom scores (Smesny et al., 2003a). Therefore, available data are suggestive that impaired niacin sensitivity as well as increased PLA2 activity and lowered levels of PUFA in red blood cell membranes indicate a subgroup of schizophrenic patients with pronounced negative symptoms.

V. Summary

At ﬁrst, empirical investigations of PGs in the ﬁeld of schizophrenia were methodically diYcult (due to the short half-life of PGs) and produced inhomogeneous results. Most direct and indirect evidence for the relevance of disturbed PG-mediated signaling to schizophrenia was integrated by the PGE1 deﬁciency hypothesis, which gained considerable importance and represented the basis for further studies of lipid biochemistry in schizophrenia. However, because the molecular mechanisms of PG-mediated signaling were not yet known, the information gained concerning the pathophysiology of schizophrenia was still limited. The ﬁrst clinical trials of PGs and their precursors as therapeutic agents revealed promising results but could obviously not fulﬁll the expectations. Accordingly, the idea that PGs themselves contribute to the pathophysiology of schizophrenia retreated into the background. In extension of the prostaglandin concept, results from a variety of studies on cerebral phospholipids, PLA2 activity, and polyunsaturated fatty acids pointed toward an alteration in membrane lipid chemistry cooccurring with schizophrenia, possibly caused by the permanent oxidative damage of phospholipids or genetically determined upregulation of divers PLA2 subtypes. However, not all patients with schizophrenia displayed such abnormalities. Evidence was increasing

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that arachidonic acid and related bioactive lipids are altered only in a subgroup of schizophrenic patients characterized by prominent negative symptoms, poor response to treatment, and poor prognosis. The implications of these ﬁndings were noted to be relevant for the understanding of a new intermediate phenotype of patients within the schizophrenia spectrum. Accordingly, the assumption of altered PG second messengers (e.g., PGD2) secondary to disturbances of phospholipid degradation led to the implementation of a niacin stimulation test, which has the potential to deﬁne this endophenotype. Studies on eicosapentaenoic acid supplementation in schizophrenia patients (Berger et al., 2002b; Emsley et al., 2002; Fenton et al., 2000) are suggestive that the availability of an easily applied skin test for phospholipid alterations and of modern methods to quantify test results are important for the implementation of new treatment options.

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MITOCHONDRIA, SYNAPTIC PLASTICITY, AND SCHIZOPHRENIA

Dorit Ben-Shachar and Daphna Laifenfeld Laboratory of Psychobiology Department of Psychiatry, Rambam Medical Center and B. Rappaport Faculty of Medicine Technion IIT, Haifa, Israel

I. Introduction II. Plasticity in Schizophrenia A. Conceptualization of Schizophrenia as a Disorder of Plasticity B. Modulation of Neuronal and Synaptic Plasticity by Neurotransmitters Involved in Schizophrenia C. Direct Evidence for Neuronal Plasticity in Schizophrenia III. Mitochondrial Dysfunction in Schizophrenia A. Mitochondrial Morphological Aberration B. Mitochondrial Oxidative Phosphorylation System Malfunction C. Abnormal Mitochondrial-Related Gene Expression IV. Mitochondria and Neuroplasticity A. Mitochondrial Function B. Mitochondria and Neuronal Activity C. Role of Mitochondrial-Regulated Factors in Neuronal Plasticity D. Schizophrenia and Abnormalities in Mitochondrial Plasticity Relevant Factors V. Conclusion References

The conceptualization of schizophrenia as a disorder of connectivity, i.e., of neuronal/synaptic plasticity, suggests abnormal synaptic modeling and neuronal signaling, possibly as a consequence of ﬂawed interactions with the environment, as at least a secondary mechanism underlying the pathophysiology of this disorder. Indeed, deﬁcits in episodic memory and malfunction of hippocampal circuitry, as well as anomalies of axonal sprouting and synapse formation, are all suggestive of diminished neuronal plasticity in schizophrenia. Evidence supports a dysfunction of mitochondria in schizophrenia, including mitochondrial hypoplasia, and a dysfunction of the oxidative phosphorylation system, as well as altered mitochondrial-related gene expression. Mitochondrial dysfunction leads to alterations in ATP production and cytoplasmatic calcium concentrations, as well as reactive oxygen species and nitric oxide production. All of the latter processes have been well established as leading to altered synaptic strength or plasticity. Moreover, mitochondria have been shown to play a role in plasticity of neuronal polarity, and studies in the visual cortex show an association between mitochondria and synaptogenesis. Finally, mitochondrial gene upregulation has been observed following synaptic and neuronal activity. This review proposes INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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that mitochondrial dysfunction in schizophrenia could cause, or arise from, anomalies in processes of plasticity in this disorder

I. Introduction

Schizophrenia is the most disabling mental disorder, aVecting 1% of the world population. Schizophrenic patients present various abnormalities of perception, thought, language, and aVect, and many of them will develop cognitive deﬁcits, as well as marked long-lasting impairments in occupational and social functioning (Green, 1996; McGlashan, 1988; McGlashan and Fenton, 1992). Despite intensive research, the etiology and the pathophysiology of the disease have remained elusive. A variety of hypotheses have been raised regarding the etiology of schizophrenia, one of the ﬁrst and still the most substantiated is the ‘‘dopamine hypothesis,’’ which postulates a malfunction of the dopaminergic system as the pathophysiological basis of the disease (Carlsson, 1988; Hietala and Syvahti, 1996; Kapur and Remington, 2001; Matthysse, 1974; Seeman, 1987; Willner, 1997). Additional neurotransmitters, such as glutamate and GABA, have also been implicated in schizophrenia (Carlsson et al., 1999a,b, 2001; Kim et al., 1980; Lewis et al., 1999). Research has converged on a new hypothesis, the neurodevelopmental hypothesis, which posits a prenatal or early postnatal cerebral aberration as the causative factor in schizophrenia (Altshuler et al., 1987; Arnold, 1999; Arnold et al., 1991a,b; Bunney et al., 1995; Chua and Murray, 1996; Conrad and Scheibel, 1987; Weinberger, 1996). This hypothesis is attractive in that it integrates data from postmortem, imaging, epidemiological, physiological, and biological studies (Altshuler et al., 1987; Arnold, 1999; Arnold et al., 1991a,b; Bunney et al., 1995; Cannon et al., 1999; Chua and Murray, 1996; Conrad and Scheibel, 1987; Marenco and Weinberger, 2000; Weinberger, 1996). One main feature of schizophrenia is that its clinical symptoms are expressed in adulthood. While this observation can be consistent with the neurodevelopmental component of the disease, it also points to the possibility of prepuberty events, secondary to the in utero genetic, epigenetic, and activity dependent pathological mechanisms taking place in the developing brain. A conceivable secondary mechanism is abnormal intrinsic- or extrinsic-dependent modulation of neuronal plasticity. Neuronal plasticity is a gradual process by which the nervous system adapts to changes in the environment by structural and functional modulations. Neuronal plasticity can be divided into two distinct processes: neurogenesis, which in the mature brain is detectable only in a limited number of brain areas, most notably the hippocampus, but apparently also in the subventricular zone as well as in subcortical white matter (Gage, 2000; Kennea and Mehmet, 2002; Nunes et al., 2003), and synaptic plasticity, which is an activity-dependent modulation

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of the pattern and strength of synaptic connections and thereby neuronal circuits. Neuronal plasticity reﬂects the interaction between neuronal ﬁring, gene expression, and protein activity, which leads to changes in synaptic eYcacy, neoritogenesis, and cell migration, as well as a slow spatiotemporal dynamic morphogenesis. These processes are associated with learning and memory, as well as with adaptive changes in emotional, cognitive, and sensorimotor function. This review suggests that the pathophysiology of schizophrenia involves abnormalities in long-term changes in neuronal plasticity, in which mitochondria could play a pivotal role. Mitochondria are the energy source driving the biochemical processes involved in various cell functions, take part in intracellular Ca2þ homeostasis, and produce reactive oxygen species (ROS). The relevance of these mitochondrial-dependent processes to synaptic plasticity is reviewed. In addition, mitochondrial dysfunction in schizophrenia, its possible role in altered neuronal plasticity and as a consequence the cognitive and behavioral anomaly characteristic of schizophrenia are discussed.

II. Plasticity in Schizophrenia

A. Conceptualization of Schizophrenia as a Disorder of Plasticity The term schizophrenia, i.e., ‘‘split mind,’’ was used ﬁrst by Bleuler (1911) and alluded to what was believed to be a disconnection between thought processes and emotional responses, suggesting the conception of schizophrenia as a disorder of connectivity dating back to the beginning of the previous century. Indeed, an illness characterized by an initial long period of subtle signs and symptoms, ﬁnally leading to full-blown malfunctioning, with reversals and exacerbations of symptoms, alongside changeable predominance of clinical symptoms, is unlikely to be attributed to a focal brain dysfunction or lesion (DeLisi, 1997). Rather, it is in line with (abnormal) waxing and waning of neuronal connections throughout an individual’s life course, the ongoing aberrations in connectivity possibly leading to a progressive disassociation between central neuronal circuits. The latter has, at times, led to the term ‘‘a disconnection syndrome’’ with reference to schizophrenia (McGlashan and HoVman, 2000). This does not preclude the conceptualization of this disorder as a neurodevelopmental one. The integrative conceptualization of schizophrenia as a neurodevelopmental disorder involving neuronal and synaptic plasticity is facilitated by the observation that signs and symptoms of schizophrenia begin to manifest only in early adulthood. Synaptic remodeling, initially occurring mainly in response to genetic and epigenetic mechanisms, is progressively more and more dependent on activity- or experience-dependent plasticity. The emotional, cognitive, and sensorimotor abnormalities in schizophrenia

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instigate ﬂawed interactions with the environment and can lead therefore to progressively abnormal synaptic modeling in line with the deterioration in symptom manifestation from early adulthood onward in schizophrenic patients.

B. Modulation of Neuronal and Synaptic Plasticity by Neurotransmitters Involved in Schizophrenia Alterations in the dopaminergic system are strongly implicated in schizophrenia (Carlsson, 1988). This is based primarily on the high correlation between the therapeutic eYcacy of antipsychotic drugs and their potency as dopamine receptor blockers (Seeman, 1987), and the ability of dopamine agonists to induce acute psychotic symptoms with marked resemblance to schizophrenia. Based on positron emission tomography (PET) studies examining blood ﬂow to the brain during a cognitive task, it has been suggested that acute psychotic episodes (positive symptoms) are associated with a hyperdopaminergic state in the mesolimbic regions, whereas negative symptoms are associated with a hypodopaminergic state in the mesocortical projections to the frontal cortex (Davis et al., 1991). A more direct approach such as PET studies of dopamine synthesis, receptor density, and amphetamine challenged release in schizophrenic brains, which were conducted in ﬁrst episode, drug-free patients, or patients in an acute exacerbation, implicated a dysfunction in dopamine metabolism, storage, release, or uptake mechanisms in the mesolimbic systems in schizophrenia (Breier et al., 1997; Laruelle et al., 1999). Further support for the modulation of dopamine activity in this disorder can be inferred from ﬁndings implicating catechol-omethyl transferase (COMT), the postsynaptic enzyme that methylates released dopamine to its ﬁnal metabolite, the homovanillic acid, as a risk factor in schizophrenia (Shifman et al., 2002; Weinberger et al., 2001). Interestingly, dopamine and dopaminergic transmission have been implicated in processes of plasticity. Evidence from animal models shows that dopamine gates and facilitates long-term potentiation (LTP) in the amygdala (Bissiere et al., 2003), as well as in the hippocampus (Li et al., 2003). Long-term potentiation and long-term depression are considered the neuronal mechanisms underlying all forms of experience-dependent plasticity. Their regulation by dopamine could, therefore, constitute a mechanism whereby neural circuit modiﬁcation in response to diVerent life experiences is altered in schizophrenic patients. Dopamine D2-like receptors, which have been speciﬁcally implicated in schizophrenia, were found to regulate synaptic transmission and LTP in the dentate gyrus (Manahan-Vaughan and Kulla, 2003). Pharmacological blockade or genetic ablation of the D2 receptor in mice led to the sprouting of dopaminergic neurons in the substantia nigra pars compacta, whereas treatment with a D2 agonist resulted in shrinkage of the terminal arbor of these neurons (Parish et al., 2002).

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The relevance of dopamine (DA) to schizophrenia not withstanding, research has focused on the involvement of glutamatergic mechanisms in the disorder. This was initially triggered by the observation that phencyclidine (PCP) ( Javitt and Zukin, 1991), ketamine, and MK801 (Krystal et al., 1994, 1999, 2000), all which, at pharmacologically relevant concentrations, act as noncompetitive antagonists of the NMDA receptor, have psychotomimetic eVects. PCP and ketamine were shown to lead to eVects resembling not only the positive symptoms of schizophrenia, but the negative symptoms and cognitive impairments as well ( Javitt and Zukin, 1991; Krystal et al., 2000). Moreover, at the physiological level, imaging studies show that PCP abusers present a hypofrontality similar to that observed in schizophrenic patients. Direct studies of glutamatergic neurotransmission in schizophrenia report a reduced concentration of glutamic acid in the cerebrospinal ﬂuid (CSF) of schizophrenic patients (Kim et al., 1980). Others have shown decreased glutamate and aspartate concentrations in the prefrontal cortex of patients with schizophrenia (Tsai et al., 1995). Postmortem studies show increases in kainate receptors in the prefrontal cortex and decreases in AMPA and kainate receptor binding in the hippocampus (Breese et al., 1995; Deakin et al., 1989; Kerwin et al., 1990). The role of N-methyl-D-aspartate (NMDA) in modulating the eYcacy of synaptic transmission is well established (for a review, see Carroll and Zukin, 2002). For example, studies reveal that Ca2þ inﬂux mediated by the NMDA receptor activates signal transduction cascades regulating the formation and modulation of synapses (Malenka and Nicoll, 1999; Mori and Mishina, 1995). It has been shown that increases in the surface density of NMDA receptors and altered NMDA receptor subunit composition accompany the tetanic stimulation of SchaVer collaterals, the latter inducing long-lasting synaptic plasticity (Grosshans et al., 2002). Similarly, long-term depression induction in pyramidal cells resulted in decreased -Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and NMDA synaptic currents, as well as a decrease in the number of functional NMDA receptors at synaptic sites (Heynen et al., 2000). Antipsychotic treatments, such as haloperidol, have been shown to facilitate glutamatergic transmission and to lead to a modulation of synaptic activity (Brene et al., 1998; De Souza et al., 1999). In all, the involvement of both of the two major neurotransmitters implicated in schizophrenia in the initiation and modulation of experience-dependent plasticity suggests that such processes are likely to accompany, if not lie at the basis of, schizophrenia.

C. Direct Evidence for Neuronal Plasticity in Schizophrenia At the cognitive level, evidence for abnormality in processes of neuronal plasticity in schizophrenia stems from deﬁcits in episodic memory observed in patients with schizophrenia (Weinberger, 1999). Episodic memory is a form of

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explicit memory that has been linked to hippocampal function and is considered the cognitive manifestation of underlying processes of plasticity. Similarly, abnormalities of cortical-evoked EEG potentials indicate malfunction of hippocampal circuitry in patients with schizophrenia, as well as in some unaVected relatives (Adler et al., 1998). Reductions in neuronal size in schizophrenic patients have been reported in the hippocampus (Arnold et al., 1995; Benes et al., 1991), as well as in the dorsolateral prefrontal cortex (Lewis, 1997). Concomitant with a lack of general gross loss of cortical neurons, gliosis, or any consistent evidence of degeneration of neurons (Dwork, 1997; Harrison, 1999; Selemon et al., 1998), such reductions may be construed as resulting from a decrease in neuronal processes and connections in these regions in schizophrenia. Indeed, direct measurement of the density of mossy ﬁber inputs to the CA3–4 subregion of the hippocampus reported a striking reduction (Goldsmith and Joyce, 1995), which was not conﬁrmed by an additional study (Adams et al., 1995). However, electron microscopy of the synaptic ultrastucture revealed an anomaly of axonal sprouting and a decrease in axon–dendrite synapses in schizophrenia (Uranova et al., 1996). It is interesting to note, in this respect, that evidence of hippocampal abnormalities suggestive of aberrations of plasticity has been found to be associated with genetic risk for the illness (Adler et al., 1998; Callicott et al., 1998), suggesting that molecular indices of plasticity constitute part of the genetic susceptibility for the disease (Weinberger, 1999). Haloperidol was found to alter spine shape as well as spine density, processes considered indicative of the ability of halperidol to modulate synaptic strength (Kelley et al., 1997). Moreover, in gerbil hippocampus, haloperidol was found to cause neurogensis (Dawirs et al., 1998), but the eVect on human neurogenesis is unknown. Finally, abnormal expression of several synaptic and plasticity markers has been reported in schizophrenic patients. Thus, reductions have been found in presynaptic proteins such as synaptophysin and synaptosomal-associated protein 25 (SNAP-25) (Eastwood et al., 1995, 1999). Similarly, mRNA expression of postsynaptic density protein 95 (PSD95), known to be involved in synaptic plasticity, was found to be reduced signiﬁcantly in the prefrontal cortex of schizophrenic patients (Ohnuma et al., 2000). Alterations in the marker for synaptic terminals, GAP43, were also reported in schizophrenia, but these have been inconsistent, with some showing increases in GAP43 (Perrone-Bizzozero et al., 1996), others showing decreases (see Weinberger, 1999), and still others ﬁnding no alterations (Honer et al., 1997). Weinberger (1999) suggested that decreased neuronal size, and reduced transcription of synaptic proteins, is a result of abnormally low pressures for synaptic remodeling and plasticity in schizophrenia. To conclude, both cognitive and behavioral manifestations of schizophrenia, as well as the established literature regarding dopamine and glutamate involvement in the disorder, support the notion of schizophrenia as a disorder of

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neuronal ‘‘miswiring.’’ Evidence directly assessing plasticity and its molecular underpinnings is lending further support to the latter conceptualization.

III. Mitochondrial Dysfunction in Schizophrenia

Several independent lines of evidence indicate mitochondrial dysfunction in schizophrenia, including mitochondrial hypoplasia, dysfunction of the oxidative phosphorylation system, and altered mitochondrial related gene expression (for review, see Ben-Shachar 2002). Indeed, the hypothesis of mitochondrial dysfunction in schizophrenia embraces most data gathered to date on this disorder. It is in line with impaired brain energy metabolism, developmental aberrations, abnormal neurotransmission and neuronal connectivity, and could account for the pattern of multifactorial inheritability in schizophrenia.

A. Mitochondrial Morphological Aberration Histological studies of the ultrastructure of the human brain are relatively sparse due to inherent diYculties in assessing postmortem specimens. However, microscopic analysis of the ultrastructure of autopsy specimens from the frontal cortex, the striatum, and the substantia nigra of schizophrenic patients all revealed morphological deformation and a reduction in the number and density of mitochondria (Kung and Roberts, 1999; Uranova and Aganova, 1998; Uranova et al., 2001). Interestingly, upon dividing the patients to drug-oV and drug-on groups, antipsychotic treatment tended to normalize mitochondrial density and volume in several brain areas (Kung and Roberts, 1999). Although the hitherto described ﬁndings are decisive, further studies with a larger sample size are needed.

B. Mitochondrial Oxidative Phosphorylation System Malfunction Alterations in the mitochondrial oxidative phosphorylation system (OXPHOS) have been observed in brain as well as blood cells in schizophrenia. The ﬁrst enzyme investigated in schizophrenic brain tissues was cytochrome c oxidase (complex IV ), which was suggested as an endogenous metabolic marker for neuronal activity (Wong-Riley, 1989). Several groups reported an increase or a decrease, as well as no change, in cytochrome c oxidase activity depending on the brain area studied (Cavelier et al., 1995; Maurer and Moller, 1997; Prince et al., 1997a). Thus, a reduction in complex IV activity was observed in most studies in the frontal cortex and in the caudate nucleus, whereas in the putamen

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there was a signiﬁcant increase with no change in brain tissue specimens from the nucleus accumbens, globus pallidus, thalamus, mesencephalon, and cerebellum. Interestingly, a more recent study demonstrated a strong negative correlation between complex IV activity and emotional and intellectual impairments, but not motor impairment, solely in the putamen (Prince et al., 2000). Substantial ﬁndings in brain and periphery in schizophrenia also implicate NADH ubiquinone dehydrogenase (complex I), which was shown to play a major role in controlling oxidative phosphorylation in synaptic mitochondria (Davey et al., 1998). In the brain, there are controversial ﬁndings regarding the activity of complex I, as it is particularly sensitive to postmortem delay (Mizino et al., 1990; Prince et al., 1998) and its detection is less sensitive in whole tissue than in isolated mitochondria. Nevertheless, a signiﬁcant reduction in NADH–cytochrome c reductase (complexes I–III) activity was observed by one study in the frontal cortex and by two others in the temporal cortex and in the basal ganglia (Maurer et al., 1997; Prince et al., 1997a; Whatley et al., 1996). The mitochondrial oxidative phosphorylation system was found to be impaired not only in brains of schizophrenic patients, but also in their platelets and lymphocytes. A small sample of schizophrenic patients showed that the activity of complex I, but not that of complex II, II–III, or V, was reduced signiﬁcantly in platelets and lymphocytes of chronic antipsychotic-treated schizophrenic patients (Burkhart et al., 1993; Whatley et al., 1998). We have reported a diseasespeciﬁc and state-dependent alteration of complex I activity, but not of complex V, in platelets in 113 schizophrenic patients (Ben-Shachar et al., 1999; Dror et al., 2002). Thus, complex I activity was increased signiﬁcantly in patients with predominant positive symptoms and reduced in patients with residual schizophrenia as compared to controls. No change was observed in patients with aVective disorders, either major depression or bipolar disorder (the depressed type). Interestingly, in the schizophrenic patients a correlation was observed between their clinical state and complex I activity, particularly with the severity of patients’ positive symptoms, as assessed by the positive and negative symptom scale (PANSS) (Dror et al., 2002). Notably, antipsychotics, typical as well as atypical, are reported to interact with the mitochondrial oxidative phosphorylation system, speciﬁcally with complex I, both in vitro and in vivo in rodents and in human (Balijepalli et al., 1999, 2001; Barrientos et al., 1998; Burkhardt et al., 1993; Maurer et al., 1997; Prince et al., 1997b, 1998; Whatley et al., 1998). In addition, strong in vivo and in vitro experimental evidence has accumulated, suggesting that dopamine, the most predominant etiological factor in schizophrenia, interacts with the mitochondrial respiratory system (Ben-Shachar et al., 1995; Berman and Hastings, 1999; Chan et al., 1994; Cohen et al., 1997; Przedborski et al., 1993). It has been shown that dopamine impairs mitochondrial membrane potential, similar to the complex I inhibitor rotenone, in B lymphocytes (Elkashef et al., 2002).

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Additional self-evident support for the dopamine interaction with mitochondria is that monoamine oxidase, the enzyme responsible for the metabolism of dopamine, is located on the outer membrane of the mitochondria. Consequently, mitochondria are a preferred target for dopamine or its metabolites.

C. Abnormal Mitochondrial-Related Gene Expression Altered activity of OXPHOS complexes can be caused either by their interaction with an endogenous or an exogenous factor (e.g., dopamine or antipsychotics, respectively) or by an alteration in the expression of the subunits, which assemble with a precise stoichiometry to give rise to functional complexes. Each complex of the OXPHOS system consists of multiple components, or subunits. Approximately 70 of those subunits are encoded by nuclear DNA (nDNA), their genes distributed randomly over the chromosomes with no obvious clustering, and 13 are encoded by mitochondrial DNA (mtDNA). Thus, interference with the expression of any of the subunits may result in a false assembly of the complex and mitochondrial dysfunction. Indeed, alterations in the expression of several subunits of both NADH dehydrogenase and cytochrome oxidase were reported. We have analyzed transcripts and protein levels of three nuclear DNA-encoded subunits of complex I in platelets and lymphocytes of schizophrenic patients and normal subjects. The 24- and 51-kDa subunits, both iron–sulfur ﬂavoproteins having catalytic properties including the site for transhydrogenation from NADH to NADþ, and the 75-kDa subunit, the largest iron–sulfur transmembranous protein. These three subunits comprise one functional unit of complex I (Hateﬁ, 1985). Both mRNA and protein levels of the 24- and 51-kDa subunits were signiﬁcantly higher in schizophrenic patients than in controls, with no change in the 75-kDa subunit (Dror et al., 2002). Preliminary ﬁndings in postmortem brain specimens from schizophrenic patients indicate that complex I subunits are similarly altered in their brain. In addition, an increase in the expression of cytochrome oxidase subunit II was observed in the postmortem frontal cortex of schizophrenic patients (Mulcrone et al., 1995). In line with the latter is the ﬁnding that chronic treatment of rats with the antipsychotic -ﬂupenthixol caused downregulation of cytochrome oxidase subunit II transcripts (Whatley et al., 1996). A further unexpected support for the dysfunction of mitochondria in schizophrenia came from studies seeking to elucidate the molecular basis for schizophrenia by scanning the expression of numerous genes in postmortem brain specimens. Already in 1995, Mulcrone and colleagues reported that in the frontal cortex, the mRNA population of schizophrenic patients diVered from that of normal subjects in ﬁve expressed cDNAs, all of which encoded mitochondrial transcripts. Four of them encoded mitochondrial rRNA and one encoded part of the amino acid sequence of cytochrome oxidase subunit II. In an elegant

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study using a cDNA microarray to compare gene expression proﬁling of several major metabolic pathways in postmortem frontal cortex samples of schizophrenic and normal subjects, only 5 out of 71 metabolic pathways showed consistent changes (all were decreased) in subjects with schizophrenia. Four of them, the mitochondrial malate shuttle system, the transcarboxylic acid cycle, and aspartate and alanine metabolic pathways, are tightly linked to the mitochondrial energy production system (Middleton et al., 2002). IV. Mitochondria and Neuroplasticity

A. Mitochondrial Function Mitochondria are the principal source of high-energy intermediates and, as such, are of enormous importance for neuron function. Neurons, like muscles, have an excitable membrane, which permits ions to enter the cell from the extracellular space through a large number of diVerent channels. Thus, to maintain the intracellular ion concentration against the concentration gradient, via Naþ/Kþ- and Ca2þ-ATPase pumps situated in the plasma and the endoplasmatic reticulum membranes, neurons require a high-energy supply. The latter process is the most costly process in energy terms in excitable cells (Nicholls and Budd, 2000). Mitochondria provide the energy needed for these processes by converting metabolites into ATP through oxidative phosphorylation, which requires a coordinated action of four respiratory enzyme complexes arranged in a speciﬁc orientation in the inner mitochondrial membrane. Electrons generated from reduced electron carriers NADH and FADH2, which are produced from the oxidation of nutrients such as glucose, are ultimately transferred through the respiratory chain to molecular oxygen. This process is coupled to proton translocation across the inner membrane, forming an electrochemical gradient, which stores energy that is then used for ATP synthesis by the ﬁfth complex, ATP synthase. Each complex of the OXPHOS is assembled from multiple protein subunits and, except for complex II, which is encoded exclusively by the nuclear genome, are all encoded by the nDNA as well as by mtDNA. B. Mitochondria and Neuronal Activity Mitochondrial involvement in the regulation of long-term structural and functional changes, which modulate synaptic connectivity, is inferred from histochemical evidence demonstrating mitochondria recruitment to the location of high-activity zones in the neuron. Mitochondria are highly mobile organelles and their movement along microtubules can be adjusted according to changes

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in neuronal activity and local energy demands. Indeed, mitochondria have been shown to move to active zones during neurotransmitter release in response to an increase in synaptic activity (Brodin et al, 1999). In addition, it has been shown that mitochondria play a key role in the establishment and plasticity of neuronal polarity by concentrating at the site of axogenesis (Matsson, 1999), which may be relevant to neurite sprouting, elongation, and growth cone motility of axons as well as dendrites. Substantial evidence for a mitochondrial role in neuronal plasticity is depicted in studies of the visual system in cat and rats, which serve as a paradigm for neuronal plasticity, in as much as anatomical and physiological development can be altered by visual experience (Sherman and Spear, 1982). Thus, in rats exposed to light as opposed to those deprived of light, optic synapses in the suprachiasmatic nucleus had larger boutons with larger mitochondria, as well as more and larger mitochondria in the postsynaptic dendrites (Guldner et al., 1997). Others have shown that in rats exposed to complex environments, synaptogenesis and an increase in the volume of the visual cortex are associated with the inﬁltration of new capillaries and mitochondria (Black et al., 1991). In addition, it has been reported that in the visual system several genes encoding mitochondrial OXPHOS complexes (several subunits of cytochrome oxidase, NADH dehydrogenase, and of ATPase as well as cytochrome b) are regulated by neuronal activity, and their expression is correlated with the extent of plasticity in the visual cortex (Hevner and Wong-Riley, 1991; Kaminska et al., 1997; Yang et al., 2001). Mitochondrial gene upregulation, primarily that of 12S rRNA, which was indicative of a generalized elevation in mitochondrial transcription, has been observed in the hippocampus following synaptic activity, both of suYcient strength to induce LTP and of reduced strength that may be related to the induction of a metaplastic state (Williams et al., 1998). Moreover, in line with synaptic activitydependent regulation of mitochondrial gene expression, and presumably function, are ﬁndings that the expression of nuclear-encoded mitochondrial genes, such as cytochrome oxidase subunit 17, propionyl-CoA carboxylase, dihydrolipoamide dehydrogenase, and coenzyme Q subunit 7, as well as heat shock protein 70, have been reported to synthesize locally in presynaptic terminals (Gioio et al., 2001). Additional evidence supporting the role of mitochondria in mediating synaptic activity is ensued from ﬁndings that the inhibition of succinate dehydrogenase, the second complex of the OXPHOS, induces a long-term potentiation of NMDA-mediated synaptic excitation, which depends on dopamine acting via D2 receptors (Calabresi et al., 2001). Finally, mitochondrial permeability transition pores and their constituents, the porin proteins, which play a signiﬁcant role in diverse cellular processes, including the regulation of mitochondrial ATP and calcium eZux, have been found to have a dynamic functional role in learning and synaptic plasticity (Albensi et al., 2000; Weeber et al., 2002). In all, these studies render a growing body of evidence for the important contribution of

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mitochondria to neuronal plasticity both to short-term modulation and to long-term phenomena. C. Role of Mitochondrial-Regulated Factors in Neuronal Plasticity Mitochondria, apart from providing the cell with energy in the form of ATP for all its essential processes, take part in the maintenance of ion gradients in general and calcium in particular. The latter are crucial for the generation of action potentials and calcium signaling processes, including neurotransmitter release, cytoskeletal dynamics, and activity-dependent regulation of gene expression. The role of mitochondria in neuronal excitability and structural complex modulation during changes in synaptic eYcacy can be attributed to their ability to generate ATP, as well as to their ability to take up or release calcium. In addition, during oxidative phosphorylation, mitochondria produce reactive oxygen species, mainly superoxide, which can then be converted to hydrogen peroxide (H2O2) by mitochondrial or cytoplasmatic superoxide dismutases. Previous studies have shown that nitric oxide (NO) can also be produced by mitochondria and can modulate ATP synthesis by inhibiting cytochrome c oxidase (Arnaiz et al., 1999; Giulivi et al., 1998). Superoxide overproduction accompanied by increased NO generation (Ceriello, 2003; Elmarakby et al., 2003) can lead to the formation of peroxynitrite, whereas H2O2, in the presence of iron or copper, can induce hydroxyl radical production, both highly reactive oxygen species. However, depending on the ratio of their intracellular concentrations, ROS and NO can also act as secondary messengers and together can either enhance or attenuate their reciprocal eVects on cell signaling (Turpaev, 2002). It is interesting to note that mitochondrial-regulated calcium, ATP, ROS, and NO and their interrelationship play a pivotal role in the regulation of neurotransmitter release from presynaptic terminals. This, together with their role in postsynaptic signaling to the nucleus, leading to alterations in a variety of proteins and thereby to the modiﬁcation of synaptic strength or plasticity, has been established by numerous studies (Knapp and Klann, 2002; Mattson and Liu, 2002; Mattson et al., 2000; Melamed-Book and RahamimoV, 1998; Meldolesi, 2001; Miller, 1998; Rizzuto, 2001; Smythies, 1997; Wieraszko, 1996; Zucker, 1999) and is beyond the scope of this review. D. Schizophrenia and Abnormalities in Mitochondrial Plasticity Relevant Factors Mitochondrial dysfunction leads to alterations in ATP production and cytoplasmatic calcium concentrations, as well as the length of its cytoplasmatic signals, ROS and NO production, and, consequently, to the modulation of

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synaptic plasticity. Despite numerous studies, ﬁndings regarding pathological abnormalities in schizophrenia are not consistent and still elusive. At the cellular level, the picture is even more obscure. However, evidence, to some extent circumstantial, has accumulated suggesting the involvement of all the aforementioned mitochondria-regulated factors in schizophrenia. Mitochondria, constituting a key factor in energy metabolism, could contribute to alterations in brain energy metabolism. Imaging studies, using diVerent imaging techniques, including positron emission tomography with ﬂurodeoxyglucose (FDG) or with 15O, functional magnetic resonance imaging (fMRI), magnetic resonance spectroscopy with 31P (31P-MRS), and single photon emission tomography (SPECT), investigated cerebral metabolic rates in schizophrenia. Most of these studies revealed decreased energy metabolism in the frontal cortex, which was termed hypofrontality (Buchsbaum and Hazlett, 1998; Gur et al., 1987; Holcomb et al., 1996; Shenton et al., 2001). Concurrently, stable deficits in cognitive functions referable to the dorsolateral prefrontal cortex, and cortical physiological abnormalities at rest but more so during the performance of tasks, have been reported consistently in studies of schizophrenia (Buchsbaum and Hazlett, 1998; Carter et al., 1998; Hazlett et al., 2000; Holcomb et al., 1996; Lahti et al., 2001; Manoach et al., 1999; Potkin et al., 2002; Weinberger et al., 1986). Alterations in brain metabolic rates, although less consistent, were also observed in other brain regions, including the temporal lobe, thalamus, and basal ganglia (Gur et al., 1987; Hazlett et al., 1999; Kinderlehrer et al., 1999; Tamminga et al., 1992). This pattern of diVused abnormality leads to the suggestion of an impairment in the fronto-striatal-thalamic circuitry in schizophrenia rather than in a speciﬁc brain region (Andreasen et al., 1997; Buchsbaum and Hazlett, 1998; Chua and McKenna, 1995). More direct evidence for ATP alterations in schizophrenia came from studies using 31P-MRS with or without chemical shift imaging, which enables the measure of ATP, phosphocreatine, and Pi. These studies showed reduced ATP in the frontal lobe and left temporal lobe of schizophrenic patients as compared to controls (Fujimoto et al., 1992; Volz et al., 2000). A state of oxidative stress has been implicated in schizophrenia. This state is often associated with processes of neurodegeneration, but there is no indication for such processes in schizophrenia. Instead oxidative stress in this disorder can be related to reductions in neuronal processes and synapses as observed in postmortem brains of schizophrenic patients. In schizophrenia there are indications of an altered oxidative stress state. Thus, in a large sample of schizophrenic patients (n ¼ 100), an increase in xanthine oxidase activity, NO levels, and unchanged thiobarbituric acid-reactive substances (TBARS) were detected in plasma. Others have reported that plasma TBARS levels were increased in schizophrenic patients in both medicated and nonmedicated patients, as well as in drug-naive patients. The levels of TBARS were found to correlate with the

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severity of the patients’ negative symptoms (Akyol et al., 2002; Mahadik et al., 2001). An increase in CSF levels of TBARS has also been reported (Pall et al., 1987; Tsai et al., 1998). An additional indication for oxidative stress in schizophrenia is altered phospholipid metabolism, which is associated with a peroxidative loss of membrane phospholipids, the polyunsaturated fatty acids (for review, see Mahadik et al., 2001). Altered membrane phospholipid metabolism was also demonstrated by brain imaging using 31P-MRS in all regions implicated in schizophrenia ( Jensen et al., 2002; Volz et al., 2000). Substantial support for the oxidative stress state in schizophrenia is altered levels of antioxidant enzymes. Thus, in most studies, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity levels were signiﬁcantly higher in the schizophrenic group compared with controls (Kuloglu et al., 2002; Mahadik et al., 2001; Tsai et al., 1998). Evidence regarding abnormal intracellular calcium and NO levels stems primarily from the glutamate dysregulation hypothesis in general and the hypofunction of the NMDA receptor in particular, which is a highly consistent ﬁnding in schizophrenia. However, additional independent lines of evidence suggest altered calcium and NO signals. Abnormal nitrergic mechanisms have been identiﬁed in brains of schizophrenic patients. Thus, schizophrenics had a distorted distribution of NOS neurons, mostly an increase, in the hippocampal formation, the neocortex, and the white matter of the lateral temporal lobe and in the cerebellum (Akbarian et al., 1993; Bernstein et al., 1998, 2001; Karson et al., 1996). Several studies report higher levels of plasma and erythrocytes NO or its metabolite, nitrite, in patients with schizophrenia. In contrast, arginase activity and manganese levels were found to be signiﬁcantly lower, suggesting that arginine–NO pathways are impaired in schizophrenia (Akyol et al., 2002; Yanik et al., 2003; Zoroglu et al., 2002). In contrast, others reported that in polymorphonuclear leukocytes, the nitrite content was reduced signiﬁcantly whereas the plasma and platelet nitrite content was similar to that of controls (Srivastava et al., 2001). Interestingly, one study reported that, in platelets, calcium-activated NOS was increased in schizophrenic patients (Das et al., 1995), which brings us to the role of calcium in schizophrenia. The role of calcium in the pathophysiology of schizophrenia has been the focus of many studies given its importance in the release of neurotransmitters and their receptor-dependent signaling, in protein synthesis, and in neurotoxic processes. Unexpectedly, despite the aforementioned, there is hardly any experimental evidence linking calcium abnormality and schizophrenia. The only hint for calcium dysregulation in schizophrenia can be reduced from studies on calcium-binding proteins (CBPs). CBPs are a family of proteins that consist of over 250 members and are found in a variety of tissue. The brain is a particularly rich source for CBPs of which parvalbumin, calbindin, calretinin, and calmodulin have been implicated in schizophrenia. Only a small number of studies have

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measured alterations in CBPs, most of which demonstrated a reduction in parvalbumin, an increase in calbindin, and no change in calretinin, with each of the CBPs distributed diVerently in the various neuron populations (for a review, see Eyles et al., 2002). The role of these proteins is to buVer intracellular calcium transients, thus aVecting depolarization as well as repolarization events. It might as well be that chronic alterations in intracellular calcium due to mitochondrial dysfunction and/or glutamate dysregulation aVect CBPs expression either by compensatory or by inhibitory mechanisms.

V. Conclusion

Neuronal plasticity underlies learning and memory that dictate adaptive or compensatory changes in emotional, cognitive, or sensorimotor integration in response to internal or external stimuli. Consequently, an impairment in processes regulating neuronal plasticity and remodeling of synapses can lead to the symptomology characteristic of schizophrenia. In addition to the clinical manifestations of schizophrenia, several additional lines of evidence support the involvement of plasticity in the pathopysiology of this disorder. First, the course of the disease, in other words, the emergence of clinical symptoms in early adulthood, points to abnormal modulation of experience-dependent plasticity as distinct from solely developmental aberrations in the cytological architecture and neurogenesis. Second, the ability of psychomimetric drugs to produce symptoms reminiscent of schizophrenia, primarily by their interaction with dopaminergic and glutamatergic systems, suggests that the abnormality observed in schizophrenia is associated with the modulation of synaptic eYcacy. Third, the abnormalities observed in mitochondrial function, which is associated with aberrations in ATP, ROS, and NO production, as well as in calcium homeostasis, all implicated in synaptic remodeling, may induce a variety of detrimental eVects, all aVecting plasticity. Finally, the neurotransmitters implicated in schizophrenia, mostly dopamine and glutamate, have a well-established role in modulating short- and long-term changes in synaptic eYcacy and in remodeling and maintaining an adaptive pattern of synaptic connectivity. The question as to the cause–eVect relationship between the aforementioned processes remains. Mitochondrial activities can be regulated by the metabolic needs of neurons and by neurotransmitter release, as well as by their receptor-induced ionic eZux and signal transduction. Thus, mitochondrial alterations in schizophrenia could be secondary to other pathologies observed in this disorder. Concomitantly, mitochondrial-regulated factors can induce reverse eVects on these same processes, leading to alterations in gene expression protein synthesis and neuronal plasticity. It is therefore diYcult to posit the mitochondrial role as

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an up/downstream process. SuYce it to say presently, mitochondria play a pivotal role in synaptic plasticity implicated in schizophrenia. Finally, the aforementioned two-way interactions can be acting synergistically to remodel the synaptic connectivity in response to either endogenous or exogenous input.

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MEMBRANE PHOSPHOLIPIDS AND CYTOKINE INTERACTION IN SCHIZOPHRENIA

Jeffrey K. Yao VA Pittsburgh Healthcare System, Pittsburgh, Pennsylvania 15206 Department of Psychiatry, University of Pittsburgh Medical Center Pittsburgh, Pennsylvania 15213 Department of Pharmaceutical Sciences, School of Pharmacy University of Pittsburgh, Pittsburgh, PA 15213

Daniel P. van Kammen Aventis, Inc., Bridgewater, New Jersey 08807 Department of Psychiatry, University of Pennsylvania Philadelphia, Pennsylvania 19104 Department of Psychiatry, Columbia University New York, New York 10032

I. Abnormal Membrane Phospholipids A. Evidence for Membrane Phospholipid Defects B. Increased Phospholipase Activities C. Increased Oxidative Stress D. Physiological Significance of Arachidonic Acid Signaling II. Disturbed Immune Function A. An Overactive ‘‘Innate’’ Immune System? B. The Blunted Th1 System C. The Activated Th2 System D. Conflicting Findings III. Polyunsaturated Fatty Acids and Cytokines IV. Stress and Immune Response A. Oxidative Stress B. Psychological Stress V. Conclusion References

Although the potential key role that lipids may have in schizophrenia is not fully understood, multiple lines of evidence to date implicate the lipid environment in the behavior of neurotransmitter systems. Decreased phospholipid polyunsaturated fatty acids (PUFAs) have been demonstrated in both brain and peripheral membranes in schizophrenia, which is consistent with the hypothesis of myelin-related dysfunction in schizophrenia. Membrane defects, such as those induced by decreased PUFAs in phospholipids, can signiﬁcantly alter a broad range of membrane functions and ipso facto behavior through multiple ‘‘downstream’’ eVects. A number of putative mechanisms have been identiﬁed INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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to explain the decreased PUFAs in schizophrenia, notably the increased turnover of phospholipids and decreased incorporation of arachidonic acid (AA) in membranes. In addition to increased oxidative stress, altered immune function may also be responsible for increased phospholipase activities. This association is particularly relevant in relation to phospholipids/PUFA, as AA can be converted to a variety of biologically active compounds, such as eicosanoids, which serve as potent messengers in regulating the inﬂammatory response, as well as endocannabinoids, which may aVect schizophrenic psychopathology. Direct evidence of immune changes in some patients with schizophrenia have come to light, particularly in the activities of several cytokines known to be altered in autoimmune dysfunction. Given the diverse physiological function of AA, the speciﬁc behavioral symptomatology of schizophrenia is related mostly to the eVect of AA changes that regulates neurodevelopment, neurotransmitter homeostasis, phosphatidylinositol signaling, and neuromodulatory actions of endocannabinoids in schizophrenia. Hence, in the current conceptualization, AA may be at a nexus point in the cascade leading to the syndrome of schizophrenia and represents a common biochemical pathway leading to the varied symptomatology of this disorder. I. Abnormal Membrane Phospholipids

Schizophrenia is a major mental disorder without a clearly identiﬁed pathophysiology. Numerous hypotheses have been proposed over the years to conceptualize the pathophysiology of schizophrenia, focusing primarily on neurotransmitter systems. However, one avenue of research that is gaining currency is the study of membrane composition and function. The membrane is a complex structure, composed primarily of phospholipids and their constituent fatty acids, that provides scaVolding for many key functional systems, including neurotransmitter receptor binding, signal transduction, transmembrane ion channels, prostanoid synthesis, and mitochondrial electron transport systems. Thus, the dynamic state of all membranes, including those of neurons and glia, is dependent on their composition, such that small changes in key phospholipids or the polyunsaturated fatty acids (PUFAs) that make up phospholipids can lead to a broad range of membrane dysfunctions. Key PUFAs in phospholipids are the n-3 (or !3) and n-6 (or !6) series, of which docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) are the most abundant in the brain. A. Evidence for Membrane Phospholipid Defects A variety of data suggest defects in phospholipid metabolism and cell signaling in schizophrenia. Those ﬁndings include (1) decreased PUFAs and altered phospholipids in plasma (Horrobin et al., 1989), red blood cells (RBC) (Assies

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et al., 2001; Glen et al., 1994; Keshavan et al., 1993; Peet et al., 1996; Ponizovsky et al., 2001; Yao et al., 1994a), platelets (Pangterl et al., 1991; Schmitt et al., 2001; Steudle et al., 1994), skin ﬁbroblasts (Mahadik et al., 1996), and postmortem brain tissues (Horrobin et al., 1991; Yao et al., 2000); (2) an increased turnover of in vivo brain phospholipid metabolites detected using 31P magnetic resonance spectroscopy (MRS) (Fukuzako, 1996; Pettegrew et al., 1991, 1993; Williamson et al., 1991); (3) a signiﬁcant correlation between RBC phospholipid PUFAs and 31 P MRS measures of phospholipid metabolites in the brain (Richardson et al., 2001; Yao et al., 2002a); (4) increased turnover of inositol phospholipids (Das et al., 1992; Essali et al., 1990; Yao et al., 1992; Zilberman-Kaufman et al., 1992) and production of second messengers (Kaiya et al., 1989; Yao et al., 1996); and (5) increased lipid peroxidation (Akyol et al., 2002; Khan et al., 2002; Mahadik et al., 1998). Reduced membrane PUFAs have been linked to the symptom severity (Glen et al., 1994; Peet et al., 1995; Ponizovsky et al., 2001; Yao et al., 1994b), development of tardive dyskinesia (Nilsson et al., 1996; Vaddadi et al., 1989), and reduced niacin-induced cutaneous ﬂushing (Glen et al., 1996; Horrobin, 1980; Hudson et al., 1995; Messamore et al., 2003; Rybakowski and Weterle, 1991). Moreover, studies have further demonstrated decreased AA and DHA levels in the RBC of ﬁrst-episode, never-medicated patients (Arvindakshan et al., 2003b; Reddy et al., in press). Taken together, these data support the notion that molecular changes in membrane phospholipids may be present prior to both clinical and biological manifestations of the disorder (Pettegrew et al., 1993).

B. Increased Phospholipase Activities A number of putative mechanisms have been identiﬁed to explain the decreased PUFA levels in schizophrenia (Yao, 2003), notably the increased breakdown of phospholipids and decreased incorporation of AA. Both oxidative stress and altered immune function may play a role in an induction of phospholipase activities. Phospholipase A2 (PLA2) is a rate-limiting enzyme responsible for the breakdown of membrane phospholipids (Dawson, 1985). In addition to being the required step for eicosanoid biosynthesis, PLA2 also plays a pivotal role in inﬂammation (Chakraborti, 2003). 1. Phospholipase A2 Increased cytoplasmic PLA2 activity has been found in serum of drug-free schizophrenic patients (Gattaz et al., 1987, 1990; Noponen et al., 1993). Such increases in serum PLA2 activity, however, were also found in patients with other psychiatric disorders (Noponen et al., 1993), raising a question about the

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speciﬁcity of this ﬁnding to schizophrenia. Subsequently, Gattaz et al. (1995) showed that intracellular membrane-bound PLA2 activity was signiﬁcantly higher in platelets of schizophrenia patients than in normal and psychiatric controls, with no signiﬁcant diVerences between normal and psychiatric controls. It is thus unlikely that the increased platelet PLA2 activity in schizophrenia results from nonspeciﬁc stressors. However, an attempt to replicate increased PLA2 activity in schizophrenia has led to a conﬂicting ﬁnding (Albers et al., 1993). Those discrepancies may be due to diVerences in the assay procedure and the heterogeneous class of extracellular PLA2. The superfamily of PLA2 is divided into three types of enzymes: Ca2þdependent cytosolic PLA2 (cPLA2), Ca2þ-dependent secretory PLA2 (sPLA2), and Ca2þ-independent PLA2 (iPLA2) (Capper and Marshall, 2001; Chakraborti, 2003). Ross et al. (1997) showed that increased iPLA2, not Ca2þ-dependent PLA2, was found in serum of patients with schizophrenia. A variety of antipsychotic drugs also inhibit PLA2 activity (Aarsman et al., 1985; Schroder et al., 1981; Taniguchi et al., 1988). The potential clinical signiﬁcance of PLA2 alterations in schizophrenia has been examined less systematically. Previously, Ross et al. (1997) found positive relationships between calcium-independent PLA2 and general psychopathology scores and positive symptoms, but not with negative symptoms. They examined PLA2 activity in chronic schizophrenic patients who were receiving long-term antipsychotic treatment and exhibiting signiﬁcant positive symptoms. Although they were not characterized as poor outcome patients, the clinical characteristics of these patients are suggestive of an unfavorable outcome. Gattaz’s laboratory has replicated their earlier ﬁndings of increased PLA2 activities in drug-free patients with schizophrenia (Tavares et al., 2003). Moreover, they demonstrated that those patients without a response to niacin had the highest PLA2 activities as compared to those with a positive response. Whether the relations between PLA2 and AA in ﬁrst-episode patients with schizophrenia will be the same or diVerent than that observed in those with severe chronic schizophrenia remains to be determined. 2. Phospholipase C In addition to PLA2, other pathways, including the phospholipase C (PLC)– diacylglycerol (DAG) lipase pathway, as well as the phospholipase D–phosphatidic acid phosphohydrolase pathway, are also involved in the release of AA from membrane phospholipids. The receptor-stimulated hydrolysis of inositol phospholipids, particularly phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), is initiated by a speciﬁc PLC (Berridge and Irvine, 1984; Nishizuka, 1984). The resulting DAG and inositol 1,4,5-triphosphate (1,4,5-IP3) led to the activation of protein kinase C (PKC) and elevation of cytosolic Ca2þ, which provides molecular links between extracellular signals and intracellular events (Kishimoto

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et al., 1980; Nishizuka, 1984). Quantitative determination of inositol phosphates provides direct evidence for PI hydrolysis by PLC in intact cells (Siess, 1989). An increased turnover of platelet PI was found in both drug-treated and drug-free patients (Das et al., 1992; Essali et al., 1990; Yao et al., 1992) but not in drug-naive patients (Essali et al., 1990). The increased production of IP3 may be due to an increase in the precursor, PI-4,5-P2, associated with a desensitization of the intracellular IP3 receptor by neuroleptics (Das et al., 1992). However, ZilbermanKaufman et al. (1992) reported an increased inositol-1-phosphatase in RBC of chronic schizophrenia patients. They interpreted that the increased enzyme activity might compensate physiologically for a deﬁciency of inositol in these patients. Furthermore, the hyperactivity of the PI signaling system in schizophrenia has also been demonstrated in the postmortem human brain ( Jope et al., 1994; O’Neill et al., 1991; Pacheco and Jope, 1996; Wallace and Claro, 1993), which is markedly diVerent from diseases with major depression and bipolar mood disorder showing a decreased activity of G-protein-mediated PI hydrolysis ( Jope et al., 1996; Pacheco and Jope, 1996).

C. Increased Oxidative Stress Much of the biochemical research focus in schizophrenia has been on neurotransmitter systems. Although the role of dopamine in the pathophysiology of schizophrenia remains preeminent, recent ﬁndings suggest instead that multiple neurotransmitter systems may be altered. In many ways, schizophrenia can be conceptualized as having a ‘‘multineurotransmitter’’ pathology. Whether these are primary or secondary to other pathological processes, such as oxidative stress and membrane dysfunction, will need to be determined. We emphasize, however, that alterations in the activity of several neurotransmitter systems can both contribute to and be modiﬁed by oxidative stress (or membrane dysfunction). 1. Activation of Phospholipase by Reactive Oxygen Species (ROS) PUFAs are highly susceptible to free radical insult and autoxidation to form peroxy radicals and lipid peroxide intermediates, the existence of which within cell membranes results in unstable membrane structure, altered membrane ﬂuidity and permeability, and impaired signal transduction. The brain, which is rich in PUFAs, is particularly vulnerable to free radical-mediated damage. Goldman et al. (1992) provided evidence that the formation of ROS is important for the activation of cellular PLA2. Later they showed that the epidermal growth factor signaling of PLA2 activation and AA release are aVected by the antioxidants, suggesting that PLA2 may be targeted by ROS (Goldman et al., 1997). However, Takekoshi et al. (1995) demonstrated that oxidized DAG are

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more eVective in activating PKC than its nonoxidized forms. The oxidized DAG is formed by the PLC-dependent hydrolysis of phosphatidylcholine hydroperoxides (Kambayashi et al., 2002). In addition, both PKC activation and protein tyrosine phosphorylation are required for the hydrogen peroxideinduced activation of phospholipase D (Min et al., 1998; Natarajan et al., 1996). Together, these ﬁndings support a role of ROS in phospholipase-mediated cell signaling (Thannickal and Fanburg, 2000). In schizophrenia, decreased levels of RBC-PUFAs (AA in particular) were associated signiﬁcantly with increased levels of plasma lipid peroxides in nevermedicated, ﬁrst-episode schizophrenia patients (Arvindakshan et al., 2003b; Khan et al., 2002). It is thus reasonable to hypothesize that increased oxidative stress may be one of the mechanisms responsible for the reduction of membrane PUFAs. 2. Multineurotransmitter Defects and Free Radical Pathology Numerous studies have shown that dopamine (DA) metabolism yields free radicals under normal physiological conditions (e.g., Cohen, 1994). A number of DA metabolic pathways exist that lead to the generation of hydroxyl radicals. DA is susceptible to autoxidation when the antioxidant defense system (AODS) is weak (Zhang and Dryhurst, 1994). Interestingly, it has been recognized that DA-mediated toxicity is also mediated through DA actions on N-methyld-aspartate (NMDA) glutamate receptors (Ben-Shachar et al., 1995; Cadet and Kahler, 1994; Michel and Hefti, 1990). There is accumulating evidence that NMDA-mediated excitotoxicity involves free radicals, such as superoxide and nitric oxide (Coyle and Puttfarcken, 1993; Patel et al., 1996). In fact, antioxidants (e.g., ascorbate and vitamin E) protect neurons against glutamate neurotoxicity (Ciani et al., 1996; MacGregor et al., 1996). Other neurotransmitters, particularly glutamate, can also induce metabolic processes that increase free radical production. Activation of NMDA receptors by glutamate stimulates PLA2 activity to release AA to act as a second messenger, which in turn can lead to the formation of free radicals (Iuliano et al., 1994). A decreased availability of AA, due either to increased PLA2 activity or to lipid peroxidation, can lead to impaired glutamatergic neurotransmission (Olney and Farber, 1995), which has been proposed as a pathogenetic mechanism in schizophrenia. A dopamine–glutamate imbalance has also been implicated in schizophrenia (Carlsson and Carlsson, 1990). Antipsychotic drugs that block dopamine receptors may also enhance glutamatergic neurotransmission. 3. Impaired Antioxidant Defense System Biological systems have evolved complex protective strategies against free radical toxicity. There are multiple pathways leading to excess free radical generation and subsequent oxidative stress. Under physiological conditions, the

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potential for free radical-mediated damage is kept in check by the antioxidant defense system, comprising a series of enzymatic and nonenzymatic components. The critical antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). These enzymes act cooperatively at diVerent sites in the metabolic pathway of free radicals. In addition to the superoxide and hydroxyl radicals, another pathway is the formation of peroxynitrite by a reaction of a nitric oxide (NO) radical and a superoxide radical. Nitric oxide can also produce hydroxyl radicals as well as nitrogen dioxide radicals. Nitric oxide is a free radical by its unpaired electron. Because NO radicals cannot produce initiation or propagation reactions, they do not generate free radical chain reactions. Elevated NO production has been linked to various neurodegenerative disorders, including Alzheimer’s disease (Norris et al., 1996; Thorns et al., 1998), multiple sclerosis (Heales et al., 1999), and Parkinson’s disease (Bockelmann et al., 1994; Gerlach et al., 1999; Hunot et al., 1996). There is increasing evidence of antioxidant defense system (AODS) deﬁcits in schizophrenia (Yao et al., 2001). The AODS is a complex, interrelated system to dampen oxidative stress and protect tissue components from free radical-mediated damage. A signiﬁcant contribution to the body’s total antioxidant capacity comes from antioxidant molecules in plasma, such as albumin, uric acid, and bilirubin. Thus, plasma is an important but complex vehicle that serves as a protective factor against oxidative damage to diVerent blood components and also distributes dietary antioxidants to the rest of the body. Signiﬁcant reductions of plasma antioxidants (e.g., albumin, bilirubin, and uric acid) are seen early in the course of schizophrenia (Reddy et al., 2003), consistent with previous ﬁndings in patients with chronic schizophrenia (Yao et al., 1998a,b, 2000b). More importantly, these reductions are observed independently of treatment, as patients were antipsychotic drug naive at entry into the study. Furthermore, these patients were physically healthy, with no evidence of liver or kidney disease or signiﬁcant calorie restrictions, suggesting that the lowered levels of plasma antioxidants are not indicative of ongoing disease processes or malnutrition. Rather, the lowered levels may be indicative of subtle changes reﬂecting either the acute-phase response (APR) (Maes et al., 1997, 2000a,b) or oxidative stress (Mahadik and Evans, 2003; Yao et al., 2001). The APR is increased in schizophrenia and is associated with a reduction in albumin (Wong et al., 1996). In addition, we have demonstrated a signiﬁcantly higher level of NO in schizophrenia brains than in those of normal and nonschizophrenia psychiatric controls (Yao et al., in press). These ﬁndings were independent of age, brain weight, postmortem interval, sample storage time, alcohol use, or cigarette smoking. Thus, elevated NO levels in schizophrenia brains lend further support for the possibility of free radical pathology in schizophrenia.

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D. Physiological Significance of Arachidonic Acid Signaling Although much of the attention by early investigators has been on the n-3 fatty acids (e.g., DHA), increasing attention is being paid to the potentially important role that AA may play in the pathophysiology of schizophrenia. In brain, AA and its metabolites are considered to be intracellular second messengers. Many neurotransmitters can potentiate AA release through a receptordependent hydrolysis of membrane phospholipids (e.g., inositol phospholipids), which suggests that the receptor-mediated AA release may participate in neuronal signal transduction (Vial and Piomelli, 1995). Therefore, the depleted AA resulting from an increased phospholipid breakdown could be a common factor that regulates prostaglandin biosynthesis, neurotransmission, and neuronal deﬁcits in schizophrenia (Peet et al., 1994). 1. Serotonin Receptor Activation There is abundant evidence that serotonin (5-HT2) receptors in the brain play a regulatory role in behavior (Leysen and Pauwels, 1990). 5-HT2 receptors stimulate the release of AA in hippocampal neurons through the activation of PLA2 that is independent of inositol phospholipid hydrolysis (Felder et al., 1990). Thus, serotonin may potentially mediate some pathophysiological processes through receptor-stimulated AA or eicosanoids. We have demonstrated that drug-free schizophrenia patients exhibit reduced physiologic responsivity mediated through the platelet 5-HT2 receptor complex, which can be modiﬁed by haloperidol treatment (Yao et al., 1996). 2. The Endocannabinoid System Another candidate neurobiological system that has received increased attention in recent years is the endocannabinoid system. 9-Tetrahydrocannabinol (THC), the psychoactive ingredient from Cannabis saliva or marijuana, has been known for centuries to cause acute euphoria, altered time perception, dissociation of ideas, paranoia, motor impairment, enhanced appetite, cognitive impairment, and occasionally hallucinations. Because of the similarities between THCinduced psychosis and many symptoms of acute schizophrenia, a possible relationship between THC use and the development of psychosis has been proposed. Two endogenous THC ligands, anandamide (Devane et al., 1992) and 2-arachidonoylglycerol (2-AG) (Sugiura et al., 1997; Stella et al., 1997), have been discovered in the brain areas known to be implicated in schizophrenic brain pathology. Both anandamide and 2-AG are derivatives of arachidonic acid. Anandamide is synthesized from phosphatidylethanolamine (PE) by the ‘‘transacylase-phosphodiesterase pathway’’ (Schmid, 2000). However, 2-AG is converted from diacylglycerols by sn-1-DAG lipase, which is mainly

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followed by the phospholipase C-mediated degradation of phosphatidylinositol. Anandamide has been shown to induce AA release and its product, prostaglandin F2 (Someya et al., 2002). Given the localization of the endogenous cannabinoid receptor (CB1) system in brain areas (i.e., cortical and limbic structures) known to be implicated in schizophrenic brain pathology (Herkenham et al., 1990, 1991), it is plausible that dysfunction of the CB1 system with endogenous ligands may be associated with the pathophysiology of schizophrenia. Moreover, there is a close interaction between CB1 and dopaminergic systems. Cannabinoid agonists such as THC and the endogenous ligands, anandamide and 2-AG, can modulate the dopaminergic system (French, 1997; Gardner and Lowinson, 1991; Sanudo-Pena et al., 1996). Indeed, initial evidence suggests elevated anandamide levels in schizophrenia patients (Leweke et al., 1999; Yao et al., 2002b), higher densities of CB1 receptors in the schizophrenia dorsolateral prefrontal cortex (Dean et al., 2001), and linkages between CB1 receptor genes and schizophrenia (Leroy et al., 2001; Ujike et al., 2002). Hence, a missing link in the PUFA/phospholipid theory of schizophrenia may have been the presence of hallucinogenic endogenous cannabinoids, a fact that can now be integrated with current hypotheses and may go a long way in relating AA activity and the clinical outcome of schizophrenia. 3. Eicosanoids The notion of altered immune function in schizophrenia has been postulated and examined for a number of years (see later). This association is particularly relevant in relation to phospholipids/PUFA, as AA can be converted into a variety of biologically active metabolites, which are collectively referred to as eicosanoids, through the concerted reactions of cyclooxygenase (COX) and lipoxygenases. Interestingly, Mu¨ller et al. (2002) reported a double-blind, addon study that the COX-2 inhibitor celecoxib decreased signiﬁcantly the total score on the positive and negative syndrome scale (PANSS) as compared to placebo. Thus, it is conceivable that immune dysfunction in schizophrenia is not just an epiphenomenon but may play a role in the pathogenetic mechanism of the disorder (Mu¨ller et al., 2002). Eicosanoids are potent messengers that modulate the inﬂammatory response of the immune system (Calder, 2001). More recently, direct evidence of immune changes in schizophrenia has come to light, particularly in the activities of several cytokines known to be abnormal in autoimmune dysfunction, even though there is no evidence of an acute brain inﬂammation or autoimmune changes in schizophrenia. Presumably, the aforementioned altered processes may induce changes in the immune system with behavioral and cellular consequences, without evidence of chronic inﬂammation.

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4. Arachidonic Acid, GAP-43, and Neurodevelopment Arachidonic acid is highly involved in the developmental process, particularly in relation to the growth-associated protein-43 (GAP-43), a key protein that contributes to dendrite growth and synaptogenesis (Benowitz and Ruttenberg, 1997). AA phosphorylates GAP-43 via protein kinase C, thus converting it to its active state, which can then modulate such processes as long-term potentiation (LTP) and axonal growth through the action of neural cell adhesion molecules (Luo and Vallano, 1995; Meiri et al., 1998; Schaechter and Benowitz, 1993). Interestingly, AA-induced GAP-43 activity is also involved in the mechanism of DA release (Ivins et al., 1993), and levels of GAP-43 itself have been shown to be elevated in schizophrenia brain (Blennow et al., 1999; Perrone-Bizzozero et al., 1996; Sower et al., 1995). Initial evidence also suggests that transgenic mice overexpressing GAP-43 display hyperlocomotive behaviors reminiscent of amphetamine psychotic animals, an eVect that is reversed by antipsychotic halperidol treatment (Routtenberg et al., 2001). Taken together, these data illustrate the fact that the AA cascade is at the core of many processes (LTP, neurite growth, glutamatergic, and DA release), which could lead to the diverse collection of symptoms observed in schizophrenia. Thus, AA dysregulation is a strong candidate for the biochemical substrate of faulty neurodevelopment in schizophrenia.

II. Disturbed Immune Function

Advances in immunology suggest that two functionally diVerent yet balanced immune systems are present in the human (Mu¨ller et al., 2000). The unspeciﬁc, ‘‘innate’’ immune system represents the ﬁrst line of defense, which consists of monocytes/macrophages, granulocytes, and natural killer cells in its cellular arm and acute-phase proteins and the complement system in its humoral arm. A person is born with an ‘‘innate’’ immune system. However, the speciﬁc, ‘‘adaptive’’ immune system consists of the cellular arm of T and B cells and the humoral arm of the speciﬁc antibodies, which is developed through the lifelong contact with pathogens. Moreover, the adaptive immune system appears to discriminate the cell-mediated cytotoxic responses from those antibody-mediated immune responses (Mosmann and Sad, 1996). Upon immune activation, native T-helper (Th0) cells are converted into either Th1 cells that mediate cytotoxic function or Th2 cells that induce an antibody-dependent immune response. Characteristically, the Th1 system produces interleukin-2 (IL-2), interferon- (IFN-), and tumor necrosis factor- (TNF-), whereas the Th2 system produces IL-4, IL-6, and IL-10.

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Cytokines are small, nonenzymatic glycoproteins that are secreted by one cell for the purpose of changing either its own functions (autocrine eVect) or those of adjacent cells (paracrine eVect) (Haddad, 2002). Administration of cytokines can lead to various psychiatric symptoms, including apathy, depression, delusions, hallucinations, paranoia, and fatigue (DenicoV et al., 1987; McDonald et al., 1987; Niiranen et al., 1988; Spath-Schwalbe et al., 1998; Walker et al., 1997). Therefore, it is possible that the altered immune system is involved in the pathophysiology of psychiatric disorders. Previously, epidemiological (Brown et al., 2000; Mednick et al., 1988; O’Callaghan et al., 1991) and genetic (Badenhoop et al., 1996; Lindholm et al., 1999; Schwab et al., 1995) studies have linked immune dysfunction to schizophrenia.

A. An Overactive ‘‘Innate’’ Immune System? Although there are no cellular and/or tissue damages resulting from abnormal immune reactions, there is a distinct humoral immune reactivity in schizophrenia (Mu¨ller et al., 2000; Schwarz et al., 2001). The unspeciﬁc, ‘‘innate’’ immune system appears to be overactivated in some patients with schizophrenia, as evident by an increase of monocytes (Wilke et al., 1996) and gamma/delta cells (Mu¨ller et al., 1998). In addition, several studies have shown increased levels of IL-6 in schizophrenia (Frommberger et al., 1997; Ganguli et al., 1994; Lin et al., 1998; Maes et al., 1995; van Kammen et al., 1999a), which might be related to the duration (Ganguli et al., 1994) and treatment resistance (Lin et al., 1998) of the disease. Moreover, high levels of the soluble IL-6 receptor (sIL-6R) were found selectively in a subgroup of schizophrenic patients with more pronounced paranoid-hallucinatory syndrome (Muller et al., 1997a,b). Following antipsychotic treatment, levels of both IL-6 and sIL-6R were reduced (Maes et al., 1995; Muller et al., 1997a,b). Thus, antipsychotic drugs may inhibit IL-6 production (Lin et al., 1998). Because the activation of monocytes and macrophages, as well as astrocytes and microglia, leads to the production and release of IL-6, increased levels of IL-6 in schizophrenia (see later) may be the consequence of activation of the ‘‘innate’’ immune system.

B. The Blunted Th1 System In contrast, the speciﬁc, ‘‘adaptive’’ immune system appears to be imbalanced in schizophrenia. There is a decreased in vitro production of IL-2 (Bessler et al., 1995; Cazzullo et al., 1998; Ganguli et al., 1989, 1995; Villemain et al., 1989; Zhang et al., 2002a), as well as a decreased production of interferon- (Arolt et al.,

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2000; Rothermund et al., 1998; Wilke et al., 1996). Both ﬁndings suggested that the Th-1 system is underactivated in schizophrenia. In normal physiological conditions, IL-2 is maintained at relatively low levels in peripheral blood. In schizophrenia, however, increased levels of IL-2 (Kim et al., 2000; Zhang et al., 2002b) and IL-2 receptors (Rapaport et al., 1993, 1994; Rapaport and Lohr, 1994) are present in serum as well as increased IL-2 levels in cerebrospinal ﬂuid (CSF) (Licinio et al., 1993; McAllister et al., 1995). In addition, McAllister et al. (1995) further demonstrated that CSF IL-2 levels were associated with a recurrence of psychotic symptoms. Relapse-prone patients had signiﬁcantly higher levels of CSF IL-2 than those patients who did not relapse, suggesting a role of CSF IL-2 than those patients who did not relapse, suggesting a role of CSF IL-2 in relapse in schizophrenia. Thus, it is possible that a decreased in vitro production of IL-2 is a consequence of overproduction of IL-2 in vivo (Ganguli et al., 1992).

C. The Activated Th2 System Several studies report increased levels of IL-6 in schizophrenia. Because the Th2 system can produce IL-6, the increased production of IL-6 can thus result from activation of either the Th2 system or the monocytes/macrophage cells (see Section II,A). Other studies demonstrating increased levels of IL-4 (Mittleman et al., 1997), IL-10 (Cazzullo et al., 1998; van Kammen et al., 1997), and IgE (Ramchand et al., 1994) further support an activation of the Th2 system in schizophrenia. Moreover, CSF IL-10 levels were signiﬁcantly correlated with negative symptoms in unmedicated patients with schizophrenia (van Kammen et al., 1997). In patients treated with haloperidol, however, a signiﬁcant correlation was found between CSF IL-10 levels and the severity of psychosis measured by the Bunney–Hamburg psychosis rating scale (van Kammen et al., 1997). Taken together, Mu¨ller et al. (2000) suggested an imbalance of the ‘‘adaptive’’ immune system with a shift to Th2-like immune reactivity in a subgroup of patients with schizophrenia. This subgroup is further characterized by more pronounced negative symptoms and poor outcome (Schwarz et al., 2001).

D. Conflicting Findings Despite the aforementioned data that support an imbalance of the ‘‘adaptive’’ immune system in schizophrenia, the respective evidence is not always conclusive. Contrary to blunted Th1 production, increased in vitro productions of IL-2 and interferon- and decreased levels of serum IL-2 were found in schizophrenia patients (Cazzullo et al., 2001; O’Donnell et al., 1996; Theodoropoulou et al., 2001). Others have reported no signiﬁcant diVerences between schizophrenia

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patients and control subjects (Baker et al., 1996; Haack et al., 1999; Wilke et al., 1996). Furthermore, several studies have failed to replicate increased circulating levels of IL-6 in schizophrenia (Baker et al., 1996; Haack et al., 1999; Monteleone et al., 1997; Shintani et al., 1991; Wei et al., 1992; Xu et al., 1994). These inconsistencies may be the result of diVerences in assay methodology, sample size, sample handling, diagnostic criteria, and comparison groups. In addition, several confounding factors, including age, gender, ethnic background, smoking, alcohol, substance abuse, and antipsychotic treatment, may also explain these discrepancies (Banks, 2000; Haack et al., 1999; van Kammen et al., 1999b).

III. Polyunsaturated Fatty Acids and Cytokines

Both n-6 and n-3 polyunsaturated fatty acids involve regulation of the inﬂammatory response system. n-6 PUFAs, particularly AA, have proinﬂammatory features, as AA is the precursor of proinﬂammatory eicosanoids, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) and increase the production of IL-1, TNF-, and IL-6 (Hayashi et al., 1998; Soyland et al., 1994; Tashiro et al., 1998). However, n-3 PUFA eicosapentaenoic acid (EPA) and DHA suppress the production of AA-derived eicosanoids, thus having anti-inﬂammatory and immunosuppressive eVects (Calder, 1998; Meydani et al., 1991). Several groups have reported that n-3 PUFA-enriched diets (e.g., ﬁsh oil) can lead to partial replacement of AA by EPA in inﬂammatory cell membranes and signiﬁcantly reduce the ex vivo production of proinﬂammatory cytokines (Calder, 1998; Endres et al., 1993; Espersen et al., 1992; Gallai et al., 1995; James et al., 2000; Meydani et al., 1991; Soyland et al., 1994). Therefore, an imbalance of n-6/n-3 PUFAs may result in an increased production of proinﬂammatory cytokines. Smith (1991) proposed that an abnormal fatty acid composition might be related to the inﬂammatory response system underlying the pathophysiology of major depression. Maes et al. (2000b) have further substantiated the role of PUFAs in predicting the response of proinﬂammatory cytokines to psychological stress. In schizophrenia, an increased breakdown of membrane phospholipids through the PLA2 reaction has been reported, as well as increased circulating levels of IL-2 and IL-6 (see earlier discussion). Interleukins have been shown to stimulate the PLA2-mediated hydrolysis of phospholipids. Evidence from the bilateral infusion of IL-6 into the rat hippocampus further supports the notion that IL-6 can activate arachidonic acid metabolic pathways in the brain (Ma and Zhu, 2000). Moreover, Yao et al. (2003) demonstrated signiﬁcant correlations between increased CSF levels of IL-6 and decreased RBC levels of PUFAs in schizophrenic patients on and oV haloperidol treatment. Taken together, these ﬁndings suggest that decreased membrane PUFAs may be related in part to an

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immune disturbance in schizophrenia, possibly resulting from increased PLA2 activity mediated through the proinﬂammatory cytokines. However, reduced levels of AA in membrane phospholipids could conceivably lead to a decreased synthesis of proinﬂammatory eicosanoids. One of the AA metabolites, prostaglandin D2, mediates vasodilatation during the inﬂammatory response. Thus, the reduced AA availability may, in part, explain a variety of clinical observations in schizophrenia that are usually ignored by the receptor-based etiological hypotheses (Horrobin, 1998). Indirect evidence for a dysregulated inﬂammatory response in schizophrenia stems from the observation of a lower risk of arthritis and other inﬂammatory diseases (Mellsop, 1972; Oken and Schulzer, 1999; Torrey and Yolken, 2001; Vinogradov et al., 1991), greater resistance to pain (Davis et al., 1979), remission of psychosis during fever (Horrobin, 1977), and decreased prostaglandin-dependent niacin skin ﬂushing (Glen et al., 1996; Horrobin, 1980; Hudson et al., 1995; Messamore et al., 2003; Rybakowski and Weterle, 1991). These eVects might be secondary to reduced eicosanoids signaling.

IV. Stress and Immune Response

A. Oxidative Stress Antioxidant status is deﬁned as the balance between antioxidants and prooxidants in living organisms (Papas, 1996). An imbalance resulting from an excessive formation of free radicals can lead to oxidative stress, and subsequently cellular toxicity. During inﬂammatory processes, inﬁltrating cells can produce large amounts of reactive oxygen species. In addition to being cytotoxic, these ROS also act as important mediators regulating various cellular and immunological processes (Dro¨ge, 2002). Under physiologically relevant concentration, hydrogen peroxide was shown to either increase the production of T-cell growth factor (Roth and Dro¨ge, 1987) or induce the gene expression of IL-2 (Los et al., 1995) and IL-6 ( Junn et al., 2000). The enhancement of IL-2 production was associated with a decrease in the intracellular glutathione (GSH) level (Los et al., 1995) and was reversed by the addition of exogenous GSH (Roth and Dro¨ge, 1991). Hehner et al. (2000) further demonstrated enhancement of T-cell receptor signaling by a shift in the intracellular GSH redox state. Taken together, these ﬁndings suggest that the intact immune system requires a delicate balance between antioxidant and prooxidant status (Dro¨ge et al., 1994). As mentioned in Section I,C,3, there is increasing evidence of perturbations in the antioxidant defense system in schizophrenia. Such an imbalanced AODS may provide the basis for an increased release of speciﬁc cytokines (e.g., increased

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levels of IL-2 and IL6), as well as membrane abnormalities that have been reported in patients with schizophrenia.

B. Psychological Stress Increasing evidence has shown that the production of proinﬂammatory cytokines such as IL-1, IL-6, and INF- may be aVected by psychological stress. Levels of in vitro production of IL-2 were increased in medical students during the examination periods (Glaser et al., 1990). Maes et al. (1998a,b) showed that the in vitro production of proinﬂammatory cytokines (IL-6, TNF-, and INF-) and IL-10 were increased signiﬁcantly by stress due to academic examination. Moreover, volunteers subjected to sleep deprivation also exhibited increased levels of plasma IL-1 and IL-2 (Moldofsky et al., 1989). Similarly, stress-induced cytokine releases were also increased in animal models. In rat studies, levels of serum IL-6 and the expression of IL-6 messenger RNA in the brain (Shizuya et al., 1997; Takaki et al., 1994), as well as brain levels of IL-1 and IL-1 mRNA (Minami et al., 1991; Nguyen et al., 1998), were enhanced by physical restraint. In a longitudinal community study assessing the relationship between chronic stress and IL-6 production, Kiecolt-Glaser et al. (2003) found that the average rate of increase in IL-6 from caregivers for a spouse with dementia was four times higher than that of noncaregivers. These authors suggest that chronic stressors may accelerate the risk of a host of age-related diseases by prematurely aging the immune response.

V. Conclusion

Although the potential key role that lipids may play in schizophrenia is not fully understood, the increasing evidence to date suggests that an altered lipid environment can have a signiﬁcant impact on the behavior of neurotransmitter systems. For example, demyelinating diseases have been considered to be associated with behavioral disturbance (Hyde et al., 1992). Multiple lines of evidence have converged to implicate oligodendroglial dysfunction with subsequent abnormalities in myelin maintenance and repair underlying the pathogenetic mechanism of schizophrenia, particularly in the more severely ill patients (for reviews see Bartzokis et al., 2003; Davis et al., 2003). The dry mass of central nervous system (CNS) myelin is characterized by a high proportion of lipid (70–85%) (Morell and Quaries, 1999). In humans, approximately 45% of total lipids in myelin or white matter are phospholipids. Thus, it is conceivable that CNS membrane phospholipids are reduced in schizophrenia, which is consistent with the hypothesis of

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Fig. 1. An overview of phospholipids turnover, arachidonic acid signaling, and schizophrenic symptomatology (adapted from Skosnik and Yao, 2003). PC, phosphatidylcholine; PI, phosphatidylinositol; PS, phosphatidylserine; PE, phosphatidylethanolamine; AODS, antioxidant defense system; NT, neurotransmitters; RO, reactive oxygen; apoD, apolipoprotein D; PLA2, phospholipase A2; PLC, phospholipase C; LOX, lipooxygenase; AA, arachidonic acid; DAG, diacylglycerol; COX, cyclooxygenase; 2-AG, 2-arachidonoyl glycerol; GAP, growth-associated protein; PGG2, prostaglandin G2; PGH2, prostaglandin H2; PGD2, prostaglandin D2; CB, cannabinoid.

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myelin-related dysfunction in schizophrenia. Further investigations are needed to conﬁrm the altered myelin-related genes as reported by Davis et al. (2003). Membrane defects, such as those induced by decreased polyunsaturated fatty acids in phospholipids, can signiﬁcantly alter a broad range of membrane (e.g., gray and white matters) functions and ipso facto behavior through multiple ‘‘downstream’’ eVects. A number of putative mechanisms have been identiﬁed to explain the decreased PUFAs in schizophrenia, including an increased turnover of phospholipids and a decreased incorporation of arachidonic acid. Both increased oxidative stress and altered immune function may be responsible for increased phospholipid breakdown. This association is particularly relevant in relation to phospholipids/PUFA because AA can be converted to a variety of biologically active eicosanoids that serve as potent messengers in regulating the inﬂammatory response. Direct evidence of immune changes in schizophrenia have come to light, particularly in the activities of several cytokines known to be abnormal in autoimmune dysfunction. Given the diverse physiological function of AA, the speciﬁc behavioral symptomatology of schizophrenia is related mostly to the eVect of AA changes that regulate neurodevelopment, neurotransmitter homeostasis, second messenger signaling, and neuromodulatory activity in schizophrenia (Fig. 1). Hence, in the current conceptualization, AA may be at a nexus point in the cascade leading to the syndrome of schizophrenia and represents a common biochemical pathway leading to the highly heterogeneous symptomatology and course of schizophrenia. Changes in membrane fatty acids not only have been associated with the severity of symptomatology, but also provide a theoretical basis for predicting a potential psychotropic eVect of PUFA supplementation. Work utilizing eicosapentaenoic acid (EPA), the molecular precursor of DHA, has shown some promise in ameliorating many of the clinical characteristics of schizophrenia (Peet et al., 1996, 2001; Peet and Horrobin, 2002; Puri et al., 2000; Richardson et al., 2000), as well as cognitive impairments associated with dyslexia and attention deﬁcit hyperactivity disorder (Richardson et al., 1999; Stordy, 1999). More recently, Arvindakshan et al. (2003a) have shown that supplementation with a combination of n-3 fatty acids (EPA/DHA, 3:2) and antioxidants (vitamins E and C) may improve the outcome of schizophrenia. While these data are promising, an EPA trial performed by Fenton et al. (2001) failed to induce beneﬁcial changes in residual symptoms, mood, or cognition as compared to placebo in patients with schizophrenia. Although the patient group in this study had a longer duration and severity of illness, these ﬁndings raise some doubt of the beneﬁcial eVect of omega-3 fatty acid treatment in schizophrenia. In short, the present review exempliﬁed multiple metabolic defects involving phospholipid and cytokine pathways in schizophrenia.

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Acknowledgments

This study was supported in part by the Highland Drive VA Pittsburgh Healthcare System and research grants from the Department of Veterans AVairs (Merit Review and Research Career Scientist Award) and the National Institute of Mental Health (MH43742, MH44841, and MH58141).

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NEUROTENSIN, SCHIZOPHRENIA, AND ANTIPSYCHOTIC DRUG ACTION

Becky Kinkead and Charles B. Nemeroff Laboratory of Neuropsychopharmacology Department of Psychiatry and Behavioral Sciences Emory University School of Medicine Atlanta, Georgia 30322

I. Introduction II. Preclinical Evidence Supporting the Role of Neurotensin in the EVects of Antipsychotic Drugs III. Clinical Evidence Supporting the Role of Neurotensin Neurotransmission in the Pathophysiology of Schizophrenia IV. NTergic Compounds as Novel Antipsychotic Drugs References

The search for the underlying pathophysiology of schizophrenia has been an active avenue of investigation since the disease was ﬁrst recognized more than 100 years ago. Although a great deal of the research has been driven by the known pharmacology of eVective antipsychotic drugs, i.e., overactivity of the dopamine system, recent advances in neurobiology provide evidence that reduced synaptic connectivity/neurotransmission may play a substantial role in this disorder. One neuropeptide long posited to play a role in the biology of schizophrenia is neurotensin (NT). Central nervous system administration of NT has been shown to produce a wide variety of eVects. Because of its close association with the dopamine (DA) system, the role of the NT system in clinical disorders hypothesized to involve DA circuits such as schizophrenia, Parkinson’s disease, and drug abuse has been closely scrutinized. In addition, NT neurotransmission has been implicated in regulation of the stress response, stress-induced gastric ulcers, temperature regulation, food consumption, and analgesia. NT also acts as a growth factor in a variety of human cancer cell lines derived from lung, colon, prostate, and pancreas. This review ﬁrst provides a background of the NT system. Second, data indicating that NT may mediate the eVects of antipsychotic drugs are summarized. Third, data implicating NT in the pathophysiology of schizophrenia are described. Finally, evidence suggesting the use of NTergic compounds as novel antipsychotic drugs are presented.

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I. Introduction

Neurotensin (NT) (N-Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-IleLeu-C) is a tridecapeptide that was ﬁrst discovered in extracts of bovine hypothalamus by Carraway and Leeman in 1973. NT is found both within the central nervous system (CNS) and in the periphery, with more than 90% of NT found in the periphery, mostly in the small intestine. In the periphery, NT is degraded rapidly by peptidases and the peptide does not permeate the blood–brain barrier in any appreciable quantities. Genes encoding NT and three NT receptors (and possibly a fourth NT receptor) have been cloned and sequenced successfully (Chalon et al., 1996; Jacobsen et al., 1996; Mazella et al., 1996, 1998; Tanaka et al., 1990; Vita et al., 1993, 1998; Watson et al., 1993; Yamazaki et al., 1996; Zsu¨rger et al., 1994). The NT gene consists of a 10.2-kb segment containing four exons and three introns and in the human genome maps to the long arm of chromosome 12 (Kislauskis and Dobner, 1990; Marondel et al., 1996). The gene encodes a 170 amino acid precursor protein containing both NT and a closely related peptide, neuromedin N (pLys-Ile-Pro-Tyr-Ile-Leu-OH). The four amino acids at the carboxy terminal of NT and neuromedin N (NN) are identical, and amino acids 8–13 of NT are essential for biological activity. Elements involved in the regulation of NT/NN mRNA expression are located in the upstream 200-bp ﬂanking region of the rat gene. In this region, several cis-regulatory elements function cooperatively to integrate multiple environmental stimuli into a concerted transcriptional response (Kislauskis and Dobner, 1990). In the rat NT/NN gene, these sites include one consensus AP-1 site, two near consensus cAMP response elements (CREs), one near consensus glucocorticoid response element, and a sequence identical to the human c-jun gene autoregulatory element ( JARE). Notably, the glucocorticoid response element is absent in the regulatory sequence of the human NT/NN gene ( Vita et al., 1993). In neurons, NT is stored in dense core vesicles and released in a Ca2þdependent manner. Within the CNS, NT released from nerve endings is neutralized primarily by cleavage of NT by several peptidases, including neutral endopeptidase 24.11 (AlmenoV et al., 1981), angiotensin-converting enzyme (ACE) (Skidgel et al., 1984), metalloendopeptidase 24.15 (Orlowski et al., 1983), and metalloendopeptidase 24.16 (Checler et al., 1986b). In brain tissue, the reported half-life of NT is approximately 15 min (Checler et al., 1986a). In addition to peptidase activity, there is some evidence that NT internalization and subsequent degradation following binding to NT receptors may play a signiﬁcant role in the termination of NT transmission (Mazella, 2001). High concentrations of NT are found in the hypothalamus, amygdala, substantia nigra, ventral tegmental area (VTA), preoptic area, nucleus accumbens,

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and olfactory tubercle, with lower concentrations in a variety of cerebrocortical areas, hippocampus, and cerebellum (Emson et al., 1985; Manberg et al., 1982b). Interestingly, the NT system has been found to be closely associated anatomically with brain dopamine (DA) systems (for review, see Binder et al., 2001c). DAergic neurons located in the ventral tegmental area that colocalize NT project to the nucleus accumbens, prefrontal cortex, and amygdala. In addition, a high density of NT ﬁbers (originating outside the midbrain) and terminals are found in the VTA. The NT system is also associated with other neurotransmitter systems. NT cell bodies and ﬁbers and moderate NT receptor binding are found in the serotonin-rich dorsal raphe, whereas NT receptors are highly concentrated in the median raphe. Additionally, NT receptors and 5-HT2A receptors are both located on DA neurons in the VTA. NT receptors are also present on cholinergic neurons projecting from the lateral dorsal tegmental nucleus (area CH6) in the brain stem to the VTA. There are currently three characterized receptors for NT: a receptor with low aYnity for NT (NTRL, NTR2, or NT2) that also binds the histamine H1 receptor antagonist levocabastine, a levocabastine-insensitive receptor with high aYnity for NT (NTRH, NTR1, or NT1), and a third NT receptor (NT3) that is located intracellularly and has been identiﬁed as the previously characterized gp95/sortilin. A fourth potential NT receptor has also been identiﬁed as the previously characterized SorLA/LR11 ( Jacobsen, 2001). Table I provides a brief summary of the biochemical characteristics and synthesis of each of the cloned NT receptors. Both NT1 and NT2 receptors are G-protein-coupled receptors with the typical seven transmembrane domain conﬁguration characteristic of these receptors. For the most part, the signaling pathway for the NT1 receptor has been fairly well characterized in vitro. NT1 receptor activation increases intracellular Ca2þ inﬂux (Memo et al., 1986; Slusher et al., 1994; Trudeau, 2000; Woll and Rozengurt, 1989), regulates cyclic AMP (Bozou et al., 1986; Slusher et al., 1994; Yamada et al., 1993), cyclic GMP (Gilbert and Richelson, 1984), phosphatidyl inositol (PI) turnover (Erwin and RadcliVe, 1993; Hermans et al., 1994; Snider et al., 1986; Watson et al., 1992), phospholipase C (Chabry et al., 1994; Hermans et al., 1992; Watson et al., 1992), protein kinase C followed by mitogen-activated protein kinases (Poinot-Chazel et al., 1996), and Naþ, Kþ-ATPase activity (Lo´pez Ordieres and Rodriguez de Lores Arnaiz, 2000). In addition to regulation of second messenger pathways, NT1 receptor activation alters the aYnity of dopamine D2 receptors via allosteric receptor–receptor interactions (Fuxe et al., 1992; Li et al., 1995; Tanganelli et al., 1993; von Euler et al., 1989) and modulates gene expression following internalization of the NT/NT1 complex (for review, see Hermans and Maloteaux, 1998). This internalization occurs on the axon terminals, perikarya, and dendrites of DA neurons in the midbrain (Beaudet et al., 1994; Faure et al., 1995a,c). In the caudate/putamen, the NT/NT1 complex is

TABLE I Biochemical Characteristics and Synthesis of Cloned NT Receptors NT receptor subtypes NT1 (NTRH, NTR1) Cloned 330 Receptor classification

Gene characteristics

NT2 (NTRL, NTR2)

Rat (Tanaka et al., 1990)

Rat (Chalon et al., 1996)

Mouse (unpublished)

Mouse (Mazella et al., 1996)

Human (Vita et al., 1993; Watson et al., 1993) G-protein-coupled seven transmembrane-spanning regions

Human (Vita et al., 1998)

Human NT1 receptor gene localized to long arm (20q13) of chromosome 20 (Le et al., 1997a) Tetranucleotide repeat polymorphism 3 kb from gene (Le et al., 1997b)

G-protein-coupled seven transmembrane-spanning regions

NT3 (gp95/sortilin)

NT4 (SorLA/LR11)

Human (Mazella et al., 1998; Zsu¨rger et al., 1994)

Human ( Jacobsen et al., 1996) Rabbit (Yamazaki et al., 1996)

Type I amino acid receptor; single transmembranespanning region

Type I amino acid receptor; single transmembranespanning region Human NT4 receptor gene mapped to chromosome 11q23-24 ( Jacobsen et al., 1996). 177-kb gene with many large introns

Human NT3 receptor gene mapped to proximal short arm of chromosome 1 (Petersen et al., 1997) hNT3-75-kb gene with many large introns, two NT3 mRNA transcripts (3.5 and 8.0 kb) expressed

Receptor size 50–60 kDa and alternative processing

Characteristics

Located on neurons

Mouse NT2 ¼ 45 kDa, 417 amino acids, alternative splice sight produces truncated receptor (282 amino acids, five transmembrane-spanning regions) expressed in spinal cord Located on neurons and glia (Nouel et al., 1999)

100 kDa; coexpression with furin (prohormone convertase) cleaves receptor to 95 kDa and changes NT affinity from 10–15 to 0.1–0.3 nM

260 kDa, smaller 100-kDa fragment also found

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Expressed in neurons, glia, and Located primarily in the adipocytes (Nouel et al., 1999). CNS with low levels of Located on intracellular vesicles expression in the testes, containing GLUT4 glucose ovaries, and lymph transporter; transported nodes. Within the CNS, to the plasma membrane expressed in neurons (Motoi, 1999). Located in response to insulin intracellularly, associated with (Morris et al., 1998) intracellular vesicles associated with perinuclear compartment. Some soluble receptor (missing cytoplasmic tail) NT triggers insertion of the receptor into the membrane from an intracellular compartment in mouse cortical neurons (Chabry et al., 1993)

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internalized exclusively by DAergic terminals (Faure et al., 1995c), whereas in the midbrain, only 88% of NT is internalized by DA neurons (Beaudet et al., 1994). Once internalized, the NT/NT1 receptor complex dissociates and is segregated into separate intracellular traYcking pathways (Boudin et al., 1998; Hermans et al., 1997). The NT1 receptor is either recycled to the cell surface or degraded in lysosomal compartments (Boudin et al., 1998; Souaze´, 2001). The internalized NT eventually moves to surround the nuclei of the cells, potentially regulating gene expression (Laduron, 1994, 1995). The cell signaling pathway of the NT2 receptor has not been clearly established (for review, see Vincent et al., 1999). In addition, it is unclear whether NT is an agonist or an antagonist at the NT2 receptor. When the rat or human NT2 receptor is expressed in Chinese hamster ovary (CHO) cells, NT acts as an antagonist, whereas both levocabastine and SR4869 (a small molecule NT1 antagonist) act as NT2 receptor agonists ( Yamada et al., 1998). When human NT2 receptors are expressed in this system, NT, NN, and levocabastine act as antagonists and the NT receptor antagonists SR142948A and SR48692 act as agonists (Botto et al., 1998; Vita et al., 1998). In vivo, however, SR142948A has been shown to block the analgesic eVects of NT in rodents, an NT eVect that has been associated with activation of the NT2 receptor (Gully et al., 1997), indicating that the NT eVects seen in CHO cells may be expression system speciﬁc. In contrast to NT1 and NT2 receptors, both the NT3 receptor and SorLA/ LR11 (the potential NT4 receptor) are type I amino acid receptors with a single transmembrane-spanning region. These receptors are members of the vacuolar protein sorting 10 protein (Vps10p) domain family of receptors and SorLA/ LR11 is also related to the low-density lipoprotein receptor family ( Jacobsen et al., 1996). Both NT3 receptor and SorLA/LR11 are synthesized as proproteins and carry an N-terminal signal peptide for translocation into the endoplasmic reticulum. Cleavage of the propeptide (approximately 50 bp) located upstream of the signal sequence is necessary for activation of the receptors ( Jacobsen, 2001; Munck Petersen et al., 1999). The cleaved propeptide can bind to the mature receptor and inhibit the binding of other ligands (including NT) ( Jacobsen, 2001; Munck Petersen et al., 1999). It is hypothesized that binding of the propeptide prevents premature binding of ligands to the receptor and protects the proreceptor during processing in the early secretory pathway (Munck Petersen et al., 1999). The NT3 receptor is highly expressed in fat, brain, and lung and is upregulated dramatically during the diVerentiation of adipocytes in vitro (Lin et al., 1997). Within the CNS, the NT3 receptor is located in neurons and glia and is believed to be involved in the sorting of lumenal proteins from the trans-Golgi to late endosomes (Mazella, 2001). The NT3 receptor may also modulate the termination of NT transmission via the regulation of NT uptake and degradation. It appears that the NT3 subtype of the NT receptor is responsible for the trophic

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eVects of NT on certain cancer cells (Dal Farra et al., 2001). Following NTmediated downregulation of NT1 and NT2 receptors, the NT3 receptor is translocated to the cell surface where it most likely scavenges NT (Mazella et al., 1998). Evidence shows that the NT1 and NT3 receptors heterodimerize at the cell surface (Martin et al., 2002). Heterodimerization of NT1 and NT3 receptors in the human adenocarcinoma cell line HT29 modulated the NT-induced phosphorylation of mitogen-activated protein kinases and the phosphoinositide turnover mediated by the NT1 receptor. SorLA/LR11 is the mammalian homologue of the head activator (HA) binding protein (HAB) in hydra ( Jacobsen et al., 1996). Both HAB and SorLA/LR11 bind HA, which in mammals is found early in development in nervous and neuroendocrine systems and is thought to stimulate cell entry into mitosis and proliferation and to stabilize cell survival and enhance neurite growth (Kajiwara and Sato, 1986; Kayser et al., 1998; Quach et al., 1992; Ulrich et al., 1996). SorLA/ LR11 is found exclusively in neurons in the CNS and is associated with cytoplasmic puncta near apical dendrites (Motoi, 1999). The unique expression pattern of SorLA/LR11 mRNA expression in brain (neurons with long processes, e.g., pyramidal cells of the cerebral cortex, hippocampus, and Purkinje cells of the cerebellum) and other organs active in morphogenesis during murine development is compatible with a role for this receptor in neural development and organ formation (Hermans-Borgmeyer et al., 1998; Kanaki et al., 1998; Motoi, 1999). The only NT receptor agonists to date are modiﬁed subfragments of the NT peptide itself (Tyler-McMahon et al., 2000). Conversely, several nonpeptide NT receptor antagonists have been identiﬁed of which SR48692 and SR142948A are the best characterized (Gully et al., 1993, 1997). Both of these receptor antagonists possess nanomolar aYnity for the NT1 receptor (Gully et al., 1993, 1997). SR142948A, however, has a 90-fold higher aYnity for the NT1 receptor than SR48692, and only SR142948A binds the NT2 receptor with nanomolar aYnity. Despite the fact that SR4892 has a low binding aYnity for the NT3 receptor (Mazella et al., 1998), there is some evidence that in cancer cell lines expressing only the NT3 receptor, SR48692 blocks NT-induced cell growth (Dal Farra et al., 2001).

II. Preclinical Evidence Supporting the Role of Neurotensin in the Effects of Antipsychotic Drugs

The CNS eVects of NT have been scrutinized closely in relationship to interactions of the peptide with the DA circuits. When administered intracerebroventricularly or directly into the midbrain, physiologic concentrations of NT oppose

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DA autoinhibition and induce a slow, long-lasting depolarization (Shi and Bunney, 1991, 1992a). Higher, potentially nonphysiologic doses of NT may also promote burst ﬁring (Mercuri et al., 1993; Sotty et al., 1998) and eventually, at very high doses, a cessation of spontaneous activity that resembles the depolarization inactivation observed after the chronic administration of antipsychotic drugs (Pozza et al., 1988; Seutin et al., 1989). DA neurons in the VTA and substantia nigra display diVerent thresholds for NT-induced depolarization (Seutin et al., 1989; Shi and Bunney, 1990) and depolarization inactivation (Shi and Bunney, 1991) with DA neurons in the VTA being more sensitive to NT than DA neurons in the substantia nigra (Myers and Lee, 1983; Pinnock and WoodruV, 1994; Seutin et al., 1989). This eVect is remarkably similar to those of atypical antipsychotic drugs that exert relatively selective eVects on the VTA. The decreased ﬁring rate of DAergic neurons in the VTA is associated with a decrease in DA release in the nucleus accumbens (Grace et al., 1997). Depolarization inactivation of DA neurons in the VTA and decreased DA transmission in the nucleus accumbens after chronic antipsychotic drug administration have been postulated to be necessary for the therapeutic eVects of antipsychotic drugs and may account for the delay in clinical eYcacy of these drugs. Acute and chronic administration of typical antipsychotic drugs also increases cell ﬁring with subsequent depolarization inactivation of DA neurons in the substantia nigra, and with corresponding initial increases or decreases of DA release in the caudate/putamen. This eVect in the caudate/putamen is not observed with atypical antipsychotic drugs. Indeed, decreases in DA transmission in the nigroneostriatal DA system are thought to be involved in the induction of extrapyramidal side eVects (EPSEs) (Grace et al., 1997). In addition to regulating DA transmission at the level of the midbrain, NT directly modulates DAergic signaling in the projection areas of these neurons. Similar to atypical antipsychotic drugs, NT opposes DA transmission in the nucleus accumbens, but not the caudate/putamen. At low doses, in vivo application of NT into the nucleus accumbens decreases DA release and increases GABA release in a TTX-sensitive manner (O’Connor et al., 1992). In contrast, NT alone has no eVect on spontaneously active cells in the caudate/putamen (Beauregard et al., 1992). Electrophysiologically, NT potentiates the inhibitory eVects of DA in 60% of the caudate/putamen cells examined (Audinat et al., 1989). In contrast to the nucleus accumbens, low-dose NT in the caudate/putamen has no eVect on DA release on its own, but blocks apomorphine-induced DA decreases (Tanganelli et al., 1989, 1994). These regionally diVerent eVects of NT on DA transmission can be explained by the heterogeneous localization of NT receptors. In the nucleus accumbens, NT receptors are located predominantly on GABAergic neurons and not on DAergic terminals. Activation of the NT receptor increases GABA release, which is believed in turn to decrease DA release via the activation of GABA receptors located on the DA terminals. In the

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caudate/putamen, NT receptors are located primarily on DA terminals and NT opposes DA autoinhibition by decreasing D2 autoreceptor signal transduction. There are several diVerent mechanisms by which NT decreases DA transmission within the mesolimbic and nigrostriatal DA pathways. At lower doses than those needed to increase DA cell ﬁring, NT antagonizes D2 receptor agonistinduced autoinhibition of DA cell ﬁring (Shi and Bunney, 1992b). This neuromodulatory eVect of NT appears to be independent of receptor/receptor interactions (implicated as a regulatory mechanism in other brain regions) and interactions at the G-protein level (Shi and Bunney, 1992a). There is some evidence that the second messenger transduction pathways for NT receptors and D2 receptors converge farther downstream at the level of the actual eVector molecule, the G-protein-coupled inward rectifying Kþ channel (Farkas et al., 1997). Intracellular cAMP and protein kinase A appear to be involved in this modulatory NT eVect (Shi and Bunney, 1992b). The neuromodulatory eVects of NT are speciﬁc for NT receptor activation and not due to a general opposition of excitation because neither glutamate nor CCK, neurotransmitters that also increase DA cell ﬁring, mimics these eVects (Shi and Bunney, 1992a). As noted previously, NT decreases the aYnity of D2 DA receptors for DA and DA receptor agonists (Fuxe et al., 1992; Li et al., 1995; Tanganelli et al., 1993; von Euler et al., 1989). The increase in Kd of D2 receptor agonist binding is due primarily to an increased dissociation rate from the high-aYnity form of the D2 receptor (von Euler et al., 1991). The exact mechanism of this eVect has not been completely elucidated, but allosteric receptor/receptor interactions between the NT receptor and D2 type receptors, as well as second messenger-dependent receptor alterations, such as phosphorylation and dephosphorylation, have been implicated (for a review, see Fuxe et al., 1992). In addition, as demonstrated by a study by Mandell et al. (1998), NT can induce changes in the Kd of D2 receptor antagonist binding similar to the eVects of noncompetitive antagonists. This eVect was observed in a mouse ﬁbroblastoma cell line transfected with the human D2 receptor lacking any NT receptors, indicating that this eVect was not mediated by NT1 activation or allosteric receptor/receptor interactions. The authors suggest that NT interacts with the D2 receptor via hydrophobic mode matches, i.e., similarities in the sequential pattern of relative hydrophobicities in the amino acid chains. This type of interaction could represent a novel mechanism for peptide/receptor modulation; however, the physiological implications of these data remain to be determined. Upon binding of NT to NT1 receptors, there is rapid ligand-induced receptor internalization (for review, see Hermans and Maloteaux, 1998). This internalization occurs on the axon terminals, perikarya, and dendrites of DA neurons in the midbrain (Beaudet et al., 1994; Faure et al., 1995b). In the caudate/putamen, NT is internalized exclusively by DAergic terminals (Faure et al., 1995b), whereas in the midbrain, only 88% of NT is internalized by DA neurons (Beaudet et al., 1994). Once internalized, the NT/NT receptor complex dissociates and is segregated into

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separate intracellular traYcking pathways (Boudin et al., 1998; Hermans et al., 1997). The NT1 receptor is either recycled to the cell surface or degraded in lysosomal compartments (Boudin et al., 1998; Souaze´, 2001). The internalized NT eventually moves to surround the nuclei of the cells, potentially regulating gene expression (Laduron, 1994, 1995). For example, after binding of NT to NT1 on DA terminals in the caudate/putamen, labeled NT is transported retrogradely to cell bodies in the substantia nigra where NT increases TH mRNA expression (Burgevin et al., 1992; Castel et al., 1991, 1994). As demonstrated in the murine neuroblastoma cell line N1E-115, NT induces tyrosine hydroxylase gene activation through the nitric oxide and protein kinase C signaling pathways (Najimi et al., 2002). Almost three decades of accumulated evidence strongly implicate the NT system in the mechanism of action of antipsychotic drugs. The ﬁrst indication that NT may act as an endogenous neuroleptic (NemeroV, 1980) was the striking similarity between the behavioral eVects of centrally administered NT and peripherally administered antipsychotic drugs (for review, see Binder et al., 2001c). Because of this similarity, it was reasonable to determine whether antipsychotic drugs produce their speciﬁc behavioral eVects by regulation of the NT system. The eYcacy of antipsychotic drugs in schizophrenia was ﬁrst demonstrated in the 1960s. Since that time, numerous antipsychotic drugs have been identiﬁed, each with a unique receptor-binding proﬁle. Nonetheless, the majority of antipsychotic drugs are antagonists at the DA D2 receptor, and there is a strong correlation between their clinical eYcacy and their aYnity for the DA D2 receptor (Creese et al., 1976; Farde et al., 1989). Antipsychotic drugs are generally classiﬁed as either ‘‘typical’’ or ‘‘atypical’’ based on their relative eYcacy in the treatment of both positive (e.g., delusions and hallucinations) and negative (e.g., amotivation, anhedonia, ﬂat aVect, and social withdrawal) symptoms of schizophrenia and their propensity for inducing EPSEs such as Parkinsonian motor disturbances, acute dystonias, and tardive dyskinesias. Neurochemical eVects shared by typical (e.g., haloperidol) and atypical antipsychotic drugs (e.g., clozapine) may well mediate the shared therapeutic eYcacy of antipsychotic drugs. In contrast, eVects that are only observed after the administration of typical antipsychotic drugs may be related solely to EPSE liability. In the same year that NT was hypothesized to be an endogenous neuroleptic, the ﬁrst report concerning the eVects of antipsychotic drug administration on NT-like immunoreactivity was published (Govoni et al., 1980). In this study, administration of the typical antipsychotic drugs haloperidol and chlorpromazine increased NT-like immunoreactivity in the nucleus accumbens and caudate/putamen in rats. This seminal study sparked considerable interest in examining the eVects of antipsychotic drug administration on virtually every aspect of the NT system (for a review, see Binder et al., 2001b). Although the speciﬁc eVects of antipsychotic drug administration on the NT system of the rat brain diVer slightly depending on the treatment protocol

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and the type of antipsychotic drug administered, there are several very consistent ﬁndings. First, all clinically eVective antipsychotic drugs examined to date modify NT neurotransmission in the rat brain. Second, regulation of NT neurotransmission in the rat brain is unique to drugs with antipsychotic eYcacy; compounds from other classes of clinically eVective psychoactive drugs (e.g., anxiolytics, antidepressants, and antihistamines), as well as clinically ineVective phenothiazines, do not eVect the NT system. Third, typical and atypical antipsychotic drugs diVerentially regulate the NT system. In general, whereas typical antipsychotic drugs regulate NT neurotransmission in both mesolimbic and nigroneostriatal neuronal systems, atypical antipsychotic drugs act preferentially on the mesolimbic NT system. This last ﬁnding has generated the hypothesis that the nigroneostriatal NT system may play a role in the classic side eVect proﬁle of typical antipsychotic drugs, i.e., extrapyramidal side eVects, whereas the mesolimbic/ mesocortical NT system may be associated with the clinical eYcacy of antipsychotic drugs. The exact role of NT neurotransmission in the side eVect proﬁle of typical antipsychotic drugs is still an active avenue of investigation. NT injected into the ventrolateral striatum induced vacuous chewing movements and potentiated antipsychotic drug-induced vacuous chewing movements (Stoessl, 1995; Stoessl and Szczutkowski, 1991). In addition, the NT receptor antagonist SR48692 decreased antipsychotic drug-induced vacuous chewing movements (Stoessl, 1995). In contrast, peripheral administration of the NT analog NT69L blocked haloperidol-induced catalepsy (Cusack et al., 2000). The ﬁnding that some alterations in the NT system are only observed following the chronic administration of antipsychotic drugs suggests that long-term changes in the NT system may mediate the clinical eYcacy associated with antipsychotic drug treatment. Chronic but not acute administration of typical and atypical antipsychotic drugs is associated with decreased NT concentrations in the prefrontal cortex and bed nucleus of the stria terminalis (Kilts et al., 1988), as well as decreased NT receptor binding in the prefrontal cortex (Giardino et al., 1990). Chronic antipsychotic drug administration also aVects NT receptor expression in other brain regions (Bolden-Watson et al., 1993; Giardino et al., 1990; Kinkead et al., 2000; Uhl and Kuhar, 1984). NT receptor binding and NT1 receptor mRNA expression are increased in the substantia nigra after chronic treatment with haloperidol (Bolden-Watson et al., 1993; Kinkead et al., 2000; Uhl and Kuhar, 1984). In contrast, NT receptor binding is decreased in the VTA and nucleus accumbens after the subchronic administration of several atypical antipsychotic drugs, including clozapine, olanzapine, quetiapine, and risperidone (Kinkead et al., 2000). In summary, the eVects of antipsychotic drugs on NT circuits occur in brain regions relevant to the pathophysiology of schizophrenia and the eVects of chronic antipsychotic drug administration are diVerent from acute administration, concordant with involvement of this peptide in the therapeutic eVects of these drugs.

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III. Clinical Evidence Supporting the Role of Neurotensin Neurotransmission in the Pathophysiology of Schizophrenia

Direct clinical evaluation of the role of NT neurotransmission in the pathophysiology of schizophrenia awaits the development of novel tools such as NT receptor ligands for functional brain imaging studies. However, because the tools necessary for in vivo evaluation of regionally speciﬁc NT transmission have not yet been developed, the ﬁeld has largely relied on postmortem studies to determine whether NT transmission is altered in this disease (for a review, see Binder et al., 2001c). To date, no consistent postmortem changes in NT peptide concentrations have been reported in schizophrenia, with the majority of studies reporting negative ﬁndings for cortical, striatal, hypothalamic, and amygdaloid areas. Two studies report increased NT concentrations in area BA 32 in the medial prefrontal cortex (Manberg et al., 1982a; NemeroV et al., 1983). No signiﬁcant alterations are seen in the number of DAergic neurons expressing NT mRNA in the ventral mesencephalon in schizophrenia (Bean et al., 1992). Changes in NT receptor binding appear to be more consistent. A 40% decrease in NT receptor binding in layer II of the entorhinal cortex was reported by Wolf et al. (1995). This ﬁnding was conﬁrmed and extended in a study by Lahti et al. (1998) demonstrating a reduced density of NT receptor binding in the caudate, cingulate, and prefrontal cortices of patients on and oV (at least 3 months) antipsychotic drugs. Uhl and Kuhar (1984) explicitly investigated the eVects of antipsychotic drugs on NT receptor density. NT receptor binding in the SN was increased in patients treated with antipsychotic drugs, an observation also observed in rats treated chronically with haloperidol. In addition to the postmortem analysis of the NT system, NT concentrations have been measured in the cerebrospinal ﬂuid (CSF) of schizophrenic patients before and after antipsychotic drug treatment. Measurement of NT concentrations in CSF is currently the only means of antemortem estimation of NT disturbances. Because there is a marked dissociation between CSF and plasma concentrations of NT, it is believed that CSF NT concentrations reﬂect CNS extracellular ﬂuid peptide concentrations and are not derived from the systemic circulation. Reduced CSF NT concentrations are the most consistently found CSF neuropeptide alteration in drug-free schizophrenic patients (Garver et al., 1991; Lindstro¨m et al., 1988; NemeroV et al., 1989; Sharma et al., 1997; Widerlo¨v et al., 1982). CSF NT concentrations are lower in a subset of schizophrenic patients compared to patients with anorexia/bulimia, premenstrual syndrome, depression, and normal controls. In a subset of schizophrenic patients with low CSF concentrations of NT, no correlation was found between CSF NT concentrations and age or duration of illness. In two studies, women were found to have

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signiﬁcantly lower CSF NT concentrations than men (Breslin et al., 1994; Garver et al., 1991), and within schizophrenic women, those with the lowest CSF NT concentrations before antipsychotic drug treatment took the longest time to respond. Decreased CSF NT concentrations also appear to be correlated with more severe psychopathology (Breslin et al., 1994; Garver et al., 1991; Lindstro¨m et al., 1988; Sharma et al., 1997). Clinically eVective antipsychotic drug treatment normalizes CSF NT concentrations in the subgroup of schizophrenic patients with lower CSF NT in most (Breslin et al., 1994; Lindstro¨m et al., 1988; Sharma et al., 1997; Widerlo¨v et al., 1982), but not all (NemeroV et al., 1989) studies, and the percentage increase in CSF NT concentration is correlated positively with the improvement in negative symptoms (Breslin et al., 1994; Sharma et al., 1997).

IV. NTergic Compounds as Novel Antipsychotic Drugs

The concatenation of data suggests possible therapeutic eVects of NT receptor agonists. Small molecule NT receptor agonists are not yet available, delaying clinical testing of these compounds. However, small molecule, blood–brain barrier-penetrating NT receptor antagonists have been developed, of which SR48692 and SR142948A are the best characterized (Gully et al., 1993, 1997). Interestingly, peripheral administration of NT receptor antagonists exert eVects on DA neurons similar to centrally administered NT and peripherally administered antipsychotic drugs. The number of spontaneously active midbrain DA neurons is increased following the acute peripheral administration of NT receptor antagonists (Gully et al., 1997; Santucci et al., 1997). Similar to NT and atypical antipsychotic drugs, NT receptor antagonists selectively exhibit mesolimbic selective activity, increasing the number of spontaneously active cells at lower doses in the VTA than in the SN (Gully et al., 1997; Santucci et al., 1997). The eVects of NT receptor antagonists on DA neuronal ﬁring in the midbrain are most likely due to eVects outside the midbrain. Direct injections of NT receptor antagonists into the PFC mimic peripheral administration of the NT receptor antagonist (Santucci et al., 1997). Similar to antipsychotic drugs, chronic administration of NT receptor antagonists induces a selective depolarization block-type inactivation of DA neurons in the VTA that can be reversed by apomorphine (Gully et al., 1997; Santucci et al., 1997). In addition to inactivity of DA neurons following chronic peripheral administration of NT receptor antagonists, there are concomitant decreases in DA release in the nucleus accumbens but not the PFC (Azzi et al., 1998), suggesting that the depolarization block reported in the VTA after chronic administration of the NT receptor antagonist is selective for DAergic projections to the nucleus accumbens. Although the biochemical and

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electrophysiologic eVects of NT receptor antagonists might render them promising antipsychotic drugs, behavioral data supporting antipsychotic-like eVects of NT receptor antagonists are lacking. In fact, acute administration of NT receptor antagonists has eVects opposite those of antipsychotic drugs in several behavioral tests used to screen for antipsychotic eYcacy (Binder et al., 2001a). The behavioral eVects of chronic NT receptor antagonist administration remain to be examined in paradigms relevant to antipsychotic drug activity. A potential therapeutic utility of NT receptor antagonists can therefore not be ruled out. However, the NT receptor antagonist SR48692 was ineVective in a randomized controlled, double-blind clinical trial in schizophrenic patients (Arvanitis et al., 2001). More recently, the hypothesis that enhanced endogenous NT neurotransmission may be involved in behaviors thought to model the psychophysiologic disturbances underlying the symptoms of schizophrenia (e.g., sensorimotor gating and selective attention) has been tested. Methodologically, the consequences of disrupting NT neurotransmission in two commonly used measures of sensorimotor gating are the latent inhibition paradigm (LI) and prepulse inhibition (PPI) of the acoustic startle reﬂex. The LI paradigm, ﬁrst presented by Lubow and Moore (1959), is a measure of attentive sensory gating. LI consists of a reduction in associative learning if the subject has ﬁrst been preexposed to the ‘‘to be conditioned stimulus’’ without consequence because the stimulus has now been categorized as nonrelevant. The PPI of the acoustic startle reﬂex is deﬁned by a decrease in the startle reﬂex induced by a strong acoustic stimulus when preceded by a weak prepulse. PPI is a measure of preattentive sensorimotor gating (for a review, see Swerdlow et al., 1994). In humans, LI and PPI are disrupted in schizophrenic patients and in patients with high schizotypal scores (for a review, see Swerdlow and Geyer, 1998). Although somewhat controversial, antipsychotic drug treatment normalizes LI in acute schizophrenic patients (Gray et al., 1995; Kumari et al., 1999; Venables, 1966; Weiner et al., 1997). Administration of psychotomimetic compounds, such as DA agonists, disrupts performance in both paradigms. These disruptions are restored by typical as well as by atypical antipsychotic drugs, but not by other clinically eVective drugs such as antidepressants or anxiolytics (for reviews, see Swerdlow and Geyer, 1998; Weiner and Feldon, 1997). Typical and atypical antipsychotic drugs, but not other psychoactive drugs, also consistently enhance LI, supporting the validity of LI as a useful animal screening test for antipsychotic drug activity (Dunn et al., 1993). NT produces eVects similar to those of antipsychotic drugs in the PPI paradigm. Similar to antipsychotic drugs, low doses of NT injected directly into the nucleus accumbens block amphetamine-induced PPI disruption, whereas a higher dose (5.0 g) enhanced the amphetamine eVects (Feifel et al., 1997a,b). Similarly, subcutaneous injections of PD149163, an NT8–13 mimetic, signiﬁcantly

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antagonized the reduction of PPI produced by amphetamine and the phencyclidine analog dizocilpine (Feifel et al., 1999). In order to determine whether the regulation of NT neurotransmission is involved in the antipsychotic drug regulation of PPI, the eVects of pretreatment with the NT receptor antagonist SR142948A on the antipsychotic drug-induced restoration of isolation rearing-induced deﬁcits in PPI were examined (Binder et al., 2001a). Isolation rearing is a nonpharmacologic means of disrupting PPI (Geyer et al., 1993), and both typical and atypical antipsychotic drugs restore isolation rearing-induced deﬁcits in PPI (Bakshi et al., 1998; Geyer et al., 1993; Varty and Higgins, 1995). The NT receptor antagonist alone had no eVect on PPI, but blocked the eVects of haloperidol, a typical antipsychotic drug, and quetiapine, an atypical antipsychotic drug, suggesting that increased NT neurotransmission may be a common component involved in the behavioral eVects of clinically eVective antipsychotic drugs. These results suggest that intact NT neurotransmission is necessary for the eVects of antipsychotic drugs in both of these paradigms. In this same study, the environmentally induced deﬁcit in sensorimotor gating in isolation-reared animals was paralleled by a decrease in NT neurotransmission in the nucleus accumbens shell, a brain region thought to be critical for the antipsychotic eVects of antipsychotic drugs. NT receptor binding was increased signiﬁcantly in the nucleus accumbens shell of isolation-reared animals compared to socially reared animals, whereas NT mRNA expression was signiﬁcantly lower. The NT system of isolation-reared rats was also found to be hyperresponsive to antipsychotic drug administration. Increasing doses of quetiapine increased NT mRNA expression in the nucleus accumbens shell in isolation-reared animals, but had no eVect on NT mRNA expression in socially reared animals. These data are consistent with the hypothesis that deﬁcits in NT neurotransmission may be linked causally to deﬁcits in sensorimotor gating and that normalization of NT neurotransmission after antipsychotic drug treatment may be involved in restoration of these deﬁcits. In addition, there is increasing evidence that NT (via the NT3 or putative NT4 receptor) may be involved during CNS development in stimulating cell entry into mitosis and proliferation, in stabilizing cell survival, and in enhancing neurite growth. Clinically, advances in understanding the role of the NT system in the pathophysiology of schizophrenia are at least partly dependent on the development of positron emission tomography and single photon emission computer tomography ligands for human NT receptor subtypes. If preclinical ﬁndings of the involvement of this peptide in schizophrenia and drug abuse hold true in humans, highpotency, peripherally administrable NTergic compounds may represent very promising novel therapeutic agents. In accordance with the report that the NT receptor antagonist SR48692 was ineVective in the treatment of schizophrenia

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in clinical trials (Arvanitis et al., 2001), the cumulative results of the last two and a half decades continue to provide extremely strong rationale for the use of NT receptor agonists in the treatment of this devastating disorder (Binder et al., 2001c). Acknowledgment

This work was supported by NIH MH-39415 and MH-63400.

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SCHIZOPHRENIA, VITAMIN D, AND BRAIN DEVELOPMENT

Alan Mackay-Sim,* Franc¸ois Fe´ron,*,{ Darryl Eyles,{,{ Thomas Burne,* and John McGrath{,x

*School of Biomolecular and Biomedical Science Griffith University Brisbane, Qld 4111 Australia {

Queensland Centre for Mental Health Research The Park Centre for Mental Health Wacol, Qld 4076 Australia {

School of Biomedical Science and x Department of Psychiatry University of Queensland Brisbane, Qld Australia

I. Introduction II. Gene–Environment Interactions III. Schizophrenia Susceptibility Genes A. Gene Array and Protein Expression Data B. Candidate Gene Studies IV. Gene–Environment Models of Schizophrenia A. The Vitamin A Hypothesis B. The Vitamin D Hypothesis C. A Nuclear Hormone Hypothesis V. Conclusion References

Schizophrenia research is invigorated at present by the recent discovery of several plausible candidate susceptibility genes identiﬁed from genetic linkage and gene expression studies of brains from persons with schizophrenia. It is a current challenge to reconcile this gathering evidence for speciﬁc candidate susceptibility genes with the ‘‘neurodevelopmental hypothesis,’’ which posits that schizophrenia arises from gene–environment interactions that disrupt brain development. We make the case here that schizophrenia may result not from numerous genes of small eVect, but a few genes of transcriptional regulation acting during brain development. In particular we propose that low vitamin D during brain development interacts with susceptibility genes to alter the trajectory of INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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brain development, probably by epigenetic regulation that alters gene expression throughout adult life. Vitamin D is an attractive ‘‘environmental’’ candidate because it appears to explain several key epidemiological features of schizophrenia. Vitamin D is an attractive ‘‘genetic’’ candidate because its nuclear hormone receptor regulates gene expression and nervous system development. The polygenic quality of schizophrenia, with linkage to many genes of small eVect, may be brought together via this ‘‘vitamin D hypothesis.’’ We also discuss the possibility of a broader set of environmental and genetic factors interacting via the nuclear hormone receptors to aVect the development of the brain leading to schizophrenia.

I. Introduction

Schizophrenia is a group of imperfectly understood brain disorders characterized by alterations in higher functions related to perception, cognition communication, planning, and motivation. The syndrome is deﬁned by the presence of hallucinations, delusions, thought disorder, and negative symptoms, such as blunted aVect and reduced speech (American Psychiatric Association, 1994). The lifetime morbid risk of schizophrenia is approximately 1 in a 100. The symptoms of the disorder usually emerge in early adulthood and while many individuals with this disorder make a good recovery, many have intermittent or persistent symptoms for decades. Unfortunately, despite optimal medication and psychosocial support, schizophrenia remains a leading contributor to the burden of disease in developed and developing countries (Murray and Lopez, 1996). Robust evidence shows that the risk of developing schizophrenia is associated with both genetic and environmental risk factors. Almost certainly schizophrenia, like many other chronic adult-onset disorders, arises from an interaction of both genetic and environmental factors, neither of which is suYcient to cause the disorder in isolation. Schizophrenia is also recognized as a ‘‘neurodevelopmental’’ disorder in which it is considered that genetic or environmental inﬂuence is predicted to occur during early brain development, in utero or soon after birth (McGrath and Murray, 2003). The neurodevelopmental hypothesis, which rests on robust evidence derived from diverse sources (see review by Weinberger and Marenco, 2003), proposes that there is an interaction between genetic and environmental factors during critical early periods of neuronal development that aVects brain development negatively. The behavioral sequelae of these early events are thought to be clinically dormant until after puberty, when maturational events lead to the emergence of the symptoms of the disorder.

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This model of gene–environment interactions acting during brain development is heuristic but it does not specify any particular genes, environments, or neurodevelopmental pathways that might be involved. Thus the hypothesis is not testable in any speciﬁc way at present, although it sets a framework for thought and experiment.

II. Gene–Environment Interactions

Gene linkage and association studies have had diYculty in identifying any obvious ‘‘schizophrenia’’ genes, although many areas of the genome have been identiﬁed as containing ‘‘hot spots’’ of association and linkage. This is usually construed as indicating that schizophrenia is a ‘‘complex’’ disease resulting from numerous genes of small eVect. Models for this type of ‘‘genetic’’ causation are Alzheimer’s disease and type II diabetes, for which several candidate genes have been identiﬁed, mutations in each of which increase the risk of the disease (Mowry et al., 1997). This is not the only model to explain the gene linkage and associations in schizophrenia. It may be a disease of great heterogeneity with multiple causes; in other words, multiple single genes that produce a similar phenotype. Examples of this are congenital deafness and epilepsy. There are also now recognized several diseases with neurological sequelae for which single gene mutations can lead to complex behavioral and cognitive outcomes, that is, a ‘‘complex’’ phenotype produced by a ‘‘simple’’ genetic alteration. Mitochondrial disorders, for example, can produce many diVerent phenotypes depending on the haplotype of the individuals—essentially ‘‘gene dosage’’ of mitochondrial genes can vary depending on the founder cells for the lineage of a particular tissue. Thus it can be possible for the nervous system to be heavily aVected, perhaps regionally, or unaVected by mitochondrial mutations, leading to multiple phenotypes. Schizophrenia is undoubtedly inherited (Levinson, 2003). The increased risks associated with family members with schizophrenia bear this out, yet the 50% concordance rate for monozygotic twins indicates some complexity in the interpretation of genetic risk. Is this concordance rate due to an extra ‘‘environmental’’ inﬂuence experienced only by one twin, incomplete penetrance of a genetic susceptibility, or a combination of both? However, the majority of individuals with schizophrenia do not have aVected family members. Does this indicate a ‘‘nongenetic’’ origin, a spontaneous mutation, a combination of genetic inﬂuences passed from two carrier parents, or possible combinations of all these? It has become clear that factors in utero and soon after birth can lead to long-term consequences for the developing child and adult (Barker, 1992).

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Of particular note here is a deeper understanding of epigenetic regulation. Epigenesis refers to stable changes in gene transcription, which do not involve mutations in the DNA. Two molecular mechanisms are thought to be responsible: DNA methylation and histone modiﬁcations ( Jaenisch and Bird, 2003). The most well understood example of epigenesis is X inactivation and gene dosage compensation where one allele is silenced through long-term modiﬁcations of DNA and the chromosome structure (Rakyan et al., 2001). It is possible therefore that early life environmental factors can alter gene expression via an epigenetic mechanism (‘‘imprinting’’), which, in turn, may have profound consequences on brain development (Butler, 2002; Jaenisch and Bird, 2003). Of interest here is a study showing that epigenetic modiﬁcations can be aVected by diet and drug treatment, that is, by the ‘‘environment’’ (Tremolizzo et al., 2002). Mouse fed l-methionine increased DNA methylation of the promoter region for reln, a gene of interest in schizophrenia (Chen et al., 2002a; Fatemi et al., 2000, 2001; Guidotti et al., 2000; Knable et al., 2001). Increased methylation of the reelin gene (reln) represses its transcription (Chen et al., 2002b). One explanation for schizophrenia discordance in monozygotic twins is that each is aVected diVerentially by ‘‘epigenetic’’ events during development (Petronis et al., 2003). In monozygotic twins discordant for schizophrenia there were signiﬁcant diVerences in DNA methylation in the 50 regulatory region of the dopamine D2 receptor, in agreement with an epigenetic explanation for schizophrenia discordance (Petronis et al., 2003). A pertinent example of the inﬂuence of epigenesis is given by Rett syndrome. Rett syndrome is a developmental disorder with severe neurodevelopmental consequences, including mental retardation and loss of purposeful hand skills ( Jellinger, 2003; Renieri et al., 2003). The syndrome is not manifest until 6–18 months of age and is associated with mild dysmorphic features, with ventriculomegally, and no obvious gliosis. Thus it has similarities to schizophrenia in having deﬁned age-of-onset and other developmental features. Eighty percent of sporadic cases and 40% of familial cases of this disease have a mutation in the MeCP2 gene. Gene carriers may have no clinical features, including an unaVected monozygotic twin. The parallels with schizophrenia are obvious—a developmental disorder of a complex phenotype that is clearly ‘‘genetic’’ in which monozygotic twins can be discordant. We are not arguing here necessarily for a single ‘‘schizophrenia’’ gene, we instead are making the point that complex diseases of developmental origin can arise from single gene mutations. Another aspect of this disease is also instructive, some of the ‘‘complexity’’ of this syndrome, its involvement in many tissues and its developmental course, arises from the nature of the MeCP2 gene (HoVbuhr et al., 2002). The MeCP2 protein binds to methylated DNA and represses the transcription of target genes ( Renieri et al., 2003). This protein is expressed ubiquitously in most tissues in development and during postnatal life. These aspects of the biology of this protein help explain the developmental

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aspects of the disease and the complexity of the phenotype: target gene expression may be repressed at speciﬁc times during development and the development of multiple tissues is aVected, including the nervous system (Akbarian, 2003; Armstrong, 2002; Renieri et al., 2003). It is possible therefore that the complex, developmentally regulated phenotype that is schizophrenia may result not from numerous genes of small eVect, but from a few genes of transcriptional regulation. The multiple genetic signals now identiﬁed (see later) may be downstream eVects of mutations in genes that inﬂuence multiple genetic pathways. Such transcriptional regulators are attractive because they provide the potential for interactions among genetic, epigenetic, and environmental regulation of phenotype during development and beyond. One example proposed for schizophrenia is transcriptional regulation via retinoic acid (vitamin A). It is argued (Goodman, 1996, 1998; LaMantia, 1999) that schizophrenia may arise from disruptions to retinoic acid signaling, and several lines of evidence relate retinoic acid signaling to schizophrenia genetics, chromosomal loci, and candidate genes (Goodman, 1998). Retinoic acid makes a plausible candidate because it has many known eVects on nervous system development. Disruption of retinoic acid signaling is thus a plausible mechanism to explain the apparent genetic complexity, developmental etiology, and clinical heterogeneity of schizophrenia. This review argues that the dysregulation of vitamin D signaling is another plausible mechanism for the etiology of schizophrenia. Like retinoic acid, the vitamin D receptor is a nuclear receptor that regulates the transcription of target genes, those with a vitamin D responsive element in the promoter sequence. Thus vitamin D signaling provides a potential conduit via which genetic, epigenetic, and environmental factors can regulate brain development (Eyles et al., 2003).

III. Schizophrenia Susceptibility Genes

After many years of genetic linkage, association, and family studies it has been concluded that schizophrenia must be ‘‘polygenic’’ with susceptibility genes distributed widely over the genome ( Levinson, 2003; Lewis et al., 2003). In recent years, several speciﬁc candidate genes have been associated with schizophrenia and some have been tested in animal models. Candidate genes and pathways have also been identiﬁed from gene expression studies of brains from persons with schizophrenia, obtained at postmortem. It is now our challenge to reconcile the neurodevelopmental hypothesis of schizophrenia with the gathering evidence for speciﬁc candidate susceptibility genes. At this time this remains a very speculative adventure but one that is worth exploring.

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A. Gene Array and Protein Expression Data The exciting possibilities of gene expression microarrays are a two-edged sword. They allow unprecedented investigations into the activities of cells of interest, but by providing such a wealth of data and allowing post-hoc analyses, the data they generate can support almost any number of hypotheses (Williams et al., 2002). There are so many genes to choose from or ﬁlter out of the gene set that one can always ﬁnd some that seem to be synaptically related, signaling related, cytoskeletal related, developmentally related, and so on. There have been several analyses of gene expression in the prefrontal cortex in schizophrenia (Hakak et al., 2001; MacDonald et al., 1996; Middleton et al., 2002; Mirnics et al., 2000). Each of these reveals numerous genes expressed at diVerent levels in schizophrenia compared to controls and each presents plausible neurobiological consequences of the observed diVerences in gene expression. Despite this, there is little agreement between studies at the level of individual genes. Some of these diVerences probably relate to technical diVerences among the studies such as the small number of individuals chosen for analysis, the postmortem interval, the microarray manufacturer, genes represented on the arrays, and statistical criteria used to judge whether gene expression was diVerent. DiVerences between studies even resulted in diVerent overall directions of gene expression diVerences in schizophrenia compared to controls: one study reported a general increase in expression (Hakak et al., 2001) whereas others reported a generally decreased gene expression in schizophrenia. Another puzzling feature is that there was little overlap between genes identiﬁed as being downregulated in the gene expression array studies and proteins downregulated in neuropathological studies of the prefrontal cortex (Knable et al., 2001), even when the gene expression studies were performed on the same group of brains from the Stanley Foundation ( Vawter et al., 2001). One exception to this was a decrease in the expression of glial ﬁbrillary acidic protein (GFAP), although decreases in this protein are not conﬁned to schizophrenia but are also seen in bipolar disorder and depression (Knable et al., 2001; Vawter et al., 2001). Another protein whose expression is agreed to change is glutamic acid decarboxylase 1-67 (GAD67), although there is dispute about the direction of the change. Expression is reduced in neuropathological studies (Knable et al., 2001; Volk et al., 2000), but increased (Hakak et al., 2001) or decreased (Mirnics et al., 2000) in gene expression studies. Decreases in GAD67, like GFAP, were not conﬁned to schizophrenia (Knable et al., 2001). One gene expression study showed that there was a subgroup of genes that were downregulated in all schizophrenia brains: a group of genes that can all be plausibly linked to the synapse (Mirnics et al., 2000) This led to the interesting hypothesis that schizophrenia may be a disease of the synapse in which synaptic relations are altered by diVerential pruning during development (Mirnics et al.,

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2001a). Like many ‘‘neurodevelopmental’’ hypotheses, including the vitamin D hypothesis, it will be diYcult to move from this heuristic statement to changes in speciﬁc developmental and genetic pathways. The hypothesis is also weakened by the fact that individual persons with schizophrenia had diVerent combinations of the synaptic genes that were downregulated—all had reductions among the subgroup of genes but there was considerable variability at the single gene level (Mirnics et al., 2000). This may reﬂect the polygenic nature of the disease. It may also simply reﬂect the underlying variability in the gene expression microarray technique and the dangers inherent in trying to make post-hoc analyses from large datasets. From the focus of review, and notwithstanding the lack of replication among the studies noted above, it is interesting to note downregulated genes that can be linked directly to neurodevelopment: RGS4, an inhibitor of G-protein-coupled receptor signaling (Mirnics et al., 2001b), which maps to 1q21-22, a Sz susceptibility locus; forkhead and PUO domain transcription factors ( Vawter et al., 2001); and reelin, glucocorticoid receptor, and Nurr transcription factor (Impagnatiello et al., 1998; Knable et al., 2001). Making a post-hoc hypothesis of schizophrenia development based on these observations is as subject to selection bias as the synaptic hypothesis discussed earlier. Given the pleiotropic nature of gene function and the time- and age-dependent ﬂuctuations in gene expression, many expected ‘‘developmental’’ genes may not be involved and many ‘‘nondevelopmental’’ genes may be crucially involved in the development and activity of neural circuits giving rise to schizophrenia.

B. Candidate Gene Studies Several publications have described a linkage to genes in putative ‘‘schizophrenia-related’’ regions of the genome. These studies describe linkage to speciﬁc genes, mostly in regions of the genome previously showing linkage to schizophrenia [neuregulin 1, dysbindin, G72, d-amino acid oxidase, regulator of G-protein signaling-4 (RGS4), catechol-O-methyl transferase (COMT), proline dehydrogenase (PRODH), and calcineurin], many of these ﬁndings have been replicated, and several studies show changes in prepulse inhibition and other ‘‘schizophrenia-like’’ behaviors in mice with deletions or mutations of these genes. Thus, unlike most of the gene array candidates, there is stronger evidence for linkage of these candidates to schizophrenia. Signiﬁcantly, like the gene array candidates, many of these genes can be linked to the synapse (Harrison and Owen, 2003), although a synaptic function is not exclusive for any of them. For example, some of them are known to be important in neurodevelopment. An area of the genome of intense interest in schizophrenia genetics is on the long arm of chromosome 22 because microdeletions in the region 22q11 are

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associated with a high rate of schizophrenia and velo-cardio-facial syndrome (reviewed by Bassett et al., 2002). There are many genes in this region and one (TBX1) is now strongly implicated in the velo-cardio-facial syndrome (Merscher et al., 2001). The search for schizophrenia-related genes in this region has identiﬁed PRODH and COMT as strong candidates. Mice with PRODH deletions have disrupted prepulse inhibition (Gogos et al., 1999) and there is signiﬁcant linkage of PRODH polymorphisms with schizophrenia ( Jacquet et al., 2002; Liu et al., 2002), although two studies have failed to replicate this linkage (Fan et al., 2003; Williams et al., 2003a). There is signiﬁcant linkage of COMT polymorphisms with schizophrenia (Bray et al., 2003; Egan et al., 2001; Kremer et al., 2003; Park et al., 2002; Shifman et al., 2002; Wonodi et al., 2003). COMT polymorphisms are also associated with panic disorder ( Woo et al., 2002), obsessive-compulsive disorder (Karayiorgou et al., 1997), and cognitive function in healthy females and in schizophrenia (Bilder et al., 2002; Tsai et al., 2003). COMT polymorphisms inﬂuence dopamine regulation in the human cortex (Akil et al., 2003), and COMT mRNA is altered in the prefrontal cortex in schizophrenia (Matsumoto et al., 2003). RGS4 was identiﬁed as a candidate in a gene expression study (Mirnics et al., 2001b) and is located in a chromosomal region linked previously to schizophrenia (Brzustowicz et al., 2000). Association and linkage analyses identiﬁed speciﬁc polymorphisms in RGS4 in some schizophrenia families (Chowdari et al., 2002). Among its other roles, RGS4 is involved in development ( Wu et al., 2000) and is expressed in speciﬁc layers of the cortex and hippocampus in the embryo (Ingi and Aoki, 2002). Mice with forebrain-speciﬁc calcineurin deletions have disrupted working memory (Zeng et al., 2001). There are also several behaviors that have homologues in schizophrenia, including hyperactivity, decreased social interaction, impaired prepulse inhibition, and impaired latent inhibition learning (Miyakawa et al., 2003). Despite the hyperactivity the animals did not show any changes in forebrain dopamine levels (Miyakawa et al., 2003), although they showed enhanced sensitivity to the locomotor stimulatory eVects of the NMDA agonist MK801 (Miyakawa et al., 2003). These results are consistent with models of schizophrenia implicating glutaminergic dysfunction (Coyle et al., 2002; Tsai and Coyle, 2002). This animal model of schizophrenia is of further interest because the expression of 10 genes related to calcineurin function was altered in microarray analysis of the forebrain in schizophrenia (Hakak et al., 2001; Miyakawa et al., 2003) and calcineurin is involved in presynaptic function, a current focus of molecular models of schizophrenia (Hakak et al., 2001; Harrison and Owen, 2003; Miyakawa et al., 2003). The calcineurin subunit is located at chromosome 8p21.3, a region linked previously with schizophrenia and there is signiﬁcant association of this subunit with schizophrenia (Gerber et al., 2003). Of interest to neurodevelopment, calcineurin signaling is required for neurotrophin-induced

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axonal outgrowth (Graef et al., 2003). High levels of calcineurin expression can also induce neuronal apoptosis (Asai et al., 1999). Another candidate gene involved in neurodevelopment is neuregulin, also located on the short arm of chromosome 8 at 8p21. Three genetic association studies have identiﬁed a neuregulin haplotype associated signiﬁcantly with schizophrenia in populations from Iceland, Scotland, and Wales (Stefansson et al., 2002; Williams et al., 2003b). The neuregulins comprise four genes with many isoforms that signal via the ErbB family of receptors (reviewed by Falls 2003). They are especially important for glial cell diVerentiation (e.g., Schmid et al., 2003). Another member of the ErbB family of ligands is the epidermal growth factor. Postnatal treatment of mice with EGF leads to disrupted prepulse inhibition, reduced motor behavior, and reduced social behavior in adulthood (Futamura et al., 2003). These changes are also suggestive of a role for EGF dysfunction in the neurodevelopment of schizophrenia (Futamura et al., 2003). Chromosome 6p24-21 is a region with linkage to schizophrenia (Lewis et al., 2003; Straub et al., 2002b). Genotyping with a map of single nucleotide polymorphisms in this region revealed a gene for dysbindin (Straub et al., 2002a). Dysbindin is a recently discovered protein, associated with dystrophin in muscle, that is expressed in synaptic terminals in the cerebellum and hippocampus (Benson et al., 2001). The linkage of dysbindin with schizophrenia was replicated once (Schwab et al., 2003) but failed replication in another study (Morris et al., 2003). Chromosome 13q34 is a region with linkage to schizophrenia. Genotyping with a map of single nucleotide polymorphisms in this region revealed genes for G30 and G72, the latter of which is a carbohydrate-binding gene that interacts with d-amino acid oxidase (Chumakov et al., 2002). Similar genotype mapping revealed an association of G30 and G72 in bipolar disorder (Hattori et al., 2003), suggesting that these genes may be involved more generally in psychosis. In discussing these interesting candidate genes it should be realized that, except for COMT, there are no or little data yet that link mutations in speciﬁc genes with speciﬁc biological outcomes. Single nucleotide polymorphisms in themselves may not alter gene function. Similarly, with the exception of calcineurin, there are little data linking speciﬁc genes to behavioral or other functions relevant to schizophrenia in animal models. To make a strong case there must be functional studies for each of these candidates in which the molecular biology of speciﬁc mutations is explored and their impact on the developing and adult animal is revealed. Despite these shortcomings, it can be agreed that the ﬁeld of schizophrenia genetics is at a very exciting juncture with many candidate genes to explore. Signiﬁcantly, data converge suYciently to suggest a plausible molecular model involving many of these genes in the synapse and synaptic transmission (Hakak et al., 2001; Harrison and Owen, 2003). Like other models of

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schizophrenia, it is too soon to speculate on whether these synaptic changes are fundamental to the etiology of the disease or result from other causative factors acting during neurodevelopment.

IV. Gene–Environment Models of Schizophrenia

In its earlier manifestations, the neurodevelopmental hypothesis of schizophrenia posited that the structural brain abnormalities seen in schizophrenia at the onset of overt symptoms resulted from a ‘‘lesion’’ during brain development. This lesion was thought to involve speciﬁc brain regions and occurred at a speciﬁc point of development ( Weinberger, 1987; Weinberger and Marenco, 2003). In favor of this model is evidence that viral or other infections during pregnancy, birth complications, and so on are associated with an increased risk of schizophrenia in the child. Animal models of this lesion hypothesis are being examined, for example, lesion of the fetal hippocampus ( Lipska and Weinberger, 2003). As described earlier, there are other possible mechanisms within the heuristic of a ‘‘neurodevelopmental model’’ that relate more directly to continuous gene– environment interactions during brain development, which provide multiple modes of disturbance, perhaps more in keeping with the heterogeneity of the clinical presentation, multiplicity of candidate genes, and diverse epidemiological evidence. Disturbance of nutritional or hormonal signaling via gene transcription provides a variable that can act during diVerent developmental periods on multiple cellular systems. Nutrients can regulate gene transcription via epigenetic regulation. For example, prenatal folate epigenetically alters the expression of certain genes in the oVspring (Finnell et al., 2002) and maternal methyl supplementation alters epigenetic variation and methylation patterns in the oVspring (Cooney et al., 2002; WolV et al., 1998). Consideration of such models in schizophrenia is important because they can link genes and environment and can provide testable hypotheses at multiple levels of the disease process: cellular, developmental, and epidemiological.

A. The Vitamin A Hypothesis It is proposed that dysregulation of retinoid (vitamin A) signaling may be important in the etiology of schizophrenia (Goodman, 1998; LaMantia, 1999). Retinoic acid is a steroid-like hormone that is derived from vitamin A. Its receptor (RAR) is a member of the family of nuclear hormone receptors whose ligands include glucocorticoids, the thyroid hormone, and sex steroids. Retinoic acid

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aVects the transcription of the many genes that have a ‘‘retinoic acid response element’’ in the promoter sequence. All-trans retinal, a related molecule, interacts with a related receptor (RXR), which heterodimerizes with several of the nuclear steroid hormone receptor family, including RAR and VDR (Barsony and Prufer, 2002; Schrader and Carlberg, 1994). Retinoic acid is essential as a paracrine and autocrine regulator that is important for the formation of the forebrain during embryonic development (LaMantia, 1999). Forebrain development is dependent on retinoic acid signaling, starting from induction by neural crest-derived cells that settle in the nasal epithelium (LaMantia, 1999) and gene mutations that prevent forebrain development disrupt this early induction of forebrain by retinoic acid. Retinoic acid has many other functions in regulating the proliferation and diVerentiation of speciﬁc cell types during later periods of brain development (Sockanathan and Jessell, 1998; Whitesides et al., 1998). Vitamin A is a required nutrient and must be available for the developing fetus from the maternal diet via her blood supply. Retinoid signaling is an interesting candidate because there are many retinoid-regulated genes in chromosomal regions associated with schizophrenia through genetic linkage and association studies (reviewed in Goodman 1998). This may be explained partly by the coincidental overlap of many retinoidregulated genes with many schizophrenia-related chromosomal regions, but the hypothesis is worth exploring because the retinoids can link an environmental factor with developmental gene regulation. Retinoid toxicity can produce several signs of schizophrenia, including enlarged ventricles, thought disorder, mental deﬁcit, and minor physical anomalies (reviewed in Goodman 1998). These signs suggest that excess vitamin A may be equivalent for brain development as are deﬁcits in vitamin D (see later). Vitamin A is a major regulator of gene transcription via the RAR, which regulates such developmental genes as the immediate early genes c-fos and c-jun, as well as growth factors/cytokines such as ciliary neurotrophic factor (CNTF) and leukaemia inhibitory factor (LIF). Vitamin A also regulates expression of the neuroﬁlament, amyloid precursor protein, MAP2, and GAP43, as well as several neurotransmitters, neurotransmitter receptors, and enzymes (reviewed in Goodman, 1998). The dopamine D2 receptor promoter region has a functional polymorphism signiﬁcantly associated with schizophrenia (Arinami et al., 1997) and contains a RARE/RXRE DNA motif that regulates dopamine expression (Samad et al., 1997) and retinoid receptor knockout mice show locomotor disturbances and reduction in the expression of dopamine receptors (Krezel et al., 1998). Among the numerous genes regulated by the RAR are a signiﬁcant number of neurotransmitters, their receptors, and their metabolic enzymes implicated in schizophrenia, including dopamine, dopamine receptors, serotonin, glutamate receptors, tyrosine– hydroxylase and dopamine -hydroxylase, nicotinic acid receptors, NMDA

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receptors, and choline-O-acetyltransferase (Goodman, 1998). Vitamin A toxicity could thus result in stimulating these neurotransmitter systems.

B. The Vitamin D Hypothesis Most of the attention addressing the biology of vitamin D has focused on its role in calcium homeostasis and subsequently its importance in bone formation and osteoporosis. More recently, cell biological studies have implicated vitamin D more generally with cell growth and diVerentiation as well as with immunomodulatory actions (Casteels et al., 1995). Additionally, evidence suggests a role for vitamin D in the developing (Eyles et al., 2003) and adult brain (Garcion et al., 2002). It is proposed that the risks of some mental disorders, such as schizophrenia and multiple sclerosis, may be increased by early vitamin D deﬁciency (McGrath, 1999, 2001). Our hypothesis is that low vitamin D during development is a risk-modifying factor for schizophrenia ( McGrath, 1999). This hypothesis is derived from epidemiological evidence that brings together several puzzling observations, each of which can be plausibly linked to a low vitamin D status of the mothers during gestation. First, there is an increased risk of schizophrenia for those born in late winter and early spring in those whose ﬁnal gestation was during the winter months (Torrey et al., 1997). This seasonality of schizophrenia births was ﬁrst linked with seasonal changes in vitamin D status in the early 1950s (Pile, 1951). Vitamin D production is strongly and consistently associated with the duration of the photoperiod, which is inﬂuenced by latitude and season (Holick, 1995; Webb et al., 1988). The implication is that low levels of sunlight in winter raise the risk of low serum vitamin D levels during the winter months, especially in regions of low winter sunlight. Low serum vitamin D levels are recorded in these regions during winter (Holick, 1995). Seasonality of birth is also seen in Australia, even in Queensland, a region of relatively high levels of winter light (McGrath et al., 1995). Serum vitamin D levels still vary with the seasons in this region, with signiﬁcant numbers of people with hypovitaminosis D (McGrath et al., 2001b). Fetal vitamin D requirements increase during pregnancy (Delvin et al., 1985), and maternal vitamin D levels fall during the third trimester, especially if this occurs during winter (MacLennan et al., 1980). Hypovitaminosis D is not uncommon, even in developed countries ( Vieth and Carter, 2001). For example, it has been reported in the United States that 12% of women of childbearing age are vitamin D deﬁcient (Looker and Gunter, 1998). Women who are veiled or with dark skin have an increased risk of hypovitaminosis D, even in sunny climates such as Australia (Grover and Morley, 2001). The optimal level of vitamin D required for pregnancy is not known, but prenatal development could be aVected by low levels of vitamin D.

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At the equator, where there is no winter season, there is seasonality of the risk for schizophrenia, with an increased risk for those born 3 months following the wet season (de Messias et al., 2001), the period during which there is more cloud cover and would have less ultraviolet radiation falling on exposed skin. Further indirect evidence is demonstrated by the increased risks of schizophrenia in phase with ﬂuctuations in measured insolation around the time of birth both in the Netherlands, a region of low overall insolation, and in Queensland, Australia, a region of relatively high insolation (McGrath et al., 2001b). Finally, in a pilot study based on banked maternal sera, the risk of schizophrenia was increased ( p ¼ 0.07) in those with very low levels of 25 hydroxyvitamin D (McGrath et al., 2003). A second series of studies providing indirect support for the vitamin D hypothesis indicate that there is a greatly increased risk of schizophrenia for the children of dark-skinned migrants who move to cold climates. Studies from the United Kingdom show a 7- to 10-fold increased incidence of schizophrenia in Afro-Caribbean migrants (Harrison, 1990). More recently, studies from the Netherlands (Selten et al., 2001), Denmark (Cantor-Graae et al., 2003), and Sweden (Zolkowska et al., 2001) have shown an increased risk of schizophrenia in migrants, especially those with dark skin. These studies support the vitamin D hypothesis because dark-skinned migrants may be at a higher risk of hypovitaminosis D because of the reduced ability of dark skin to manufacture vitamin D and because of reduced exposure to sunlight through wearing more clothing and staying indoors to avoid the unfamiliar cold climate (Holick, 1995). Consequently, these migrant mothers have a higher risk of low vitamin D during pregnancy and their oVspring a higher risk for schizophrenia. The risk of schizophrenia is also raised in ﬁrst-generation migrants (Cantor-Graae et al., 2003), thus suggesting that exposure to hypovitaminosis D after migration and before the onset of illness may also increase the risk of schizophrenia in susceptible individuals. A third series of studies revealed an increased risk of schizophrenia for those living in urban compared to those in nonurban areas. The increased risk for this factor is 30–35% (Marcelis et al., 1998; Pedersen and Mortensen, 2001a). In support of the vitamin D hypothesis is an increased risk of vitamin D deﬁciency in the city compared to the country, thought to be a consequence of reduced exposure to sunlight in built environments (Holick, 1995). Thus the increased risk of schizophrenia is a consequence of the maternal risk of hypovitmainosis D in the city. The increased risk associated with an urban birth also extends into urban living during childhood where there is a dose-eVect of urban living on the risk of subsequent schizophrenia (Pedersen and Mortensen, 2001b). This is consistent with an extended time window for the eVect of low vitamin D on the risk of schizophrenia. It is noted that other plausible explanations may explain the aforementioned observations. The vitamin D hypothesis is not exclusive in this. For example, it was proposed that schizophrenia may result from early life exposure to infectious

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agents, either as a direct eVect on brain development or indirectly via cytokine activation pathways (Buka et al., 2001; Yolken and Torrey, 1995). Early life exposure to infectious agents may also underpin key epidemiological features of schizophrenia such as season of birth, the urban–rural gradient, and the increased risk in second-generation migrants. Importantly, these hypotheses advance science by directing research along particular paths. In the case of the vitamin D hypothesis, this has led us to investigate the biological plausibility of vitamin D as a factor that can regulate brain development, surely a prerequisite for its involvement in schizophrenia. We have investigated this biological plausibility in several ways: (1) by studying the eVect of in utero vitamin D deﬁciency on the neonatal rat brain, (2) by investigating the eVect of developmental vitamin D deﬁciency on the adult rat brain and on adult behavior, (3) by investigating the eVect of vitamin D on cultured neurons from rat embryo, and (4) by investigating the timing of vitamin D receptor expression in embryonic brain. Our data provide strong evidence that vitamin D contributes signiﬁcantly to brain development and that vitamin D deﬁciency during development can have long-term eVects on the adult brain and may aVect the risk of various disorders (Garcion et al., 2002; McGrath, 2001). 1. Biology of Vitamin D Vitamin D3 is a circulating hormone produced by the action of sunlight on the skin; ultraviolet B radiation acts on a cholesterol metabolite in the epidermis to produce previtamin D. Hydroxylation in the liver and then in the kidney creates the most active form of the vitamin, 1,25-dihydroxyvitamin D3 (vitamin D3). Circulating vitamin D3 stimulates calcium uptake through the gut epithelium and thus helps regulate calcium metabolism. Vitamin D3 may also be available in tissues independently of circulating levels of the hormone. In the brain, for example, both the hydroxylating enzymes (vitamin D3 25-hydroxylase and 25-hydroxyvitamin D31-hydroxylase) and the inactivating enzyme (vitamin D3 24-hydroxylase) are present. This suggests that vitamin D3 could act locally as an autocrine or paracrine factor (Garcion et al., 2002; McGrath et al., 2001a; Miller and Portale, 2000). Vitamin D3 is a nuclear transcription regulator acting via a nuclear hormone receptor. The vitamin D receptor (VDR) is present in the brain (Musiol et al., 1992; Prufer et al., 1999; Stumpf and O’Brien, 1987; Stumpf et al., 1982), including at least the hippocampus in humans (Sutherland et al., 1992). The VDR is present in the developing rat brain ( Veenstra et al., 1998) and, given its role as a nuclear transcription factor, may aVect brain development, a hypothesis we have been investigating vigorously. 2. Vitamin D and the Brain Accumulating evidence shows that vitamin D can act on the brain, in adults as well as during development. Early studies using autoradiographic techniques demonstrated vitamin D binding in the rat brain and spinal cord (Stumpf and

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O’Brien, 1987; Stumpf et al., 1982). More recently, immunohistochemical techniques in the rat have revealed vitamin D receptors in the nucleus and cytoplasm of cells in many brain regions, including the neocortex, hippocampus, thalamus, piriform cortex, hypothalamus, amygdala, and cerebellum (Prufer et al., 1999). In the adult rat hippocampus, vitamin D receptors can be found on both neurons and glia (Langub et al., 2001), and vitamin D treatment may be neuroprotective in vivo (Landﬁeld and Cadwallader-Neal, 1998) and in vitro (Brewer et al., 2001). In the hypothalamus, the vitamin D receptor is colocalized in some oxytocin neurons (Prufer and Jirikowski, 1997). Nerve growth factor synthesis is potently stimulated by vitamin D (Wion et al., 1991) and induces oligodendrocytes to express mRNA for NGF and p75 (Baas et al., 2000). Intraventricular injection of vitamin D in the adult rat induced a potent upregulation of NGF mRNA in the hippocampus and cortex (Saporito et al., 1993). Vitamin D upregulates the expression of glial cell line-derived neurotrophic factor (GDNF) in a glioma cell line (Naveilhan et al., 1996) and stimulates its release from these cells ( Verity et al., 1999). It upregulates TGF2 mRNA expression in neuroblastoma cells ( Veenstra et al., 1997) and NT3 and NT4 mRNA in astrocytes (Neveu et al., 1994a). The molecular and cellular eVects of vitamin D on neural tissues have been reviewed (Garcion et al., 2002). Vitamin D upregulates mRNAs for a variety of growth factors and cytokines in astrocytes and glioma cells: NGF (Neveu et al., 1994b), neurotrophin 3 (Neveu et al., 1994a), colony stimulating factor 1 (CSF-1) (Zhu et al., 2002), macrophage colony stimulating factor (M-CSF), and LIF (Furman et al., 1996). Administered intraperitoneally it increases GDNF expression in the striatum (Sanchez et al., 2002). In adult mouse brain it upregulates TGF and IL-4 expression. Vitamin D is neuroprotective ( Wang et al., 2000, 2001), and in various neural tissues, vitamin D increases glutathione levels, -glutamyl transpeptidase, and parvalbumin levels; reduces neurotoxicity resulting from ischemia, diabetic neuropathy, and 6-hydroxydopamine; and reduces inﬂammation and the pathophysiology associated with experimental autoimmune encephalopathy (reviewed in Garcion et al., 2002). Vitamin D deﬁciency in adult rats signiﬁcantly increases dopamine, DOPAC, and noradrenaline content in the cortex and dopamine content in the hypothalamus (Baksi and Hughes, 1982), although these changes may be due to reduced calcium levels as a result of vitamin D deﬁciency (Baksi and Hughes, 1982). 3. Vitamin D and Brain Development From the foregoing it is clear that vitamin D has signiﬁcant actions in the nervous system and could potentially act during brain development. The vitamin D receptor is present in the embryonic rat brain ( Veenstra et al., 1998), and its protein and mRNA expression increases dramatically from embryonic day 17 (Burkert et al., 2003). In the normal brain, the increase in expression of the VDR from embryonic day 17 onward is paralleled by a declining rate of mitosis

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(Burkert et al., 2003). When hippocampal neurons from brains of embryonic day 18 are grown in vitro, vitamin D promoted diVerentiation and inhibited mitosis and induced the expression of NGF (Brown et al., 2003). Additionally, vitamin D has known actions on cellular pathways aVecting diVerentiation, redox pathways, and inducible nitric oxide synthase (Garcion et al., 2002). These multiple cellular pathways provide plausible mechanisms whereby reductions in vitamin D levels could aVect the developing brain. What are the consequences of vitamin D deﬁciency on brain development? This was assessed in neonatal rats whose mothers were made vitamin D deﬁcient by eliminating vitamin D from the diet and eliminating ultraviolet B radiation from the lighting in the animal holding room. The eVects on the brains of the oVspring were dramatic (Eyles et al., 2003). Vitamin D deﬁciency changed the size and shape of the neonatal brain, altered growth factor expression, and altered cell proliferation. After vitamin D deﬁciency the brains of the neonatal rats were 30% longer and were larger in overall volume, with 200% larger lateral ventricles and a thinner cortex (Eyles et al., 2003). Vitamin D deﬁciency increased the number of mitotic cells in the dentate gyrus, hypothalamus, and basal forebrain, without aVecting cell density or apoptosis (Eyles et al., 2003). This suggests that the higher rate of mitosis in vitamin D-depleted neonatal rats represents a ‘‘less mature’’ brain in which normal development is restrained by the lack of vitamin D signaling. Finally, vitamin D3 deﬁciency reduced the levels of expression of NGF by 17%, GDNF by 25%, and p75, the low-aYnity neurotrophin receptor, by 30% (Eyles et al., 2003). These observations conﬁrm that vitamin D3 deﬁciency can have very large eVects on the developing brain, some of which, like enlarged ventricles (Lawrie and Abukmeil, 1998) and cortical thinning (Selemon et al., 1995), are associated with schizophrenia. Assessed by its eVects on many cell types, vitamin D is ‘‘antiproliferative,’’ driving cells out of the cell cycle into diVerentiation and apoptosis (Baudet et al., 1998; Mathiasen et al., 1999; Menard et al., 1995; O’Connell et al., 1997; Vandewalle et al., 1995; Weinreich et al., 1996). Vitamin D downregulates the expression of cyclins, which are proteins that govern the cell cycle (Kawa et al., 1997). Our observations are consistent with these same actions occurring in brain development. Removal of the antiproliferative vitamin D increased cell proliferation, reduced diVerentiating signals (NGF and GDNF), and reduced apoptotic signals (via reduced expression of p75) (Eyles et al., 2003). The vitamin D hypothesis of schizophrenia posits that altered vitamin D signaling in brain development will have long-term consequences on brain function by altering the trajectory of brain development. We have shown that vitamin D deﬁciency in utero alters the brain at birth, but would this have long-term consequences for brain development? Evidence shows that it does. Animals that developed in conditions of vitamin D deﬁciency had altered brains as adults even when later development occurred in conditions of normal

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vitamin D availability (unpublished observations). Animals depleted of vitamin D in utero but restored to vitamin D-suYcient diets and lighting at birth or at weaning still had brains with larger ventricles when examined at 10 weeks of age. As well as this gross morphological diVerence, more subtle cytoarchitectural diVerences were observed with increased cell densities in the cortex and hippocampus. There was a reduced expression of mRNA for several synaptic and cytoskeletal genes and reduced levels of expression of NGF and GDNF (unpublished observations). Vitamin D deﬁciency in utero alters brain development and leaves enduring changes in the adult brain but does it lead to altered behaviors consistent with schizophrenia? There is no evidence yet that it does. Animal models of schizophrenia emphasize reduced sensorimotor gating as a measurable behavior that can be observed in persons with schizophrenia and in animals. This is usually measured as reduced prepulse inhibition of the acoustic startle response (BraV et al., 2001; Swerdlow et al., 2000). Reduced prepulse inhibition is observed in rats whose dopamine and other neurotransmitter systems are altered in ways consistent with changes seen in the brain in schizophrenia. Reduced prepulse inhibition is seen in other developmental models of schizophrenia (Lipska et al., 1995) and in some genetic models whose gene alterations mimic some of those in candidate schizophrenia susceptibility genes (Miyakawa et al., 2003). When animals are deprived of vitamin D during development, they showed no reductions in prepulse inhibition of acoustic startle (unpublished observations). These observations indicate that in itself, developmental vitamin D deﬁciency may not be suYcient to alter adult behavior. This is consistent with a gene–environment interaction for the manifestation of schizophrenia such that vitamin D deﬁciency during development acts in concert with a susceptible genetic background. This would be expected when comparing the prevalence of vitamin D deﬁciency in the population (over 10%) with the lower prevalence of schizophrenia (1%). How might developmental vitamin D deﬁciency interact with genetic susceptibility to manifest as schizophrenia? To answer this we need to understand a little about the biology of vitamin D and its actions via the vitamin D receptor. 4. Vitamin D and Gene Transcription Vitamin D aVects gene transcription via a nuclear hormone receptor. VDR is a member of the nuclear receptor family that includes receptors for ligands such as retinoic acid, glucocorticoids, thyroid hormone, and the sex hormones. The VDR regulates transcription by forming heterodimers with other nuclear hormone receptors, most principally with the retinoid X receptor whose cognate ligand is 11-trans retinol, a vitamin A derivative (Barsony and Prufer, 2002). VDR and the thryroid hormone receptor (TXR) compete for RXR such that formation of the TXR–RXR heterodimer represses gene transcription by VDR by sequestering RXR (Raval-Pandya et al., 1998; Schrader and Carlberg, 1994).

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The VDR regulates gene transcription via a vitamin D response element in the promotor region of certain genes, such that it may either up- or downregulate gene transcription and its action can be independent on binding of vitamin D to the VDR (Barsony and Prufer, 2002). The full spectrum of genes regulated directly by vitamin D is not yet established, but they include many genes acting on the nervous system (Garcion et al., 2002). By regulating fundamental genes involved in cell cycle control, diVerentiation, and apoptosis, the cellular eVects of vitamin D are very broad and the consequences of altered vitamin D signaling are diYcult to predict. The danger in this is that it potentially makes the vitamin D hypothesis simply another heuristic explanation without predictive power. The challenge for the future is to explore further the biology of the candidate schizophrenia susceptibility genes and their relationships with cellular pathways regulated by vitamin D and the VDR.

C. A Nuclear Hormone Hypothesis This model of early life hormonal or nutritional regulation of gene transcription is not unique to vitamin D and vitamin A. As indicated earlier, the nuclear hormone receptor family mediates the gene transcription actions of hormones (glucocorticoids, sex steroids, and thyroid hormone) and nutrients (vitamin D, vitamin A, and fatty acids). Neonatal disruptions to any of these pathways can alter the sexual behavior of adult rats (Csaba and Gaal, 1997; Csaba and Karabelyos, 2001; Csaba et al., 1995; Karabelyos and Csaba, 1997; Mirzahosseini et al., 1996). It is thus possible that early life signaling through any of these pathways may have signiﬁcance for schizophrenia. Weinberger (1987) proposed that prenatal stress is a risk factor for schizophrenia. Experimental evidence from animal models suggests that elevated glucocorticoid levels in the fetus alter maturational aspects of dopaminergic neural systems ( Weinberger, 1987; Weinstock, 2001), whereas epidemiological evidence indicates that the oVspring of mothers who were exposed to a stressor during the later part of gestation are at an increased risk of developing schizophrenia later in adult life (Koenig et al., 2002). Whereas there are many possible mechanistic pathways, such as sensitization of the hypothalamus–pituitary–adrenal axis to glucocorticoids, it is also possible that an early life disruption of glucocorticoids (both increased and decreased) could alter brain function permanently. It is well recognized that hypothyroidism during development disrupts brain formation severely, leading to severe cognitive deﬁcits in the adult (Anderson, 2001). Familial thyroid disease is associated with schizophrenia (DeLisi et al., 1991, 2000), and hypothyroidism alters the expression of molecules reported to be disrupted in the brains of patients with schizophrenia such as two proteins responsible for normal cortical lamination, reelin and Dab 1 (Alvarez-Dolado et al., 1999).

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Neonatal hypothyroidism also reduces expression of the neurotrophins, BDNF and NGF, and the neurotrophin receptor p75NTR (Alvarez-Dolado et al., 1994; Neveu and Arenas, 1996). Expression of these neurotrophin-signaling molecules is also reduced after developmental vitamin D deﬁciency (Eyles et al., 2003). As indicated earlier, the nuclear hormone receptors interact with each other, forming heterodimers with each other and competing for heterodimerization with the retinoid X receptor (Kraus and Wong, 2002). The binding of nuclear hormone receptors to promoter sequences in their regulated genes can vary depending on homodimerization and heterodimerization and the presence or absence of ligands (Kraus and Wong, 2002). The outcome may be activation or repression of gene transcription varying with the presence or absence of the cognate ligand, and these actions are mediated in part by histone-modifying enzymes such as histone methyltransferase and acetylase (Kraus and Wong, 2002). The complexity of this regulation is still being recognized, but it is clear that it can be considered ‘‘multiplex,’’ with parallel regulation of transcription of many downstream genes. It is likely therefore that any alteration in signaling via one of these nuclear hormone receptors will aVect the balance of signaling via the other hormone receptors. Given this complexity, there may not be a ‘‘vitamin D hypothesis,’’ a ‘‘vitamin A hypothesis,’’ or a ‘‘glucocorticoid hypothesis’’ in isolation of the others: schizophrenia may result from a more general disturbance in nuclear hormone signaling.

V. Conclusion

From the studies outlined in this review, there is a growing list of genes whose expression is altered in schizophrenia and genes with haplotypes associated with schizophrenia. This is a fruitful time for the schizophrenia research community— there are many leads to explore and many hypotheses to be tested. It is even possible to draw together gene expression data (Mirnics et al., 2001a) and some of the candidate genes (Harrison and Owen, 2003) into models that focus on the synapse as a major site of disruption in schizophrenia. Such models should be explored further. In the future they may lead to molecular and cellular models of the brain circuits aVected in schizophrenia and they have potential in identifying new targets for pharmacological intervention. Despite the increasing number of ‘‘schizophrenia susceptibility genes,’’ a genetic and cellular description of the etiology of schizophrenia is still a long way oV. There are not yet plausible genetic and molecular pathways that can explain how the adult brain circuits come about in schizophrenia. The challenge for any neurodevelopmental hypothesis is to link adult brain function with dysregulation of brain development due to speciﬁc genes and speciﬁc environments.

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We have made a case for vitamin D deﬁciency as a regulator of brain development and a risk factor for schizophrenia. As a contributor to the biological etiology of schizophrenia, it is attractive because it acts at the level of gene transcription and thus provides a mechanism for interactions between genetic susceptibility and the developmental environment. Vitamin D is also an attractive candidate because it acts at many places in the genome and thus provides a basis for the many chromosomal regions identiﬁed by genetic studies as regions of schizophrenia susceptibility. Vitamin D is also an attractive candidate because it appears to explain several key epidemiological features of schizophrenia. As a regulator of diVerentiation and gene transcription, vitamin D certainly provides a plausible candidate for gene–environment interactions with wide-ranging eVects on the development of the brain. There is still a lot of scope for exploration of this hypothesis. Vitamin D signaling through its nuclear receptor can aVect many genes and gene pathways and thus accommodates the polygenic nature of this disease with many genes of small eVect whose inﬂuence may be brought together via the ‘‘vitamin D hypothesis.’’ The possibility is raised that this hypothesis is one facet of a wider ‘‘nuclear hormone’’ hypothesis linking a broader set of environmental and genetic factors with the development of the brain and with schizophrenia.

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POSSIBLE CONTRIBUTIONS OF MYELIN AND OLIGODENDROCYTE DYSFUNCTION TO SCHIZOPHRENIA

Daniel G. Stewart and Kenneth L. Davis Mount Sinai School of Medicine Department of Psychiatry New York, New York 10029

I. Introduction II. The Changing Role of Glia III. Background on Oligodendroglia and Myelin A. Oligodendroglia Development B. Function of Oligodendrocytes and Myelin IV. Disconnectivity in Schizophrenia V. Imaging Evidence for White Matter Involvement in Schizophrenia A. Magnetic Transference Imaging B. DiVusion Tensor Imaging VI. Demyelinating Diseases and the Symptoms of Schizophrenia VII. Age-Related Changes in Normal Aging VIII. Expression of Myelin-Related Genes in Schizophrenia A. Myelin-Associated Glycoprotein B. 20 -30 Cyclonucleotide, 30 -Phosphodiesterase C. Myelin and Lymphocyte Protein D. ErbB3 E. Transferrin F. Gelsolin G. Linkage Studies at Myelin-Related Gene Regions IX. Direct Examinations of Myelin and Oligodendroglia in Schizophrenia X. Mechanistic Considerations XI. Summary and Future Directions References

I. Introduction

Multiple lines of evidence point to an involvement of white matter in schizophrenia. Microarray data, white matter imaging techniques, ultrastructural studies, quantitative stereologic analyses, and similarities between schizophrenia and other demyelinating conditions all point to possible oligodendroglial dysfunction and an associated structural and functional impairment in myelin that can be proposed as a possible contributing mechanism in the etiology of schizophrenia. Although evidence at this point is strong for a role for white matter in schizophrenia, the exact mechanism by which oligodendroglia and myelin become INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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dysfunctional is unknown, and the nature of the contributions of such dysfunction in the schizophrenic syndrome remains speculative. This chapter reviews accumulating data that argue for a serious consideration of white matter dysfunction in schizophrenia.

II. The Changing Role of Glia

It has been a widely accepted position that myelin surrounds neurons and provides insulation, allowing electrical conduction to occur over the nodes of Ranvier at speeds increased greatly from the conduction speeds of unmyelinated axons. Oligodendroglia have long been understood as the cells that produced myelin. Recently, however, the understanding of the role of both myelin and oligodendroglia has been expanded dramatically. Glial cells in the central nervous system (CNS) can be divided into two classes: macroglia and microglia. Macroglia can be further separated into astrocytes and oligodendrocytes, and microglia are considered to have a macrophage-like role in the CNS. Macroglial cells have been discovered to have a role in the integration of neuronal signals and to act as modulators of synaptic activity (Araque et al., 1999; Haydon, 2001; LoTurco, 2000; Parpura and Haydon, 2000; Smith, 1994). Although much of the early work in understanding the role of glial cells as neuronal modulators was applied to the study of astrocytes, oligodendrocytes have begun to be considered in a similar light. Oliodendrocytes have been discovered to have a role in regulating the microenvironment around neurons (Ludwin, 1997) and playing a trophic/survival role for neurons (Cheng et al., 1998), as well as contributing to neuronal development itself (Sanchez et al., 2000). It appears to be the case that astrocytes, oligodendrocytes, and neurons engage in a symbiotic dance at the area surrounding the synapse and that each contributes to the function and stability of the other. For example, astrocytes have been found to be wrapped about synaptic terminals (Grosche et al., 1999; Ventura and Harris, 1999) and to modulate neurons through calcium-linked glutamate release. Oligodendrocytes have proven to be exquisitively sensitive to ﬂuctuations in glutamate concentrations surrounding neurons and have been shown to have a role in reuptake and thus in the management of glutamate levels to which neurons are exposed (Levy et al., 1998; Zerangue and Kavanaugh, 1996). Neurons have been shown to give direct synaptic output via glutamate to oligodendrocyte precursors (Bergles et al., 2000). Thus, each of these CNS components exists in a graceful and meticulous interdependence with the others, and the dysfunction of one of these is likely to have a signiﬁcant impact on the others. The possible contributions of astrocytes to psychiatric illness have been reviewed elsewhere (Araque et al., 1999; Cotter et al., 2001; Gallo and Ghiani, 2000; Haydon,

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2001). As explained later, oligodendroglia and myelin have been implicated in the schizophrenic syndrome and are the focus of the remainder of this review. III. Background on Oligodendroglia and Myelin

Oligodendrocytes have been divided into categories on a variety of criteria: the number of processes (Baumann and Pham-Dinh, 2001), morphology, size and thickness of their myelin sheaths (Butt et al., 1995), cytoplasmic or nuclear variations, or on response to staining techniques (Mori and Leblond, 1970). At present, however, it seems that the only clear distinction that can be made is that there are at least two types of oligodendrocytes: those that make myelin and those that have been named ‘‘satellite’’ oligodendrocytes (Baumann and Pham-Dinh, 2001), which are perineuronal and may perform regulatory functions with respect to neurons. A. Oligodendroglia Development Oligodendrocytes express a variety of markers as they progress from progenitor to mature cell, including nestin, proteolipid protein, platelet-derived growth factor receptor, monoclonal antibody A2B5 and 04, carbonic anhydrase II, and transferrin, to name a few (Baumann and Pham-Dinh, 2001). Of these, transferrin is thought to be relevant to the study of schizophrenia, as explained later in the section on the speciﬁc myelin-related genes found to be regulated abnormally in schizophrenia. Oligodendrocyte cells migrate through the CNS during their development (Small et al., 1987), and their ﬁnal number is controlled by selective apoptosis (Barres, et al., 1994). As oligodendrocytes develop, they respond to signals for diVerentiation including but not limited to platelet-derived growth factor (PDGF), neurotrophin-3 (NT-3), glial growth factor (GGF), and insulin-like growth factor I (IGF-I). PDGF is a survival factor for oligodendrocytes (Grinspan and Franceschini, 1995), synthesized in both neurons and astrocytes (Bogler et al., 1990; Mayer et al., 1993). Astrocytes are believed to synthesize NT-3—at least in the optic nerve of rats—and NT-3 has been shown to promote oligodendrocyte survival (Barres et al., 1992, 1994a) in vitro, as well as to promote the regeneration of oligodendrocytes and remyelination in a model of rat spinal cord (McTigue et al., 1998). GGF is a neuroregulin that is a survival factor for oligodendroglia (Canoll et al., 1999). When absent, oligodendrocytes fail to develop, at least in spinal cord tissue (Vartanian et al., 1999). Oligodendrocytes are also responsive to IGF-I. Receptors for IGF-I have been found on oligodendrocytes, and IGF-I has been shown to stimulate oligodendrocyte growth, induce oligodendrocyte maturation,

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regulate myelin gene expression, impact the amount of myelin produced by oligodendrocytes (McMorris and Dubois-Dalcq, 1988), and prevent oligodendrocyte apoptosis (Mason et al., 2000). Interestingly, in mice that have been altered genetically to overexpress IGF-I, there is an increase in both the percentage of myelinated axons and the thickness of myelin sheaths (Beck et al., 1995; Carson et al., 1993). Likewise, in IGF-I null mice, hypomyelination, decreased numbers of oligodendrocytes, and decreased numbers of neurons (in direct proportion to the decrease in oligodendrocytes) have been found (Cheng et al., 1998). This reliance on IGF-I for oligodendrocyte survival, and consequently neuronal survival, might be of particular importance to schizophrenia, where IGF-I has been implicated in genetic studies as related to psychosis (Niculescu et al., 2000), implicated indirectly in the seasonality of birth investigations (reviewed in Moises et al., 2002), and found to be altered in some cases by antipsychotic medications (Melkersson et al., 2001). It is clear that oligodendroglial development is a complicated process in which any number of subtle alterations in inﬂuencing factors might have relatively profound eVects on myelination and on oligodendroglial or neuronal survival. Numerous neurotransmitters relevant to schizophrenia have been found to impact the development of oligodendrocytes. Glutamate has been shown to have an active role in oligodendrocyte development and survival (Gallo et al., 1996). Oligodendrocytes have both AMPA and kainate receptors, and oligodendrocyte proliferation can be inhibited by non-NDMA glutamate receptor agonists in culture (Gallo et al., 1996), suggesting that glutamate has an inhibitory role in the proliferation of oligodendrocytes. Furthermore, glutamate has been shown to have an antimitotic eVect on oligodendrocytes in cerebellar slice cultures (Yuan et al., 1998) and, even more interesting, is the fact that these glutamate eVects seem to be AMPA speciﬁc, whereas astrocyte proliferation is not aVected under similar conditions (Yuan et al., 1998). GABAA receptors have been found on oligodendrocytes, but the role of these receptors remains to be described. Dopamine D3 receptors have been found on oligodendrocyte precursors located primarily in the corpus callosum but not on mature oligodendrocytes and appear to modulate the timing of oligodendrocyte maturation and myelination (Bongarzone et al., 1998). Dopamine D2 receptors have also been found on immature oligodendrocytes in the corpus callosum (Howard et al., 1998); however, their role has not been elaborated.

B. Function of Oligodendrocytes and Myelin Data suggest that myelin itself clusters sodium channels at the nodes of Ranvier during axonegenesis and has a role in the development, regulation, and maintenance of axons, as well as an inhibitory role in axonal growth and

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regeneration (Baumann and Pham-Dinh, 2001). Oligodendroglia have been found to have functional inﬂuences on neurons. Oligodendroglia have now been shown to produce signiﬁcant radial expansion in axons and to impact neuroﬁlament accumulation and spacing (Sanchez et al., 2000). In fact, the expansion and maturation of neurons contacted by oligodendroglia seem to occur largely before myelination has begun, at least in a subset of neurons that proceed to full maturation in knockout mice who have a deﬁciency in myelination, but not in oligodendroglial maturation (Sanchez et al., 1996). This oligodendroglial contact with neurons was apparently critical to radial axon growth (Sanchez et al., 1996). Conversely, axonal signals appear to promote oligodendroglial maturation and myelination (Barres and RaV, 1993; Hardy and Reynolds, 1993; Kidd et al., 1990; Valat et al., 1988), and neuron-to-oligodendrocyte signaling is considered to be responsible for regulating the number of oligodendrocytes necessary to myelinate a speciﬁc population of axons (Matute et al., 1997). Oligodendroglia development is regulated by direct cell-to-cell interactions (Wang et al., 1998), as well as by secreted factors (Barres et al., 1994), and it has been suggested that the development of the nervous system, its regeneration, and its synaptic plasticity are dependent on neuronal–glial interactions (Bahr and BonhoeVer, 1994; Keynes and Cook, 1995; Tessier-Lavigne and Goodman, 1996; Yang et al., 1999). Evidence for communication between hippocampal neurons and oligodendrite precursor cells has been discovered (Bergles et al., 2000). This communication is modulated by glutamate via AMPA receptors and generates inward currents in these immature oligodendrites, suggesting that rapid, excitatory signaling pathways exist and are related to oligodendrite functioning. Oligodendritic processes are in intimate contact with neurons at both pre- and postsynaptic structures (Gallo and Ghiani, 2000), suggesting a functional and intimate relationship between oligodendrites and neurons. Given the importance of oligodendroglia and myelin to the functioning of neurons, it seems reasonable to consider the possible contributions that glial dysfunction would have to a disease with such diVuse and often equivocal structural and functional impairments as schizophrenia.

IV. Disconnectivity in Schizophrenia

Schizophrenia has been described as a disconnectivity syndrome (Andreasen et al., 1998; Arnold, 1999; Bullmore et al., 1998; Davis et al., 2003; Friston, 1996; Friston and Frith, 1995; Gallhofer et al., 1999; Gruzelier, 1999; Harrison, 1999; Liddle, 1996; McClure et al., 1998; McGlashan and HoVman, 2000; Pearlson, 1999). In this conceptualization, more important than the function or dysfunction of any particular area of the brain, the connections between regions are seen as integral to understanding pathology. This is not to say, of course, that

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particular brain regions are not seen as paramount in the understanding of schizophrenia, but rather that the key to understanding the dysfunction of these critical areas may lie, at least partially, in understanding how the connections of these brain regions to each other may be impaired. Thus, the one-structure–onefunction model of neuroscientiﬁc understanding is reformulated as a neural network model where impairments in one area of the brain can have far-reaching consequences both in a wide radius and signiﬁcantly downstream from the original insult. The evidence for disconnectivity in schizophrenia rests in brain imaging studies, neurochemical studies, altered synaptic expression, and alterations in neuronal density. Imaging studies that have focused on prefrontal connectivity have revealed a disconnection in schizophrenia on fMRI and PET scanning (Andreasen et al., 1997; Frith and Dolan, 1996). By focusing on neuropsychological tasks (such as working memory), along with simultaneous functional imaging, schizophrenia patients have been shown to have at least a functional disconnectivity between various brain regions, including thalamocortical projections (Schlosser et al., 2003), as well as connections to and from the dorsolateral prefrontal cortex (DLPFC) and temporal cortex (Andreasen et al., 1998; Erkwoh et al., 1999; Frith et al., 1995; Lawrie et al., 2002; Manoach et al., 1999, 2000; Schlosser et al., 2003; Spence et al., 2000). Comparative deﬁcits were sometimes found in siblings (Callicott et al., 2003) and even preliminarily in oVspring (Keshavan et al., 2002) of schizophrenia patients as well. Although these ﬁndings are not universal, diVerences in methodology and technical precision might account for the inconsistent ﬁndings (for a review, see Manoach, 2003). Neurocytochemical evidence for altered connectivity proceeds from abnormalities both in synaptic protein expression (synaptophysin and synapsin 1) and in proteins expressed by neurons (GAP-43 and NAA). Synaptophysin is a synaptic protein present ubiquitously on presynaptic vesicles (Tcherepanov and Sokolov, 1997; Wiedenmann and Franke, 1985) and has become a marker of synaptic density (Eastwood et al., 1994, 1995; Hamos et al., 1989; Masliah et al., 1990; Wiedenmann and Franke, 1985; Williamson et al., 1991). Synapsin 1 (1A and 1B) is found in all types of nerve terminals ( Jahn and Sudhof, 1994), and deﬁcits in synapsin in knockout mice have been shown to lead to short-term plasticity deﬁcits similar to the auditory gating deﬁcit of schizophrenia (Rosahl et al., 1993). Levels of both synaptophysin and synapsin 1 have been found to be increased in the temporal cortex of schizophrenia patients below age 75 compared to controls and to be similar to controls in schizophrenia patients above 75 years old (Tcherepanov and Sokolov, 1997), suggesting that synaptic elimination may be abnormal in schizophrenia (Feinberg, 1982) or that pruning may be delayed (Tcherepanov and Sokolov, 1997). Alternatively, a survival/selection eVect or other abnormality may be operating to explain these abnormalities in synaptic mRNAs in schizophrenia (Tcherepanov and Sokolov, 1997). Importantly, correlations between levels of synaptic mRNAs, proteins, and synaptic density measured by

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electron microscopy have been found (Eastwood et al., 1994; Moore and Bernstein, 1989; Okada et al., 1994; Walaas et al., 1988), although these correlations have not yet been examined directly in schizophrenic brains. Interestingly, both normal (Gabriel et al., 1997) and decreased levels (Eastwood and Harrison, 1995) of synaptophysin have been reported in temporal regions in schizophrenia. Numerous other brain areas have also been associated with altered synaptic protein expression in schizophrenia. In the hippocampus, decreased levels of synaptophysin have been reported (Eastwood and Harrison, 1995, 1999), as have normal levels (Browning et al., 1993; Young et al., 1998). In this last investigation, examination of the granule cell layer of the dentate gyrus of the hippocampus has revealed a regionally speciﬁc increased expression of synaptophysin. In other regions, synaptophysin has been found to be decreased in the brains of schizophrenics in the left thalamus (Landen et al., 1999) and prefrontal cortex (PFC) (Glantz and Lewis, 1997; Karson et al., 1999), and evidence for increases in synaptophysin has been found in the anterior cingulate gyrus (Gabriel et al., 1997). Discrepancies in levels of synaptic proteins within and between brain regions highlight that these synaptic abnormalities may be region speciﬁc (Tcherepanov and Sokolov, 1997) and support the notion that neuronal disconnectivity in schizophrenia is somehow related to altered synaptic protein expression in these regions. High levels of GAP-43 are expressed in developing neurons (PerroneBizzozero et al., 1996), but these levels decrease sharply in neurons in which mature synaptic connections have been established ( Jacobson et al., 1986), except in hippocampal and association cortices, where high levels persist in adulthood in human brains. It has been suggested that GAP-43 expression in these regions in humans may be associated with synaptic plasticity (Akers et al., 1985; Lovinger et al., 1985) and may mark circuits in which novel information acquisition, processing, or storage occurs (Perrone-Bizzozero et al., 1996). Expression of GAP-43 is increased preferentially in the brains of schizophrenics in visual association and frontal cortices (Perrone-Bizzozero et al., 1996), suggesting a failure of normal regulatory cues within the cortex of schizophrenia patients. Interestingly, as mentioned earlier, levels of synaptophysin are decreased selectively compared to controls in the same areas (Perrone-Bizzozero et al., 1996), although not universally found (Gabriel et al., 1997). N-Acetylaspartate (NAA) is considered a marker of neuronal integrity. Decreases in NAA have been described in schizophrenia (Auer et al., 2001; Bertolino et al., 1996, 1998a,b, 1999; Block et al., 2000; Braus et al., 2002; Callicott et al., 1998, 2000a,b; Cecil et al., 1999; Choe et al., 1994; Gimenez et al., 2003) in numerous brain regions, including the thalamus, DLPFC, hippocampus, temporal lobe, basal ganglia, and anterior cingulate. These NAA decreases are not universally found, however (Bartha et al., 1999; Delamillieure et al., 2000). Measures of NAA have been shown to be altered in schizophrenia patients who have impairments in procedural memory (Gimenez et al., 2003) and verbal learning (Hagino et al., 2002). Perhaps even more signiﬁcantly, lower prefrontal NAA

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concentrations have been correlated with more severe negative symptomatology in schizophrenia (Callicott et al., 2000a), and lower prefrontal NAA has also been shown to strongly predict D2 receptor availability in the striatum when examined in schizophrenic patients (Bertolino et al., 2000). Antipsychotic treatment may impact these NAA levels (Bertolino et al., 2001) but may not (Choe et al., 1996). Choline, often considered a marker of myelin or white matter, has been shown to fall oV more rapidly than NAA in the hippocampus of aging schizophrenia patients when compared to controls, suggesting that myelin may suVer more than neurons themselves in aging schizophrenia patients (Maier and Ron, 1996). Another marker of disconnectivity could lie in the signiﬁcantly higher neuronal density reported in a number of brain regions of schizophrenics (Casanova, 1997; Daviss and Lewis, 1995; Pakkenberg, 1993; Selemon et al., 1995, 1998; Selemon and Goldman-Rakic, 1999). Schizophrenics have been shown to have an increase in neuronal density in the PFC, occipital cortex (Selemon et al., 1995, 1998; Williams and Rakic, 1988), and temporal lobes (Pakkenberg, 1993), but interestingly, without a reduction in cell number. Although not demonstrated universally (Anderson et al., 1996), the brains of schizophrenics have been shown to have an increased cell density in deep white matter along with a maldistribution of neurons in deeper white matter PFC (Akbarian et al., 1996; Kirkpatrick et al., 1999). This increased neuronal density could be explained by any combination of several ﬁndings. Somal size has been found to correlate with dendritic arborization, and reduced somal sizes have been found in the cortex of schizophrenics (Rajkowska et al., 1998), which could have a role in the hypoconnectivity seen in schizophrenia (Akbarian et al., 1996). Dendritic spines have also been found to be reduced in schizophrenics (Roberts et al., 1996). Increased neuronal density with decreased arborization and fewer synaptic contacts would be expected to be associated with decreased cortical volume, a widely reported result (Andreasen et al., 1994; Brown et al., 1986; Lim et al., 1996; Pakkenberg, 1987; Selemon and Goldman-Rakic, 1999; Sullivan et al., 1996; Ward et al., 1996; Zipursky et al., 1992). Increased neuronal density, decreased dendritic processes, and abnormal functional activation found in schizophrenia suggest that the integrity of white matter tracts—the anatomical and functional connection between brain regions—is somehow compromised. Indeed, as newer methodologies and technologies have been applied to anatomical studies of white matter, abnormalities in the white matter of schizophrenic brains have begun to emerge.

V. Imaging Evidence for White Matter Involvement in Schizophrenia

Early imaging techniques failed to ﬁnd alterations in white matter structures; however, these techniques had particularly low resolving power for examining white matter. In more recent examinations, the brains of patients with

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schizophrenia have been found to have decreases in global white matter volumes (Cannon et al., 1998; Wright et al., 2000). Similarly, global white matter decreases have been described in comparisons between schizophrenia patients and unaVected siblings (Cannon et al., 1998). A meta-analysis of numerous studies that derived white matter volumes from measures of ventricular brain ratios has found a small reduction in the global white matter volume in schizophrenia (Wright et al., 2000). Regional changes in white matter have been revealed as well. Numerous studies have found reductions in the white matter of the PFC (Breier et al., 1992; Buchanan et al., 1998; Sanﬁlipo et al., 2000; Sigmundsson et al., 2001), and in two of these investigations, white matter PFC reductions were associated with greater negative symptoms (Sanﬁlipo et al., 2000; Sigmundsson et al., 2001). Given that the PFC ﬁgures centrally in so many conceptualizations of schizophrenia, white matter changes in this region are particularly important. Other more diVuse white matter hyperintensities have been described in other investigations (HulshoVPol et al., 2000; Rivkin et al., 2000; Sachdev and Brodaty, 1999) in schizophrenia patients and in patients with late-life psychosis (Lesser et al., 1991; Miller et al., 1986, 1989, 1991; Tonkonogy and Geller, 1999), although these ﬁndings are not universally found (Howard et al., 1995; Krull et al., 1991; Symonds et al., 1997). These ﬁndings are summarized in Table I. The relative paucity of white matter ﬁndings in schizophrenia using magnetic resonance imaging (MRI) could be due in part to the limitations of the method itself rather than the lack of abnormalities. Indeed, as reﬁnements have been made to MRI techniques, abnormalities in white matter have been revealed in schizophrenia.

TABLE I Summary of White Matter (WM) Structural Findings on MRI in Schizophrenia Global

" WM hyperintensities

Global WM volumes

# WM volume

Regional

Ø WM volume difference Ø prefrontal WM volume difference

Corpus callosum

# WM volume (temporal and frontal) # WM volume Neg. Sx # Volume

Persaud et al. (1997) (n ¼ 48) Sachdev et al. (1999) (n ¼ 25) Hulshoff et al. (2000) (n ¼ 9) Cannon et al. (1998) (n ¼ 75) Wright et al. (2000) (n ¼ 126) Lim et al. (1999) (n ¼ 10) Sanfilipo et al. (2000) (n ¼ 53) Buchanan et al. (1998) (n ¼ 18) post hoc Sigmundsson et al. (2001) (n ¼ 27) Sanfilipo et al. (2000) (n ¼ 13) Woodruff et al. (1997) (n ¼ 313 meta)

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A. Magnetic Transference Imaging Magnetic transfer imaging (MTI) is a technique that gives an indirect visualization of protons bound to macromolecular structures, such as myelin and cell membranes, to create a magnetization transfer ratio (MTR), a putative index of myelin (Foong et al., 2000a). The ﬁrst MTI study in schizophrenia, in which 25 schizophrenia patients and 30 controls were examined using a region of interest (ROI) approach, revealed a signiﬁcant group decrease in the myelin or axonal membrane integrity in both right and left temporal regions in schizophrenics (Foong et al., 2000a). Variables such as age, duration of symptoms, schizophrenic symptomatology, and soft neurological signs did not account for this reduction in MTR. In the left and right frontal regions of schizophrenics, a similar trend was seen, although these frontal ﬁndings failed to reach statistical signiﬁcance. In a separate voxel-by-voxel analysis, signiﬁcant MTR reductions were found in inferior frontal, middle frontal, inferior and middle temporal, and superior occipital gyri, particularly in frontal and temporal regions (Foong et al., 2001). MTR changes were again not related to other clinical variables of age, duration of illness, and current dose of antipsychotic medication. Decreased MTR was found to be related to negative symptom severity in left parietal and temporo-occipital areas, and when patients with high total negative symptom scores were analyzed separately, a further trend toward more diVuse cortical MTR reductions was observed. Overall, this decrease in MTR in cortical white matter supports the notion of compromised axonal or myelin integrity in schizophrenia (Foong et al., 2000a,b).

B. Diffusion Tensor Imaging Another technique that has been applied to schizophrenia is diVusion tensor imaging (DTI). This new type of MRI imaging measures the sum of the vectors of water diVusion within axons or myelin sheaths, and this sum represents the coherence of the structures within a given region, called anisotropy. Tissue that is aligned along a similar axis will have high anisotropy, whereas tissue with a less uniform orientation will have low anisotropy (Lim et al., 1999). This technique has been applied to the study of white matter structures, given the directional aspect of myelin wrapped around axons, and as would be suspected, axons and myelin aligned in the same plane have a higher anisotropy than structures found in multiple planes. The resulting anisotropy can be understood as a measure of the coherence of white matter in that region. Other information gathered from DTI is a measure of the diVusion of water molecules within normal and abnormal white matter, with damaged white matter having a greater diVusivity than normal (Rugg-Gunn et al., 2001; Ulug et al., 1999). This measure helps describe

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the structural integrity of white matter (Taber et al., 2002). DTI is also the only technique for the in vivo investigation of white matter tracts—tractography— although true three-dimensional representations of connections between regions are still under development (Pearlson, 1999). Although DTI is not yet able to address the heterogeneity of anisotropy within normal white matter based on location, it is hoped that new developments will be able to control for regional anatomical diVerences within white matter structures. Decreased coherence along white matter tracts—decreased anisotropy—has been found in schizophrenic brains in the PFC, temporoparietal, parietooccipital regions, splenium, cingulum, posterior capsule, and adjacent occipital white matter (Agartz et al., 2001; Buchsbaum et al., 1998; Lim et al., 1999; Shihabuddin et al., 2000). Decreased global white matter anisotropy is consistent with the prolonged white matter transverse relaxation times found in schizophrenia (PfeVerbaum et al., 1999) and underscores the apparent diVuse nature of the loss of coherence in white matter tracts in schizophrenia. Decreased anisotropy has been found in all currently published studies save one, but MRI scanning techniques in that series had reduced sensitivity and resolution compared to other studies (Steel et al., 2001). More recently, decreases in inferior frontal anisotropy have been found to be related to increased ratings of negative symptoms (SANS) in schizophrenia patients (Wolkin et al., 2003), suggesting that measures of anisotropy may well be correlated with worsening functional status in schizophrenia patients. This small, preliminary study appears to have had insuYcient power to show more widespread relationships between functional status and frontal anisotropy, which suggests that with more subjects, their ﬁndings would have been even more robust. Some insight into the meaning of decreased anisotropy derives from the investigation of anisotropy in other neuropsychiatric conditions. Decreases in anisotropy are associated with demyelinating diseases, including the leukodystrophies (Ito et al., 2001), multiple sclerosis (Filippi et al., 2001), and HIV (Filippi et al., 2001a; Pomara et al., 2001) (see later), suggesting that DTI is providing a measure of myelin integrity and further implicating myelination in the pathophysiology of schizophrenia. The decreases in anisotropy seen in schizophrenia support the notion that the elements of white matter—myelin and oligodendroglia—are abnormal in schizophrenia, creating at least a functional if not structural obstacle to cortico-cortical and cortical–subcortical interaction that would clearly be compatible with some of the symptoms of schizophrenia, particularly the cognitive deﬁcits. If the decreased anisotropy shared by schizophrenia and some demyelinating diseases actually reﬂects a shared abnormality in myelin, then some overlap in symptoms should be expected between schizophrenia and demyelinating diseases that aVect cortical regions. Such data can be found in the symptomatology of metachromatic leukodystrophy (MLD) and multiple sclerosis (MS).

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VI. Demyelinating Diseases and the Symptoms of Schizophrenia

Metachromatic leukodystrophy is an autosomal recessive disease caused by defects in arylsulfatase A or its sphingolipid activator protein B (saposin B). Either defect leads to an accumulation of sulfatides in both central and peripheral nervous systems, with consequent demyelination. MLD usually begins with demyelination in the frontal lobes, particularly the periventricular white matter and corpus callosum. The late-onset form (adolescent to young adulthood) of MLD is rare, but notably often presents with symptoms of acute schizophrenia (Alves et al., 1986; Cerizza et al., 1987; Finelli, 1985; Fukutani et al., 1999; Hageman et al., 1995; Hyde et al., 1992; Schaumburg et al., 1975; Shapiro et al., 1994; Skomer et al., 1983) followed by progressive mental deterioration with or without focal neurologic symptoms. Psychotic symptoms occur in up to 53% of cases of adolescent and early adult-onset MLD; more importantly, psychotic symptoms are reported only in cases presenting in adolescence and young adulthood (Hyde et al., 1992), paralleling when schizophrenia appears as a diagnostic entity. MLD patients suVer from a host of psychotic symptoms, including auditory hallucinations, thought fragmentation, catatonic posturing, bizarre gesturing, poor concentration, inappropriate aVect, and poor insight (Hyde et al., 1992). Although most case reports of adult-onset MLD report psychiatric symptoms as a behavioral disturbance, cases of predominantly negative symptomatology have also been described (Cerizza et al., 1987; Finelli, 1985). Interestingly, in presentations in which psychosis appears, it is present only during the early stages when the disease is conﬁned mostly to the frontal lobes. Later, as demyelination spreads through the rest of the forebrain, the psychosis disappears and is replaced by sensorimotor neurological signs and dementia (Hyde et al., 1992). This is consistent with the distinctive pattern of brain changes observed in schizophrenia and a distribution of white matter abnormalities paralleling this pattern. Indeed, widespread neuropathology, like that observed in the later stages of MLD, is not characteristic of schizophrenia and may therefore be inconsistent with the types of brain changes required to produce the core symptoms of schizophrenia. Furthermore, these cases are typically characterized on CT scan by atrophy, ventricular enlargement, and bilateral white matter hyperintensities, tending to the frontal and parietal lobes (Alves et al., 1986; Finelli, 1985; Hageman et al., 1995; Skomer et al., 1983). Myelin sheath thickness is reduced in many patients, predominantly in the large ﬁbers, but this is not a universal ﬁnding (Hageman et al., 1995). Decreased anisotropy has been demonstrated in X-linked adrenoleukodystrophy, a disease closely related to MLD, with a gradation across zones of demyelination similar to the histopathological ﬁndings of Schaumberg (Schaumberg et al., 1975; Ito et al., 2001). That decreases in anisotropy were consistent with

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histologically observed changes in myelin that were demonstrated in other studies further suggests that loss of anisotropy is a strong correlate of loss of myelin integrity. Cognitive changes are common in late-onset MLD. Although no large subject investigations have been undertaken (likely due to the rarity of this form of MLD), patients with late-onset MLD have been described to have a marked diVerence between verbal IQ and performance IQ of up to 10 points or more and diYculty in mathematical performance (Fukutani et al., 1999; Shapiro et al., 1994). Visual perception and nonverbal reasoning were impaired severely, as was visual short-term memory (Shapiro et al., 1994) and visuospatial processing ( Johannsen et al., 2001). Tests of encoding, delayed recall (Shapiro et al., 1994), and memory function (Fukutani et al., 1999) also showed deﬁcits. Further severely impaired domains were reaction time, omission errors, and sustained attention (Fukutani et al., 1999; Johannsen et al., 2001; Shapiro et al., 1994). Finally, executive functioning was impaired (Shapiro et al., 1994), including abstract thinking and problem solving (Fukutani et al., 1999); however, verbal ﬂuency has been described as preserved in some reports (Shapiro et al., 1994) and impaired in others (Fukutani et al., 1999). Preserved in these patients were domains such as reading achievement, general language function, and verbal repetition (Shapiro et al., 1994), including tests of aphasia, apraxia, and agnosia (Fukutani et al., 1999). Motor function varied between relatively intact to markedly severe ( Johannsen et al., 2001; Shapiro et al., 1994). These cognitive changes in late-onset MLD suggest a pattern of dementia with combined frontal and white matter cognitive changes (Shapiro et al., 1994) that has a great deal of overlap with the cognitive changes seen in schizophrenia, especially the group of schizophrenics who have a chronic, progressive course. The tendency of MLD to parallel the psychiatric and cognitive presentations of schizophrenia makes it a model psychosis and suggests that dysfunction in myelin may be an important functional and neuroanatomical element in schizophrenia. Multiple sclerosis is a disease in which demyelination occurs in unpredictable patches throughout the CNS, an unpredictability that accounts for the tremendous variations in presentation in both neurological and psychiatric symptoms. A variety of psychiatric symptoms are reported in the literature; however, psychosis is rarely found in MS (Beatty, 1993). Thus, the most informative data with applicability to schizophrenia are the relationship between the distribution of demyelination in brain and the behavioral symptoms of patients with MS. Although MS patients with psychiatric symptoms were found to have the same number of lesions on MRI as a psychiatrically uncompromised MS group, those with the presence of psychiatric symptoms had more of their lesions located in the temporal lobe (Honer et al., 1987). In contrast, Reischies and colleagues (1988) demonstrated that the severity of psychiatric symptoms correlated with the extent of frontal demyelination, rather than with demyelination in other

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brain regions. The diVerence in the incidence of psychosis between MLD and MS highlights two important points. First, timing appears to be a critical factor in determining the overt symptomatology of the diseases. When MLD has its onset in late adolescence/early adulthood, the same as for schizophrenia, it is often accompanied by psychosis; MS, however, has its peak incidence later than the typical age of onset for schizophrenia. Second, the location of the white matter lesions appears to impact the illness presentation. In MLD, that demyelination begins in the frontal lobes and is accompanied by psychotic symptoms early in its presentation, often mistaken for schizophrenia initially, oVers strong support for the idea that disconnectivity aVecting frontal brain regions is relevant to schizophrenia and to abnormalities of myelin. MS, however, is associated with psychotic symptoms less frequently, a function of the variability of lesion location and its frequent early sparing of frontal and temporal regions. The magnetization transfer ratio has been found to be reduced in MS patients (Dousset et al., 1998; Gass et al., 1994; Thorpe et al., 1995) and to correlate with axonal loss as conﬁrmed in portmortem analyses (van Walderveen et al., 1998). Decreased anisotropy has also been demonstrated in the white matter lesions of MS patients, but more impressively, decreased anisotropy has also been demonstrated in the normal-appearing white matter of MS patients (Filippi et al., 2001), suggesting that changes in myelin and axonal integrity that are too subtle for MRI visualization are observable on DTI. Analogously, one could speculate that decreases in anisotropy in schizophrenia may reﬂect functional or anatomical changes too subtle for many current visualization techniques, even MTI. Multiple sclerosis patients also display cognitive dysfunction (Arnett et al., 1994; Comi et al., 1995; Demaree et al., 2000; Dousset et al., 1998; Foong et al., 1997; Gaudino et al., 2001; Hohol et al., 1997; Medaer et al., 1987; Peyser et al., 1990; Rao et al., 1989, 1991; Reischies et al., 1988; Sperling et al., 2001; Swirsky-Sacchetti et al., 1992), often related to lesion volume (Comi et al., 1995; Honer et al., 1987; Hohol et al., 1997; Medaer et al., 1987; Rao et al., 1989; Reischies et al., 1988; Sperling et al., 2001; Swirsky-Sacchetti et al., 1992), that impressively overlaps the cognitive dysfunction of both MLD and schizophrenia. The neuropsychological deﬁcits of MS vary depending on the areas aVected by demyelination, and thus, it is not possible to adequately describe the nature of MS deﬁcits in cognition as a group. Instead, analyses tend to correlate cognitive ﬁndings with regional areas of demyelination. In general, total lesion area has been described as the best overall predictor of whether there will be cognitive dysfunction (Rao et al., 1989; Swirsky-Sacchetti et al., 1992), and deﬁcits have been reported more frequently on measures of recent memory, sustained attention, abstract/conceptual reasoning, verbal ﬂuency, and visuospatial problem solving (Rao et al., 1989; Swirsky-Sacchetti et al., 1992). In general, MS patients are impaired less frequently on measures of language and immediate and remote memory (Rao et al., 1991).

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When examined regionally, left frontal lobe involvement tended to correlate with impaired abstract problem solving, memory, and word ﬂuency (SwirskySacchetti et al., 1992); and when analyzed along with parietal lesion volume, signiﬁcant impairment was found on tests of sustained attention, processing speed, and verbal memory (Sperling et al., 2001). Interestingly, frontal involvement predicted impairment on conceptual reasoning tasks when compared to MS patients who had the same total lesion area but no frontal involvement. Left parietooccipital involvement best predicted deﬁcits in verbal learning and complex visual-integrative skills (Swirsky-Sacchetti et al., 1992). Importantly, periventricular and frontal lesions were the most important for predicting the presence of psychological symptoms. Cognitive deﬁcits, as expected, worsened with disease progression or with more severe forms of the illness (Comi et al., 1995; Gaudino et al., 2001; Hohol et al., 1997). Furthermore, disentangling the impact of each region on cognitive performance remains more a goal than a practice (Foong et al., 1997). However, the congruence between the neuropsychological proﬁles of MLD, MS, and schizophrenia is striking.

VII. Age-Related Changes in Normal Aging

Signiﬁcant age-related changes in white matter are remarkably coincident with the onset of schizophrenia (for a review, see Davis et al., 2003). Brieﬂy, ongoing axonal growth and myelination (Yakovlev and Lecours, 1967) cause ongoing linear white matter expansion through age 20 (Giedd et al., 1999), and cortico-hippocampal connections continue to myelinate throughout life (Benes, 1989; Benes et al., 1994). Furthermore, myelination is ongoing in adolescence and adulthood, with frontal and temporal lobes being the last cortical areas to complete the myelination process (Goldman-Rakic and Selemon, 1997; Haug, 1987; Huttenlocher, 1979; Huttenlocher and de Courten, 1987; Peters et al., 1994; Terry et al., 1987), and the fact that these lobes are developing during the time when schizophrenia typically has its onset is compelling. White matter, in fact, increases through middle age, and the age-related white matter volume expansion for schizophrenia patients during this time is less robust than for controls (Bartzokis et al., 2001, 2003). Frontal lobe white matter volumes have been shown to decrease over time in schizophrenia (Ho et al., 2003). In this investigation, which constitutes the largest longitudinal study of its type to date, patients were followed for a mean of 3 years, predominantly early in their course of their illness. The severity of this loss of frontal lobe white matter volume was correlated with both poor outcome and severity of negative symptoms and, importantly, no such eVects were seen with frontal gray matter volumes. This supports the idea that there may be something

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awry in the second large-scale wave of myelination and that this abnormality may lead to a progressive loss of frontal lobe brain tissue volume rather than the expected increase in frontal lobe brain tissue volume during this time period. Although there has been considerable controversy surrounding the cognitive functioning in aging schizophrenic patients, some studies have demonstrated unequivocally that there is at least a subgroup of schizophrenic patients who have a progressive decline in their cognitive functioning over time (Davis et al., 1998; Harvey et al., 1999, 1999a; Keefe et al., 1989), although other groups report no such progression (for a review, see Goldberg et al., 1993). In one group (Davis et al., 1998), a total of 1103 patients have been diagnosed and assessed with longitudinal data collection consisting of assessments at baseline and then at 15, 46, and 60 months. In this 6-year longitudinal assessment (comparing schizophrenic, normal, and Alzheimer’s subjects), proportional rates of cognitive decline to a CDR score of 2 were compared among the three groups using Z tests. These comparisons demonstrated dramatic decreases in cognitive functioning in the schizophrenic patient group beginning at approximately age 65. These data are consistent with the idea that schizophrenia has a progressive cognitive component, at least in this chronically hospitalized and relatively treatment-refractory population. This drop in cognitive functioning is markedly similar to the types of cognitive failure seen in the so-called demyelinating diseases as described earlier and, coupled with the emerging white matter structural ﬁndings in schizophrenia, begins to suggest that myelin-related changes may be responsible, at least in part, for this cognitive decline.

VIII. Expression of Myelin-Related Genes in Schizophrenia

DNA microarray analysis—a technique that provides a quantitative measure of transcriptional gene expression—has been performed in the postmortem dorsolateral prefrontal cortex of 12 schizophrenic and 12 control patients (Hakak et al., 2001). An analysis of over 6500 genes was performed using homogenized tissue from the PFC of schizophrenia patients, which was compared to PFC tissue from control brains. Only 7 genes were found to be downregulated in the brains of schizophrenia patients in this sample, and 6 of those genes were related to myelin—myelin-associated glycoprotein (MAG), CNP, myelin and lymphocyte protein (MAL), gelsolin (GSN), ErbB3 (also called HER3), and transferrin ( Tf ). These decreases were reexamined using mRNA RT-PCR, and directionality was conﬁrmed for the change in expression. Interestingly, a linear discriminate function analysis of those 6 genes between schizophrenia patients and controls had an area of overlap, even though there was a statistically

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signiﬁcant discrimination of the two groups; however, when schizophrenia patients were compared to controls using all 35 of the myelin-related genes in the original microarray, a large point of nullity arose between the schizophrenic brains and those of the controls. Various other investigations have conﬁrmed one or more of these genes as downregulated (Bailer et al., 2000; Ekelund et al., 2000; Gurling et al., 2001). These diVerent brain collections, which reﬂect diVerent ages, diVerent disease severities, and diVerent quantitative techniques, make a compelling argument for the validity of these ﬁndings. The myelin-related genes that were discovered to be downregulated in schizophrenic patient’s brains have wide-ranging functions, most of which have been described only tentatively in the literature. These include signaling for and targeting of the formation and compaction of myelin, maintaining oligodendrites and myelination, apoptosis of oligodendrites, and promoting and/or inhibiting myelination in oligodendrites. The genes aVected in this cohort of schizophrenic patients are summarized in Table II and are discussed later.

A. Myelin-Associated Glycoprotein Myelin-associated glycoprotein, a member of the immunoglobulin superfamily (Arquint et al., 1987; Lai et al., 1987; Salzer et al., 1987), is a minor but important component of myelin. MAG is expressed only by myelin-forming cells (Quarles, 1983; Trapp et al., 1989) when oligodendrites initiate contact with axons ( Johnson et al., 1989) and is involved in the initiation of myelination in the CNS (Bartsch et al., 1997; Montag et al., 1994). In mature myelin, MAG is found in periaxonal regions of myelinated axons and in the paranodal regions of myelin sheaths (Bartsch et al., 1989; Martini and Schachner, 1986; Trapp et al., 1989), suggesting that it has a role in maintaining interactions between oligodendrites and axons (Sternberger et al., 1979), and MAG has been inferred to mediate oligodendrite– oligodendrite and oligodendrite–neuron adhesion (Poltorak et al., 1987). Furthermore, MAG interacts with other extracellular matrix components, is believed to have an inﬂuence in regulating the properties of sodium channels at the nodes of Ranvier (Srinivasan et al., 1998), and has been shown to enhance the survival of oligodendrocytes (Gard et al., 1996). MAG, it has been suggested, has a positive inﬂuence on the caliber of myelinated axons (Yin et al., 1998), and the integrity of CNS myelin may in part be dependent on the presence of MAG (Lassmann et al., 1997). MAG can have either a growth-promoting or a growth-inhibiting inﬂuence on neurite outgrowth, depending on the environment (Yang et al., 1999), and it has been noted that MAG may provide a trophic signal to oligodendrites, without which oligiodendroglial deterioration occurs (Weiss et al., 2000). In MAG-deﬁcient mice, a variety of ultrastructural abnormalities have been observed in the CNS as well as abnormal oligodendrites–axonal interactions

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TABLE II Myelin-Related Genes Found Downregulated in Schizophrenia Myelin-related gene

Function

Myelin and lymphocyte protein

Found in compact myelin May signal myelin compaction Organizes and stabilizes myelin via adhesion High-affinity receptor for NGF1b Critical for oligodendrite survival and funtion Related to myelin targeting Myelinating cells are unresponsive to this factor if mature (unless injured) Very early expression in myelination process Associated with myelin formation and maintenance May help target myelin basic protein to compact myelin (modulates cell surface morphology) Early marker of oligodendrite differentiation Believed to upregulate myelin production Intraparechymal injection Upregulates myelin production in rats Enables myelin-deficient rats to synthesize MBP and to myelinate axons Modulates ion channel activity to change the shape of dendritic spines Involved in the apoptosis of oligodendrites and retraction of myelin Affects glutamate excitotoxicity Regulator between myelinating cells and axons: growth inhibitor and/or promoter Expressed early when oligodendrites first interact with axons Homology with neural cell adhesion molecule Loss of MAG associated with redundant myelination and inability to form mature myelin MAG knockout mice make normal myelin, but lose both myelin and small-diameter axons as they age

HER3 (ErbB)

20 -30 Cyclonucleotide, 30 -phosphodiesterase

Transferrin

Gelsolin

Myelin-associated glycoprotein (MAG)

(Montag et al., 1994; Schachner and Bartsch, 2000), including delayed myelination (Montag et al., 1994) and hypomyelination (Bartsch et al., 1997) of optic nerves. Interestingly, hypomyelination in the optic nerve of MAG-deﬁcient mice was not associated with a loss of oligodendrites (BiYger et al., 2000), suggesting that this hypomyelination may be related to an impaired oligodendrite–axonal interaction. In MAG-deﬁcient mice, an increased number of unmyelinated axons was found compared to that of wild-type mice (BiYger et al., 2000; Montag et al., 1994), as well as an increase in the number of small-caliber unmyelinated axons

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(Bartsch et al., 1997; BiYger et al., 2000), indicating that MAG is important for the initiation of myelination. MAG-deﬁcient mice have morphologically abnormal myelin sheaths, lacking a well-developed cytoplasmic collar, containing redundant myelin and noncompact areas of myelin, periaxonal areas of degeneration, and dystrophy of distal oligodendrite processes (Bartsch, 1996; BiYger et al., 2000; Lassmann et al., 1997; Li et al., 1994; Montag et al., 1994). MAG has been suggested to stabilize the myelin–axon interface (Filbin, 1995; Quarles and Trapp, 1984; Salzer et al., 1990) and thus to be an important factor in the maintenance and ultimate survival of mature myelin. Furthermore, because both axons and myelin have been found to be lost in older MAG knockout mice, it has been suggested that MAG plays a role in the stability of axons as well (Lassmann et al., 1997; Li et al., 1998; Yin et al., 1998) because MAG-null mice display a ‘‘dying-back oligodendropathy’’ at an 8-month oligodendroglia age (Lassmann et al., 1997) that was similar to that described for toxic or immunemediated diseases in humans ( Ludwin and Johnson, 1981; Rodriguez and Scheithauer, 1994; Rodriguez et al., 1993), in which downregulation of myelinrelated gene expression in oligodendrites is typical ( Jordan et al., 1989; Rodriguez, et al., 1994). In MAG-null mice, levels of MBP and myelin were observed to be normal; however, levels of 20 ,30 -cyclic nucleotide 30 -phosphodiesterase (CNPase) and N-CAM were found to be reduced in whole brain, myelinated axons, and myelin. Because CNP is localized selectively in oligodendrocytes, this decrease suggests that there are oligodendroglial abnormalities ( Weiss et al., 2000), supporting the notion that oligodendrite injury is occuring in these mice, with resultant liberation of these transmembrane proteins. Interestingly, these MAG-deﬁcient mice demonstrate an age-dependent dysfunction in myelin that parallels the cognitive decline in aging chronic and relatively treatment-refractory schizophrenia patients. B. 20 -30 Cyclonucleotide, 30 -Phosphodiesterase The gene 20 -30 cyclonucleotide, 30 -phosphodiesterase (CNP), located on chromosome 17q21, is a transmembrane protein in oligodendrite membrane sheets (Lintner and Dyer, 2000). CNP is synthesized very early in the myelination process by oligodendrites (O’Neill et al., 1997; Vogel and Thompson, 1988), is not present in compact myelin, but is present on the cytoplasmic membranes of developing oligodendrites (Braun et al., 1988; Trapp et al., 1988). CNP may play a role in the modulation of cell surface morphology through its interactions with the plasma membrane and actin-based cytoskeleton (De Angelis and Braun, 1996a,b; Gravel et al., 1996), where it has a role in oligodendroglial membrane expansion and migration (De Angelis and Braun, 1996). CNP may also have a role in intracellular communication and signal transduction cascades (De Angelis and Braun, 1994).

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C. Myelin and Lymphocyte Protein The myelin and lymphocyte protein is one of four identiﬁed transmembrane domain proteins expressed in myelin (for a review, see Frank, 2000). Its expression parallels myelinogenesis; however, MAL is expressed late in the process of myelination in the CNS when myelin compaction occurs (Frank et al., 1999). In mature myelin, MAL is found predominantly in compact myelin, in close localization with structural proteins (MBP and PLP) along myelin sheaths (Frank et al., 1998) and has been found to be in close proximity to and likely in functional contact with myelin glycosphingolipids, particularly sulfatide, and MAL may be associated with the transport of sulfatide to the myelin membrane (Frank et al., 1998; Kim et al., 1995). MAL has been suggested to play a role in organizing and stabilizing microdomains in myelin membranes and possibly contributing to myelin membrane adhesion (Coetzee et al., 1998; Koshy and Boggs, 1996; Stewart and Boggs, 1993). In humans, the MAL gene has been assigned to the long arm of chromosome 2q13 (Alonso et al., 1988).

D. ErbB3 ErbB3 (also called HER3) is a receptor-protein tyrosine kinase that trandsduces neuregulins and is believed to be involved in cell survival, migration, and diVerentiation in both neural and nonneural cells (for a review, see Adlkofer and Lai, 2000). NRG-1, a neuregulin, may be required for the establishment of the oligodendroglial lineage in cells in the developing forebrain (Corfas et al., 1995; Vartanian et al., 1994, 1999). NRG-1 has been found to promote the diVerentiation of oligodendroglial precursors (Raabe et al., 1997; Vartanian et al., 1994), to promote the maintenance and growth of radial glial ﬁbers, and to play a role in the survival of oligodendroglial cells in vitro (Canoll et al., 1996; Raabe et al., 1997). It is possible that decreased expression of ErbB3 may limit the function of NRG-1 and hence have a detrimental eVect on oligodendroglial diVerentiation, signaling, or survival. Importantly, NRG-1 has been found to be associated with schizophrenia in a number of separate populations and samples (Law, 2003; Stefansson et al., 2002, 2003a,b; Williams et al., 2003; Yang et al., 2003).

E. Transferrin Transferrin, an iron-transport protein, is not speciﬁc to the central nervous system; however, it has been demonstrated to be structurally and functionally diVerent from the iron-transporting forms of the protein outside the central

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nervous system and is involved in the life of oligodendrocytes (Bloch et al., 1985), including having a role in myelin formation. The expression of Tf is correlated with oligodendrite development (Cassia et al., 1997) and with myelin formation (Escobar Cabrera et al., 1997), and Tf is believed to be synthesized by oligodendrocytes (Bloch et al., 1985; Connor and Fine, 1987; Connor and Menzies, 1996). When the blood–brain barrier forms and neural cells can no longer utilize the transferrin that originates in the liver, oligodendrocytes take over the production of transferrin, which has been shown to have both a survival and a trophic impact on neurons (Espinosa de los Monteros et al., 1989). In fact, oligodendrocytes are the predominant iron-containing cells in the brain, and oligodendrocytes rich in iron are particularly abundant within white matter tracts, although not all oligodendrocytes along white matter tracts are (Connor and Menzies, 1996). Iron is involved in myelin production as a cofactor in lipid and cholesterol biosynthesis and may be involved in oxidative metabolism within oligodendrocytes. In hypotransferrinemic mice, a 30% reduction in myelin thickness is observed (Dickinson and Connor, 1994), and the amount of Tf expressed correlates better with the amount of myelin than with the number of oligodendroglia (Abney et al., 1981). Oligodendroglia contain Tf receptors (Espinosa de los Monteros and Foucaud, 1987; Giometto et al., 1990), and injection of apotransferrin in rats allows Tf-negative mutant oligodendrocytes to synthesize MBP and to myelinate axons (Escobar Cabrera et al., 1997).

F. Gelsolin Gelsolin is a cytosolic protein expressed in high concentrations in developing oligodendrocytes (Lena et al., 1994) and is speciﬁcally enriched in myelin-forming cells (Tanaka et al., 1993). Additionally, gelsolin is expressed in neurons (Furukawa et al., 1997; Kwiatkowski et al., 1988a,b) and is concentrated in neural growth cones (Tanaka et al., 1993). Gelsolin binds to and severs actin when activated by Ca2þ (Cooper et al., 1987; Janmey and Stossel, 1987; Yin et al., 1981). The attachment of gelsolin to actin causes actin depolymerization, which, in turn, modulates Ca2þ inﬂux through NMDA receptors in hippocampal neurons (Furukawa et al., 1997). Using a gelsolin-deﬁcient knockout mouse, it has been demonstrated that neurons that lack gelsolin have an increased Ca2þ response to glutamate with a resultant increase in neuronal excitotoxicity (Furukawa et al., 1997). It is likely that the increased excitotoxicity is secondary to deﬁcient actin depolymerization, as administering an alternative actin-depolymerizing protein to gelsolin-negative mice prevented the excitotoxic neuronal injury in these mice. The downregulation of gelsolin in schizophrenia may contribute to ongoing glutamate-induced injury in neurons and in oligodendroglia.

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G. Linkage Studies at Myelin-Related Gene Regions A number of studies have found associations between schizophrenia and the chromosomes on which these myelin genes reside with markers somewhat near these six genes (see Davis et al., 2003). Exploration of these datasets is a potentially informative way to determine the importance of some of these genes in the heritability of schizophrenia. MAG is on chromosome 19q13.1, and support has been found for its involvement in schizophrenia in three studies (Bailer et al., 2000; Faraone et al., 1998; Levinson et al., 1998). Support for GSN, on chromosome 9q33–34, is found in ﬁve studies (Kaufmann et al., 1998; Levinson et al., 1998; Park et al., 1991; Riley et al., 1997; Stober et al., 2000). MAL, located at 2cenq13, also has genetic support in six studies (Faraone et al., 1998; Gurling et al., 2001; Levinson et al., 1998; Mowry et al., 2000; Stober et al., 2000; Williams et al., 1999). Tf is on chromosome 3q21, and support for it has been found in six studies (Bailer et al., 2000; Brzustowicz et al., 2000; Ekelund et al., 2000; Gurling et al., 2001; Levinson et al., 1998; Stober et al., 2000). ErbB3 on chromosome 12q13 has support in four studies (Ekelund et al., 2000; Gurling et al., 2001; Levinson et al., 1998; Stober et al., 2000). CNP is located on chromosome 17q21 and has been found to be associated with schizophrenia in four studies (Bailer et al., 2000; Ekelund et al., 2000; Gurling et al., 2001; Levinson et al., 1998). This gene was examined using a single nucleotide polymorphism and was found to be associated with schizophrenia ( p ¼ 0.02) in a case–control study of 368 patients (O’Donovan, 2002, personal communication). As schizophrenia is a complex multigenic disease, the suggestive series of LOD scores for these myelin-related genes is consistent with a role for myelin in schizophrenia. Given both the heterogeneity of schizophrenia and the lack of ﬁne resolution in most genome scans, that many of these myelin-related genes have been associated with schizophrenia lends support to the likelihood that a myelin-related process and an abnormality in oligodendroglial function play some role in schizophrenia. The downregulation of these myelin-related genes is likely to impair myelin integrity as well as the function and even survival of oligodendroglia. If this is the case, then studies that examine oligodendroglia should show reductions in either the structure of myelin sheaths or the number of oligodendroglia in patients with schizophrenia.

IX. Direct Examinations of Myelin and Oligodendroglia in Schizophrenia

Postmortem examination of the brains of schizophrenia patients is fraught with diYculties related to the postmortem index (PMI), which is essentially a measure of the amount of time from death until ﬁxation of brain tissue, to the

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pH of the brain at the time of ﬁxation (often related to the cause of death as well as the PMI), and to the method of sectioning of the tissue itself. In addition to tight inclusion criteria applied to the brains chosen for analysis, many of the problems of postmortem examination are limited by the use of control brains matched on each of the potential confounds listed earlier. This approach assumes that the brains of schizophrenia patients undergo similar processes as well as degrade at the same rate as control brains after death, and no evidence yet suggests that this is not the case. Thus, postmortem examinations of brain tissue with the use of controls is felt to be a reasonable method for investigating myelin and oligodendroglial structure. Examination of myelin sheaths in the brains of schizophrenia patients has yielded ultrastructural abnormalities (Deicken et al., 1994; Keshavan et al., 1998; Miyakawa et al., 1972; Oifa and Uranova, 1991; Uranova, 1988; Uranova et al., 2001a,b). The myelinated ﬁbers extending from the PFC and caudate nucleus are altered, with a loss of compactness of the myelin sheath lamellae, abnormal inclusion bodies, and concentric lamellar bodies (Uranova et al., 2001a). More recently, oligodendroglial cells have been found to demonstrate reactive, progressive, and regressive changes in the brains of schizophrenia patients (Uranova et al., 2001b). Reactive changes seen included swelling of cytoplasm and organelles, nuclear ectopia, heterochromatin clumping, and an accumulation of a lipofuscin-like material. None of these changes were observed in the control brains. Progressive changes were noted only rarely; however, regressive changes were found in both the PFC and the caudate nucleus in schizophrenic brains and included shrunken nuclei, nuclear chromatin clumping, nuclear pyknosis, condensation, cell swelling, and vacuolization of endoplasmic reticulum. These changes, again, were seen only rarely in the brains of controls. These changes are remarkably similar to the changes seen in MAG knockout mice (Lassmann et al., 1997), although whether these similar changes result from similar processes or represent a common ﬁnal pathway of independent dysfunctions remains to be determined. As mentioned earlier, the functioning of oligodendroglia, astrocytes, and neurons is now considered to be intimately interdependent, and interestingly, astrocytes in the schizophrenia brains examined in the aforementioned studies demonstrated reactive changes as well. Furthermore, astrocytes were often found engulﬁng oligodendroglial cells that had been subject to regressive changes, further underscoring the interplay of these cell types (Uranova et al., 2001b). Oligodendroglial density has also been discovered to be reduced in schizophrenia by as much as 21% in the PFC (Rajkowska et al., 2001). In fact, a postmortem study using rigorous stereologic counting methods found decreased total numbers of oligodendrocytes in schizophrenic brains (Hof et al., 2003). Reductions in the total number of oligodendrocytes by 28% in layer III of PFC area 9 and a reduction of white matter by 27% in the underlying white matter were

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observed in the brains of schizophrenics based on CNP immunohistochemistry and Nissl-stained materials. Comparable results were obtained when individual variations in the volume of the region were taken into account and densities were calculated. Furthermore, analysis of the spatial distribution of oligodendrocytes in the superior frontal white matter revealed a more isotropic arrangement in schizophrenic cases as well as correlations between spatial distribution and numbers of oligodendrocytes in both control and schizophrenics. These results point to a severe pathology of oligodendrocytes in schizophrenia and provide a quantitative cellular correlate of the white matter changes observed by brain imaging in vivo—showing reduced anisotropy in schizophrenic patients—and support the evidence that genes encoding myelin-related proteins exhibit reduced expression in schizophrenia (Hakak et al., 2001). Abnormalities in or death of oligodendroglia could readily lead to abnormalities in myelin integrity, including myelin initiation, deposition, compaction, and maintenance (Bartsch et al., 1997; Canoll et al., 1996; Corfas et al., 1995; De Angelis and Braun, 1996; Gard et al., 1996; Filbin, 1995; Hakak et al., 2001; Hof et al., 2003; Koshy and Boggs, 1996; Montag et al., 1994; Quarles, 1983; Salzer et al., 1990; Sternberger et al., 1979; Stewart and Boggs, 1993; Vartanian et al., 1994, 1999; Weiss et al., 2000). These microscopic changes further support the notion that myelin and oligodendroglia are abnormal in schizophrenia. With the wealth of data accumulating to implicate white matter components in schizophrenia, one must consider the question: what could be causing this insult to oligodendroglia and myelin?

X. Mechanistic Considerations

Both genetic and environmental insults may contribute to oligodendroglial and myelin dysfunction in schizophrenia. Genetic evidence from microarray examinations continues to receive replication; however, with so many diVerent myelin-related genes being impacted, one has to entertain the notion that there is either some hitherto undiscovered upstream genetic insult that is causing the downregulation of these genes or that there is an environmental insult that may be impairing the functioning and survival of oligodendrocytes and thus causing a downregulation in myelin-related gene expression. Of course the possibility remains that the deﬁcits in oligodendrocytes and myelin are the result of the early disease process and do not represent an early contributing cause; however, whether an environmental or upstream genetic insult starts the process of oligodendroglial dysfunction does not negate the potential role for white matter impairment in schizophrenia. One potential environmental insult to oligodendroglia that has some support in the literature is hyperglutamatergia. Oligodendrocytes express functional

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ionotropic glutamate receptors of AMPA, NDMA, and kainate classes, although AMPA and kainate receptors are the predominant types (Sanchez-Gomez and Matute, 1999). Oligodendroglial cells have AMPA receptors that lack the GluR2 subunit, and the kainate GluR6 subunit is edited slightly in oligodendroglia, rendering both receptors more permeable to Ca2þ (Burnashev, 1996). Overactivation of these receptors in vivo is excitotoxic to oligodendroglia (Matute et al., 1997; McDonald et al., 1998; Sanchez-Gomez and Matute, 1999), and the susceptibility of oligodendrocytes to the toxic inﬂuences of glutamate is age dependent—immature oligodendrocytes are insensitive to glutamate-medicated excitotoxic damage (Choi et al., 1987; Frandsen and Schousboe, 1990); however, when more mature oligodendrocytes are examined, they are found to be highly vulnerable to AMPA-induced death (McDonald et al., 1998). The suggestion has been made that the polymorphic variations in GluR receptors may increase vulnerability to glutamate-induced excitotoxicity, leading to demyelination (Green et al., 1998; for a review, see Matute et al., 2001). Furthermore, overactivation of oligodendroglial glutamate receptors is excitotoxic and causes oligodendrocyte death (Matute, 1998; Matute et al., 1997; McDonald et al., 1998; Sanchez-Gomez and Matute, 1999; Yoshioka et al., 1996). Interestingly, a slow, chronic infusion of kainate caused more damage to oligodendrocytes than an acute infusion (Matute, 1998). Thus, the health of oligodendroglia, which is at least partially dependent on glutamate modulation and signaling, plays a role in diseases of myelination (Matute, 1998) and may play a signiﬁcant role in schizophrenia. Furthermore, glutamate transporters exist in oligodendroglia and presumably regulate glutamate concentrations at levels that avoid glutamate-induced excitotoxicity (Levy et al., 1998; Zerangue and Kavanaugh, 1996). Thus, oligodendroglia are in a pivotal position regarding glutamate-induced excitotoxicity. Glutamate-induced excitotoxicity is believed to result from a loss of neuronal Ca2þ homeostasis. The activation of metabotropic glutamate receptors increases intracellular Ca2þ, which may subsequently trigger a process of cytotoxicity via activation of a number of Ca2þ-sensitive mechanisms (Choi, 1992; Siesjo, 1993; Verity, 1992). The vulnerability of oligodendroglia to the excitotoxic eVects of glutamate has obvious and profound downstream eVects on neural transmission. Not only are oligodendroglia responsible for maintaining myelination by regulating synaptic glutamate concentrations, they can inﬂuence the excitotoxicity of glutamate toward neurons, with obvious implications in considering the pathophysiologic mechanisms of schizophrenia. Glutamate has been hypothesized to have a role in schizophrenia (Olney and Farber, 1995b) through its action on multiple receptor systems. Psychotic symptoms have previously been related to hypo-NMDA receptor function; however, data suggest that the picture may be more complex than a simple hypoglutamatergic state in the schizophrenic brain.

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The NMDA glutamate receptor has been demonstrated to be related to the emergence of psychotic symptoms when blocked by phencyclidine, ketamine, or other NMDA receptor antagonists, causing functional NMDA receptor hypoactivation (Bogerts et al., 1985; Brown et al., 1986; Falkai and Bogerts, 1986; Falkai et al., 1988; Jeste et al., 1998; Olney and Farber, 1995a,b; Scheibel and Kovelman, 1981). However, the downstream eVects of NMDA antagonists may actually increase the release of glutamate (Anand et al., 2000; Deakin et al., 1990; Lovinger and McCool, 1995; Moghaddam et al., 1997). Consistent with the notion that hyperglutamatergia may be the actual functional consequence of the administration of NMDA antagonists is the fact that lamotrigine, a drug that blocks the release of glutamate, reverses the psychosis-like symptoms of ketamine in healthy normal subjects (Anand et al., 2000). Furthermore, these same NMDA antagonists, when given to rats, cause corticolimbic neurodegeneration (Hargreaves et al., 1993; Olney et al., 1989, 1991) that spreads into the anterior cingulate, parietal, temporal, entorhinal and piriform cortices, hippocampus, and amygdala (Allen and Iversen, 1990; Corso et al., 1994, 1997; Fix et al., 1993; Sharp et al., 1994; Wozniak et al., 1993), an anticipated consequence of a hyper not hypoglutamatergic state. It has been suggested that hyper- and hypoglutamatergic conditions may coexist in the schizophrenic brain (Olney and Farber, 1995a). In one model, glutamatergic neurons in the retrosplenial cortex provide inhibitory control over other excitatory pathways by the activation of GABAergic neurons, which, in turn, inhibit downstream excitatory pathways and provide a negative feedback loop for the regulation of retrosplenial cortex neurons themselves. Hypofunctioning of these retrosplenial cortex neurons would decrease inhibitory control over both downstream excitatory pathways and retrosplenial cortex activation, leading to chaotic and chronic overactivation of glutamatergic neurons in the retrosplenial cortex and potentially in frontal brain regions. Chronic increased ﬁring in these downstream excitatory pathways may provide the mechanism for glutamateinduced excitotoxicity, with resulting oligodendrocyte and neuronal dysfunction. The excitotoxic eVects of blocking NMDA receptors are also age dependent. Prepubertal rats are insensitive to the neurotoxic action of NMDA antagonists, but beginning in puberty, they become fully susceptible to such damage (Farber et al., 1992, 1995). Analogously, ketamine-induced psychotic reactions are rare in prepubertal human children, but in young and middle-aged adults, they can occur in as many as 50% of patients (Marshall and Longnecker, 1990). These age-related properties of glutamate to induce excitotoxicity have a seductive congruity with the rarity of schizophrenic-like psychosis in children and in early and midadolescence. However, if hyperglutamatergia is a precursor to oligodendroglial dysfunction and perhaps myelin maintenance and repair in schizophrenia, it would be useful to this notion to establish, at the very least, the biological plausibility of hyperglutamatergia in some regions of schizophrenic brains.

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Indeed, direct evidence for increased glutamate activity in schizophrenia comes from the study of glutaminase (PAG), an enzyme involved in the formation of glutamate. PAG has been found to be increased fourfold in the PFC of schizophrenic’s brains (Dracheva et al., 2001; Gluck et al., 2002), and these postmortem glutaminase levels in the PFC were found to correlate signiﬁcantly with poorer antemortem cognitive performance GABAergic neurons have been studied extensively in schizophrenia, with a growing consensus of diminished GABAergic input in critical cortical regions. A decrease in the density of interneurons in the anterior cingulate cortex of schizophrenics has been described along with an increase in GABAA receptor binding in the same regions (Benes et al., 1992, 1996). These data would suggest the possibility of increased glutamatergic outﬂow from the PFC to other regions of the brain in schizophrenia as a consequence of a loss of GABAergic inhibition. A decrease in GABAergic terminal ﬁelds in the PFC of schizophrenics has been suggested by immunocytochemical studies (Akil and Lewis, 1996; Lewis et al., 1997) and is paralleled by ﬁndings of decreased GAD67 mRNA, which may even be occurring independent of GABAergic neuronal changes (Akbarian et al., 1995; Guidotti et al., 2000; Volk et al., 2000). GABAergic neurons, particularly interneurons and chandelier cells, are reduced in the cortex in schizophrenia (Benes et al., 1992, 1996; Lewis et al., 1997; Woo et al., 1998). The role of these neurons in providing inhibitory control to excitatory brain areas lends support to the idea that decreased GABA interneurons lead to increased glutamatergic activity and a hyperexcitatory state in other brain areas.

XI. Summary and Future Directions

Evidence continues to accumulate that implicates oligodendroglia and myelin involvement in schizophrenia. Imaging studies, microarray analyses, and ultrastructural examinations taken together provide reasonable evidence to support ongoing research into white matter in schizophrenia, as evidenced by the NIMH’s support of the Conte Center for Neuroscience of Mental Disorders at Mount Sinai, which has funded a 4-year investigation into oligodendroglial and myelin involvement in schizophrenia. Further quantiﬁcation and qualiﬁcation of oligodendroglial abnormalities at the structural level, expanding the number of myelin-related genes examined in the brains of schizophrenia patients, applying genetic markers of myelin-related genes in a population genetics model, continuing the structural and functional imaging of patients with schizophrenia across the life span, and correlating behavioral abnormalities with both imaging and postmortem brain examination of oligodendroglial activity are some of the goals of this center. Ultimately, and no doubt with contributions not only from other

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laboratories but from other ﬁelds as well, research in this area will approach a point where therapeutic interventions can be considered in light of accumulated ﬁndings. References

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BRAIN-DERIVED NEUROTROPHIC FACTOR AND THE PLASTICITY OF THE MESOLIMBIC DOPAMINE PATHWAY

Oliver Guillin,* Nathalie Griffon,* Jorge Diaz,y Bernard Le Foll,* Erwan Bezard,z Christian Gross,z Chris Lammers,* Holger Stark,x Patrick Carroll,k Jean-Charles Schwartz,*,y and Pierre Sokoloff* *Unite´ de Neurobiologie et Pharmacologie Mole´culaire, INSERM U 573 Centre Paul Broca, 75014 Paris, France y Laboratoire de Physiologie, Universite´ Rene´ Descartes, 75006 Paris, France z Basal Gang, Laboratoire de Neurophysiologie CNRS UMR 5543, 33076 Bordeaux, France x Institut fur Pharmakologie und Toxikologie Universitat Bonn, 53113 Bonn, Germany k Unite´ de De´veloppement et Pathologie du Motoneurone Spinal INSERM U 382, 13288 Marseille CEDEX 09, France

I. Introduction II. Brain-Derived Neurotrophic Factor (BDNF) Controls D3 Receptor Expression during Development III. BDNF Triggers Ectopic D3 Receptor Expression and Behavioral Sensitization in Denervated Rats IV. Normalization of Dopamine D3 Receptor Function Attenuates Dyskinesia Induced by Levodopa V. BDNF and Dopamine D3 Receptor in Reactivity to Drug Cues VI. BDNF and Dopamine D3 Receptor in Stress, Depression, and Schizophrenia VII. Conclusions References

I. Introduction

Brain-derived neurotrophic factor (BDNF ), like other neurotrophins, was initially regarded as being responsible for neuronal proliferation, diVerentiation, and survival, following its neuronal uptake and retrograde transport to the soma (Thoenen, 1995). A more diverse role for BDNF as an extracellular transmitter has, nevertheless, been inferred from observations that it is transported anterogradely (Altar et al., 1997; von Bartheld et al., 1996), released upon neuron depolarization, and triggers rapid intracellular signals (Altar and Di Stefano, 1998; Thoenen, 1995) and action potentials in central neurons (Kaﬁtz et al., 1999) via intracellular transduction of its high-aYnity membrane receptor TrkB (Blum et al., 2002). BDNF can alter fast synaptic transmission by speeding up the INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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development of excitatory and inhibitory synapses ( Vicario-Abejon et al., 1998), but also by modulating synaptic eYcacy (Huang et al., 1999; Lohof et al., 1993). In particular, BDNF is necessary for the induction and maintenance of hippocampal long-term potentiation (Figurov et al., 1996; Korte et al., 1995; Kovalchuk et al., 2002; Patterson et al., 1996). Some observations suggest a role of BDNF in nociception (Kerr et al., 1999), mechanosensation (Carroll et al., 1998), and learning (Linnarsson et al., 1997; Minichiello et al., 1999). However, little is known about the consequences of BDNF-induced synaptic plasticity on physiological functions. Converging evidence implicates the dopamine D3 receptor in the physiopathology and treatment of Parkinson’s disease (PD) (Bezard et al., 2003; Bordet et al., 1997), schizophrenia (Schwartz et al., 2000), drug addiction (SokoloV et al., 2001), and depression (Lammers et al., 2000). Moreover, a partial D3 receptor agonist selectively reduces cocaine-seeking behavior in rats (Pilla et al., 1999), levodopa-induced dyskinesia (Bezard et al., 2003), and hyperactivity induced by a ‘‘psychogenetic drug’’ (Leriche et al., 2003). All antipsychotics with proved clinical eYcacy bind with high aYnity to the D3 receptor (SokoloV et al., 1992). In animals, chronic administration of neuroleptics upregulates the D2, but not the D3 receptor (Fishbum et al., 1994; Le´vesque et al., 1995; Tarazi et al., 1997), and there is no tolerance to D3 receptor blockade after repeated administration of haloperidol (Le´vesque et al., 1995). Whereas tolerance accounts for the progressive reduction in parkinsonian-like side eVects occurring at the beginning of treatment, an eVect presumably related to D2 receptor upregulation, it does not seem to occur at the level of the dopamine receptor subtype(s) involved in the antipsychotic activity, as this activity does not diminish upon long-term treatment. In rat brain, the D3 receptor density is highest in the islands of Calleja and in the shell part of the nucleus accumbens (Diaz et al., 1995; Le´vesque et al., 1992), which receives its dopaminergic innervation from the ventral tegmental area and other innervations from the cerebral cortex, hippocampus, and amygdala (Pennartz et al., 1994; Zahm and Brog, 1992). The shell of the nucleus accumbens projects indirectly to entorhinal and prefrontal cortices and participates in a neuronal circuitry subserving the control of emotion, motivation, and reward ( Willner and Sheel-Kru¨ger, 1991). In the human brain, D3 receptor distribution is similar, yet less restricted, with a signiﬁcant expression in the striatum, which controls movement, and in cortical areas processing sensorimotor information (Susuki et al., 1998). In postmortem brain, D3 receptor density is lowered in Parkinson’s disease (Ryoo et al., 1998), and the intensity of this loss is correlated with the loss of response to dopaminergic drugs ( Joyce et al., 2002). The D3 receptor expression is elevated in drug addiction (Staley and Mash, 1996). In animals, D3 receptor expression is, respectively, down- and upregulated by repeated stress and antidepressant treatments (Lammers et al., 2000).

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This review shows that BDNF is a factor controlling D3 receptor expression and thus the plasticity of dopaminergic neurons. Moreover, we show that the D3 receptor in animal models of Parkinson’s disease and drug-seeking behavior is a target for correcting the consequences of BDNF-induced abnormal behavior seen in these models.

II. Brain-Derived Neurotrophic Factor (BDNF) Controls D3 Receptor Expression during Development

In adults, expression of the D3 receptor in medium-sized neurons of the nucleus accumbens, but not in granule cells of the islands of Calleja, is highly dependent on dopaminergic innervation. Ablation of the aVerent neurons by unilateral 6-hydroxydopamine (6-OHDA) results in a dramatic decrease in D3 receptor density in the ipsilateral nucleus accumbens (Le´vesque et al., 1995). This paradoxical change (the D2 receptor is upregulated under these circumstances) was shown to depend on the lack of an anterogradely transported factor from dopaminergic neurons, distinct from dopamine itself and its known peptide cotransmitters, and which is released upon dopamine neuron activation (Le´vesque et al., 1995). Among the candidate factors for regulating D3 receptor expression, BDNF was particularly attractive, as it is expressed by dopamine neurons (Seroogy et al., 1994). BDNF immunoreactivity is prominent in the shell of the nucleus accumbens of normal rats (Conner and Lauter, 1997), and its receptor TrkB colocalizes with the D3 receptor (Guillin et al., 2001). Moreover, BDNF and D3 receptor expressions, which are both very low at birth, increase in parallel during postnatal development (Fig. 1). A local infusion of BDNF reverses the 6-OHDAinduced decrease in D3 receptor gene expression, indicating that exogenous BDNF compensates for the loss of dopamine neurons (Guillin et al., 2001). We have examined the eVect of a BDNF-null mutation on D3 receptor expression in developing mice. In wild-type BDNF þ/þ mice, D3 receptor binding and mRNA in the shell of the nucleus accumbens increase sharply from postnatal days 9–14 (P9–P14) to P17–P23, whereas in homozygous BDNF / mice, D3 receptor binding and mRNA are low at P9–P14 and do not increase at later stages (Fig. 4). Moreover, D3 receptor expression is unaVected by BDNF gene mutation in the islands of Calleja (Fig. 1), a region where TrkB is not expressed. These results show that BDNF is required for the normal development of D3 receptor expression in the shell of the nucleus accumbens. The BDNF gene mutation does not impair the early development of dopamine neurons (Emfors et al., 1994), nor their later development, as tyrosine hydroxylase, a marker of these neurons, was not signiﬁcantly aVected by the lack of BDNF (Guillin et al., 2001). This suggests that BDNF acts directly on

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Fig. 1. Impaired D3 receptor expression in developing BDNF / mice. (a) Autoradiography showing D3 receptor binding obtained with 125I-labeled 7-OH-PIPAT in wild-type BDNF þ/þ or BDNF / mice at postnatal day 23. (b) Animals at P9–P10 or P14–P23 were analyzed. Means SEM of values from 4 to 11 animals. *P < 0.01 versus BDNF þ/þ littermates.

D3 receptor-expressing neurons rather than indirectly via an eVect on the development of dopamine neurons. Moreover, BDNF deprivation selectively reduces expression of the D3 receptor and not that of the homologous dopamine D1 and D2 receptors (Guillin et al., 2001), which are not or only marginally downregulated by 6-OHDA lesions (Bordet et al., 1997).

III. BDNF Triggers Ectopic D3 Receptor Expression and Behavioral Sensitization in Denervated Rats

In unilaterally 6-OHDA-lesioned rats (PD-like rats), repeated administration of levodopa, leading to extraneuronal dopamine formation, triggers D3 receptor overexpression not only in the shell of the nucleus accumbens, but also in the denervated striatum, a brain structure in which D3 receptor expression is hardly detectable (Bordet et al., 1997). During levodopa treatment of 6-OHDA-lesioned rats, infusion into the denervated striatum of IgG-TrkB, a selective BDNF antagonist (Cabelli et al., 1997), impairs induction of both D3 receptor mRNA and protein expression (Fig. 2). This indicates that BDNF is necessary for this process.

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Fig. 2. Levodopa-induced D3 receptor expression in 6-OHDA-lesioned rats requires BDNF. The asterisk denotes the lesioned side. (a) In situ hybridization signals of D3 receptor mRNA in animals receiving continuous infusions into the striatum of IgG (control animal, left) or IgG-TrkB, a BDNF antagonist (right) for 7 days and levodopa for 5 days. (b) Quantitative analysis of pictures as in a, *P < 0.05 and **P < 0.01 versus IgG-treated animals.

This D3 receptor overexpression has been shown to be responsible for the development of behavioral sensitization to levodopa, i.e., a progressive enhancement of responsiveness, which appears as an increased number of levodopa-induced rotations. The development and extinction of behavioral sensitization parallels D3 receptor expression in the striatum during the treatment with levodopa and after its cessation (Fig. 3a). Moreover, the increase in the number of rotations is blocked by a preferential D3 receptor antagonist (Bordet et al., 1997) and induced by a selective partial D3 receptor agonist (Pilla et al., 1999). Infusion of IgG-TrkB dose dependently inhibits behavioral sensitization (Fig. 3b), indicating that behavioral sensitization is triggered by BDNF. Striatal BDNF in fact originates mainly from cortical neurons (Altar et al., 1997). In agreement, cortical ablation partially impairs the induction of D3 receptor overexpression in the striatum and behavioral sensitization, indicating that both processes require the participation of corticostriatal neurons. Levodopa also induces BDNF mRNA in the frontal cortex in the 6-OHDA-lesioned side, mainly in cortical deeper layer V containing pyramidal cell bodies and in layer VI, which

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Fig. 3. (a) Parallel changes in D3 receptor binding and levodopa-induced rotations in hemiparkinsonian rats during and after repeated administration of levodopa. Rats with a unilateral 6-OHDA-induced lesion of the ascending mesencephalic dopaminergic pathways placed 3 weeks earlier received levodopa (50 mg/kg, ip, twice daily) for up to 15 days and were challenged with a single same dose of levodopa. (b) Rotations were recorded after the first and last L-DOPA administrations in 6-OHDA-lesioned animals receiving increasing doses of IgG-TrkB during L-DOPA treatment. *P < 0.05 versus first L-DOPA injection.

send projections to various subcortical areas, notably various striatal and accumbal areas (Berendse et al., 1992). This eVect depends critically on activation of a dopamine D1 or D5 receptor (Guillin et al., 2001) and is consistent with the presence of D1 receptors on cortical pyramidal cells (Huang et al., 1992) that project to subcortical areas, and with the observation that stimulation of a D1 or D5 receptor under similar circumstances phosphorylates CREB (Cole et al., 1994), a factor that activates BDNF gene transcription (Shieh et al., 1998; Tao et al., 1998). We conclude that the induction of D3 receptor expression in the striatum is triggered by a D1/D5 receptor stimulation-dependent elevation of BDNF in cortico-striatal neurons, a process prominent in the 6-OHDA-lesioned side as compared to the control side, which accounts for the induction of D3 receptor expression restricted to the lesioned side. Moreover, BDNF-induced D3 receptor

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expression causes a more pronounced disequilibrium in responsiveness to dopamine between the two sides, which leads to enhanced rotational behavior.

IV. Normalization of Dopamine D3 Receptor Function Attenuates Dyskinesia Induced by Levodopa

Parkinson’s disease manifests several symptoms, such as akinesia, rigidity, and tremor, which result from the lack of the brain neurotransmitter dopamine (Homykiewicz, 1963). Substitution treatment of PD, e.g., by levodopa, initially reduces motor symptoms, but eventually induces, in most patients, debilitating and pharmacoresistant involuntary movements, i.e., dyskinesia, presumably resulting from an excessive response to dopamine (Bezard et al., 2001). This excessive response to dopamine results from a process of behavioral sensitization to the drug (Bezard et al., 2001). Enhanced responses to levodopa in PD-like rats could reﬂect either the progressive motor recovery occurring at treatment initiation or the development of levodopa-induced dyskinesia (LID) in long-term treated PD patients (Cotzias et al., 1969). This cannot be assessed in PD-like rats because these animals do not develop typical LID. We have used monkeys treated with 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), which destroys dopamine neuron terminals and cell bodies and produces a variety of PD-like symptoms, including akinesia and rigidity (Burns et al., 1983). Moreover, long-term treatment of these monkeys with levodopa elicits dyskinesia, of which both the repertoire and the severity are not distinguishable from LID occurring in PD patients (Bezard et al., 2001). D3 receptor binding has been measured in the brain basal ganglia. MPTP alone produces a severe loss of D3 receptor binding in the caudate nucleus (Bezard et al., 2003), a brain structure involved in associative locomotion. This eVect is compensated by treatment with levodopa in MPTP-intoxicated animals without LID (Fig. 4). However, in MPTP-intoxicated monkeys with LID, D3 receptor binding is higher than in nondyskinetic monkeys in the putamen and the internal part of the globus pallidus and is even higher than in normal monkeys (Bezard et al., 2003). Moreover, D3 receptor-binding levels in the putamen correlate with the occurrence and severity of LID. These results show that PD-like symptoms and LID are accompanied by down- and upregulation of D3 receptor expression, respectively, while such a correlation does not exist for either D1 or D2 receptors under comparable experimental conditions (Bezard et al., 2001, 2003). In addition, the occurrence of dyskinesia does not correlate with the severity of the lesion (Bezard et al., 2003), which indicates that the level of D3 receptor expression is an accurate marker of dyskinesia.

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Fig. 4. Dyskinesia is accompanied by D3 receptor overexpression. (Top) Typical receptor autoradiograms obtained with 125I-labeled 7-OH-PIPAT, a selective D3 receptor radioligand, in the brain from a typical normal monkey (control), untreated MPTP-intoxicated monkeys (MPTP), and nondyskinetic and dyskinetic MPTP-intoxicated monkeys treated by levodopa (L-DOPA). Cd, caudate nucleus; GPe, external part of globus pallidus; GPi, internal part of globus pallidus; Pu, putamen. (Bottom) Quantitative analysis of autoradiograms. Results are mean SEM of values in mCi/mg. *P < 0.05 vs control; #P < 0.05; ##P < 0.01 vs nondyskinetic.

The changes in D3 receptor expression are likely to reﬂect ﬂuctuations in D3 receptor function, as is the case in PD-like rats in which such changes are responsible for alterations of motor responses (Bordet et al., 1997; Guillin et al., 2001; Pilla et al., 1999). To test this hypothesis, we used a selective partial agonist, BP 897, as a stabilizing agent, which could limit neurotransmitter ﬂuctuations in either direction, i.e., maintain substantial but blunt excessive receptor stimulations (Bezard et al., 2003). BP 897 was administered to dyskinetic MPTP-intoxicated monkeys in combination with levodopa using a randomized, crossed-over, placebo-controlled design in which the animals were treated repeatedly (4–12 times by each treatment) and evaluated for LID and PD-like symptoms by experimenters unaware of the treatment received (Bezard et al., 2003). BP 897 attenuated LID by 66%

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Fig. 5. A partial D3 receptor agonist, but not D3 receptor antagonists, reduced LID without aVecting the therapeutic eVects of levodopa. LID (a), PD-like symptoms (b), and locomotor activity (c) as a function of time in MPTP-intoxicated monkeys treated with placebo, the D3 receptor partial agonist BP 897 administered at an optimal dosage, or the D3 receptor antagonist nafadotride or ST 198 administered at a minimal dosage. D3 receptor-acting agents significantly aVected both the dyskinesia disability score and the Parkinson disability score over the time course. *P < 0.05 vs placebo. (d) On-time duration measured from locomotor activity counts over a 5-h period of time. * P < 0.05 vs placebo.

(calculated from area under curve, see Fig. 5a), but had almost no inﬂuence on the therapeutic eVect of levodopa, i.e., it did not reverse the improvement of PD-like symptoms (Fig. 2b). Both hyperkinesia (i.e., not true dyskinesia and thus not rated as one) and choreic/athetoid movements were improved. The kinetics of activity counts (Fig. 5c) highlighted this characteristic pharmacological behavior by showing the ability of BP 897 to undermine the levodopa-induced activity around the dyskinesia threshold, deﬁned from a correlation with clinically assessed LID. In addition, the monkey receiving repeated administrations of BP 897 (12 times) did not show any obvious signs of decline of the antidyskinetic eVect along treatments nor reappearance of Parkinson-like symptoms. Nafadotride, a D3 receptor-preferring antagonist, or ST 198, a novel highly D3 receptor-selective

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antagonist (Bezard et al., 2003; Weber et al., 2001), elicited a reduction of LID similar to that obtained with BP 897 (Fig. 5a), which was, however, accompanied by a reappearance of PD-like symptoms (Fig. 5b). We conclude that the worsening of PDlike symptoms is produced by the antagonism of levodopa at the D3 receptor. Results indicate that attenuation of LID, leaving the therapeutic eVect of levodopa intact, can be obtained by administration of a D3 receptor-selective partial agonist.

V. BDNF and Dopamine D3 Receptor in Reactivity to Drug Cues

Environmental stimuli associated previously with drug taking are key factors in drug addiction because they acquire secondary reinforcing properties able to maintain drug-seeking behavior or to induce relapse (Goldberg and Gardner, 1981; O’Brien et al., 1992b). Lesion studies (Whitelaw et al., 1996) in rodents and brain imaging in humans (Childress et al., 1999) have indicated that conditioned eVects of drug involve the mesolimbic dopamine pathway. Moreover, dopamine neuronal activity (Mirenowicz and Schultz, 1996) and dopamine release (Fontana et al., 1993) are induced by an unpredictable presentation of reward-associated cues. Behavioral sensitization (the enhancement of behavioral response to the drug) has been suggested to be partly determined by the association of the drug with a particular context. Such context-dependent behavioral sensitization is considered to take part in the development of compulsive drug-seeking behaviors (Robinson and Berridge, 2001). Hyperresponsiveness to drug-associated cues and context-dependent behavioral sensitization might be related to hypersensitive postsynaptic dopaminergic receptors. Among them, the dopamine D3 receptor appears as a critical target because its highest expression is concentrated in the shell part of the nucleus accumbens, in which dopamine release is preferentially triggered by drugs (Di Chiara and Imperato, 1988; Imperato et al., 1986). Moreover, modulation of the dopamine D3 receptor expression controls behavioral sensitization (Bordet et al., 1997; Guillin et al., 2001), a selective partial agonist inhibits drug-seeking behavior (Pilla et al., 1999), and BDNF, the factor that controls D3 receptor expression, increases the behavioral response and conditioned reward to cocaine (Horger et al., 1999). We have used classical Pavlovian procedures to assess the role of the control of the dopamine D3 receptor by BDNF in the expression of a drug-conditioned response (Le Foll et al., 2002, 2003). We have shown that drug-free mice or rats reexposed to an environment, initially novel and associated repeatedly with cocaine or nicotine administration, which display hyperactivity, have increased dopamine D3 receptor expression in the shell of the nucleus accumbens and no change in the level of dopamine D2 and D1 receptor expression (Figs. 6B, 6C, and 6D) (Le Foll et al., 2002,

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2003). In the same animals, BDNF mRNA was increased in the ventral tegmental area (Fig. 6A), the brain area from which BDNF released in the nucleus accumbens is synthesized (Conner and Lauter, 1997). Interestingly, upregulation of the dopamine D3 receptor did not result directly from repeated cocaine administrations, but from the association of these administrations with a particular environment (Le Foll et al., 2002). Furthermore, we have used two D3 receptor-selective compounds with distinct chemical structures, BP 897 and SB-277011-A, to assess the role of this upregulation in the control of drug cue-conditioned hyperactivity. These two compounds inhibited cocaine or nicotine cue-induced hyperactivity (Figs. 6E, 6F, 6G, 6H, and 6I) (Le Foll et al., 2002, 2003). The eVects of BP 897 were

Fig. 6. (Continued )

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Fig. 6. Cocaine or nicotine cue-conditioned hyperactivity is accompanied by BDNF and D3 receptor overexpression and is inhibited by D3 receptor-selective partial agonist (BP897) or antagonist (SB-277011-A). (A) In situ hybridization signals for BDNF mRNA in the ventral tegmental area of mice treated with cocaine or saline in the paired or unpaired environment. *P < 0.05 vs saline. (B) Autoradiographic signals of D3 receptor binding measured with [125I ]trans-7-OH-PIPAT of rats treated with saline (empty columns) or nicotine (filled columns). D3 receptor bonding was increased significantly in rats exposed to the nicotine-paired environment. *P < 0.05 vs saline in the paired environment. (C and D) In situ hybridization signals for dopamine D1 (D1R), D2 (D2R), or D3 (D3R) receptor mRNA in the shell (AccSh) or the core (AccCo) of the nucleus accumbens and the dorsal striatum (CdPu) of mice treated with cocaine or saline in the paired (D) or unpaired environment (C). (E) EVects of the D3-selective partial agonist BP 897 at increasing indicated dosage. Results are mean SEM of counts for horizontal locomotor activity. *P < 0.05, **P < 0.01 vs cocaine–pretreated mice receiving saline on the test day. (F) Same data as in E for BP 897 at 1 mg/kg, expressed by temporal sets of 5 min. *P < 0.05 vs saline-pretreated mice, xP < 0.05, xxP < 0.01 vs cocaine-pretreated mice. (G) EVect of BP 897 (1 mg/kg) or SB-277011-A (10 mg/kg), a D3 receptor antagonist, administered alone or in combination with cocaine cue-induced hyperactivity. *P < 0.05, **P < 0.01 vs saline-treated animals. (H) EVect of BP 897 (1 mg/kg) on nicotine-conditioned activity. Saline or nicotine-conditioned rats were pretreated before exposure to the paired environment with saline (empty columns) or BP 897 (filled columns). BP 897 inhibited locomotor activity elicited by nicotine-associated cues, but had no eVect on saline-treated rats. *P < 0.01 vs nicotine-conditioned rats receiving saline on the test day. ( I ) EVect of SB-277011-A on nicotine-conditioned activity. SB-277011-A inhibited horizontal locomotor activity induced by nicotine-associated cues at 3 and 10 mg/kg. *P < 0.05, **P < 0.01.

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exerted selectively on drug cue-induced hyperactivity and not on salinepretreated animals (Figs. 6E, 6F, and 6H), indicating the absence of motor eVects that could alter the response (Le Foll et al., 2002, 2003). Moreover, other stereotypical behaviors or modiﬁcation of rearing in mice did not appear concomitantly upon inhibition of cocaine-conditioned hyperactivity, which indicates that the treatment has ablated the locomotor eVects of cocaine cues rather than altering the pattern of the response. We propose that drug cue-induced hyperactivity results at least partly from an upregulation of BDNF in the ventral tegmental area, which in turn induces an upregulation of the dopamine D3 receptor in the nucleus accumbens. The shell of nucleus accumbens also receives innervations from areas highly enriched in BDNF (Conner and Lauter, 1997). These include the hippocampus, lesions of which also downregulate D3 receptor expression (Flores et al., 1996) ( presumably by a mechanism similar to that described here), and the basolateral amygdala, from which BDNF is also transported anterogradely (Conner and Lauter, 1997). These brain areas, and notably the amygdala ( Whitelaw et al., 1996), process conditioned aspects of the environment, such as contextual drug takingassociated cues, the reactivity to which is controlled by the D3 receptor (Pilla et al., 1999). We suggest that, via the aforementioned pathways and mechanisms, progressive changes in BDNF expression occurring during repeated drug taking might alter dopamine responsiveness and induce drug-conditioned responses, a key process in drug addiction (O’Brien et al., 1992a). In agreement, intraaccumbal infusions of BDNF induce enhancement of behavioral sensitization to cocaine and responding for cocaine-related stimuli in rats (Horger et al., 1999) and D3 receptor expression is elevated in accumbens of cocaine addicts (Staley and Mash, 1996). Modulation of drug cue-conditioned activity by selective dopamine D3 compounds suggests that these pharmacological tools could be a novel therapeutic approach to help drug addiction by disrupting conditioning to environmental stimuli (Le Foll et al., 2002, 2003).

VI. BDNF and Dopamine D3 Receptor in Stress, Depression, and Schizophrenia

Several lines of evidence suggest that BDNF could be the target for antidepressant drugs. BDNF mRNA is reduced by stress (Smith et al., 1995) and elevated by chronic treatment with norepinephrine and several antidepressant drugs, including speciﬁc serotonin receptor inhibitors (SSRIs) and electroconvulsive therapy (ECT) (Nibuya et al., 1995). Furthermore, a single bilateral infusion of BDNF into the dentate gyrus of the hippocampus is eYcacious in two behavioral models predictive for the antidepressant eVect: learned helplessness and forced swim test

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(Shirayama et al., 2002). Clinical evidence also supports a role of BDNF in depression. Hippocampal BDNF immunoreactivity was shown to be increased in postmortem brains of mood disorder patients treated with antidepressant medication at the time of death versus antidepressant-untreated subjects (Chen et al., 2001), and serum levels of BDNF are decreased in antidepressant-untreated depressed patients versus antidepressant-treated patients (Shimizu et al., 2003). Chronic treatment with antidepressant drugs in rats produces a variety of changes in dopaminergic neurotransmission, most notably a sensitization of behavioral responses to agonists acting at dopamine D2/D3 receptors within the nucleus accumbens ( Willner, 1997). Similar results have been obtained after chronic ECT (Barkai et al., 1990; Nomikos et al., 1991; Smith and Sharp, 1997). Hence, we asked whether these changes in dopamine responsiveness could be attributable to changes in D3 receptor expression. We found that D3 receptor binding or mRNA expression in the shell of nucleus accumbens was decreased by stress but increased after chronic, but not acute, antidepressant treatments, including monoamine uptake inhibitors, a monoamineoxidase inhibitor, and ECT (Lammers et al., 2000). These results suggest that enhanced dopamine neurotransmission through this receptor participates in the adaptive changes leading to antidepressant activity. In conclusion, these results suggests that D3 receptor expression and function are downregulated in stress and, possibly, depression, secondary to the downregulation of BDNF in hippocampus, and that these changes are reversed by antidepressant treatments. Schizophrenia, assumed to result from a neurodevelopmental disorder, is marked by neuroanatomical abnormalities, such as ventricle enlargement ( Weinberger, 1987) possibly related to a defect in neuroepithelium proliferation. A role for the D3 receptor in this pathological process might be inferred from the selective expression of this receptor in the neuroepithelium during the prenatal period (Diaz et al., 1997) and is supported by genetic studies (Dubertret et al., 1998; Williams et al., 1998). In addition, at later stages of development and in adulthood, D3 receptor expression in neurons is controlled positively by BDNF, the level of which is elevated in the cortex of patients with schizophrenia (Takahashi et al., 2000). This may explain the overexpression of the D3 receptor found in these patients (Gurevich et al., 1997).

VII. Conclusions

The regulatory mechanism controlling D3 receptor expression diVers markedly from that occurring in various neurotransmission systems, in which the level of receptor density and sensitivity is controlled primarily by the endogenous ligand (Laufer and Changeux, 1989; Schwartz et al., 1978). These observations,

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together with those showing that BDNF modulates synaptic transmission, strengthen the view that BDNF elicits adaptive changes in cerebral neurotransmission, demonstrating a role of this neurotrophin in the plasticity of dopaminergic neurons. Modulation of dopamine responsiveness by BDNF could be an important determinant in the etiopathology and/or treatment of several conditions implicating dopamine. Thus, the progressive treatment-dependent elevation of corticostriatal BDNF, which may occur in Parkinson’s disease, as it does in 6-OHDA-lesioned rats, is a previously unraveled mechanism possibly accounting for the enhanced response to levodopa that underlies its initial beneﬁcial eVect, but leads to the abnormal movements often present in long-term treated patients. The eVects of drug-induced cues are crucial for relapse in humans and probably reﬂect the progressive acquisition by these cues of an aVective valence, often thought to be associated with incentive motivation properties (O’Brien et al., 1992a,b). Overexpression of the dopamine D3 receptor elicited by BDNF could be one of the molecular mechanisms by which environmental stimuli are associated with drug taking. More generally, the control of dopamine D3 receptor expression by BDNF could aVect the dopamine-mediated response, which indicates that BDNF has a role in the plasticity of the mesolimbic dopamine pathway.

Acknowledgments

We thank V. Mignon for technical assistance. O.G. received grants from Fondation pour la Recherche Me´dicale and Lundbeck Foundation. This work was supported by grants from the European Commission (Fifth Framework Programme) and the National Institute on Drug Abuse.

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S100B IN SCHIZOPHRENIC PSYCHOSIS

Matthias Rothermundt, Gerald Ponath, and Volker Arolt Department of Psychiatry, University of Muenster D-48149 Muenster, Germany

I. Background and Hypotheses II. Origin and Molecular Structure of S100B III. In Vitro/Cell Culture Experiments A. Target Proteins B. Cytoskeleton Modification C. Protective Properties D. Deleterious EVects E. Interaction between S100B and the Cerebral Immune System IV. Morphology Studies (Animal Experiments) A. Induced Brain Lesions B. EVects of Lifelong S100B Overproduction and Aging V. Serotonergic Regulation VI. Functional Studies VII. Clinical Studies in Schizophrenic Patients VIII. Conclusions References

Recent ﬁndings have strengthened the hypothesis that a dysfunction of neuronal synapses and dendrites is relevant for the pathogenesis of schizophrenia. It might be present during neurodevelopment as well as in degenerative and regenerative processes of the mature brain. S100B, a small, Ca2+-binding, astrocytic protein, plays an important role in modulating the proliferation and diVerentiation of neurons and glia cells. It is involved in the regulation of cellular energy metabolism and interacts with many immunological functions of the brain. This review addresses ﬁndings from cell cultural and animal experiments potentially pertinent for the pathogenesis of schizophrenic psychoses. Morphological and functional data are analyzed and clinical studies reporting alterations of S100B concentrations in schizophrenic patients are reviewed. Evidence and limitations of the available studies are pointed out and promising future research strategies are outlined.

I. Background and Hypotheses

For many years, two major hypotheses on the pathogenesis of schizophrenia have been discussed. The neurodevelopmental hypothesis suggests that schizophrenia is INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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a disorder based on disturbances in the early development of neurons and glial cells starting in the second trimenon of intrauterine life. It is supported by several pieces of evidence: (a) the presence of minor physical anomalies; (b) the presence of neurological, cognitive, and behavioral dysfunction long before illness onset; (c) a course and outcome of the illness itself that is predominantly incompatible with a classical degenerative disorder; (d) the presence of ventricular enlargement and decreased cortical volume at onset of symptoms, if not earlier; (e) the presence and nature of cytoarchitectural abnormalities (such as neuron density, number and morphology, dendritic arbors and spines, synapse-related proteins); and (f) the absence of postmortem evidence of neurodegeneration comprising gliosis as a sequelae of a classic degenerative mechanism (Arnold, 1999; Harrison, 1999; Marenco and Weinberger, 2000). The second and for a long time opposing approach was called the neurodegenerative hypothesis. More than 100 years ago Kraepelin and Alzheimer were convinced that schizophrenia was an organic disease involving the destruction of neural tissue. This hypothesis fell behind when researchers failed to consistently demonstrate a destruction of neurons and glial cells or gliosis as a consequence of a passed neurodegenerative process. The implementation of imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI), as well as improved methodologies and new techniques in postmortem studies, however, has conﬁrmed that neurodegeneration is present in at least a subgroup of schizophrenic patients. There are convincing data showing the progression of cerebral volume reduction during the course of disease (DeLisi, 1999; Gur et al., 1998; Pearlson and Marsh, 1999; Shenton et al., 2001). A classic neurodegenerative mechanism involving the loss of neurons and development of gliosis, however, appears unlikely to be relevant for schizophrenia. Brains from patients who had suVered from schizophrenia mostly show unchanged numbers of neurons but a reduction of neuronal cell size and neuropil that accounts for the loss of brain volume (Harrison, 1999; McGlashan and HoVman, 2000; Powers, 1999; Selemon and Goldman-Rakic, 1999). A reduction of neuropil involves compromised cell structure and a decrease of neuronal connectivity, resulting in a presumed loss of functional communication between neurons. Beyond that, several studies reported changes in synaptic proteins and their gene expression (Glantz and Lewis, 2000; Harrison and Eastwood, 2001; Karson et al., 1999; Kung et al., 1998; Mirnics et al., 2001). These ﬁndings support a diminution or dysfunction of dendrites, neurites, and synapses in schizophrenia. Several authors have endeavored to integrate the neurodevelopmental and the neurodegenerative aspects into a comprehensive pathogenetic hypothesis of schizophrenia. Lieberman and colleagues (1997) postulated that an initial developmental insult to the central nervous system (CNS) (stage 1) does not result in psychosis until a combination of developmental and dysregulatory events occur causing neurochemical sensitization (stage 2), which can then lead to the formal onset of

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illness, which, if persistent or recurrent, can progress to a self-limiting degenerative phase (stage 3), manifested by persistent morbidity, treatment resistance, and clinical deterioration. Bartzokis (2002) based his integrative view on the assumption that the brain is in a constant state of change in development and maturation continuing into middle age followed by degeneration. From adolescence until middle age the brain normally is in a constant state of well-regulated structural and functional change. Multiple genetic and environmental factors interfere with the developmental process, resulting in a dysregulation of the complementary changes occurring in gray and white matter. This dysregulation in development results in an insuYcient capacity to maintain the temporal synchrony of neural networks. Bartzokis stressed the contributory role of myelination to synchronous brain function. Woods (1998) considered schizophrenia a progressive developmental disorder. Developmental mechanisms such as apoptosis and pruning continue to go awry over many years without resulting in excessive gliosis. Neuropathological ﬁndings implicating prenatal pathology and the imaging ﬁndings of excessive extracerebral CSF space that must have resulted from brain volume loss occurring after brain volume expansion was complete could be explained by this hypothesis. Based on gene expression patterns discovered via microarray technology, Mirnics and colleagues (2001) proposed that schizophrenia is a disease of the synapse. Defects in the gene expression of presynaptic secretory pathway elements (PSYN group and G-protein regulators) might aVect the extended postnatal developmental process of synapse formation and pruning. Impaired synaptic transmission in schizophrenic patients is present probably in a subset of synapses from early life, but the exuberant synaptic connections that form during the third trimester of gestation and early childhood compensate for a functional synaptic curtailing. The exuberant synapses are pruned by late adolescence, uncovering the existing synaptic impairment. Inadequate synaptic activity might then lead to overpruning and manifestation of the symptoms of schizophrenia. The ﬁnding that neuregulin was identiﬁed as a candidate gene for schizophrenia (Stefansson et al., 2002, 2003) provides additional support for the synapse hypothesis. Neuregulin is a peptide involved in the regulation of proliferation and diVerentiation in neurons and glia. It mediates between electrical neural activity and molecular components by regulating the expression of ion channel receptors or transmitter release in synapses. Neuregulin is a key molecule regarding dynamic changes in the synapse, as it is involved in the coordination of excitatory and inhibitory neurons (Ozaki, 2001). Neurodevelopmental as well as neurodegenerative processes always aVect synapses and dendrites. Synapses are constantly set up and removed throughout the whole life of an individual with varying intensity depending on genetic and environmental factors. Focusing on the molecular mechanisms that regulate the plasticity of dendrites and synapses therefore oVers a promising approach to

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improve the understanding of pathophysiological processes in schizophrenia. In this context it is indispensable not to conﬁne oneself to the investigation of neuron pathology, but rather include the interaction between neurons and glial cells. Research has approved the decisive role of glial cells in processes involved in neuroplasticity. The astrocytic protein S100B has a major impact on degenerative and regenerative mechanisms in neuronal and glial cells. It evolves autocrine and paracrine action balancing proliferation and diVerentiation of neurons and astrocytes. Molecular, cellular, and animal experiments have been undertaken to clarify the structure and function of S100B. In addition, clinical investigations have helped in understanding the importance of this protein for the pathophysiology of schizophrenia. This chapter reviews the scientiﬁc ﬁndings on S100B that are relevant for the pathogenesis of schizophrenia and outlines future research perspectives.

II. Origin and Molecular Structure of S100B

S100B is a member of the S100 family. This family of proteins was termed ‘‘S100’’ because it was soluble in 100% saturated ammonium sulfate solution (Moore, 1965). S100B is an acidic protein with a molecular mass of 21 kDa existing as a homodimer consisting of two subunits. The two monomers are conﬁgured in a twofold axis of rotation and are held together by disulﬁde bonds (Beaudeux et al., 1999; Drohat et al., 1996, 1998; Kilby et al., 1996; Matsumura et al., 1998; Smith et al., 1998). The disulﬁde-linked form of S100B appears to be required for fully functional neurotrophic action (Winningham-Major et al., 1989), whereas S100B mutants lacking one or both of the cysteine residues induce the activation response in glia but not in neurons (Koppal et al., 2001). S100B is a Ca2+-binding protein of the EF hand type (helix–loop–helix) with four Ca2+-binding sites (Heizmann, 1999; Isobe and Okuyama, 1978). It also binds Cu2+ at four binding sites (Nishikawa et al., 1997) and Zn2+ at six to eight binding sites (Donato, 1991; Heizmann, 1999; Scha¨fer and Heizmann, 1996; Zimmer et al., 1995), and such binding inﬂuences the Ca2+-binding capacity of the protein (Heizmann and Cox, 1998). The gene encoding S100B in humans is located on chromosome 21q22.3 (Allore et al., 1988) at a distance of 100–140 kb from the chromosome terminus (Reston et al., 1995). S100B is produced primarily by astrocytes and exerts autocrine and paracrine eVects on glia, neurons, and microglia (Adami et al., 2001; Vives et al., 2003). Therefore, S100B can be found in all three cell types. However, so far it is unknown whether neurons and microglia cells expressing S100B secrete the protein, and the mechanism of secretion has not been identiﬁed. It is thought to be released from glial cells via a mechanism similar to that governing the secretion or release of other factors, such as ciliary

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neurotrophic factor, interleukin-1a and 1b, or human endothelial growth factor (Barger et al., 1992). S100B is signiﬁcant for the development of many disorders and clinical conditions aVecting the brain (for a review see Rothermundt et al., 2003). This review focuses on the signiﬁcance of S100B in schizophrenia. A summary of ﬁndings from in vitro and in vivo animal experiments pertaining to the pathophysiology of schizophrenia is given, followed by a presentation of clinical S100B ﬁndings in schizophrenic patients.

III. In Vitro/Cell Culture Experiments

Many target proteins and mechanisms of action of S100B have now been identiﬁed from cell culture and in vitro experiments. S100B is involved in the regulation of energy metabolism in brain cells. It modulates the proliferation and differentiation of neurons and glia. Furthermore, it interacts with many immunological functions of the brain. Quite clearly, S100B exerts a proliferative eVect as long as it is kept within the cells at physiological levels. However, once it is secreted or released, its local concentration then dictates its beneﬁcial or detrimental eVects. Nanomolar concentrations appear to exert neuroprotective eVects, whereas micromolar concentrations produce neurodegenerative or apoptosis-inducing eVects.

A. Target Proteins The major intracellular function of S100B is to block the phosphorylation of protein kinase C (PKC) target proteins in a Ca2+ concentration-dependent manner (Table I). S100B inhibits the phosphorylation of several neuronal proteins, which are involved in transmitter release, ion channel modulation, and plasticity such as the myristolated alanine-rich C kinase substrate (MARCKS) (Albert et al., 1984; Sheu et al., 1995), MARCKS-like retinal phosphoprotein p80 (Pozdnyakov et al., 1998), GAP-43 (neuromodulin) (Lin et al., 1994; Sheu et al., 1994, 1995), and neurogranin (Sheu et al., 1995). S100B also inhibits the phosphorylation by PKC of proteins involved in cytoskeleton formation such as glial ﬁbrillary acidic protein (GFAP) (Bianchi et al., 1994), vimentin (Ziegler et al., 1998), annexin VI (Garbuglia et al., 2000), caldesmon (Polyakov et al., 1998; Skripnikowa and Gusev, 1989), proteins (Baudier et al., 1987, 1988), actin capping protein CapZ (Ivanenkov et al., 1995; Kilby et al., 1997), and p53 (Baudier et al., 1992; Lin et al., 2001; Rustandi et al., 1998; Scotto et al., 1998; Wilder et al., 1998). However, for most of these proteins the functional correlates remained

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TABLE I S100B Protein-Dependent Inhibition of Protein Phosphorylationa Target protein

Functional correlates (in vitro)

Reference

Annexin VI

Blocks the ability of S100B to inhibit intermediate filament assembly

Garbuglia et al. (2000)

Caldesmon

Reversal of caldesmon-dependent inhibition of actomyosin ATPase activity

Pritchard and Martson (1991); Skripnikowa and Gusev (1989)

GAP-43 (neuromodulin)

Unknown

Lin et al. (1994); Sheu et al. (1994, 1995)

GFAP, vimentin

Inhibits intermediate filament assembly

Ziegler et al. (1998)

MARCKS MARCKS-like retinal phosphoprotein p80

Unknown

Albert et al. (1984); Pozdnyakov et al. (1998); Sheu et al. (1995)

Neurogranin

Unknown

Sheu et al. (1995)

p53

Protection of p53 from thermal denaturation and aggregation, stimulation of p53-dependent cell growth arrest and apoptosis, inhibition of p53-dependent transcription activation via disruption of the p53 tetramer

Baudier et al. (1992); Rustandi et al. (1998); Scotto et al. (1998); Wilder et al. (1998);

proteins

Unknown

Baudier et al. (1987, 1988)

a

Modified from Donato (2001).

unknown until now. Available data suggest that S100B may play a role in signal transduction by linking the elevation of the intracellular calcium concentration to the phosphorylation state of the target protein. This could represent a means to regulate speciﬁc steps in the signaling pathways in which the respective target protein is involved (Donato, 2001). S100B appears to be involved in the regulation of the energy metabolism of brain cells (Table II), as it has been shown to stimulate the enzymatic activity of fructose-1,6-bisphosphate aldolase (Zimmer and Van Eldik, 1986) and phosphoglucomutase (Landar et al., 1996). The activity of other enzymes such as twitchin kinase (Heierhorst et al., 1996), Ndr (Millward et al., 1998), and membrane-bound guanylate cyclase (GC) (Duda et al., 1996; Margulis et al., 1996; Pozdnyakov et al., 1997, 1998; Rambotti et al., 1999) is also increased by S100B. Ndr is involved in regulation of the cell cycle and membrane-bound GC in the dark adaptation of photoreceptors. The function of the stimulatory eVect of S100B on twitchin kinase remains to be clariﬁed. With exception of membrane-bound GC, there is

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TABLE II S100B Protein-Dependent Regulation of Enzyme Activitya Enzyme

Effect

Suggested function

Fructose-1, 6-bisphosphate aldolase

Stimulation

Regulation of energy metabolism

Zimmer and Van Eldik (1986)

Membrane-bound guanylate cyclase

Stimulation

Dark adaptation of photoreceptors

Duda et al. (1996); Margulis et al. (1996); Pozdnyakov et al. (1997, 1998); Rambotti et al. (1999)

Phosphoglucomutase

Stimulation

Regulation of enery metabolism

Landar et al. (1996)

a

Reference

Modified from Donato (2001).

no information at present about the localization of sites on S100B protein that recognize these enzymes or their regulatory proteins (Donato, 2001).

B. Cytoskeleton Modification S100B inﬂuences the integrity of the cytoskeleton (Table III). It inhibits the assembly of microtubules via the sequestration of tubulin and stimulation of the Ca2+ sensitivity of preformed microtubules (Donato, 1985, 1988; Sorci et al., 1998). It also inhibits the assembly and stimulates the disassembly of type III intermediate ﬁlaments (IF) via the sequestration of unassembled intermediate ﬁlament subunits (Bianchi et al., 1993, 1994; Garbuglia et al., 1996). Astrocytes in the brain of mutant mice expressing a much reduced amount of S100B exhibit larger amounts of GFAP intermediate ﬁlaments, a ﬁnding that has been put in relation to the ability of S100B to inhibit GFAP IF assembly (Ueda et al., 1994a,b). S100B reverses the caldesmon- and calponin-dependent inhibition of actomyosin ATPase activity (Fujii et al., 1994; Pritchard and Martson, 1991; Skripnikowa and Gusev, 1989; Wills et al., 1993).

C. Protective Properties Secreted glial S100B exerts trophic or toxic eVects depending on its concentration. At nanomolar concentrations, S100B in vitro stimulates neurite outgrowth in cerebral cortex neurons and dorsal root ganglia of the embryonic chick (Van Eldik et al., 1991; Winningham-Major et al., 1989) and enhances the survival of neurons in various systems (neurons, dorsal root ganglia, and Schwann cells from

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TABLE III S100B Protein-Dependent Cytoskeleton Modificationsa Cytoskeleton element

Suggested function

Reference

Caldesmon

Reversal of caldesmon-dependent inhibition of actomyosin ATPase activity

Fujii et al. (1994); Prichard and Martson (1991); Skripnikowa and Gusev (1989)

Calponin

Reversal of calponin-dependent inhibition of actomyosin ATPase activity

Wills et al. (1993)

Type III intermediate filaments

Inhibition of assembly and stimulation of disassembly via sequestration of unassembled intermediate filament subunits

Bianchi et al. (1993, 1994); Garbuglia et al. (1996)

Astrocyte specific type III intermediate filaments

Inhibition of GFAP IF assembly

Ueda et al. (1994a,b)

Microtubules

Inhibition of assembly via sequestration of tubulin and stimulation of Ca2+ sensitivity of preformed microtubules

Donato (1985, 1988); Sorci et al. (1998)

a

Modified from Donato (2001).

embryonal chicks; primary astrocytes from newborn rats; C6 glioma cells) during development (Bhattacharyya et al., 1992; Ueda et al., 1995; Van Eldik et al., 1991; Whitaker-Azmitia et al., 1990) and after damage from glucose deprivation in hippocampal neurons from rats (Barger et al., 1995). Concentrations of S100B greater than 100 nM cause the expression of the proinﬂammatory cytokine interleukin-6 (IL-6) in fetal rat cortical neurons and its secretion by neurons via the activation of nuclear factor B (NF-B) (Li et al., 2000). These ﬁndings suggest that S100B may be a neurotrophic factor during development and nerve regeneration. S100B decreases cell death and the loss of mitochondrial function resulting from glucose deprivation in hippocampal neurons from rats (Barger et al., 1995) and protects neurons (from embryonic chick and neonatal rat) against glutamateand staurosporin-induced damage in vitro (Ahlemeyer et al., 2000). Colchicininduced apoptosis of N18 neuroblastoma cell cultures is prevented by the addition of S100B (Brewton et al., 2001). In chick embryonal neurons, transfected N18 mouse neuroblastoma, and C6 rat glioma cells, the prosurvival activity of extracellular S100B and the ability of this protein to stimulate neurite outgrowth appear to depend on the nuclear translocation of NF-B and upregulation of the expression of the antiapoptotic factor Bcl-2 in target neurons (Alexanian and

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Bamburg, 1999; Huttunen et al., 2000). This mechanism of action reported in transfected cells apparently depends on the binding of S100B to the receptor for advanced glycation end (RAGE) products (Huttunen et al., 2000). However, it remains to be shown whether S100B can also interact with endogenous RAGE or whether this interaction is limited to transfected cells. Furthermore, it is so far unclear whether S100B depends on RAGE to evolve its action or whether other pathways exist in parallel. In addition to its paracrine eVects on neurons, nanomolar levels of S100B exert an autocrine eVect on astrocytes so that glial proliferation is stimulated in rat C6 glioma cells in vitro (Selinfreund et al., 1991). Extracellular S100B stimulates the phosphorylation of ERK1/2 in primary neonatal rat astrocytes (Goncalves et al., 2000), a ﬁnding consistent with the stimulatory eVect of S100B on astrocyte proliferation.

D. Deleterious Effects Micromolar levels of extracellular S100B in vitro may have deleterious eVects. Exposure to S100B was shown to increase the expression of -amyloid precursor protein and its mRNA in neuronal cultures (Li et al., 1998). However, a chronic elevation of S100B is not suYcient for inducing the formation of -amyloid plaques in the mouse brain (Yao et al., 1995). -Amyloid stimulates the synthesis of both S100B mRNA and S100B protein in astrocyte cultures (Pena et al., 1995). On this basis, secreted S100B is currently viewed as a cytokine, which, like interleukin-1 (IL-1) (Brenneman et al., 1992), promotes neuronal survival at low concentrations, but might be hurtful at high levels. However, in vivo experiments are needed to prove the relevance of these mechanisms in a more complex setting. S100B induces apoptosis in PC12 cells, B104 neuroblastoma cells, and primary embryonic rat neurons in vitro (Hu et al., 1997; Mariggio et al., 1994). In transfected cells, micromolar concentrations interact with RAGE and cause an elevation of reactive oxygen species, cytochrome C release, and activation of the caspase cascade (Huttunen et al., 2000). Bcl-2 is then downregulated, a ﬁnding that is consistent with the observation that downregulation of Bcl-2 is necessary for S100B to cause neuronal apoptosis in human neuronal precursor NT2/D1 cells (Wang et al., 1999). S100B induces the expression of inducible nitric oxide synthase (iNOS) mRNA and elevation of its activity in primary rat neonatal astrocytes (Hu et al., 1996; Petrova et al., 2000). Cocultured neurons were seen to die, probably due to NO diVusion (Hu et al., 1997). S100B-induced apoptosis in PC12 cells has also been attributed to a S100B–dependent increased conductance in L-type Ca2+ channels (Mariggio et al., 1994), as well as upregulation of a set of genes implicated in apoptosis (c-fos, c-jun, bax, bcl-x, p-15, and p-21) (Fulle et al., 2000).

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E. Interaction between S100B and the Cerebral Immune System Several environmental factors potentially accounting for the pathogenesis of schizophrenia involve the immune system. Intrauterin or postnatal infections with neurotropic viruses or the activation of an endogenous retrovirus activate parts of the immune system. Furthermore, some authors hypothesize that an autoimmune mechanism might contribute to pathogenic mechanisms in schizophrenia (for a review, see Rothermundt et al., 2001a). Therefore, the interface between S100B and the cerebral immune system should be considered. Extracellular S100B causes neuronal expression and secretion of IL-6 by inducing the nuclear translocation of N-BF in fetal rat cortical neurons (Li et al., 2000). S100B in micromolar concentrations synergizes with interferon- in stimulating NO secretion from cultured microglia cells (Adami et al., 2001; Petrova et al., 2000), suggesting that S100B might contribute to neuropathological changes in the course of neurodegeneration and/or brain inﬂammatory diseases by the activation of microglia as well. Interferon- causes a downregulation of S100B mRNA expression in BV2 microglial cells; treatment of BV2 cells with S100B results in increased IFN-induced expression of iNOS mRNA and secretion of NO (Adami et al., 2001). Although the BV-2 microglial cell line expresses RAGE (Hori et al., 1995), no correlation between S100B binding to RAGE and stimulation of microglial iNOS and/or release of cytokines by microglia has been presented. Treatment of astroglia cultures from the adult human cerebral temporal lobe with interleukin-1 increases the content of S100B and decreases GFAP (Davies et al., 2000). Dexamethasone causes a biphasic response of S100B mRNA in rat neonatal hippocampal astrocytes. After 24 h S100B mRNA was increased signiﬁcantly, whereas after 96 and 120 h there was a decrease. Concerning S100B protein concentrations, there was no increase after 24 h, but a decrease corresponding to mRNA levels after 96 and 120 h (Niu et al., 1997).

IV. Morphology Studies (Animal Experiments)

A. Induced Brain Lesions After unilateral lesioning of the entorhinal cortex in rats, McAdory and colleagues (1998) observed an upregulation of S100B in astrocytes of the ipsilateral dentate gyrus during sprouting and reactive synaptogenesis. Hinkle et al. (1997) reported an increased bilateral S100B mRNA expression 24 h after mild unilateral cortical contusion in rats in the cortex and hippocampus. Cerebrospinal ﬂuid (CSF) S100 showed peak concentrations 7.5 and 60 h after traumatic cortical injury or focal cerebral ischemia in rats (Hardemark et al., 1989a). Serum

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S100B was shown to be increased between 0 and 24 h after severe cortical injury in rats, a value that returned to normal after 48 h. In this experiment, S100B serum levels did not reﬂect the severity of the trauma (Rothoerl et al., 2000a). The increased S100B protein levels in CSF and serum could be caused by an active secretion of S100B or the consequence of astrocytic destruction with the release of S100B into the extracellular space due to membrane leakage. In cats, Li and colleagues (1998), however, detected no change in S100 expression within 6 h after severe brain trauma. An infusion of anti-S100B in rat ventricles resulted in a signiﬁcant decrease in synapse number (Wilson et al., 1998).

B. Effects of Lifelong S100B Overproduction and Aging Friend and colleagues (1992) established a S100B transgenic mouse. Four transgenic lines carried between 8 and 110 copies of the human S100B gene. The number of gene copies correlated with the expression of human S100B and no diVerences between male and female animals were detected. Tissue and cell speciﬁcity were maintained. These animals showed an increased staining of MAP-2, which suggests an increased rate of dendritic maturation among such mice. As the transgenic animals get older, the increase in MAP-2 staining compared to normals is no longer evident. A trend toward dendritic loss is then observed (Whitaker-Azmitia et al., 1997). In such transgenic mice, signs of accelerated development followed by premature aging were also demonstrated. The animals showed a loss of dendrites and an increase in cells revealing cell body staining indicative of the presence of neuroﬁbrillary tangles and cytoskeletal collapse. Adult animals of another strain of transgenic mice with S100B RNA production two- to sevenfold above normal (two transgenic lines, correlation between gene copies and S100 mRNA expression, tissue and cell speciﬁcity maintained) demonstrated increased levels of axonal sprouting markers, including neuroﬁlament L, phosphorylated epitopes of neuroﬁlament H and M, and -tubulin. Immunocytochemistry displayed alterations in astrocyte morphology and axonal sprouting, especially in the dentate gyrus (Reeves et al., 1994). Aging is clearly associated with an increased expression of S100B protein and mRNA in rats (Kato et al., 1990). In senescence-accelerated mice, S100B protein and mRNA are increased signiﬁcantly (GriYn et al., 1998a). Homozygous APPV717F transgenic mice overexpressing a human -amyloid precursor protein minigene encoding a familial Alzheimer’s disease mutation show an increased expression of S100B and its mRNA (Sheng et al., 2000). However, a chronic elevation of S100B does not alter APP mRNA expression or promote -amyloid deposition in the brains of aging transgenic mice (Reeves transgenic mouse; Reeves et al., 1994) with a lifelong overproduction of S100B (Yao et al., 1995).

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V. Serotonergic Regulation

Convincing evidence from in vitro and in vivo animal studies shows that serotonin is involved in the regulation of cellular S100B release via the 5-HT1a receptor. As shown in cell culture studies, 5-HT1a agonists stimulate S100B release from primary rat neonatal astrocytes (Whitaker-Azmitia et al., 1990). Application of a 5-HT1a agonist or S100B to hippocampal neurons from adult mice increases synaptophysin reactivity (Nishi et al., 1996). S100B antisense mRNA was shown to block the serotonergic nerve sprouting of hippocampal neurons into transplanted C6 glioma cells induced by S100B (Ueda et al., 1995). In S100B knockout mice, however, the distribution of serotonergic ﬁbers and the content of serotonin were comparable to that of wild-type controls (Nishiyama et al., 2002b). This indicates that even though S100B is regulated via serotonin and evolves some inﬂuence on serotonergic ﬁbers, it is apparently not essential for the neurite extension of serotonergic neurons. These ﬁndings from cell culture experiments are supplemented by the following results from in vivo animal experiments. (1) In adult animals, lesioning of 5-HT nerve terminals by parachloramphetamine (PCA) causes a reduction of S100B in various brain regions (parietal and temporal cortex, temporal pole, hippocampus, and hypothalamus) in rats. This results in a signiﬁcant decrease of MAP-2, a marker for dendritic integrity that is also involved in the modulation of cytoskeleton formation (Whitaker-Azmitia et al., 1995). (2) Application of a 5-HT1a antagonist during synaptogenesis leads to decreased synaptic density (Mazer et al., 1997; Wilson et al., 1998). (3) The loss of S100B can be reversed by treatment with a 5-HT1a agonist (Azmitia et al., 1995). (4) Also, an increase of serotonin levels by ﬂuoxetine administration increased S100B concentrations (Haring et al., 1993; Manev et al., 2001). (5) Ethanol exposure in utero was shown to impair the development of serotonergic neurons in fetal rats by a reduction of S100B. Addition of a 5-HT1a agonist prevented the ethanol-induced decrease of S100B (Eriksen et al., 2000). (6) In adrenalectomized rats, the immunoreactivity of S100B in the dentate gyrus is also reduced dramatically. These eVects were reduced within 72 h by ipsaspirone, a 5-HT1a receptor antagonist (Huang et al., 1997). In schizophrenia it is hypothesized that the development of cognitive symptoms might partly be attributed to a dysregulation of 5-HT1a receptors. The receptor proﬁle of newer antipsychotic medications (quetiapine, ziprasidone, aripiprazole) includes the 5-HT1a receptor in addition to dopaminergic and other serotonergic receptors. There is hope that a 5-HT1a agonistic action might have a positive inﬂuence on the cognitive deﬁcits of schizophrenic patients. A change in the S100B concentration induced by 5-HT1a stimulation might contribute to this eVect.

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VI. Functional Studies

Considerable eVort was undertaken to characterize the functions of S100B in the brain using in vitro and in vivo experiments. Hippocampal long-term potentiation (LTP) is thought to be a physiological correlate of long-term memory (Swanson et al. 1982). It is a long-lasting enhancement of synaptic eYcacy evoked by repetitive aVerent stimulation. Because LTP has been shown to be a calcium-dependent process, it appears reasonable to consider that S100B might be involved in the regulation of that process. Lewis and Teyler (1986) were able to show that after addition of anti-S100 antibodies to rat hippocampal brain slices, a repetitive stimulation of the aVerents failed to produce LTP. Gerlai and colleagues (1995) demonstrated an overall decreased LTP in hippocampal slices from two diVerent strains of S100B transgenic mice carrying 8 and 70 copies of the human S100 transgene (Friend transgenic mouse; Friend et al., 1992). A more detailed analysis showed that an increase of long-term depression (LTD) was responsible for the decreased LTP. Nishiyama and colleagues (2002a) demonstrated increased LTP in S100B knockout mice resulting in enhanced spatial and fear memory. Perfusion of hippocampal slices with recombinant S100B proteins reversed the levels of LTP in the mutant slices to those of the wild-type slices. Gromov and colleagues (1992) showed that formation of an alimentary-conditioned reﬂex is accompanied by an increase of S100 in rat brains. The intracisternal administration of S100 antiserum led to a disruption of the developed skill, as was also shown by Karpiak et al. (1976) for a maze-learning task. O’Dowd and colleagues (1997) demonstrated that the application of S100B antiserum in chicks before or immediately after training on an avoidance task caused amnesia. Infusion of S100B to the hippocampus of rats immediately after training in a step-down inhibitory avoidance task (footshock) facilitated long-term memory in a dose-dependent manner (Mello e Souza et al., 2000). S100B transgenic mice with multiple copies of the human S100B gene (Friend transgenic mouse, mainly strains 5 and 8 with 8 and 70 copies of the human S100; Friend et al., 1992) showed a range of defects such as femalespeciﬁc hyperactivity, lack of habituation to novelty, and a reduced T-maze spontaneous alternation rate. These dysfunctions resemble behavioral manifestations of hippocampal dysfunction (Gerlai and Roder, 1993, 1995; Gerlai et al., 1993, 1994). These mice also appear to have impaired short-term memory, as they demonstrate diVerences in spatial and temporal exploratory pattern compared to controls (Roder et al., 1996a). In the water maze they show normal memory function. However, a single alteration of an environmental stimulus (water temperature) signiﬁcantly attenuated the performance of the transgenic mice. According to Roder et al. (1996b), this might demonstrate

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a hippocampal dysfunction, as brain temperature has been shown to be a factor in hippocampal-dependent learning. In another experiment, Gerlai and Roder (1996) performed cognitive tasks on young and old S100B transgenic mice (Friend transgenic mouse, strains 5 and 8 with 8 and 70 copies of the human S100; Friend et al., 1992). Threemonth-old mice showed an impairment in a spatial task reﬂecting a hippocampal dysfunction, whereas 16-month-old mice were statistically indistinguishable from their normal counterparts. This result does not support a progressive eVect of increased S100B. However, they also showed that age, independently of the transgene, impairs spatial learning, spares nonspatial learning and reference memory, but leads to behavioral rigidity. Others demonstrated a dementia-like cognitive proﬁle regarding hippocampus-based cognitive functions in S100B transgenic mice (Friend transgenic mouse strain 8 with 70 copies of human S100; Friend et al., 1992), which included impaired spatial and nonspatial memory or learning deﬁcits (Whitaker-Azmitia et al., 1997; Winocur et al., 2001). Both memory qualities are identiﬁed with hippocampal function but represent quite diVerent memory processes and serve diVerent purposes. The behavioral characterization of S100B knockout mice with no production of S100B is insuYcient so far. They were shown to be viable, fertile, and did not exhibit overt behavioral abnormalities up to 12 months of age (Xiong et al., 2000). Studies focusing on cognitive tasks in S100B null mice have not been published yet. These ﬁndings from functional studies indicate that S100B is involved in cognitive functions such as spatial and nonspatial memory and learning. Apparently, a balanced concentration of S100B in the synapses and the extracellular space is needed for normal memory and learning abilities. Too much or too little S100B disturbs the process of acquiring and recalling information.

VII. Clinical Studies in Schizophrenic Patients

The ﬁrst study concerning S100 in schizophrenia was originally designed to investigate antibodies against brain tissue in these patients. S100 was used as a marker for brain cells (neurons and astrocytes). Jankovic and colleagues (1980, 1982) performed tests on local Arthus and delayed hypersensitivity reactions against S100 in schizophrenic patients. About 75% of schizophrenic patients showed positive reactions compared to 3–7% of normal controls. On the basis of their ﬁndings, the authors hypothesized that T-cell immunity might be involved in the development of psychiatric disorders. However, these ﬁndings may indicate either that an increased permeability of the blood–brain barrier (BBB) allows relatively higher amounts of brain-derived S100 to arrive in the

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blood or that the disorder causes a higher expression of S100 in the brain, which in case of a normal BBB leads to a relatively normal, but absolutely increased appearance of S100 in the blood inducing an immune reaction. In the ﬁrst published study measuring S100B in the serum of schizophrenic patients, 20 patients at various stages of disease were investigated (Wiesmann et al., 1999). All patients were on neuroleptic medication. A signiﬁcantly increased serum concentration of S100B was found in schizophrenic subjects compared to matched healthy controls. No correlation between S100B and age at onset or duration of illness was seen. However, S100B levels tended to be higher in patients with residual symptomatology and with long-term continuous psychotic symptoms without reaching statistical signiﬁcance potentially due to a lack of statistical power. Two years later, the same group published a study of 26 patients suVering from an acute episode of paranoid-type schizophrenia (Rothermundt et al., 2001b). No patients had received any psychotropic medication for at least 6 months prior to examination; 7 patients were drug naive. All patients were examined twice: upon admission to the hospital (unmedicated) and after 6 weeks of neuroleptic treatment. Upon admission, the S100B plasma level in schizophrenic patients was signiﬁcantly higher compared to the matched healthy controls. After 6 weeks of treatment the level of signiﬁcance was no longer reached. However, there was a signiﬁcant positive correlation between the negative subscale score of the positive and negative syndrome scale (PANSS) and the S100B concentration after 6 weeks. In the generally accepted division of psychiatric symptoms into separate categories, hallucinations, delusions, disorganized thinking, excitement, grandiosity, suspiciousness, ideas of persecution, and hostility are called ‘‘positive symptoms.’’ ‘‘Negative symptoms’’ include blunted aVect, emotional withdrawal, poor rapport, passive social withdrawal, diYculty in abstract thinking, lack of spontaneity and ﬂow of conversation, and stereotyped thinking. The negative symptoms are frequently linked with cognitive impairment. Negative symptoms and cognitive deﬁcits are not only present in an acute stage of disease, but rather persist and regularly impair the functional outcome of the disorder. In the cited study, intraindividual diVerences between negative subscale scores upon admission and after treatment were signiﬁcantly correlated with the S100B concentration after 6 weeks, indicating that little change or even deterioration of the negative symptomatology was associated with high S100B levels. Patients with S100B levels that were higher than mean levels of the healthy controls plus two standard deviations showed signiﬁcantly higher PANSS negative scores after 6 weeks of treatment than patients with lower S100B upon admission, whereas upon admission the PANSS negative scores did not diVer between the groups. After 6 weeks of treatment, 7 patients still had S100B levels above mean levels of the healthy controls plus two standard deviations. These patients demonstrated signiﬁcantly less improvement in the PANSS total and negative subscale scores

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after treatment compared to patients with lower S100B levels after 6 weeks of treatment. Continuously increased S100B levels were associated with the persistence of negative symptoms. In 2000 a Brazilian group presented a study of 23 schizophrenic outpatients all medicated with antipsychotic drugs; 16 were on clozapine (Gattaz et al., 2000). Citrate plasma was used to measure the S100B plasma concentration. The concentration of S100B was signiﬁcantly lower in the schizophrenic patients. No correlations were found between S100B levels and age, psychopathology scores, duration of illness, number of hospitalizations, and the dose of antipsychotic drugs in chlorpromazine equivalents. It is remarkable that the S100B concentrations measured in the citrate plasma of healthy controls (0.55 mg/liter) were almost 10 times higher than the S100B concentrations measured in serum of healthy controls (0.066 mg/liter) by the same group using the identical assay (Lara et al., 2001). According to the manufacturer’s instructions, the assay is designed to measure S100B in serum or CSF. They advise not using EDTA plasma. Even though the manufacturer does not comment on the use of citrate plasma, it must be considered that the assay may not be applicable in measuring S100B in plasma. Therefore, one has to be cautious interpreting the results of this study. In 2001 the same group published data on 20 medication-free schizophrenic patients, 6 outpatients, and 14 patients who had just been admitted for inpatient care (Lara et al., 2001). The serum of the patients contained signiﬁcantly higher concentrations of S100B than that of the matched healthy controls. No correlations between S100B levels and PANSS total, positive subscale, or negative subscale scores could be detected. The total group showed a signiﬁcant negative correlation with illness duration. However, when one outlier with an especially high serum level (0.603 mg/liter), the only drug-naive patient, was excluded from analysis, the negative correlation with illness duration was not signiﬁcant any more. Schroeter and colleagues (2003) reported increased S100B serum concentrations in schizophrenic patients treated with antipsychotic drugs for 3 weeks while untreated patients showed normal values. Patients with deﬁcit schizophrenia had higher S100B concentrations than nondeﬁcit subtypes. The authors conclude that treatment with antipsychotic agents might increase S100B levels. This ﬁnding is inconsistent with the reports of Rothermundt and coworkers (2001b) who showed a decrease of S100B in initially unmedicated schizophrenic patients after six weeks of antipsychotic treatment. Therefore, the inﬂuence of antipsychotic medication on S100B serum levels in schizophrenic patients remains to be clariﬁed. Recently, Rothermundt et al. (2004) were able to demonstrate in a large sample that the persistence of negative symptoms in schizophrenic patients is associated with a continuously elevated S100B concentration for 24 weeks after an acute episode. Patients with initially elevated S100B concentrations showed a signiﬁcantly slower improvement of psychopathology (PANSS total and negative subscale scores) under standardized treatment than patients with normal S100B.

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In summarizing the results of clinical studies on S100B in schizophrenic patients, it is likely that patients suVering from schizophrenia show increased S100B serum concentrations in the acutely psychotic stage of disease. Furthermore, this increase remains in those patients who develop a residual state with relevant negative symptoms, whereas S100B normalizes in recovering patients. Neuroleptic medication most likely cannot be regarded as a confounding factor, as these ﬁndings have been reported in medicated and nonmedicated patients. S100B as a rather small protein of only 21 kDa in the dimeric form can pass through the BBB easily. In healthy individuals, as well as in patients with various neurological diseases, serum measures have been proven to valuably reﬂect the S100B concentration in the CSF (Reiber, 2001). Moreover, S100B serum levels may image brain metabolism even more reliably than CSF levels because due to the natural CSF ﬂow, lumbar CSF reﬂects only disorders taking place in the brain tissue that are less than 30 mm away from the lateral ventricles and the third ventricle (Reiber, 2001). However, the relationship of S100B in CSF and serum of schizophrenic patients has not yet been published and needs to be appended. S100B has a very high brain speciﬁcity. The only known condition where serum S100B is not necessarily originated from the brain is major soft tissue damage, such as after polytrauma or cardiac surgery. The S100B released from soft tissue is most probably not originated from S100 homodimers, but is rather from other S100 species that contain the subunit in a heterodimer. Unfortunately, all available assays used to measure S100B contain antibodies that are speciﬁc for the monomer and not for the functionally relevant dimer. This problem will be solved as soon as an antibody speciﬁc for the homodimer is created. To prove the biological relevance of S100B ﬁndings in schizophrenia, we should not conﬁne the studies to psychopathology evaluations. Investigations combining S100B measurements with established parameters such as neurocognitive functions, evoked potentials, or structural and functional imaging should be undertaken.

VIII. Conclusions

There is convincing evidence that pathological processes involving synapses and dendrites may be relevant for the pathogenesis of schizophrenia. The various genetic and environmental factors known to be involved in the development of schizophrenic psychoses cause a disturbance of the physiological function of

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synapses and dendrites. This disturbance may take place during the development of the brain and continue in the mature brain, inﬂuencing degenerative and regenerative mechanisms. S100B regulates the balance between proliferation and diVerentiation in neurons and glial cells, thus playing a major role in developmental as well as in degenerative, apoptotic, regenerative, and protective processes. The cellular pathways of how S100B evolves its regulative action are only partly known; mechanisms involved in the regulation of the production and release of S100B also need further investigation. Of special interest concerning the impact of S100B on the pathogenesis of schizophrenia is the fact that animals with too much or too little S100B develop cognitive deﬁcits in spatial learning and working memory. Cognitive deﬁcits are also present in many patients suVering from schizophrenia. Main domains of cognition that are disrupted signiﬁcantly include attention, executive function, verbal and visuospatial working memory, and learning and memory. They are relevant not only in the acute stage of disease, but often remain in remission and/or a residual state. Cognitive deﬁcits are an important factor in the frequently impaired functional outcome of the disorder (Elvevag and Goldberg, 2000; Heinrichs and Zakzanis, 1998; Sharma and Antonova, 2003). An impaired 5-HT1a activity in schizophrenia is suspected to be involved in the development of cognitive dysfunction. Because S100B is also regulated via 5-HT1a receptors, one might hypothesize that S100B in schizophrenia is involved in the development of cognitive deﬁcits. This hypothesis is supported by the clinical ﬁnding that schizophrenic patients with persisting negative symptoms, including cognitive dysfunction, showed constantly elevated S100B serum concentrations. The fact that increased S100B concentrations in schizophrenics have been reported repeatedly in independent samples and by diVerent research groups encourages further clinical research. This should focus on the combination of S100B measurements with other methods, such as cognitive testing and various imaging techniques (magnetic resonance spectroscopy (MRS), structural and functional magnetic resonance tomography (MRT)) or evoked potentials to prove the biological relevance of increased S100B concentrations. We also need further clariﬁcation whether the increased S100B concentrations detected in schizophrenic patients with persistent negative symptoms indicate an ongoing degenerative process or whether the upregulation of S100B is a sign for an attempt of the brain to counter a destructive process such as inﬂammation. The availability of S100B knockout and transgenic mice oVers excellent opportunities to study the various regulatory molecular pathways of S100B in in vitro and in vivo experiments. The knowledge achieved from basic research experiments could then be transferred to clinical investigations. This combined approach oVers good opportunities for a considerable contribution to the unraveling of the pathogenesis of schizophrenia.

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Oct-6 TRANSCRIPTION FACTOR

Maria Ilia Institute of Psychiatry Department of Neuroscience London SE5 8AF, United Kingdom

I. POU Domain Proteins II. Oct-6 A. Transcriptional and Regulatory Properties B. Expression Profile in Peripheral Nervous System C. Role in Oligodendrocyte Development D. Developmental Expression Profile in Central Nervous System E. Cellular Compartmentilization F. Expression Profile in Schizophrenia III. Conclusions References

A variety of neurological disorders may have their origin during development of the central nervous system. Defects consistent with abnormal brain development have been reported in schizophrenia. These include faulty neuronal migration, altered spatial neuronal distribution, and the absence of signiﬁcant gliosis. These abnormalities in the adult are allocated to developmental processes and thus indicate a developmental basis for schizophrenia. Our way toward understanding schizophrenia pathology has been to consider the genes that regulate normal brain development. One such family of genes is the POU family of homeobox transcription factors. This chapter reviews POU domain proteins, focusing on the POU III domain gene, Oct-6, along with its potential relevance to schizophrenia.

I. POU Domain Proteins

POU domain proteins represent a variant family of homeodomain-containing transcription factors. Members of this family are expressed in the nervous systems of a wide range of vertebrate and invertebrate species. This family is characterized by a bipartite DNA-binding domain that has not been observed in other homeodomain relatives and is referred to as the POU domain (Herr et al., 1988). The initials are taken from the four proteins ﬁrst seen to have such INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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domains: Pit-1 (also called GHF-1), a pituitary-speciﬁc factor that activates the genes encoding growth hormone, prolactin, and other pituitary proteins; Oct-1, a ubiquitous protein that recognizes a certain 8-bp sequence called the octamer box; Oct-2, the B-cell-speciﬁc protein that recognizes the octamer box and activates immunoglobulin genes; and UNC-86, a nematode gene product involved in determining neuronal cell fates. POU domain proteins are interesting transcriptional regulators on the basis of their protein structure and their developmental expression proﬁle. First, the mechanism by which these factors bind to DNA involves tethering of two independently folded DNA-binding highly conserved helix–turn–helix motifs (Fig. 1). This recognition helix is referred to as the tryptophan–phenylalanine–cysteine (WFC) region, which consists of 15–18 amino acids. Within the WFC region, 12 residues are identical in POU domain proteins but not in other homeodomain proteins. Both N- and C-terminals are characterized by a cluster of basic amino acids (Wegner et al., 1993). In particular, the POU gene family consists of a

Fig. 1. The three-dimensional structure of the Oct-1 POU domain bound to DNA. In addition to binding DNA, the POU domain provides surface-exposed interaction interfaces recognized by multiple transcriptional activators (see text). Modified from Ryan and Rosenfeld (1997). (See Color Insert.)

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Fig. 2. Structure of the POU domain. The POU-specific domain has four -helices (cylinders), two of which include a region of basic amino acids (+++). The POU homeodomain has three helices. The two domains are separated by a linker region varying from 14 to 25 amino acids among the family members. Modified from Herr and Cleary (1995). (See Color Insert.)

150–160 amino acid unique bipartite DNA-binding domain (Herr and Cleary, 1995). This domain is composed of a POU-speciﬁc (POUs) and POU homeodomain (POUh) separated by a short linker region (Mandemakers et al., 1999; see Fig. 2). Both subdomains contain helix–turn–helix motifs that associate directly with the two components of bipartite DNA-binding sites. In other words, the POU domain contains two structurally independent domains that cooperate functionally as a DNA-binding unit. This mechanism has been reviewed in detail by Herr and Cleary (1995). Second, POU genes, while highly homologous in the POU domain, are distributed on several diVerent chromosomes (see Table I). POU domain genes have been described in organisms as divergent as Caenorhabditis elegans (Burglin et al., 1989), Drosophila (Thali et al., 1988), Xenopus (Agarwal et al., 1991), zebraﬁsh (Johansen et al., 1993; Spaniol et al., 1996), and humans, but have not yet been identiﬁed in plants or fungi. In all species these genes seem to carry out essential functions for organ development and cellular diVerentiation. More than 13 mammalian POU genes have been identiﬁed and most tend to be widely expressed throughout the developing nervous system, becoming restricted to deﬁned neural populations in the adult (Alvarez-Bolado et al., 1995; Frantz et al., 1994; He et al., 1989; Ilia et al., 2003; Suzuki et al., 1990). The 15 known mammalian POU domain genes have been classiﬁed into six diVerent classes, and the ﬁrst amino-terminal basic 11/12 amino acids of POUh are identical in each class (Mandemakers et al., 1999). The 15–18 amino acids of the WFC motif in the carboxy terminus of POUh are also particularly well conserved between classes (Wegner et al., 1993). For expression patterns, see Table I. From all POU domain factors, the POU III subfamily is of great interest in that they are expressed in the mammalian central nervous system (CNS) and are implicated in forebrain patterning and cortical development. One of these genes, Oct-6, has been shown to be upregulated in the schizophrenic brain (Ilia et al., 2002).

TABLE I The POU Gene Family in Mammals Expression Subfamily class

Gene

Chromosome

I

Pit1; GFH1

16 (mouse)

II

Oct1; OTF1

1 (mouse þ human) 7 (mouse) 19 (human) 9 (mouse) 1 (mouse)

Oct2; OTF

III

Skn1a; Oct11 Brn1

Brn2; N-Oct3 4 (mouse) 474 Brn4; RHS2; N-Oct4 Tst1; SCIP; Oct-6

X (mouse)

Neural tube except telencephalon Developing epidermis Nervous system widespread Nervous system widespread Neural tube Blastocyst, ES cells, nervous system

14 (mouse)

Nervous system widespread

Brn3

V

Oct3/4; Oct5 17 (mouse) 6 (human)

Sprm1 Brn5; Emb

Neural tube, pituitary Widespread

4 (mouse)

IV

VI

Developmental

13 (mouse) 15 (mouse)

ES þ EC cells, ectoderm primordial germ cells, testis and ovary Nervous system

Adult Pituitary Widespread CNS, lympoid, cells, thymus, testis Skin, thymus, testis CNS; kidney CNS; glioblastoma; neurobladtoma CNS-forebrain CNS neurons, myelinating glia, testis CNS (retina, sensory ganglia), spleen Oocytes

Function

Reference

Development of the anterior pituitary gland Gastrulation, apoptosis

Xia et al. (1993)

Proliferation of maturing B cells; neonatal lethal Wound healing Positional patterning neocortical neuron development Neural induction; hypothalamic development Neural induction; hearing sense Schwann cell differentiation; control in peripheral myelination; respiration Neurite outgrowth, development of retinal ganglion cells; development of hair cells in the inner ear (null mice are deaf ) Pluripotency

Siracusa et al. (1991); Wilson et al. (1993) Xia et al. (1993) Sumiyama et al. (1998)

Testis Genetic fitness Brain, kidney, skeletal muscle, testis

Hsieh et al. (1990)

Xia et al. (1993) Xia et al. (1993) Rohdewohld and Gruss (1992) Xia et al. (1993)

Scholer et al. (1990); Takeda et al. (1992)

Xia et al. (1993) Xia et al. (1993)

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II. Oct-6

A. Transcriptional and Regulatory Properties Oct-6, also known as Tst-1 and SCIP (suppressed cAMP-inducible protein), belongs to the class III subfamily of POU domain transcription factors along with Brn-1, Brn-2, and Brn-4. The Oct-6 POU domain shares approximately 95% homology with that of Brn1 and 2 (Blanchard et al., 1996; Sock et al., 1999). The Oct-6 locus (designated Otf6 or Pou3f1) is located on the distal arm of chromosome 4, and the entire transcriptional unit is contained within a 3-kb EcoRI fragment, coding for a protein of approximately 449 amino acids with a molecular mass of 45 kDa ( Jensen et al., 1998). Unlike other POU family members, the POU III subfamily consists of intronless genes and contains two long A-rich stretches within the genomic sequence encoding the 30 -untranslated region. The promoter and 50 region of the gene coincides with a 1-kb CpG island, which is undermethylated in both expressing and nonexpressing cells (Mandemakers et al., 1999). It has a strong basal promoter with an atypical TATA box (TTAA) at position 23 relative to the start site and a canonical CAAT box at approximately 80. The protein structure of Oct-6 is still lacking; however, given the overall homology with Oct-1, one can model the Oct-6 POU domain on the Oct-1 crystallographic coordinates to get an estimate of the speciﬁc side chains sticking out and the ones involved in DNA contacts. The activation domain of Oct-6 lies within the N-terminal of the protein between amino acids 115 and 157, a region rich in glycine–alanine residues (Meijer et al., 1990; Monuki et al., 1993). Studies have shown that Oct-6 binds to and activates octamer site-dependent promoters (Meijer et al., 1990; Suzuki et al., 1990). DiVerent binding aYnities were observed with perfect octamer sites containing distinct ﬂanking sequences, indicating that the ﬂanking sequences are also important determinants of binding aYnity for Oct-6 (Meijer et al., 1990). Oct-6 also binds to ﬁve distinct sites in the myelin-speciﬁc P0 promoter with a consensus sequence of GA(A/T)T(T/A)ANA, which appears unrelated to the octamer site (He et al., 1991). The rat GnRH promoter contains three Oct-6-binding sites that appear to conform to the consensus derived from the Oct-6-binding sites in the P0 promoter (Wierman et al., 1997). Oct-6 also binds with high aYnity to two sites in the JC virus promoter, but only one of the sites confers transcriptional activation. Although they are AT rich, neither site bears any similarity to octamer sites nor the P0 consensus site (Wegner et al., 1993). As a transcription factor, Oct-6 encodes a nuclear localization signal (NLS) and thus exerts its function in the nucleus. NLSs are recognized and bound by proteins such as importin and are short and rich in positively charged amino acids. The NLS for Oct-6 is located in a highly conserved region preceding helix

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1 of the POUh domain (Sock et al., 1996) and is composed of a single contiguous stretch of positively charged amino acids. In this respect, it is similar to the NLS of the SV40 large antigen T antigen. In the latter, regulation of nuclear cytoplasmic transport is controlled in part by phosphorylation at key residues in the NLS. It is plausible that the same is true for Oct-6, as its NLS overlaps with a potential protein kinase A (PKA) phosphorylation site. Like the NLS itself, this phosphorylation site is strongly conserved in other POU proteins and has been shown to be a target for the phosphorylation for Pit-1 and Oct-1 (Sock et al., 1996). The upstream regulatory region for the mouse Oct-6 gene contains at least eight consensus-binding sites for Sp1 and Krox20 family members and at least two consensus AP2-binding sites. The promoter contains no tissue-speciﬁc regulatory elements (Herr and Cleary, 1995). The region responsible for directing Schwann cell-speciﬁc expression has been mapped to a 4.3-kb fragment approximately 12 kb downstream of the start site and is conserved in the human Oct-6 gene, with which it shares approximately 94% sequence homology (Tobler et al., 1993). This ‘‘Schwann cell-speciﬁc enhancer’’ (SCE) (Mandemakers et al., 2000) also contains putative CREB-binding sites and may be responsible for the observed cAMP inducability of Oct-6 in cultured Schwann cells (Jessen and Mirsky, 1998; Monuki et al., 1989). In this system, Oct-6 is also induced on administration of cycloheximide (a protein synthesis inhibitor), suggesting that cAMP acts by relieving suppression of Oct-6 transcription (Monuki et al., 1989). Oct-6 can interact with the large T antigen from the human papovavirus JC virus, and this interaction seems to lead to transcriptional activation on the early and late promoters from the JC virus (Renner et al., 1994, 1996; Wegner et al., 1993). The amino-terminal 82 residues of the large T antigen and the POU domain of Oct-6 mediate this interaction, but for a functional eVect on transcription, the N-terminal transactivation of Oct-6 is also required. This functional eVect is speciﬁc for Oct-6 because while Oct-6 is also capable of interacting with the large T antigen, this interaction is not transcriptionally eVective because of diVerences in the transactivation domains of Brn-1 and Oct-6 (Sock et al., 1999). Interestingly, Oct-6 can also interact with other proteins via the POU homeodomain to regulate transcription independently of DNA binding. For example, Oct-6 has been reported to repress the P0 (protein zero) promoter and there are four to ﬁve potential binding sites of reasonable aYnity within the distal part of the P0 promoter (He et al., 1991; Monuki et al., 1990, 1993). Nevertheless, repression of the P0 promoter by Oct-6 in transient transfections was essentially independent of binding to these sites (Monuki et al., 1993). Moreover, Oct-6 has been shown to activate expression of the 3 nicotinic acetylcholine receptor (Yang et al., 1994). This activation is not dependent on the Oct-6-binding sites found upstream of the gene. In addition, mutations in the Oct-6-coding region that prevent the factor from binding to DNA with high aYnity do not obliterate 3 activation. These results suggest that regulation by Oct-6 is achieved

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via protein–protein interactions between Oct-6 and a speciﬁc complement of transcription factors that act directly on the promoter.

B. Expression Profile in Peripheral Nervous System The best-characterized role of Oct-6 is in Schwann cell development in the peripheral nervous system (PNS) where it has been considered to be a repressor of Schwann cell gene expression and/or diVerentiation. Together with the zinc ﬁnger protein Krox 20, Oct-6 is directly associated with the processes that control the development and diVerentiation of Schwann cells. Mice deﬁcient in Oct-6 myelination are either absent or severely delayed (Bermingham et al., 1996; Jaegle et al., 1996). However, knocking out Oct-6 is postnatally lethal, as mutant mice die due to respiratory failure. It is notable that in mice that survive, myelination eventually occurs, resulting in the delayed formation of apparently normal myelin ( Jaegle et al., 1996). This result could be either because the gene is involved in the timing of myelination or because a related but unidentiﬁed POU domain gene expressed later in development can compensate for the loss of Oct-6 and enable myelination to proceed, albeit with a delay. Oct-6 mRNA and protein can be detected in Schwann cell precursors, rising to a peak in early postnatal life. OCT-6 protein is clearly detectable in the nuclei of most myelinating Schwann cells during the ﬁrst week of active myelination (Blanchard et al., 1996; Scherer et al., 1994; Zorick and Lemke, 1996). Many nonmyelinating Schwann cells, particularly in the cervical sympathetic trunk, continue to express OCT-6 at relatively low levels in adult life (Blanchard et al., 1996). While these ﬁndings indicate that Oct-6 function is necessary for myelination to proceed, other cotransfection experiments using an N-terminal deletion of Oct-6 that acts as a dominant-negative inhibitor of Oct-6 repression of a 1.1-kb rat (P0) promoter have raised the possibility that Oct-6 acts to delay or suppress myelination. In transgenic mice, Oct-6 causes PNS myelination, taken as evidence that Oct-6 represses the progression from promyelinating Schwann cells to myelinating Schwann cells (Weinstein et al., 1995). However, Schwann cells in Oct-6-null mice show delayed diVerentiation, and their expression of myelin-related mRNAs, including those of P0 and Mbp, was not higher than in wild-type mice (Bermingham et al., 1996; Jaegle et al., 1996). These results contradict the idea that Oct-6 represses the expression of these genes in Schwann cells. To resolve these observations, Oct-6 has been hypothesized to activate genes required for Schwann cell diVerentiation but to repress terminal diVerentiation ( Jaegle and Meijer, 1998; Mirsky and Jessen, 1996; Zorick and Lemke, 1996). In this scheme, Oct-6-null mice lack both functions, whereas Oct-6 inhibits only the repression function. Therefore, in myelinating glial cells, the precise regulated expression

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of Oct-6 during development is responsible for the timely onset of myelination and might be a crucial factor in myelin disease pathogenesis.

C. Role in Oligodendrocyte Development Unlike any other homeodomain gene, Oct-6 is expressed in both Schwann cell and oligodendrocyte lineages. In the CNS, Oct-6 is expressed in select neurons and in cells of the oligodendrocyte lineage (Bermingham et al., 1996; Collarini et al., 1992). In the latter, Oct-6 expression is detectable in oligodendrocyte precursors, but is downregulated in mature oligodendrocytes. Myelination within the CNS of Oct-6-deﬁcient animals proceeds normally (Bermingham et al., 1996; Jaegle and Meijer, 1998), as does diVerentiation of Oct-6-deﬁcient oligodendrocytes in culture (unpublished data). Although it is expressed in the myelinating lineages of the CNS and PNS, of sequence similarity between Brn-1, Brn-2, and Oct-6 and their virtually indistinguishable DNA-binding speciﬁcity, it seems plausible that these three proteins can functionally substitute for each other in oligodendrocyte development. Whereas knockout experiments failed to reveal an obvious eVect on oligodendrocyte development, interesting observations were made after CNS expression of an Oct-6 transgene under the control of 58 ﬂanking MBP sequences ( Jensen et al., 1998). A severe neurologic syndrome characterized by action tremors, recurrent seizures, and premature death develops that on an ultrastructural level correlates with abnormal, precocious myelination comparable to what has been observed in the PNS of transgenic mice overexpressing a truncated form of Oct-6 under the control of the P0 promoter (Weinstein et al., 1995). Although interpretation of the phenotypes of these transgenic animals is complicated, they further support the conclusion from knockout experiments that Oct-6 has an important role in the development of myelinating glia. Bermingham and colleagues (2002) identiﬁed ﬁve genes that are downregulated in the absence of the Oct-6 in sciatic nerve via representational diVerence analysis (RDA). Only one of these genes, the fatty acid transport protein P2, was known previously to be expressed in Schwann cells. The other two genes, CRP1 and CRP2, are members of the cysteine-rich protein subclass of LIM-only proteins and may participate in cytoskeletal changes that accompany the transition from a promyelinating Schwann cell to a myelinating Schwann cell (Bermingham et al., 2002). Finally, two more genes, dendrin, originally found in dendrites, and Tramdorin1/mPAT2, a proton-dependent transporter of small amino acids, have both been reported to be downregulated by Oct-6; nevertheless, their function both in dendrites and in myelinating Schwann cells remains to be determined. Again, the majority of these genes regulated by Oct-6 do not seem to be any of the major myelin genes apart from P2. The identiﬁcation of target genes for Oct-6 is currently the focus of intense investigations.

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D. Developmental Expression Profile in Central Nervous System In the developing CNS, studies in the rodent dorsal telencephalon have shown that Oct-6 expression is turned on by young neurons of the cerebral cortical anlage as they migrate from the ventricular zone to their ﬁnal position in the cortical plate (Alvarez-Bolado et al., 1995; Frantz et al., 1994; He et al., 1989; Suzuki et al., 1990). However, less is known about the expression pattern of Oct-6 in the mature CNS. In the existing literature, sex and species-related discrepancies raise questions regarding the normal expression of the gene in the adult CNS. Evidence shows that in the postnatal brain, Oct-6 expression is mostly retained by certain subpopulations of neurons in layer 5 of the cerebral cortex and in the CA1 ﬁeld of the hippocampus (Alvarez-Bolado et al., 1995; Frantz et al., 1994). In addition, He et al. (1989) observed Oct-6 mRNA in cortical layers 2 and 3 and also in granule cells of the cerebellum. The slight inconsistencies in these studies raised the question whether the expression proﬁle of Oct-6 in the adult brain is sex linked or species speciﬁc. Because He et al. (1989) used male adult rats and Alvarez-Bolado et al. (1994), Frantz et al. (1994), and Suzuki et al. (1990) used unknown sex adult rats, the possibility of species- or gender-speciﬁc Oct-6 regulation cannot be assessed from these studies. In a report where Oct-6 expression was examined at progressive stages of adulthood in the mouse brain, it was shown that the expression of this POU homeodomain gene on both mRNA and protein levels is widespread in the forebrain of adult mice and that the Oct-6 þ cells are predominantly neuronal (Ilia et al., 2003). It was not until later during adulthood [ postnatal week (PW ) 24] that the more restricted expression of Oct-6 in cortical layers 2, 3, and 5 and in the CA1 hippocampal region reported previously in adult rats was observed (Frantz et al., 1994). Moreover, this expression pattern was transient, as it became attenuated in older animals and lost by PW 30. Therefore, in contrast to the impression generated by earlier studies, Oct-6, for the ﬁrst 5 months or so of adult life, is largely a pan-neuronal marker in the telencephalon. The transience of the laminar-speciﬁc pattern of expression is noteworthy given the use of Oct-6 as a lamina marker in experimental situations where lamina fate or organization has been disrupted (Mallamaci et al., 2000; Nakagawa et al., 1999; Tole and Grove, 2001). The aforementioned study was followed by another report in which we examined whether there are sex-related diVerences in Oct-6 expression (Ilia et al., 2003b). In situ hybridization experiments in female mice brains revealed that Oct-6 mRNA in female mouse brains was widely expressed in the young adult CNS and that its expression was attenuated at later stages of development. These observations are in agreement with our ﬁnding in the male mouse brain; however, we noticed that the restricted laminar expression of Oct-6 appears earlier in the female brain than in the male brain. (Ilia et al., 2003b). These ﬁndings

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Fig. 3. High Oct-6 immunoreactivity in the CA1 (B, arrows) and CA3 (C, arrows) regions of a schizophrenic specimen, whereas there is no or very little expression in similar regions of a matched control (A). Oct-6 staining is predominantly cytosolic.

suggest a gender-speciﬁc pattern in the expression of Oct-6 in the rodent brain. Interestingly there is evidence that Oct-6 is activated by an estrogen-dependent enhancer (Renner et al., 1996). Endogenous Oct-6 levels have been shown to increase following the addition of 17-estradiol, an estrogen receptor ligand (Renner et al., 1996). Regulation of Oct-6 by estrogen could therefore account for the diVerences observed in its expression pattern between male and female rodents. Finally, the attenuation of Oct-6 expression in the cortical anlage of male and female mice (Fig. 3) suggests that this transcription factor may play a role in regulating gene expression during brain aging (Ilia et al., 2003b). However, its sustained expression in the cerebellum of adult and old mice of both genders implies that Oct-6 by itself cannot compensate for the functional decline occurring in aging cerebellum, implicating the requirement of additional regulatory proteins. Taken together, these results show that Oct-6 expression occurs in a genderand age-dependent manner. This may be attributed to sex-speciﬁc organization of the brain and to progressive functional decline observed during brain aging.

E. Cellular Compartmentilization Oct-6 encodes a nuclear localization signal (Sock et al., 1996) and therefore OCT-6 protein should be localized in the nucleus. Developmental studies revealed that OCT-6 has a much more complex pattern of cellular localization than expected, being both nuclear and/or cytoplasmic across diVerent cerebral and hippocampal subpopulations. This pattern changes with developmental time and, as adulthood progresses, expression appears to be exclusively cytoplasmic. In the early stages of adulthood, OCT-6 is localized predominantly in the nucleus of cerebral neurons, with the exception of the pyramidal layer of the hippocampus where OCT-6 immunoreactivity was seen in the pyramidal dendrites. These data suggest that OCT-6 is sequestered in the cytoplasm of some neurons. Cytoplasmic sequestration is a recognized method of transcription factor control,

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as with NF-B and the glucocorticoid receptors (Bauerle and Baltimore, 2002). These proteins are retained in the cytoplasm by complex formation with I-B and hsp90, respectively, but no such mechanism has been described for OCT-6. The homeobox transcription factors EMX1 and EMX2 have also been shown to be localized in apical dendrites of neurons of the cortical plate (Chan et al., 2001; Mallamaci et al., 1998), although their function in those locations is also unknown. Interestingly, as adulthood progresses, OCT-6 expression is excluded from the nuclear core in layer 5 neurons and appears to be restricted to the perinuclear cytoplasm. This expression pattern is not unique for OCT-6, as it has been reported for another POU domain transcription factor, Oct-1, in aging human diploid ﬁbroblast cultures (Imai et al., 1997). In that study, OCT-1 immunoreactivity became localized to the nuclear periphery of ﬁbroblasts during the aging process. Imai and colleagues (1997) also showed that the aging-associated gene collagenase seemed to be depressed by the dissociation of OCT-1 protein from the nuclear periphery, which led to the suggestion that OCT-1 may be part of a repressive aging mechanism by anchoring nuclear matrix attachment regions onto the nuclear periphery. It is therefore possible that the OCT-6 in aging layer 5 neurons could be the result of breakdown of the nuclear transport or retention machinery in these cells.

F. Expression Profile in Schizophrenia Evidence shows that Oct-6 expression is upregulated in the frontal and temporal lobes of schizophrenic specimens, whereas there is limited expression of Oct-6 in matched controls (Ilia et al., 2002). However, Western blot analysis revealed no diVerences in levels of -catenin expression between the two groups (Ilia et al., 2002). -Catenin protein has been shown previously to be expressed at similar levels in schizophrenic and control brains (Beasley et al., 2001). These data appear to indicate unequivocally that Oct-6 is expressed at greater levels in schizophrenic versus control brain (Fig. 1). To our knowledge, this is the ﬁrst reported example of a transcription factor being expressed diVerentially in schizophrenic versus control brain. We also believe it to be the only example of a protein that is undetectable in control brain being expressed so demonstrably in schizophrenic tissue. Other examples of diVerential expression have, of course, been demonstrated, but these have all been relatively subtle quantitative changes (for a discussion, see Harrison, 1999). These ﬁndings indicate that Oct-6 may be useful as a neuropathological marker in schizophrenia. This disorder has been linked with numerous pathologies, such as enlarged lateral ventricles and changes in the cellular architecture and circuitry in the frontal and temporal lobes of the cerebral cortex. Nonetheless, there is not yet any single pathophysiological marker that unequivocally distinguishes schizophrenic tissue from normal.

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The aforementioned observations also beg to question how a transcription factor with a predominantly developmental role may be associated to schizophrenia. In the ﬁrst instance, the notion that genes responsible for the patterning of the cerebral cortex might be relevant to the development of schizophrenia seems unlikely. Schizophrenia appears during the third decade of life, well after the developmental events that sculpt the cerebrum are complete. Nonetheless, the neurodevelopmental hypothesis has become prominent. This hypothesis states that an early insult to the developing brain could result in brain changes that are ﬁxed, nonprogressive, and lie dormant until manifest in adolescence or early adulthood (Lewis and Murray, 1987; Weinberger, 1987). The successful impact of this hypothesis lies in its capacity to explain many of the known features of schizophrenia (see Table II). The predominant role of Oct-6 is believed to be developmental. It is expressed in the embryonic stem cells and the mouse inner cell mass (Meijer et al., 1990; Suzuki et al., 1990). As development progresses, Oct-6 is expressed in the embryonic telencephalon but is downregulated during early postnatal development so that expression in the adult is maintained only in layer 5 and some supragranular neurons of the cerebral cortex and the CA1 region of the hippocampus (Alvarez-Bolado et al., 1995; Frantz et al., 1994). Its expression continues to fall with age and becomes undetectable in older adult rodents as in the control human specimens used in the schizophrenic study (Ilia et al., 2002). Oct-6 is known to activate certain neurotransmitter receptors, such as the nicotinic acetylcholine receptor subunit genes (Liu et al., 1999) and the acetylcholine 3 receptor (Yang et al., 1994). It is expressed during the phase of neuronal migration, fate determination, and axonal outgrowth. Nonetheless, the role of Oct-6 in neuronal development is poorly understood. Schizophrenia, however, has been linked with many developmental brain abnormalities, such as faulty neuronal migration (Arnold and Trojanowski, 1996; Lewis and Murray, 1987), altered spatial neuronal arrangement (Akbarian et al., 1993), and the absence of signiﬁcant gliosis (Arnold and Trojanowski, 1996). It is plausible that Oct-6 may play a role in the mechanism that is responsible for these developmental changes in schizophrenia. In rodents, Oct-6 is broadly expressed only during development. Assuming this is true in humans as in rodents, Oct-6 upregulation in schizophrenic brain may be because either Oct-6 expression was never lost in individuals who were to go on to develop the disease or Oct-6 was subsequently upregulated prior or subsequent to the onset of the disease. These alternatives will be diYcult to distinguish in human patients, but animal studies may be able to cast light on the factors that may prolong or reactivate Oct-6 expression and that may therefore have a role in the expression in schizophrenia. However, evidence shows that Oct-6 may regulate the expression of neuronal nicotinic acetylcholine receptor (nAChR) subunits, which in turn are involved in psychiatric disorders. In particular, expression of the 7-nicotinic receptor subunit is decreased in schizophrenia. Multiple nAChRs are expressed early in

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TABLE II Basis for the Neurodevelopmental Hypothesis of Schizophrenia Reference Morphological abnormalities Enlarged ventricles 5–8% reduction in cortical and hippocampal volume Cortical dysplasia Neuronal loss Neuroimaging Increased ventricular volume Reduction in cortical gray matter Hypofunctionality of frontal lobes Epidemiology Childhood behavioral and motor defects Obstetric complications Association with maternal flu virus Correlation with season of births

Ward et al. (1996)

Crow et al. (1989); McCarley et al. (1999); Pantelis et al. (2003); Shelton et al. (1988)

Cooper (1992); Jones et al. (1994); McNeil (1995)

CNS development, suggesting a morphogenetic role (Agulhon et al., 1998, 1999). Several POU domain transcription factors are coexpressed with neuronal nAChR in the medial habenula, one of the best-characterized brain nuclei in terms of nAChR function (Deneris et al., 2000). In the vertebrate genome, genes encoding the 4, 3, and 5 subunits are clustered in a 50-kb region (Deneris et al., 2000). Evidence suggests that Oct-6 may activate the expression of 3 and 4 (Deneris et al., 2000; Yang et al., 1994). Activation of the 3 promoter in PC12 cells does not require upstream Oct-6-binding sites nor the amino terminus of Oct-6, a situation similar to Oct-6-mediated repression of the Po promoter in SCs (Deneris et al., 2000). There is no evidence yet that Oct-6 may regulate the a7 nAChR, which is mainly linked to schizophrenia. Nonetheless, the malfunction of both of these genes may be part of the developmental pathogenesis of schizophrenia. Moreover, ﬁndings for gender-associated Oct-6 expression in the rodent brain (Ilia et al., 2003b), together with evidence for Oct-6 regulation by an estrogen receptor enhancer (Renner et al., 1996), are of interest considering that estrogen has been shown to protect the brain against neurodegenerative diseases. A potential neuroprotective eYcacy of estradiol was suggested by clinical studies reporting that estrogen treatment can decrease the incidence and delay the onset of neurodegenerative processes such as occur in Alzheimer’s and Parkinson’s diseases and that it can signiﬁcantly reduce the risk of stroke. The mechanisms responsible for these eVects are unknown, although numerous potential mechanisms have been

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proposed (for a review, see Garcia-Segura et al., 2001). Taking into account that Oct-6 is upregulated in schizophrenia, then Oct-6 could be an integral part of the mechanism through which estradiol may exert its neuroprotective eVects. A further possibility that still remains to be elucidated is whether Oct-6 could be turned on in schizophrenic subjects as a result of their antipsychotic medication. Evidence suggests that this is not the explanation. Chlorpromazine (CPZE) values taken a month prior to death varied from 0 to 4000 across the schizophrenic group, yet we observed no diVerence in Oct-6 expression among schizophrenic subjects (Ilia et al., 2002). This indicates that Oct-6 expression does not vary with exposure to neuroleptic treatment and suggests that medication is unlikely to be the cause of Oct-6 expression. In addition to this, current research has pointed out that the therapeutic eYcacy of the most commonly used antipsychotics such as clozapine may be due to their ability to inhibit viral replication ( Jones-Brando et al., 1997). In addition to this study, another report by Lillehoj and colleagues (2000) presented evidence for a link between retroviral replication and ﬁrst-episode schizophrenia by measuring antibodies to diVerent primate retroviruses in the sera of individuals undergoing their ﬁrst episode of the disease. Given the synergistic relationship between Oct-6 and a large T antigen from the human papovavirus JC virus to promote viral replication (Renner et al., 1994), then Oct-6 could be a potential cofactor between retroviral replication and schizophrenia. Another interesting observation was cytoplasmic OCT-6 staining in pyramidal cell layer neurons of the hippocampus and in the dentate granule cell layer of the dentate gyrus. In the temporal lobe of schizophrenic specimens, OCT-6 staining was more prominent in the CA2, CA3, CA4, and in the granule cell layer of the dentate gyrus than staining in the CA1. This ﬁnding suggests that Oct-6 may be associated with changes in neuronal subpopulations and circuitry that are known to occur in schizophrenia. In support of this, it has been reported that there is increased MAP-2 (microtubule-associated protein)-immunoreactive dendritic length in the CA1, CA4, and subicular regions in schizophrenic specimens (Cotter et al., 2000). Similarly, the large abnormal neurons in focal cortical dysplasia (FCD) that express traits of neurons and glial cells are interpreted as a result of erroneous neuronal diVerentiation during development. In FCD there is also OCT-6 upregulation in the cytoplasm of the large abnormal neurons that are characteristic of the disease (unpublished observations). The cytosolic localization of OCT-6 in the schizophrenic brain is interesting because under nonpathological conditions, OCT-6 is found predominantly in the nucleus (Ilia et al., 2003a), in line with its role as a transcription factor. Oct-6 is normally sequestered to the nucleus because of a nuclear localization sequence in the POU domain of the protein (Sock et al., 1996). Why is then a transcription factor expressed in the cytoplasm of hippocampal neurons in schizophrenic specimens? What is the nature of this gene and is the activity of this gene itself a regulated process? Perhaps by creating an active cytoplasmic reservoir, the cell can respond

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more rapidly and accurately to an extracellular signal. Alternatively, cytoplasmic OCT-6 could be the result of breakdown of the nuclear transport or retention machinery in these cells. Then again, the OCT-6 protein could be sequestered in the cytoplasm through speciﬁc interactions with other proteins (Bauerle and Baltimore, 2002), as occurs with NF-B and glucocorticoid receptor transcription factors. These proteins are retained in the cytoplasm through complex formation with I-kB or hsp90, respectively. Thus, the cytosolic localization of OCT-6 found here suggests the existence of a mode of regulation not previously anticipated for Oct-6 or the POU domain proteins in general. These considerations are important if we are to elucidate the importance of Oct-6 expression for the schizophrenic brain.

III. Conclusions

Oct-6 is a member of the POU-III family of homeodomain proteins, whose primary role is thought to be developmental. In the brain, it is expressed in young migrating cortical neurons in rodents, but is turned oV gradually postnatally such that expression is absent in older adults. Oct-6 also plays a role in peripheral myelination, and some of the myelin genes that it regulates are known. Almost nothing is known, however, concerning neuronal gene regulation by Oct-6. Oct-6 is of interest in schizophrenia because of its long-lived ectopic expression in neurons in the brains of schizophrenic patients. Oct-6 is not detectable in adult brain, but neurons of both frontal and temporal lobes in schizophrenia express high levels of this protein. Given the developmental role of Oct-6, this may be an important link to the proposed neurodevelopmental basis of schizophrenia. While this review was written, we have replicated our ﬁnding of Oct-6 upregulation in schizophrenia from a series of the Stanley Foundation consortium. In addition, preliminary data from the same specimen series indicate that Oct-6 is also upregulated in the brains of patients diagnosed with bipolar disorder and manic depression. This suggests that Oct-6 may be a functional marker of psychosis.

Acknowledgments

I thank Professor Jack Price for his constant support and input. I am also grateful to Professor I. Everall, Dr. D. Uwanogho, and Dr. D. Liolitsa for their comments on the manuscript and to Dr. Dies Meijer for kindly providing us with the Oct-6 antiserum. This work was supported by the Stanley Foundation, European Commission, the Medical Research Council, and the Biotechnology and Biological Sciences Research Council.

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Siracusa, L. D., Rosner, M. H., Vigano, M. A., Gilbert, D. J., Staudt, L. M., Copeland, N. G., and Jenkins, N. A. (1991). Chromosomal location of the octamer transcription factors, Otf-1, Otf-2, and Otf-3, defines multiple Otf-3-related sequences dispersed in the mouse genome. Genomics 10, 313–326. Sock, E., Enderich, J., Rosenfeld, M. G., and Wegner, M. (1996). Identification of the nuclear localization signal of the POU domain protein Tst-1/Oct6. J. Biol. Chem. 271, 17512–17518. Sock, E., Enderich, J., and Wegner, M. (1999). The J domain of papovaviral large tumor antigen is required for synergistic interaction with the POU-domain protein Tst-1/Oct6/SCIP. Mol. Cell Biol. 19, 2455–2464. Spaniol, P., Bornmann, C., Hauptmann, G., and Gerster, T. (1996). Class III POU genes of zebrafish are predominantly expressed in the central nervous system. Nucleic Acids Res. 24, 4874–4881. Sumiyama, K., Washio-Watanabe, K., Ono, T., Yoshida, M. C., Hayakawa, T., and Ueda, S. (1998). Human class III POU genes, POU3F1 and POU3F3, map to chromosomes 1p34.1 and 3p14.2. Mamm. Genome 9, 180–181. Suzuki, N., Rohdewohld, H., Neuman, T., Gruss, P., and Scholer, H. R. (1990). Oct-6: A POU transcription factor expressed in embryonal stem cells and in the developing brain. EMBO J. 9, 3723–3732. Takeda, J., Seino, S., and Bell, G. I. (1992). Human Oct3 gene family: cDNA sequences, alternative splicing, gene organization, chromosomal location, and expression at low levels in adult tissues. Nucleic Acids Res. 20, 4613–4620. Thali, M., Muller, M. M., DeLorenzi, M., Matthias, P., and Bienz, M. (1988). Drosophila homoeotic genes encode transcriptional activators similar to mammalian OTF-2. Nature 336, 598–601. Tobler, A., Schreiber, E., and Fontana, A. (1993). The human Oct-6 POU transcription factor lacks the first 50 amino acids of its murine counterpart. Nucleic Acids Res. 21, 1043. Tole, S., and Grove, E. A. (2001). Detailed field pattern is intrinsic to the embryonic mouse hippocampus early in neurogenesis. J. Neurosci. 21, 1580–1589. Ward, K. E., Friedman, L., Wise, A., and Schulz, S. C. (1996). Meta-analysis of brain and cranial size in schizophrenia. Schizophr. Res. 22, 197–213. Wegner, M., Drolet, D. W., and Rosenfeld, M. G. (1993a). POU-domain proteins: Structure and function of developmental regulators. Curr. Opin. Cell Biol. 5, 488–498. Wegner, M., Drolet, D. W., and Rosenfeld, M. G. (1993b). Regulation of JC virus by the POUdomain transcription factor Tst-1. Implications for progressive multifocal leukoencephalopathy. Proc. Natl. Acad. Sci. USA 90, 4743–4747. Weinberger, D. R. (1987). Implications of normal brain development for the pathogenesis of schizophrenia. Arch. Gen. Psychiat. 44, 660–669. Weinstein, D. E., Burrola, P. G., and Lemke, G. (1995). Premature Schwann cell differentiation and hypermyelination in mice expressing a targeted antagonist of the POU transcription factor SCIP. Mol. Cell. Neurosci. 3, 212–229. Wierman, M. E., Xiong, X., Kepa, J. K., Spaulding, A. J., Jacobsen, B. M., Fang, Z., Nilaver, G., and Ojeda, S. R. (1997). Repression of gonadotropin-releasing hormone promoter activity by the POU homeodomain transcription factor SCIP/Oct-6/Tst-1: A regulatory mechanism of phenotype expression? Mol. Cell. Biol. 17, 1652–1665. Wilson, G. L., Najfeld, V., Kozlow, E., Menniger, J., Ward, D., and Kehrl, J. H. (1993). Genomic structure and chromosomal mapping of the human CD22 gene. J. Immunol. 150, 5013–5024. Xia, Y. R., Andersen, B., Mehrabian, M., Diep, A. T., Warden, C. H., Mohandas, T., McEvilly, R. J., Rosenfeld, M. G., and Lusis, A. J. (1993). Chromosomal organization of mammalian POU domain factors. Genomics 18, 126–130. Yang, X., McDonough, J., Fyodorov, D., Morris, M., Wang, F., and Deneris, E. S. (1994). Characterization of an acetylcholine receptor alpha 3 gene promoter and its activation by the POU domain factor SCIP/Tst-1. J. Biol. Chem. 269, 10252–10264. Zorick, T. S., and Lemke, G. (1996). Schwann cell differentiation. Curr. Opin. Cell Biol. 6, 870–876.

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NMDA RECEPTOR FUNCTION, NEUROPLASTICITY, AND THE PATHOPHYSIOLOGY OF SCHIZOPHRENIA

Joseph T. Coyle and Guochuan Tsai Department of Psychiatry Harvard Medical School McLean Hospital Belmont, Massachusetts

I. II. III. IV. V. VI. VII.

Introduction NMDA Receptors EVects of Dissociative Anesthetics Postmortem Studies of Glutamatergic Markers in Schizophrenia Glutamate Receptor-Associated Genes Preclinical Studies on NMDA Receptor Hypofunction Clinical Trials of NMDAR Modulators in Schizophrenia A. Open Trials of Glycine B. Glycine Placebo-Controlled Trials C. d-Cycloserine D. d-Serine and Sarcosine E. Clozapine and the NMDA Receptor VIII. NMDA Receptor and Neuroplasticity IX. Conclusion References

I. Introduction

Schizophrenia is a disorder that aVects approximately 1% of adults with little variation in prevalence throughout the world. It aVects multiple domains, resulting in hallucinations and delusions (positive symptoms), lack of initiative, emotional impoverishment, and poor social skills (negative symptoms), and impairments in memory and problem solving (cognitive symptoms). Although positive symptoms wax and wane, negative symptoms and cognitive impairments are more enduring, cause greater disability, and correlate with cortical atrophy/ ventricular enlargement (GoV and Coyle, 2001). The age of symptomatic onset is typically late adolescence and early adulthood with approximately 70% of aVected individuals remaining disabled for the rest of their lives. This chronic course accounts for the fact that schizophrenia is the seventh most costly illness to society in terms of both cost of care and loss of income (Rice, 1999; Rupp and Keith, 1993). INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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Schizophrenia is highly heritable (80%) with the risk in ﬁrst-degree relatives of an aVected individual 12-fold greater than that of the general population (Harrison and Owen, 2003). The lack of complete concordance in identical twins (approximately 60%) indicates that environmental and epigenetic factors play a role in the phenotype, with most evidence pointing toward perinatal insults (Tsuang, 2000). Schizophrenia likely involves complex genetics with multiple risk alleles of small eVect interacting to produce the phenotype. Given the variation in phenotypic manifestations of the disorder, it is likely that certain genes may contribute to the cognitive aspects of schizophrenia, whereas others might account for the psychotic dimension. In fact, linkage studies have yielded a number of sites in the human genome that are associated with a substantially increased risk for schizophrenia (Harrison and Owen, 2003). The hypothesis that has dominated thinking about the pathophysiology of schizophrenia for the last four decades is the ‘‘dopamine hypothesis.’’ It was based on the ﬁndings that dopamine-releasing stimulants such as amphetamine cause psychosis and that antipsychotics act by blocking D2 dopamine receptors (Snyder, 1981). The limitation of this hypothesis is that negative symptoms, cognitive impairments, and cortical atrophy are poorly responsive to typical antipsychotic medications such as haloperidol (Meltzer, 1997). That additional processes might be involved was further supported with the introduction of clozapine, a drug with weak D2 dopamine receptor aYnity but one that could have remarkable therapeutic eVects, especially on negative symptoms, in schizophrenic subjects who responded poorly to typical antipsychotics. This review addresses an alternative hypothesis for the pathophysiology of schizophrenia that has accrued considerable supportive evidence over the last decade—the core symptoms of schizophrenia result from hypofunction of the NMDA receptor (R). This chapter reviews critically the evidence supporting the hypothesis from NMDAR antagonist challenge studies, postmortem neurochemical/gene expression analyses, genetic studies, and the results of clinical trials with agents that enhance NMDAR function.

II. NMDA Receptors

NMDARs are members of a larger family of glutamate-gated cation channels (iGluRs) that mediate fast excitatory neurotransmission in brain. Glutamate is the major excitatory neurotransmitter in brain, accounting for 40% or more of all synapses in the central nervous system (CNS). iGluRs are further subdivided into AMPA/kainite receptors and NMDARs. AMPARs (GluR1–4) are the primary mediators of excitatory postsynaptic currents (EPSCs). Although NMDARs

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(NR1; NR2A–D) may contribute to a varying degree to the EPSC, they serve a more fundamental role in coincidence detection because of the unusual biophysical feature of being voltage dependent (Tsien, 2000). Thus, at resting membrane potential, the channel of the NMDAR is blocked by Mg2þ; the blockade is removed by membrane depolarization primarily through the activation of AMPARs. Another unusual characteristic of the NMDAR is that in addition to the binding site for the agonist, glutamate, there is a second site on the NR1 subunit to which glycine and/or d-serine bind (Sheinin et al., 2001). The glycine modulatory site (GMS) must be occupied for glutamate to open the channel. The aYnity of the GMS varies from 0.01 to 3 M depending on the NR2 subunit composition of the NMDAR. The availability of d-serine depends on the activities of serine racemase (SR), which generates d-serine from l-serine, and the degrading enzyme d-amino acid oxidase (DAAO) (Mothet et al., 2000). An inverse relationship exists in brain regions between the amount of d-serine and the expression of DAAO such that the cerebellum has low d-serine and high DAAO. The availability of synaptic glycine is determined by the activity of the glycine transporter, GlyT1 (Kim et al., 1994). Both SR and GlyT1, as well as the glutamate transporters that protect against excitotoxicity (EAAT 1 and 2), are expressed exclusively in astrocytes, pointing to the vital role played by astrocytes in modulating glutamatergic neurotransmission, especially involving NMDAR (Berger et al., 1999; Coyle and Schwarcz, 2000). Studies in acute slice preparations from the hippocampus and the prefrontal cortex indicate that the GMS is not saturated by endogenous glycine/d-serine (Berger et al., 1998; Bergeron et al., 1998; Chen et al., 2003). The precise mechanisms responsible for regulating synaptic glycine and d-serine are poorly understood but likely reinforce the coincidence detection role of NMDAR. The channels of the NMDAR are large so as to conduct Ca2þ readily, which activates a variety of intracellular transducers that ultimately regulate gene expression. The activation of NMDAR during high presynaptic glutamatergic activity results in a permanent increase in synaptic eYcacy known as long-term potentiation (LTP) (Tsien, 2000). The inﬂux of Ca2þ through NMDAR during LTP causes the recruitment of AMPAR to the synapse from intracellular stores. In addition to the functional plasticity associated with LTP, persistent hyperactivity through a glutamatergic synapse can cause structural changes, such as the elaboration of new postsynaptic spines via NMDAR activation, thereby further strengthening synaptic connections (Leuner et al., 2003). NMDAR activation has trophic eVects (see later), especially during development, with the inactivity of NMDAR resulting in neuronal apoptosis (Ikonomidou et al., 2001). Conversely, excessive activation of extrasynaptic NMDAR causes neuronal injury and ultimately death through a pathologic process known as excitotoxicity (Coyle et al., 2002a).

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III. Effects of Dissociative Anesthetics

The motivating force behind the hypothesis that schizophrenia may result from hypofunction of a subset of NMDA receptors is the expanding body of evidence on the behavioral, neurocognitive, and physiologic eVects of the dissociative anesthetics, which are use-dependent noncompetitive inhibitors of the NMDA receptors. Historically, the fact that phencyclidine could cause a syndrome in normal individuals that closely resembled schizophrenia and that it could exacerbate the symptoms of individuals suVering from schizophrenia was appreciated soon after its introduction in the early 1960s (Itil et al., 1967; Luby et al., 1959). Javitt and Zukin (1991) can be credited with ﬁrst linking this schizophrenia-like state induced by phencyclidine and its antagonism of NMDA receptors. Dissociative anesthetics act as use-dependent, noncompetitive inhibitors of the NMDAR binding to a site within the channel. Subsequent studies in normal individuals under laboratory conditions demonstrated that the infusion of subanesthetic doses of ketamine produced a syndrome characterized by withdrawal, blunted aVect, psychomotor retardation, delusions, and illusions (Krystal et al., 1994). Furthermore, ketamine impaired performance on a number of cognitive tasks that require frontal cortex and/or hippocampal involvement, including the Wisconsin card sorting test, verbal declarative memory, delayed word call, and verbal ﬂuency in a manner similar to what is observed in schizophrenia (Newcomer et al., 1999). Schizophrenic patients in remission were particularly sensitive to the psychotomimetic eVects of ketamine (Malhotra et al., 1997b). What is extremely important about these studies is that these impairments were observed under conditions in which the individuals exhibited virtually normal performance on the minimental state exam, an instrument sensitive to dementia and delirium, as is the case for subjects with schizophrenia. The logical implication of these ﬁndings is that a discrete subpopulation of NMDARs are aVected by the low dose of ketamine. In other words, if all or the majority of NMDARs were blocked, this would represent an anesthetic dose of ketamine. In addition to mimicking the neuropsychologic manifestations of schizophrenia, subanesthetic doses of ketamine can also reproduce some of the physiologic abnormalities associated with schizophrenia. Normal subjects attenuate their startle reﬂex when presented with an antecedent warning tone, a phenomenon known as prepulse inhibition (PPI). Subjects with schizophrenia generally fail to adapt (BraV et al., 2001). Low-dose ketamine results in impaired PPI in experimental animals, which can be reversed by antipsychotics. Schizophrenic subjects exhibit abnormal cortical event-related potentials (ERP). In normal subjects given ketamine, abnormal ERPs are associated with psychotic experiences (Umbricht et al., 2002). Some subjects with schizophrenia exhibit abnormalities with eye tracking, which can be reproduced in controls by low-dose ketamine

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(Avila et al., 2002). Schizophrenic subjects have been shown to have an enhanced release of subcortical dopamine with amphetamine provocation in positron emission tomographic studies. A similarly enhanced release of dopamine has been documented in controls receiving low-dose ketamine (Kegeles et al., 2000). While acute administration of low-dose ketamine results in elevation in frontal cortical blood ﬂow (Vollenweider et al., 1997), chronic use of phencyclidine has been shown to result in ‘‘hypofrontality’’ in functional imaging studies analogous to what is observed in chronic schizophrenia (Cochran et al., 2003; Hertzmann et al., 1990; Wu et al., 1991). Administration of ketamine to schizophrenic patients who are clinically stable causes a reemergence of delusions, hallucinations, and thought disorder characteristic of the individual’s disorder (Lahti et al., 1995; Malhotra et al., 1997a). The patients generally report the experience as quite aversive in contrast to individuals who abuse dissociative anesthetics, consistent with a peculiar sensitivity of individuals with schizophrenia. Clozapine but not haloperidol attenuates the symptoms induced by ketamine in subjects with schizophrenia (Malhotra et al., 1997a), which supports the inference that clozapine enhances NMDAR function (see Section VII,E).

IV. Postmortem Studies of Glutamatergic Markers in Schizophrenia

Starting over a decade ago, investigators began studies of glutamate receptor expression in postmortem brains obtained from subjects with schizophrenia. Three approaches have been employed: ligand binding, which identiﬁes the major iGluR families; subunit protein measurement by Western blots or immunocytochemistry, which measures speciﬁc components of these receptors; and measurement of the mRNA encoding these subunits by Northern blots or in situ hybridization (for review, see Coyle et al., 2002b). Because of evidence that receptor traYcking, phosphorylation, and allosteric modulators aVect their function, the precise relationship between these indices and the functional status of synaptic iGluRs remains unclear. Notably, the most consistent ﬁnding of abnormalities in glutamate receptors in schizophrenia have centered on the kainate receptor. Decreased binding of [3H]kainic acid was observed in the hippocampus and perihippocampus by Kerwin et al. (1988). In situ hybridization also showed decreased levels of mRNA for GluR6 and KA2 in the hippocampus but not in the cerebellum. Immunocytochemical studies revealed decreased expression of GluR5, 6, and 7 in the stratum radiatum and moleculare of Ca2/3 and portions of Ca1 of the hippocampal formation (Benes et al., 2001a). In another study, in situ hybridization revealed decreased mRNA for GluR7 and KA2 in the superior frontal gyrus, whereas

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another showed a selective decline in KA2 in the prefrontal cortex (Porter et al., 1997). As the KA2 subunit confers high-aYnity binding for [3H]kainic acid, this latter ﬁnding was consistent with reduced [3H]kainic acid receptor binding observed in this region. Despite the variation in methods and tissue sources, there seems to be consistent evidence of a reduced expression of kainic acid receptors in brain regions relevant to schizophrenia (Meador-WoodruV et al., 2001). Similar signiﬁcant decreases in AMPA receptor subunits have been reported in schizophrenia. In situ hybridization has revealed reductions in GluR1 in the dentate gyrus and Ca3/4 region of the hippocampal formation, whereas GluR2 was reduced in the parahippocamus. Polymerase chain reaction (PCR) also revealed reduced GluR2 mRNA in the medial temporal cortex (Eastwood et al., 1995; Harrison et al., 1991; Meador-WoodruV and Healy, 2000). Immunocytochemical analysis showed lower GluR1 in the parahippocampus and lower levels of GluR2/3 throughout the hippocampal formation, especially in the parahippocampus (Eastwood et al., 1997). Notably, acute treatment with MK 801 reduced the expression of GluR3, GluR4, and mGluR3 in rat cortex (Storvik et al., 2003). Considering the homology between the schizophrenic-like symptoms associated with the dissociative anesthetics, ﬁndings on NMDAR in corticolimbic regions have been surprisingly modest and inconsistent (Meador-WoodruV and Healy, 2000). Thus, ligand-binding studies for the PCP site with the NMDAR channel have revealed no consistent alterations in either the hippocampus or the cerebral cortex. No absolute diVerences in mRNA levels for any of the NR1 or NR2 subunits were observed in the prefrontal cortex in schizophrenics in one study, although another reported a reduction of NR1 transcripts in the superior frontal cortex (Akbarian et al., 1996; Gao et al., 2000). In contrast, PCR measurements indicated an elevation of NR1 in the dorsolateral prefrontal cortex but not of NR2A/B. As functional and structural imaging studies revealed abnormalities in thalamic nuclei in schizophrenia (Highley et al., 2003; Kemether et al., 2003). Ibrahim et al. (2000) have carried out extensive analysis of glutamatergic markers in this region using in situ hybridization. They found reduced levels of mRNA encoding NR1, NR2B/2C, GluR1, GluR2, and KA2. Consistent with a reduced expression of NMDAR subunits, speciﬁc binding of the NMDAR modulatory ligands, [3H]ifenprodil, which is NR2B selective, and [3H] MDL 105, 519, which binds to the NMDAR glycine modulatory site, was also reduced. Furthermore, expression of the glutamate vesicular transporter DNPi was reduced signiﬁcantly. In contrast, the astrocyte-associated glutamate transporters, EAAT 1 and EAAT 2, were elevated signiﬁcantly in the thalamus in schizophrenia. In aggregate, these alterations would be associated with reduced glutamatergic neurotransmission.

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V. Glutamate Receptor-Associated Genes

Despite searches by several groups, none have found a signiﬁcant association of markers within or near the genes encoding NR1 or NR2 subunits and the risk for schizophrenia with one exception. Itokawa et al. (2003) reported an association of an expanded GT repeat in the promoter region of NR2A with chronic schizophrenia. However, ﬁndings regarding genes that encode for proteins that aVect the NMDAR have been more promising. Glutamate carboxypeptidase II (GCPII) catabolizes the neuropeptide N-acetyl-aspartylglutamate (NAAG), which is an mGluR 3 agonist (Lea et al., 2001; Wroblewska et al., 1997) and antagonizes NMDA receptor function in CA1 at the GMS (Grunze et al., 1996). The activity of GCPII was reported to be reduced in the prefrontal cortex, temporal cortex, and hippocampus in schizophrenia (Tsai et al., 1995). A reduced expression of GCPII has been replicated in a cohort of aged schizophrenics by DNA microarray analysis (Hakak et al., 2001). In another study, quantitative PCR has conﬁrmed reduced GCPII mRNA in the prefrontal cortex in a cohort of predominantly African-American schizophrenic subjects as compared to matched controls (S. Bahn, personal communication). A translocation, T (1:11) (Q42.1, Q14.3), which is associated with an increased risk of schizophrenia (Semple et al., 2001), aVects the chromosomal region encoding GCPII. Notably, an allelic variant of mGluR3 (GRM3), at which NAAG acts, has been associated with an increased risk for schizophrenia (Marti et al., 2002; Weinberger, personal communication). A mutation at 13q 34 that is associated with schizophrenia aVects a gene encoding G 72, a primate-speciﬁc modulator of DAAO. The mutation appears to increase the activity of DAAO, which would decrease the levels of the GMS agonist, d-serine (Chumakov et al., 2002). Furthermore, four single nucleotide polymorphisms (SNPs) for DAAO have been associated with schizophrenia in Canadian pedigrees (Chumakov et al., 2002). Consistent with these ﬁndings on DAAO, the serum levels of d-serine are reduced in schizophrenia (Hashimoto et al., 2003). Finally, the endogenous GMS antagonist kynurenic acid is elevated in the prefrontal cortex in schizophrenia (Schwarcz et al., 2001). The NMDAR sits in a nexus of nearly 80 interacting proteins in the postsynaptic density, including PSD-95 (Husi et al., 2000). At least three studies have demonstrated a linkage for schizophrenia with the gene encoding neuregulin 1 (8p11-p21) (Stefansson et al., 2002; Williams et al., 2003; Yang et al., 2003). In mice homozygous for a null mutation of neuregulin 1, NMDAR expression is reduced. Dysbindin (6p22.3) is a component of dystrophin complex, which also comprises the postsynaptic complex. Linkage at the dysbinden gene has been reported for schizophrenia (Schwab et al., 2003; Straub et al., 2002).

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Fig. 1. Schematic representation of a glutamatergic synapse identifying modulators of NMDA receptor function implicated in schizophrenia. Red arrows denote pathways that enhance NMDAR function, whereas blue arrows denote pathways that attenuate NMDAR function. Glutamate is an agonist at AMPA, NMDA, and mGluR3 receptors. NAAG is an agonist at the mGluR3 receptor that inhibits glutamate release and inhibits NMDAR in a glycine-sensitive manner. GCPII catabolizes NAAG, thereby decreasing NMDAR inhibition. GlyT1 removes glycine from the GMS, thereby attenuating NMDAR function. d-Serine, synthesized by serine racemase, is an agonist at the GMS. d-Amino acid oxidase (DAAO) degrades d-serine, thereby attenuating NMDAR function. The activity of DAOO is stimulated by G72. Kynurenate amino transferase (KAT) synthesizes kynurenic acid, an NMDAR antagonist at the GMS. Neuregulin and dysbinden may aVect NMDAR function indirectly through association with the postsynaptic density. Abnormalities in the expression of GCPII, NAAG, d-serine, and kynurenic acid have been reported in schizophrenia. Gene association studies have implicated mGluR3, DAAO, G72, neuregulin, and dysbinden in schizophrenia. (See Color Insert.)

Taken together, these ﬁndings from postmortem studies and gene linkage analysis point to abnormalities associated with schizophrenia that individually or in concert would disrupt NMDAR function (see Fig. 1). Several converge on the GMS of the NMDAR, including GCPII, DAAO, and G72. The diYculties in ﬁnding associations with NMDAR genes themselves may speak to the critical role of the receptor, as mice homozygous for a null mutation of NR1 do not survive (Mohn et al., 1999). Similarly, in Alzheimer’s disease (AD), although the pathologic disposition of amyloid precursor protein (APP) is likely the cause of the disorder, mutations in the gene encoding APP represent an exceedingly rare cause of AD (Kowalska, 2003).

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VI. Preclinical Studies on NMDA Receptor Hypofunction

Whereas acute treatment with dissociative anesthetics increases dopamine release in the prefrontal cortex and subcortical structures as measured by in vivo dialysis (Mathe et al., 1999; Moghaddam et al., 1997), chronic administration results in decreased dopamine release in the frontal cortex but not in subcortical structures ( Jentsch et al., 1998). As elevated subcortical dopamine release has been implicated in the positive symptoms (psychosis), the hypofunction of frontocortical dopaminergic projections with chronic NMDAR blockade is more consistent with the negative symptoms and cognitive impairments in schizophrenia (Castner et al., 2000). Chronic treatment of experimental animals with PCP and ketamine also sensitizes the animals to the behavioral eVects of subsequent exposure to stimulants, stress, and NMDA receptor antagonists, all of which exacerbate schizophrenia ( Jentsch et al., 1998; Lannes et al., 1991). As in the clinical studies, the subanesthetic doses associated with these behavioral responses must clearly aVect only a subpopulation of NMDAR. Studies by Moghaddam et al. (1997) have demonstrated that the behavioral eVects of dissociative anesthetics are associated with an elevated release of glutamate in the prefrontal cortex, consistent with a disinhibition of cortical glutamatergic neurons. The PCP-induced behaviors, as well as the elevated release of glutamate, were shown to be reversed by the mGluR2/3 agonist LY 379268, which inhibits glutamate release (Moghaddam and Adams, 1998). The disinhibition of cortical glutamatergic systems with subanesthetic doses of dissociative anesthetics thus likely represents a selective antagonism at the NMDAR on GABAergic interneurons. Recombinant DNA strategies have also been exploited to probe the role of NMDAR hypofunction in behaviors homologous to schizophrenia. Mohn et al. (1999) developed a mouse that was NR1 hypomorph, expressing only 5% of the normal levels of NR1. The hypomorphs exhibited hyperactivity, impaired social behaviors, and no mating. While the hyperactivity alone responded to treatment with haloperidol, clozapine, a drug that is eVective in reducing negative symptoms in a subpopulation of typical antipsychotic nonresponders, not only reduced the hyperactivity and improved social behaviors, but also signiﬁcantly increased successful mating. Hyperactivity has long been considered a surrogate for positive symptoms. The impaired social behavior and reduced mating aVected selectively by clozapine may be homologues of negative symptoms of schizophrenia, again suggesting that NMDAR hypofunction may more faithfully reﬂect the full range of symptoms of schizophrenia. However, a mutation ‘‘knocked’’ into the NR1 gene encoding for the GMS to impair NMDA receptor function is also associated with hyperactivity and other behavioral disturbances that did not respond to antipsychotics (Ballard et al., 2002).

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As suggested by these ﬁndings by Moghaddam et al. (1997), the NMDAR on corticolimbic GABAergic interneurons may be diVerentially more sensitive to inhibition by dissociative anesthetics than glutamatergic pyramidal cells, which are disinhibited. Consistent with this inference, Grunze et al. (1996) found that CA1 GABAergic interneurons were 10-fold more sensitive to the canonical NMDAR antagonist aminophosphono valeric acid (APV) and by GMS antagonist NAAG than the pyramidal neurons in a study of SchaVer collateral-induced LTP. In acute slice preparations from the limbic cortex, Li et al. (2002) found that MK 801 disinhibited pyramidal cell glutamatergic neurotransmission, consistent with a diVerential sensitivity of the GABAergic NMDAR to dissociative anesthetics. This convergence on GABAergic NMDAR dovetails with a compelling list of ﬁndings from several laboratories pointing to a loss or downregulation of expression of discrete populations of cortical GABAergic neurons (Coyle, 1996; Coyle et al., 2003). For example, in prefrontal cortex glutamic acid decarboxylase (GAD), 67 neurons were lost and GAD67 mRNA expression was reduced in schizophrenia (Guidotti et al., 2000; Volk et al., 2000). In the GABA transporter (GAT), another GABAergic presynaptic marker, expression was also reduced in the same sectors (Volk et al., 2001). Conversely, GABA-A receptors have been reported to be increased, a changed predicated on reduced presynaptic activity (Benes et al., 1996; Volk et al., 2002). These very same alterations occur in rat cortex as a consequence of chronic treatment with the NMDAR antagonist, MK 801 (Paulson et al., 2003).

VII. Clinical Trials of NMDAR Modulators in Schizophrenia

A. Open Trials of Glycine If a subpopulation of NMDA receptors are hypofunctional in schizophrenia, thereby accounting for positive symptoms, negative symptoms, and speciﬁc cognitive impairments, then treatments that enhance NMDA receptor function should have eVects on these symptoms. The GMS has attracted the greatest interest as a drug target because preclinical studies indicate that agonists at this site can reverse the behavioral eVects of dissociative anesthetics, but are not neurotoxic ( Javitt et al., 1999). Furthermore, electrophysiologic studies have provided compelling evidence that the GMS is not saturated by either glycine or d-serine, endogenous agonists, as the potent GlyT1 inhibitor N-[3-(40 -ﬂuorophenyl)-3-(40 phenylphenoxy)propyl]sarcosine (NFPS) augments NMDAR responses in CA1 pyramidal cells with SchaVer collateral stimulation in the acute hippocampal slice (Bergeron et al., 1998). Moreover, in vivo NFPS augments LTP in the dentate gyrus and enhances prepulse inhibition of the acoustic startle response, which is impaired in schizophrenia (Kinney et al., 2003).

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The initial open label studies on the eVects of glycine were motivated not primarily by the NMDA receptor hypothesis, but rather by the proposal that schizophrenia may involve abnormalities of single carbon metabolism. In a brief letter, Waziri (1988) reported the eVects of a trial of 5–25 g/day of glycine in patients with chronic schizophrenia who were poorly responsive to neuroleptics and socially impaired. Four patients were reported to demonstrate deﬁnite salutary responses, resulting in a reduction in antipsychotic doses. A subsequent open trial used a lower dose of glycine (10.8 g/day) in patients with chronic schizophrenia who were receiving typical antipsychotics. Three of ﬁve patients exhibited moderate reductions in brief psychiatric rating scale (BPRS) total scores accompanied by modest reductions in scale for the assessment of negative symptom (SANS) total scores, a validated index of negative symptoms (Rosse et al., 1989). Another open clinical trial with somewhat higher glycine doses (15 g/day) in patients who responded poorly to antipsychotics revealed two out of the six subjects also exhibited a greater than 30% reduction of the BPRS and a third with a 14% reduction (Costa et al., 1990). The responders were treated longer than the nonresponders. In aggregate, these open trials and clinical observations suggested that glycine might reduce symptoms, particularly negative symptoms, in patients suVering from chronic schizophrenia who were poorly responsive to typical antipsychotic medications.

B. Glycine Placebo-Controlled Trials Speciﬁcally motivated by the NMDAR hypothesis of schizophrenia, Javitt and colleagues (1994) carried out a series of placebo-controlled studies of highdose glycine ranging from 30 to 60 g/day (0.4–0.8 g/kg/day) added to typical antipsychotics (Table I). Their initial study consisted of 14 patients with chronic schizophrenia and prominent negative symptoms who were randomized to placebo or glycine for 8 weeks and then crossed over ( Javitt et al., 1994). A signiﬁcant reduction in negative symptoms but not positive symptoms or general psychopathology was observed in those patients who initially received glycine or were crossed over to glycine from placebo. As the glycine cohort, which was extended to an additional 8 weeks of glycine, did not exhibit further improvement, maximal therapeutic eVects of glycine likely occurred within the initial 8-week period. The number of subjects in this study was small. However, the 17% decrement in negative symptoms appeared unequivocal ( p < 0.001). Notably, those treated with glycine initially and then crossed over to placebo did not deteriorate, suggesting that the hypothesized enhanced NMDA receptor function produced a persistent alteration. A replication study with a cohort of patients with a chronic course (greater than 5 years) and negative symptom scores in the 70th percentile used 60 g/day of glycine in a placebo-controlled crossover design (Heresco-Levy et al., 1996).

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TABLE I Effects of Glycine Modulatory Site Agonists or Glycine Transporter 1 Inhibitor in Schizophrenic Patients Receiving Antipsychotics: Placebo-Controlled Studies Agent

Mechanism

Design

N a Negative Cognitive Positive

Reference

Glycine Glycine

Agonist Agonist

Parallel 14 Crossover 11

Sb S

S S

— —

Glycine

Agonist

Parallel

22

S

S

—

d-Cycloserine

Partial agonist Partial agonist Partial agonist Agonist GlyT1 inhibitor

Dose finding Parallel

9

S

S

—

Javitt et al. (1994) Heresco-Levy et al. (1996) Heresco-Levy et al. (1999) Goff et al. (1995)

47

S

—

—

Goff et al. (1999)

Parallel

24

S

NTc

—

Parallel Parallel

28 38

S S

S S

S S

Heresco-Levy et al. (2002) Tsai et al. (1998) Tsai et al. (2003)

d-Cycloserine d-Cycloserine d-Serine Sarcosine a

Number of subjects. Significant ( p < 0.05). c Not tested. b

Signiﬁcant reductions in negative symptoms, general psychopathology, and total positive and negative symptom scale (PANSS) scores without eVects on positive symptoms were observed during glycine treatment. In a more extensive double-blind study of 0.8 g/kg/day of glycine in a similarly impaired cohort of schizophrenic patients receiving antipsychotic medications, factor analysis revealed gradual but robust improvement in negative symptoms, cognitive symptoms, and depression but not in positive symptoms as measured by the PANSS (Heresco-Levy et al., 1999). Maximal eVects of glycine were not achieved until 4 to 6 weeks of treatment. Those receiving glycine and subsequently placed on placebo did not deteriorate. Metabolic analyses indicated that the improvement in negative symptoms ( p ¼ 0.002), cognition ( p ¼ 0.01), and depression ( p ¼ 0.002) on PANSS correlated inversely with pretreatment serum glycine levels, a ﬁnding that was replicated in the d-cycloserine clinical trials.

C. d-Cycloserine The next GMS examined was d-cycloserine, a drug that has been used to treat tuberculosis for 40 years and has a history of neurocognitive side eVects. d-Cycloserine was shown in Xenopus expression studies of the NMDA receptor

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to act as a partial agonist at the GMD with approximately 60% of the eYcacy of glycine (Sheinin et al., 2001). Conversely, it inhibited the eVects of saturating concentrations of glycine by 40–50%. Thus, d-cycloserine behaves as an agonist or an antagonist depending on the degree of occupancy of the modulatory site by the full agonists, glycine and/or d-serine. d-Cycloserine also inhibits serine racemase, the enzyme that synthesizes d-serine (Cook et al., 2002). The combination of this action and the partial agonist property of d-cycloserine would predict a U-shaped dose–response curve. In an initial dose-ﬁnding study in which d-cycloserine was added to typical antipsychotics in a cohort of patients with chronic schizophrenia characterized by prominent negative symptoms, GoV et al. (1995) found a U-shaped dose response in a dose range from 5 to 250 mg/day. At 50 mg/day, a 21% reduction in negative symptoms and signiﬁcant improvement on a cognitive task were observed. The loss of response at higher doses is consistent with the prior ﬁndings of Cascella et al. (1994), who observed no eVect of 250 mg/day in schizophrenics receiving antipsychotics. In a dose-ﬁnding study with schizophrenic patients not treated with antipsychotics, van Berckel et al. (1996) also observed a signiﬁcant reduction in negative symptoms but no eVects on positive symptoms at 100 mg/day of d-cycloserine with loss of eVect at 250 mg/day. A subsequent larger parallel double-blinded, placebo-controlled study of d-cycloserine at 50 mg/day in 46 schizophrenic patients who were receiving stable doses of typical antipsychotics for at least 4 months and had prominent negative symptoms revealed a 23% reduction in negative symptoms, particularly associated with an improvement in blunted aVect, after 8 weeks of treatment (GoV et al., 1999). The eVect size of 0.8 was virtually identical to that observed in the large glycine trial. Nevertheless, in this study, no eVects on cognition were observed. Heresco-Levy et al. (2002) also studied d-cycloserine (50 mg/day) in a 6-week placebo-controlled crossover study in 24 patients with chronic schizophrenia who were on stable doses of typical antipsychotics, resperidol or olanzapine. They observed a signiﬁcant 15% reduction in negative symptoms regardless of the antipsychotic type. In the smaller trials with d-cycloserine, GoV et al. (1996) found a highly signiﬁcant inverse correlation between serum glutamate levels at baseline and improvement in negative symptoms with 50 mg of d-cycloserine. The improvement in these symptoms correlated with changes in serum glycine during treatment. In their d-cycloserine trial, Heresco-Levy et al. (2002) also found that those patients with lower glycine levels exhibited the greatest response in negative symptoms. Nevertheless, the large parallel placebo-controlled trial by GoV et al. (1999b) did not replicate these ﬁndings with regard to serum glycine, although d-cycloserine concentrations correlated negatively with a change in serum glutamate and positively with serum HVA.

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D. d-Serine and Sarcosine In contrast to d-cycloserine, d-serine is a full agonist at the GMS, is approximately 3-fold more potent than glycine, and crosses the blood–brain barrier more readily than glycine. d-Serine is not a substrate for the glycine transporter (GlyT1), but rather is catabolized by DAAO. Tsai et al. (1998) carried out a double-blind, parallel placebo-controlled trial of d-serine (30 mg/kg/day) on 28 patients with chronic schizophrenia who were poorly responsive to typical antipsychotic medications. Six weeks of treatment with d-serine resulted in signiﬁcant reductions in negative symptoms, cognitive symptoms, and positive symptoms. Cognitive improvement assessed by the PANSS was corroborated by a signiﬁcantly better performance on the Wisconsin card sort test, which engages the frontal lobe and is impaired in schizophrenia. d-Serine treatment resulted in a 50-fold increase in serum d-serine levels, whereas the levels of glycine, glutamate, and aspartate were unchanged. Symptom improvement observed after 6 weeks of d-serine treatment correlated signiﬁcantly with serum d-serine levels. Exacerbation of extrapyramidal side eVects was not observed with d-serine treatment. Sarcosine (N-methylglycine) is a potent inhibitor of the glycine transporter GlyT1. Tsai et al. (2003) carried out a parallel design, double-blind placebocontrolled trial of sarcosine (2 g/day) in 38 schizophrenic patients. Patients were on stable doses of antipsychotics with two-thirds receiving resperidol. After 6 weeks of treatment with sarcosine, patients exhibited robust reductions in negative and positive symptoms and improvement in cognition. The only property that glycine, d-serine, d-cycloserine, and sarcosine are known to have in common is their ability to activate directly or indirectly at the GMS on the NMDAR. The placebo-controlled clinical trials with these agents have consistently demonstrated improvement in negative symptoms, and most have shown improvement in cognitive symptoms. d-Serine and sarcosine, however, also reduced positive symptoms in patients receiving concurrent antipsychotics. This eVect may be due to the greater potency of d-serine and sarcosine versus glycine and the ceiling of 60% eYcacy of d-cycloserine.

E. Clozapine and the NMDA Receptor Clozapine is the one antipsychotic that appears to beneﬁt patients poorly responsive to typical antipsychotics and improves negative symptoms in particular (Meltzer, 1997). The speciﬁc eVects of clozapine resemble those aspects of the schizophrenia syndrome that are most consistently responsive to the GMS agonists. In a study looking for potential additive eVects, GoV et al. (1996) carried out a blinded dose-ﬁnding trial with d-cycloserine in 10 patients maintained

NMDA RECEPTOR FUNCTION

505

on clozapine. Unexpectedly, there was a signiﬁcant dose-related exacerbation of negative symptoms with a 21% increase at 50 mg/day. This exacerbation induced by d-cycloserine in clozapine-treated schizophrenic patients was replicated in a second trial (GoV et al., 1999a). In other words, the dose-dependent exacerbation of negative symptoms in clozapine responders was the mirror image of the dose-dependent improvement in negative symptoms in patients receiving typical antipsychotics. In contrast, three double-blind, placebo-controlled studies of full agonists at the GMS [glycine (30 and 60 g/day) and d-serine (30 mg/kg/ day)] in schizophrenic patients receiving stable doses of clozapine revealed no signiﬁcant treatment eVect on negative symptoms as rated by BPRS scores or SANS (Evins et al., 2000; Potkin et al., 1999; Tsai et al., 1999). Serum studies revealed levels of glycine and d-serine comparable to those observed previously in schizophrenic patients treated with typical antipsychotics who had exhibited a signiﬁcant reduction in negative symptoms. Power analyses indicated that signiﬁcant eVects would have been observed in the clozapine responders if the eVect size of 0.8 from previous studies of glycine or d-serine obtained in these negative studies. The most parsimonious explanation for these ﬁndings is that clozapine, unlike other antipsychotics, causes some alteration that results in full occupancy of the relevant GMS on the NMDA receptor, which accounts for its eVects on negative symptoms and cognition. This hypothesis is consistent with the fact that neither glycine nor d-serine, full agonists, provide an additional beneﬁt for negative symptoms in clozapine-treated patients. In contrast, d-cycloserine exacerbates negative symptoms in clozapine-treated patients because it should behave as an antagonist only if the GMS is fully occupied. Neurophysiologic studies in frontocortical slices from rats indicate that clozapine enhances NMDA receptor responses of pyramidal cells and prevents phencyclidine-induced blockade (Ninan et al., 2003). Furthermore, Sur et al. (2003) reported that desmethyl clozapine, a metabolite of dozapine associated with a clinical response, directly activates muscarinic M1 receptors, which enhance hippocampal NMDAR responses.

VIII. NMDA Receptor and Neuroplasticity

The hypothesis that NMDAR hypofunction contributes to the pathophysiology of schizophrenia has generally been formulated in terms of the acute or subacute manifestations of the disorder. However, one of the most robust ﬁndings in schizophrenia research is the reduction in cortical volume, which may aVect the frontal and temporal cortex diVerentially (Kuperberg et al., 2003; Selemon et al., 2002; Wright et al., 2000). While reduced cortical volume is observed by the time

506

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of symptomatic onset of the disorder, it appears to be progressive, at least early in the course of the disorder (Kasai et al., 2003; Thompson et al., 2001). The loss of cortical volume does not appear to reﬂect a comparable loss of neurons. The absence of reactive gliosis has long been used as an argument against neuronal degeneration in the mature cortex in schizophrenia (Benes, 1993). Except for a subtle possible loss of GABAergic interneurons in discrete layers of the cortex or sectors of the hippocampus, stereologic cell-counting techniques have not revealed a signiﬁcant loss of neurons (Benes et al., 2001b; Selemon et al., 1998). An alternative explanation would be atrophy of neurons themselves. Limited data lend support to this interpretation, as cortical pyramidal cells have been reported to be smaller with less complex dendritic trees and fewer spines (Glantz and Lewis, 2000; Pierri et al., 2001, 2003; Selemon et al., 1998). These atrophic changes at the neuronal level are not inconsistent with NMDAR hypofunction. Increased NMDAR function has been associated not only with functional plasticity, but also with structural plasticity, including the elaboration of spines (Leuner et al., 2003). Conversely, Olney and Farber (1995) have also shown in a series of studies that the treatment of adult animals with NMDAR antagonists results in neuronal damage in the limbic cortex manifested by the induction and expression of heat shock proteins, a process that in part appears to result from reduced GABAergic tone (Lan et al., 1997; Li et al., 2002). There is increasing evidence of reinforcing interactions between NMDAR and trophic factors such as brain-derived neurotrophic factor (BDNF) (Lu, 2003). Activation of NMDAR induces the expression of BDNF (Fumagalli et al., 2003; Xiong et al., 2002), as this is prevented in an activity-dependent model in which NMDAR subunit epsiolon has been inactivated by a null mutation (Kitamura et al., 2003). Furthermore, BDNF acts in synergy with NMDAR activation to cause transcriptiondependent LTP (Kovalchuk et al., 2002; Messaoudi et al., 2002). BDNF both increases NMDAR function acutely (Levine and Kolb, 2000) and increases NMDAR subunit expression chronically (Glazner and Mattson, 2000). Consistent with this, memory formation is impaired moderately in humans with a less eVective allelic variant of BDNF (Egan et al., 2003). Whether this variant is a risk gene for schizophrenia remains unclear. Notably, loss of BDNF is associated with impaired LTP in experimental animals (Korte et al., 1995). Postmortem studies of BDNF expression in the cortex suggest that it is reduced in schizophrenia ( Weikert et al., 2003).

IX. Conclusion

Converging ﬁndings from the similarities in clinical features of NMDAR antagonists and schizophrenia, postmortem ﬁndings, results of genetic association studies, and the therapeutic eVects of drugs that enhance NMDAR function

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507

provide a convincing case for the NMDAR hypofunction hypothesis for schizophrenia. Given the central role of the NMDAR in mediating not only neuroplasticity but actual neurotrophic eVects, it is conceivable that persistent NMDAR hypoactivity would account for the loss of cortical volume in schizophrenia. Studies carried out thus far with GMS agonists have been of relatively brief duration—2 to 8 weeks—which is likely to be insuYcient to address more gradual neurotrophic eVects. For example, the clinical response to clozapine, especially regarding negative symptoms, has been described as gradual, requiring many weeks to months (Meltzer, 1997). Inasmuch as some of the eVects of clozapine may be mediated through the NMDAR, it is reasonable to speculate that prolonged treatment with GMS agonists such as d-serine or GlyT1 uptake inhibitors might promote corrective neuroplasticity, especially if the treatment were given in the context of a cognitive rehabilitation program directed at improving memory and problem solving in schizophrenia. In the absence of evidence of neuronal degeneration in schizophrenia (Benes et al., 2003), it does not seem farfetched to envision alternative treatments that promote neurotrophic eVects that may be associated with enhanced NMDAR function, resulting in reversal of the cognitive impairments, negative symptoms, and the associated cortical atrophy.

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INDEX

A AA. See Acids Acetylocholine (ACh), 94–95 Acetylocholinesterases, 94 Acids aminophosphono valeric (APV), 500 arachidonic (AA), 255, 298, 304–316, 313 c-aminobutyric (GABA), 129 dihomogammalinolenic (DGLA), 255 docosahexaenoic (DHA), 258–259 eicosapentaenoic (EPA), 255 gamma linoleic (GLA), 257 homovanillic (HVA), 138 retinoic, 360–362 Acids, polyunsaturated fatty (PUFA), 258–260 cytokines and, 309–310 levels of, 297–298, 313 Acquired immunodeﬁciency syndrome (AIDS), 7–8. See also Human immunodeﬁciency virus Acute-phase response (APR), 303 AD. See Alzheimer’s Disease Adenosine diphosphate (ADP), 256 Afro-Caribbeans, 363 Aging, 395–396, 455 Agonists, BP 897, 432–434, 433f AIDS. See Acquired immunodeﬁciency syndrome AKAP. See Protein(s) Alzheimer’s Disease (AD), 6–7, 112, 483, 498 Amino acids, 26, 238 NAA, 27, 28t–29t schizophrenia and, 27 AMPAkines, 37–39, 38t a-MSH. See Hormones Amygdala, 125, 136, 139 Analgesia, 112 Anandamides, 304–315 ANC. See Archineostriatum caudale Anesthetics, dissociative, 494–495 Anhedonia, 98

Animals, knockout/wild-type, 96–97 Anisotrophy, 391, 392–393 Ankyrin (ANK), 232–233 Antioxidant defense system (AODS), 302–303, 313 Antipsychotics, 280–281, 302. See also Drug(s) Anxiety, 112, 132, 135–137, 139 AODS. See Antioxidant defense system Apathy, 98 apoE. See Receptor(s) Apoptosis, 22, 206, 453 APP. See Protein(s) APR. See Acute-phase response APV. See Acids Arabidopsis, 76 ARC. See Protein(s) Archineostriatum caudale (ANC), 5 Astrocytes, 454 ATF. See Transcription factor, activating Australia, 362–363 Autism, 132, 134–135

B BDNF. See Brain derived neutrophic factor Behaviors, 126–127, 126f human, 221–222 learning/memory and, 241–243 Blotting immuno, 185–186 Northern, 185–186 Western, 58 BPRS. See Brief psychiatric rating scale Brain. See also Amygdala; Cerebellum; Cortex; Hippocampus; Schizophrenia cholinergic neurons and, 94–95, 100 function, 2, 48–49, 202 hippocampus of, 1–2, 4, 23, 36 human, 96 ischemia, 231 mid, 334 517

518 Brain (cont.) plasticity in, 4, 25f, 29–36, 30t–31t, 32t–33t, 35t postmortem, 78–79, 258 presynaptic proteins and, 179–185, 180t–184t, 186t, 187t–192t regions of, 1–2, 48–49 structure, 144 vitamin D and, 364–367 Brain derived neutrophic factor (BDNF), 111, 126f, 209, 506 D3 receptor and, 426–438, 428f, 429f, 430f, 432f–433f, 435f–436f, 437–438 depression and, 437–438 role of, 425–426 schizophrenia and, 437–438 stress and, 437–438 Brain development, 2, 48–49. See also Serotonergic systems; Serotonin serotonin and, 111–113, 118–120, 118f, 131–149 synaptic plasticity and, 111, 113, 143–148 vitamin D and, 365–367 Brief psychiatric rating scale (BPRS), 501

C Caenorhabditis elegans, 473 Calcium dendrites and, 11–12, 142 inﬂux, 83 levels, 2–3, 286 mobilization, 4–5 neuropils, 11–12 schizophrenia and, 286–287 spines and, 11–12 CaMKII. See Protein kinase type II, calcium calmodulin-dependent cAMP. See Cyclic adenosine monophosphate Cancers, 76 Cannabinoid receptor (CB1) system, 305 Cannon-Spoor, H.E., 56 Catalase (CAT), 303 Catechol-o-methyl transferase (COMT), 276, 357–359 Causations, 74 CB. See Cerebellum CB1. See Cannabinoid receptor system CBPs. See Protein(s) Cell(s)

INDEX

Chinese hamster ovary (CHO), 332 glial, 382 growth, 128 mammal, 120–124, 121f–122f natural killer, 306 red blood (RBC), 258, 298–299 Schwann, 477 T, 458 Cerebellum (CB), 212f Cerebrospinal ﬂuid (CSF), 26–27, 138, 338–339 cGMP. See Cyclic guanidine monophosphate Chlorpromazines, 21 CHO. See Cell(s) Cholinesterases, 94 Chromatins, 76 Chromosomes, 237, 359, 402 Clopazines, 504–505 CNP. See Gene(s) CNS. See Nervous system, central Cocaine, 132, 436f COMT. See Catechol-o-methyl transferase Conditioning, fear, 136–137 Conte Center for Neuroscience of Mental Disorders, 407 Cortex, 125 frontal (FC), 212f neuropils, 1–2 prefrontal (PFC), 9–10, 127, 185, 230–231, 356, 387 somatosensory (SI), 5, 131 visual, 130–131 CREB. See Proteins, cAMP response element-binding CSF. See Cerebrospinal ﬂuid Cyclic adenosine monophosphate (cAMP), 95, 124, 262 Cyclic guanidine monophosphate (cGMP), 129 Cycling, 24 Cytokines, 313, 361, 454 PUFA and, 309–310 schizophrenia and, 297, 307–309 Cytoplasms, 403 Cytosines, 76–78, 205–206

D DA. See Dopamine DAAO. See Degrading enzyme D-amino acid oxidase

INDEX

DAG. See Diacylglycerol D-cycloserines, 37–39, 38t, 502–503 Degrading enzyme D-amino acid oxidase ( DAAO), 493 Delusions, 97 Dementia, 94 Dendrites, 2–3, 447–448. See also Spine (s) calcium and, 11–12, 142 remodeling of, 131 Denmark, 363 Dentate gyrus ( DG), 125, 132, 241 Depression 5-HTRs and, 139–141 BDNF and, 437–438 D3 receptor and, 437–438 Depression, long-term ( LTD), 23, 208 LTP and, 23 neuroplasticity and, 23–25, 25f PSD-95/SAP90 and, 229–230 synaptic plasticity and, 49, 93, 100 Dermatology, 261–262, 264–265, 363 Dexamethasone, 454 DG. See Dentate gyrus DGLA. See Acids DHA. See Acids Diacylglycerol (DAG), 300 Diacylglycerols, 304 Disconnectivity, 385–388 Disease(s). See also Schizophrenia Alzheimer’s (AD), 6–7, 112, 483, 498 demyelinating, 392–395 Down’s Syndrome as, 7, 148–149 Huntington’s ( HD), 8, 231–232 Parkinson’s ( PD), 327, 336, 431–434, 432f–433f, 483 RETT Syndrome as, 354 Disorders affective, 112, 132–136, 143 behavioral, 132 eating, 112 learning, 132 neurological, 471 neuropathological, 6–7 progressive developmental, 447 stress-related, 132 variety of, 1 Disorders, neuropsychiatric, 9–11 Homer proteins and, 241–242 PSD-95/SAP90 and, 230–232

519

Disorders, psychiatric, 112–113, 132, 140–141 MAP kinases and, 213–214 DNA changes, 86 cytosine methyltransferases in, 76–78 methylation, 75–77, 87 mitochondrial (mtDNA), 281, 282 non, 85 nuclear (nDNA), 281, 282 proteins, 76, 78 role of, 74–75 Dopamine (DA), 21, 93, 495 D3 receptor, 426–438, 428f, 429f, 430f, 432f–433f, 435f–436f hypotheses, 211–212 interactions, 133 LTP and, 276–277 metabolites of, 138, 302 neurons, 334 neurotransmitter, 98 NT and, 333–335 receptors, 99 6-hydroxy (6-OHDA), 427–430, 429f, 430f system, 327, 329 Doppler ﬂowmetry, 264 Down’s Syndrome, 7, 148–149 Drosophila, 473 Drosophila melangoster, 76 Drug(s) action of, 241–242 addiction, 242 antidepressant, 137–138, 140–141 antipsychotic, 302, 333–337, 339–342 cues, 434–437, 435f–436f psychiatric, 111–113 serotonergic, 112, 132, 135 SERT and, 141–143 treatments, 140–141 D-serines, 37–39, 38t, 504 DSM IV diagnosis, 145, 261 DTI. See Imaging Dynamins, 224 Dysfunctions, 112 E Ectopia, nuclear, 403 Edema, 264 Eicosanoids, 305

520 Embryogenesis, 111, 125 Endocannabinoid system, 304–305 Enzymes, N-acetyl-a-linked acidic depeptidase (NAALADase), 27, 223 EPA. See Acids Epigenetics, 354 Aristotelian theory of, 86 biochemical processes in, 75–78 CpG islands and, 77 deﬁnition of, 73–74, 85–87 DNA cytosine methyltransferases in, 76–78 DNA methylation in, 75–77, 87 evolution and, 75 genes and, 74–75 genotypes and, 74–75 inhibitory synapses and, 82–83 methylation in, 77–78 phenotypes and, 74–75 PSD and, 80–82, 83–84 psychiatry and, 86–87 synaptic plasticity and, 78–85 transmitter receptors and, 84–85 Epimutations, 75–76 EPSCs. See Excitatory postsynaptic currents ERK. See Kinase(s), mitogen-activated protein ERP. See Event-related potential Erythema, 264 Estradiol, 483 Estrogen, 3–4, 136, 139, 483–484 Event-related potential (ERP), 494 Evolution, 75 Excitability, 101 Excitatory postsynaptic currents (EPSCs), 492–493 Excitotoxicity, 22 F FC. See Cortex FCD. See Focal cortical dysplasia FDG. See Flurodeoxyglucose Females, 3 5-HIAA, 138 5-HT. See Serotonin 5-HTP. See Tryptophans Fluoxetines, 140 Flurodeoxyglucose (FDG), 285

INDEX

f MRI. See Imaging Focal cortical dysplasia (FCD), 484 Freed, W. J., 56 G GABA. See Acids GABARAP. See Protein(s) GAD. See Glutamic acid decarboxylase GAP-43. See Protein(s) GC. See Guanylate cyclase GCPII. See Glutamate carboxypeptidase II Gene(s) 20 -30 cyclonucleotide, 30 -phosphodieterase (CNP), 399 array, 356–357 candidate studies, 357–360 coding, 237–238, 260 DDM1, 76 dendrin, 478 in development, 86 dysbinden, 497 epigenetics and, 74–75 expression, 47–48, 57–64, 61f–63f, 281–282 functions, 73 GAD67, 78 GT receptor-associated, 497–498, 498f immediate early (IEG), 241 imprinting, 76–77 mitochondria, 281–282 myelin-related, 396–402, 398t regulation, 243 schizophrenia and, 353–360, 369–370 synaptic, 47 Tramdorin 1/mPAT2, 478 transcription, 206, 367–368 VAMP, 177 Genetics, 85 Genotypes, 74–75 Gephyrins, 82 GFAP. See Protein(s) GGF. See Growth factor(s) GK. See Guanylate kinase-like domains GKAP. See Protein(s) GLA. See Acids Glucocorticoids, 136 Glucogenesis, 126f, 127 Glutamate (GT), 302 GRIP, 80, 223, 225–226 levels, 26

INDEX

receptors, 4–5, 19, 23–24, 25f, 132–133, 223, 495–496 schizophrenia and, 25–39, 28t–29t, 30t–31t, 32t–33t, 35t, 38t, 211 synapses, 19, 23–24 Glutamate carboxypeptidase II (GCPII), 497 Glutamic acid decarboxylase (GAD), 3–4, 129 Glutaminase (PAG), 407 Glutathione, intracellular (GSH), 310 Glutathione peroxidase (GSH-Px), 303 Glycine(s), 37–39, 38t, 500–501 modulatory site (GMS), 493, 497 placebo-controlled trials, 501–502, 502t GPCRs. See Receptor(s) Granulocytes, 306 GRIP. See Protein(s) Growth factor(s), 361 glial (GGF), 383 insulin-like (IGF), 383–384 neutrophins and, 120 platelet-derived (PDGF), 383 responses to, 205 serotonin, 114, 118f, 120–124, 121f–122f GSH. See Glutathione, intracellular GSH-Px. See Glutathione peroxidase GSN. See Protein(s) GT. See Glutamate Guanylate cyclase (GC), 450–451, 451t Guanylate kinase-like (GK) domains, 227–228, 228f, 235 H HA. See Protein(s) Hallucinations, 20, 22, 97 HC. See Hippocampus HD. See Huntington’s Disease Hemperly, J. J., 56 Heterodimers, 206 Heterogeneity, 29 Hippocampus (HC) of brain, 1–2, 4, 23, 36 effects in, 46, 125, 212f schizophrenia and, 230–231 HIV. See Human immunodeﬁciency virus HIVE. See Human immunodeﬁciency virus encephalitis Homodimers, 206 Hormones, 112. See also Serotonin levels of, 136

521

a-melanocyte-stimulating (a-MSH), 137 HPA. See Hypothalamic-pituitaryadrenal axis HRH. See Mouse, heterozygous reeler Human immunodeﬁciency virus (HIV), 7–8. See also Acquired immunodeﬁciency syndrome Human immunodeﬁciency virus encephalitis (HIVE), 7–8 Huntington’s Disease (HD), 8, 231–232 Huntintin, 231 HVA. See Acids Hyde, T.M., 56 Hydrolysis, 94, 309–310 PI, 300–301 signal transduction pathway, 126 Hydroxylation, 364 Hypothalamic-pituitary-adrenal (HPA) axis, 135 I IAR. See Immunoautoradiography ICD-10 criteria, 261 IEG. See Gene(s) IFN-c. See Interferon-c IGF. See Growth factor(s) IL. See Interleukin Imaging, 434 diffusion tensor (DTI), 390–391, 394 evidence, 388–391, 389t functional magnetic resonance (f MRI), 285 magnetic resonance (MRI), 102, 389, 389t, 394 magnetic transfer (MTI), 390 studies, 386, 496 Immunoautoradiography (IAR), 58 Immunohistochemistry, 404 Immunology, 306 conﬂicts, 308–309 overactive innate system in, 307 stress and, 310–311 Th1 system, 307–308 Immunoreactivity, 336 In situ hybridization histochemistry (ISHH), 57–58, 60, 230–231 Inhibitors, 94 COX, 261–262 MAOI, 137 PCPA, 128–129

522

INDEX

Inhibitors (cont.) serotonin reuptake (SSRIs), 140–141 iNOS. See Synthase, inducible nitric oxide Inositol triphosphate (IP3), 121f Interferon-c (IFN-c), 306 Interleukin (IL), 306, 454 Intrauterins, 454 IP3. See Inositol triphosphate Irritability, 139 ISHH. See In situ hybridization histochemistry Isoforms, 237–238, 238f J JNK. See Kinase(s), mitogen-activated protein K Ketamines, 25–26, 494–495 Kinase(s), 201 C, 80 protein, 224 protein A (PKA), 476 protein C (PKC), 121f, 300 stress-activated protein (SAPK), 205 tyrosine, 63 Kinase(s), mitogen-activated protein (MAP) cascades, 202, 203–209, 204f c-jun N-terminal (JNK), 201, 203, 204f, 205, 210f, 213 CNS and, 207–209 extracellular signal-regulated (ERK), 201, 203–205, 204f, 209–212, 210f, 213–214 p-38, 203, 205–206, 210f, 213 phosphatases (MKPs), 206 psychiatric disorders and, 213–214 schizophrenia and, 209–213, 210f, 212f transcription factor targets and, 206–207 Kleinman, J. E., 56 L LAL. See Long attack latency Latent inhibition paradigm (LI), 340 Learning, 287 aversive, 136 behavior and, 241–243

disorders, 132 LTP/neuropils and, 4–5, 34, 36 serotonin and, 115t–117t, 118f, 132–134 spine and, 4–5, 34 synaptic plasticity and, 49, 100–101 Lederberg, J., 85 Levodopa, 429–430, 429f, 430f Levodopa-induced dyskinesia (LID), 431–434, 432f–433f LI. See Latent inhibition paradigm LID. See Levodopa-induced dyskinesia Lithium, 140 Long attack latency (LAL), 136 Long-term potentiation (LTP), 4–5, 7, 208. See also Memory DA and, 276–277 LTD and, 23–25, 25f PSD-95/SAP90 and, 229–230 requirements of, 27 synaptic plasticity and, 49, 93, 100–101 LSD. See Lysergic acid diethylamide LTD. See Depression, long-term LTP. See Long-term potentiation Lubec, G., 6–7 Lysergic acid diethylamide (LSD), 113–114, 128 psychosis, 145–147 M Macrophages, 306 MAG. See Myelin MAGuK. See Protein(s) MAL. See Protein(s) Malar thermal circulation index (MTCI), 263 Mammals cells of, 120–124, 121f–122f methylation in, 78 MAP. See Kinase(s), mitogen-activated protein MARCKS. See Myristolated alanine-rich C kinase substrate Mass spectrometry, 80 McClintock, Barbara, 76, 87 M-chlorophenylpiperazine (mCPP), 137 Medial dorsal (MD), 9 Memory, 36, 49, 100, 101, 287 aversive, 136 behavior and, 241–243

INDEX

episodic, 277–278 LTP and, 132 neocortical, 132 serotonin, 115t–117t, 118f, 132–134 Mental retardation, 132, 148–149 Metabolism, 285, 302 Metabolites, 27 Metachromatic leukodystrophy (MLD), 391–394 Methylation cytosine, 76–78 DNA, 75–77, 87 in mammals, 78 transcription, 77–78 Mianserins, 140 Microdomains, 226 Microglia, 454 Mitochondria, 273 function of, 282, 287–288 genes, 281–282 morphological aberration of, 279 neuronal activity and, 282–284 neuroplasticity and, 282–287 plasticity of, 284–287 schnizophrenia and, 273, 279–287 Mitochondrial oxidative phosphorylation system (OXPHOS), 279–281, 282 MKPs. See Kinase(s), mitogen-activated protein MLD. See Metachromatic leukodystrophy Molecule, neural cell adhesion (NCAM), 53–57, 55t ATF-1, 120, 121f CREM, 120, 121f levels, 102 Molecules, synaptic cell adhesion (SynCAM), 81 Monamines, 138 Monoamine oxidase A (MAOA), 131 Monocytes, 306 MOPEG. See 3-methoxy-4-hydroxyphenylglycol Mouse, heterozygous reeler (HRM), 64 MPTP. See 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine MRI. See Imaging MS. See Multiple sclerosis MTCI. See Malar thermal circulation index mtDNA. See DNA MTI. See Imaging Multiple sclerosis (MS), 391–395

523

Myelin, 447. See also Disease(s) associated glycoprotein (MAG), 396–399, 398t development of, 383–384 examinations of, 402–404 function of, 384–385 related genes, 396–402, 398t schizophrenia and, 381–407, 398t Myristolated alanine-rich C kinase substrate (MARCKS), 449–450, 450t N NAA. See N-acetylaspartate NAAG. See N-acetyl-aspartylglutamate NAALADase. See Enzymes, N-acetyl-a-linked acidic depeptidase N-acetylaspartate (NAA), 387–388 N-acetyl-aspartylglutamate (NAAG), 497 nAChR. See Receptor(s) National Institute of Mental Health (NIMH), 407 NCAM. See Molecule, neural cell adhesion nDNA. See DNA Neromedin N (NN), 328 Nervous system, central (CNS), 94 effects of NT, 333–334 function of, 111–113, 149 MAP kinases and, 207–209 Oct-6 proteins and, 479–480, 480f Nervous system, peripheral (PNS), 477 Netherlands, 363 Neuregulins (NRGs), 359, 400, 447 Neurexins, 81 Neurochemical sensitization, 446–447 Neurocytochemicals, 386 Neurodevelopment, 306, 445–446, 483t Neurogenesis, 131, 139 Neuroligins, 81 Neuron(s) activity of, 282–284 cholinergic, 94–95, 100 cortical, 429 DA, 334 GABAergic, 79, 407 inter, 3 migration of, 53–54 morphology of, 1–2 NOS, 286

524 Neuron(s) (cont.) periventricular hypophysial dopaminergic (PHDA), 137 postsynaptic, 100 presynaptic, 84 serotonergic, 114, 118f, 120, 121f signals from, 202 Neuropil(s). See also Disease(s), Alzheimer’s calcium and, 11–12 components, 6 cortex and, 1–2 dendrites/plasticity/spines and, 1–4, 6, 11–12, 121, 142 HD and, 8 HIV and, 7–8 learning/long-term potentiation and, 4–5, 34, 36 schizophrenia and, 9–11 Neuroplasticity. See also Plasticity, Neuronal; Schizophrenia disorder of, 19–20, 36, 39 LTD and, 23–25, 25f mitochondria and, 282–287 NMDA and, 505–507 schizophrenia and, 19–20, 23–25, 25f substrates of, 23–25, 25f Neurotension (NT), 327 antipsychotic drugs and, 333–337 CNS and, 333–334 DA and, 333–335 -ergic compounds, 339–342 neurotransmission, 338–339 receptors, 328–29, 330t–331t, 332 schizophrenia and, 336 Neurotransmitters, 20, 112, 202, 209 cholinergic, 94–95 defects of multi, 302 functions of, 128–130 muscarinic, 101–102 in schizophrenia, 276–277 Neutrophins, 120, 203 Niacin dermatology and, 261–262, 264–265 psychopathology and, 265 schizophrenia and, 260–265 tests, 260–265 Nicotine, 132, 436f NIMH. See National Institute of Mental Health Nissl-stained materials, 404

INDEX

Nitric oxide (NO), 284–286, 287, 303 NLS. See Nuclear localization signal NMDA. See Receptors, N-methyl-D-aspartate NN. See Neromedin N NO. See Nitric oxide Noradrenalines, 138 NRGs. See Neuregulins NT. See Neurotension Nuclear localization signal (NLS), 475–476 Nucleus, median raphe´, 133 O Oligodendrocytes development of, 383–384 function of, 384–385 schizophrenia and, 381–385, 402–407 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 431–433, 432f–433f Open reading frame (ORF), 238 Organelles, 403 Organophosphates, 94 ORS. See Spectroscopy Osteoporosis, 362 OXPHOS. See Mitochondrial oxidative phosphorylation system P PAG. See Glutaminase PANSS. See Positive and negative syndrome scale Parkinson’s Disease (PD), 327, 336, 431–434, 432f–433f, 483 Pathology, free radical, 302 Pathology, synaptic cortical, 59–61, 61f–63f of schizophrenia, 47–64, 51t, 55t, 61f–63f PCP. See Phencyclidine PD. See Parkinson’s Disease PDGF. See Growth factor(s) PDZ domains, 232–233 PE. See Phosphatidylethanolamine PFC. See Cortex PGs. See Prostaglandins PHDA. See Neuron(s) Phencyclidine (PCP), 21, 25–26, 201 rat model, 211–213, 212f Phenotypes, 74–75

INDEX

Phobias, 112 Phospholipase A2 (PLA2), 258–260, 299–300, 301 activation by ROS, 301–302 C (PLC), 121f, 300–301 Phosphatidylethanolamine (PE), 304 Phosphoinositide (PI), 126 Phospholipids, 255 abnormal membrane, 298–306 defects of, 298–299 increased activities of, 299–301 membrane, 257–260 schizophrenia and, 257–260, 297–306, 309–313, 312f Phosphorylation CREB, 3 mechanisms, 222 PKA, 3, 4, 81 PI. See Phosphoinositide PICK. See Protein(s) PKA. See Kinase(s) PKC. See Kinase(s) PLA2. See Phospholipase Plasticity, 1, 74, 86. See also Neuroplasticity in brain, 4, 25f, 29–36, 30t–31t, 32t–33t, 35t deﬁnition of, 113 developmental, 114–128, 115t–117t, 121f–122f, 126f estrogen and, 3–4 modulation of, 115t–117t, 125–128, 126f normal, 2–3 schizophrenia and, 25f, 29–36, 30t–31t, 32t–33t, 35t, 275–276 spine, 2–3 use-dependent, 101 Plasticity, neuronal, 113. See also Neuroplasticity mitochondria and, 282–287 process, 274–275, 287–288 schizophrenia and, 277–279, 287 Plasticity, synaptic altered, 61–64, 61f–63f, 128–131, 143–148 brain development and, 111, 113, 143–148 deﬁntion of, 222–223 disorders of, 221–222 epigenetics and, 78–85 5-HT1AR in, 124–125 gene expression and, 57–64, 61f–63f LTD and, 49, 93, 100 LTP and, 49, 93, 100–101

525

models, 130–131 presynaptic protein and, 57–58 PSD-95/SAP90 and, 229–230 reelin and, 58–64, 61f–63f, 78–79 role of, 93–103 schizophrenia and, 21–23, 47–64, 51t, 55t, 61f–63f, 143–48, 276–277 serotonin and, 128–131, 137–143 PLC. See Phospholipase PMI. See Postmortem index PNS. See Nervous system, peripheral Polysialylated NCAM (PSA-NCAM), 53–57, 55t, 123 Positive and negative syndrome scale (PANSS), 459–460, 502 Positron emission tomography (PET), 134 Postmortem index (PMI), 402–403 Postsynaptic density (PSD), 80–82, 244. See also Proteins, scaffolding AMPA receptors and, 223–225 assembly of, 228–229 CaMKII and, 224–225 deﬁnition of, 222–223 epigenetics and, 80–82, 83–84 function/structure of, 223–227 Homer proteins and, 237–243, 238f microdomains and, 226 95/SAP90, 223–232, 228f NMDA and, 223–225 organization, 83–84 proteins, 34, 35t, 80 receptor diffusion and, 226–227 SAP 97 and, 234–237, 235f Shank/ProSAP proteins and, 232–234, 232f POUh. See Protein(s) POUs. SeeProtein(s) PPI. See Prepulse inhibition Prepulse inhibition (PPI), 340–341, 494 PRO. See Proline-rich regions Proline dehydrogenase (PRODH), 357–358 Proline-rich (PRO) regions, 232–233 Prostaglandins (PGs), 255, 266 hypotheses, 256–260 niacin tests and, 260–265 schizophrenia and, 255–265 Protein(s) adhesion, 81 A-kinase-associated/anchoring (AKAP), 81 amyloid precursor (APP), 498, 499

526 Protein(s) (cont.) anchoring, 80 calcium-binding (CBPs), 186–187 cAMP-dependent, 3 chromosomal, 76 CNP, 396 cytoskeleton-associated (ARC), 127–128, 147, 222, 225 DNA, 76, 78 drebrin as, 6–7 ErbB3, 396, 400 expression data, 356–357 -43, growth-associated (GAP-43), 2–3, 129, 278, 306, 387 GABA-A R-associated (GABARAP), 82 gelsolin (GSN), 396, 401 glial ﬁbrillary acidic (GFAP), 449–450, 450t glutamate receptor-interacting (GRIP), 80, 223, 225–226 GTP binding, 49, 95 guanylate kinase-associated (GKAP), 81, 225 head activator (HA) binding, 333 HER3, 396, 400 interact with C kinase (PICK), 80 MAP2, 10, 12 MBD, 77 membrance-associated GuK (MAGuK), 80–81, 224, 235–236 mRNAs of, 58, 176, 185, 193t–194t, 195 myelin and lymphocyte (MAL), 396, 400 Oct-1, 472 Oct-2, 472 PDZ, 237 phosphatases, 224–225 Pit-1, 472 POU domain, 471–473, 472f, 473f, 474t POU homeodomain (POUh), 473, 473f POU-speciﬁc (POUs), 473, 473f PSD, 34, 35t, 80 receptor-associated, 24, 36 SAP 97, 234–237, 235f schizophrenia and, 49–57, 51t, 55t SCIP (suppressed cAMP-inducible), 475 Shank/ProSAP, 232–234, 232f Sidekick, 81 signaling, 80 synaptic, 47–48, 58 synaptic GTPase-activating (SynGAP), 225

INDEX

synaptosomal-associated (SNAP 25), 177, 278 synthesis, 2–3 Tst-1, 475 UNC-86, 472 vacuolar, 332 Protein kinase type II, calcium calmodulindependent (CaMKII), 3, 11–12, 224–225 Proteins, cAMP response element-binding (CREB), 3, 120, 121f, 207, 209 Proteins, Homer, 80–82, 223 function of, 239–241 gene regulation and, 243 induction/implication, 241–242 neuropsychiatric disorders and, 241–242 structure of, 237–239, 238f Proteins, Oct-6, 471 cellular compartmentilization of, 480–481 CNS and, 479–480, 480f PNS and, 475–477 properties of, 475–477 role of, 478 schizophrenia and, 481–485, 483t Proteins, presynaptic. See also Schizophrenia brain regions and, 179–185, 180t–184t, 186t, 187t–192t complexins as, 177 families of, 175–179 microarrays and, 195, 381 NSF as, 177 Rab3a as, 178 studies, 185, 193t–194t, 195 synapsins as, 178 synaptophysins as, 62f–63f, 102, 176–77 synaptotagmins as, 178 syntaxin as, 177, 178f VAMP as, 177 Proteins, S100B, 445, 462 cytoskeleton modiﬁcation of, 451, 452f effects of, 453 origin/molecular structure of, 448–449 properties of, 451–453 serotonin and, 456 studies on, 454–55, 457–461 target proteins of, 449–451, 451t Proteins, scaffolding, 63, 80–82, 244 PSD, 221–224 PSD-95/SAP90 as, 223–232, 228f Pruning, differential, 356–357 PSA-NCAM. See Polysialylated NCAM

INDEX

PSD. See Postsynaptic density Psychiatry, 86–87 Psychopathology, 265 Psychosis, 9–10, 145–146. See also Schizophrenia onset of, 259–260 PUFA. See Acids, polyunsaturated fatty R Radiation, ultraviolet (UVR), 364 RBC. See Cell(s) RDA. See Representational difference analysis Reactive oxygen species (ROS), 284–285, 287 phospholipase activation by, 301–302 Receptor(s) apolipoprotein E (apoE), 62 channel, 95 dopamine, 99 dopamine D3, 426–438, 428f, 429f, 430f, 432f–433f, 435f–436f G protein-coupled (GPCRs), 95–96, 203–204 GABA, 132–133, 334, 407 glutamate, 4–5, 19, 23–24, 25f, 132–133, 223, 495–496 muscarinic, 93–99, 100–103 nicotinic acetylchloline (nAChR), 482–483 NT, 328–329, 330t–331t, 332 related (RXR), 361 schizophrenia and, 25f, 29–36, 30t–31t, 32t–33t, 35t serotonin, 111, 113–114, 115t–117t thyroid hormone (TXR), 367 trafﬁcking, 84–85 transmitter, 84–85 trkB, 120, 121f tyrosine kinase (TKRs), 203 very low density lipoprotein (VLDL), 62–63 vitamin D (VDR), 364, 367–68 Receptors, AMPA, 23–25, 25f, 29–36, 32t–33t, 35t binding of, 80–81, 129 movements of, 83–84 PSD and, 223–224 sensitivity to, 126f, 127 Receptors, N-methyl-D-aspartate (NMDA), 492 activation of, 3–5 binding of, 129

527

channels, 11–12, 203, 493 clopazine and, 504–505 expressions of, 27, 29–36, 30t–31t, 35t, 83 glycine/D-serine site of, 37–39, 38t, 504 hypofunction, 499–500 neuroplasticity and, 505–507 pharmacological regulation of, 23–25, 25f PSD and, 223–224 schizophrenia and, 212, 406, 500–505, 502t Reelin schizophrenia and, 58–64, 61f–63f synaptic plasticity and, 58–64, 61f–63f, 78–79 Regulator of G-protein signaling-4 (RGS4), 357 Representational difference analysis (RDA), 478 Retinoids. See Vitamin A Retrotransposons, nonviral, 76 RETT Syndrome, 354 RGS4. See Regulator of G-protein signaling-4 Ritanserins, 141 ROS. See Reactive oxygen species RXR. See Receptor(s) S SAL. See Short attack latency SAPK. See Kinase(s) Schizophrenia. See also Brain; Brain Development; Proteins, presynaptic aging and, 395–396 amino acids and, 27 BDNF and, 437–438 calcium and, 286–287 CSF and, 26–27, 138, 338–339 cytokines and, 297, 307–309 D3 receptor and, 437–438 demyelinating diseases and, 392–395 disconnectivity in, 385–388 etiology of, 20, 97–99, 202, 274–275, 351–353, 491–492 GAP-43 and, 49–53, 51t gene expression and, 47–48, 57–64, 61f–63f, 281–282 genes and, 353–360, 369–370 glia and, 381–382 glutamatergic dysfunction in, 25–39, 28t–29t, 30t–31t, 32t–33t, 35t, 38t glutamatergic markers in, 495–496 hypotheses, 21–22, 211–12, 352, 357, 368–369, 445–447

528 Schizophrenia (cont.) MAP kinases and, 209–213, 210f, 212f mitochondrial dysfunction in, 273, 279–287 models of, 360–369 muscarinic receptors in, 93–99 myelin and, 381–407, 398t, 447 NCAM and, 53–57, 55t neurodegenerative hypothesis of, 446 neurodevelopmental hypothesis of, 445–446, 483t neuronal plasticity and, 277–279, 287 neuropils and, 9–11 neuroplasticity and, 19–20, 23–25, 25f neurotransmitters in, 276–277 niacin tests and, 260–265 NMDA and, 212, 406 NMDA receptors and, 212, 406, 500–505, 502t Oct-6 proteins and, 481–485, 483t oligodendrocytes and, 381–385, 402–407 pathogenic hypothesis of, 446 PCP and, 25–26 PGs and, 255–265 pharmacological treatment of, 36–39, 38t, 145–148 phospholipids and, 257–260, 297–306, 309–313, 312f plasticity and, 21–23, 25f, 29–36, 30t–31t, 32t–33t, 35t presynaptic protein and, 57–58, 175–195, 178t, 180t–184t, 185t, 187t–192t, 193t–194t PSD-95/SAP90 and, 230–231 receptors and, 25f, 29–36, 30t–31t, 32t–33t, 35t reelin and, 58–64, 61f–63f, 79 S100B and, 445–462, 450t, 451t, 452t SAP97 and, 9237 semaphorin 3A in, 58–59, 61–64, 61f–63f serotonergic hypothesis of, 145–148 spine and, 9–11 symptomatology of, 312f synaptic plasticity and, 21–23, 47–64, 51t, 55t, 61f–63f, 143–148 treatment of, 98 vitamin A and, 360–362 vitamin D and, 351, 362–370 white matter in, 388–391, 389t Scopolamines, 101–102 Serine racemase (SR), 493

INDEX

Serotonergic systems distribution/projection of, 114, 118f growth factors and, 114, 118f, 120–124, 121f–122f manipulation of, 128–131 signaling dysfunction of, 131–149 Serotonin (5-hydroxytryptamine, 5-HT) affective disorders and, 132–136, 143 anxiety and, 135–137 autism and, 134–135 brain development and, 111–113, 118–120, 118f, 131–149 depletion, 123–124, 128–130, 133 discovery of, 113–114 Down’s Syndrome and, 148–149 drugs, 112, 132, 135 learning and, 115t–117t, 118f, 132–134 levels, 132 memory and, 115t–117t, 118f, 132–134 mental retardation and, 148–149 receptors, 111, 113–114, 115t–117t, 122f, 124–28, 126f, 304 role of, 114–128, 115t–117t, 121f–122f, 126f S100B and, 456 stress and, 135–137 synaptic plasticity and, 128–131, 137–148 transporter (SERT), 129, 135, 141–143 SERT. See Serotonin Serum response element (SRE), 207 Serum response factor (SRF), 207 SH3. See Src homology 3 domains Shanks, 81 Shim, K. S., 6–7 Short attack latency (SAL), 136 SI. See Cortex SIE. See Sis-inducible enhancer Signal transducer and activator of transcription (STAT), 207 Single nucleotide polymorphisms (SNPs), 146 Sis-inducible enhancer (SIE), 207 6-OHDA. See Dopamine SNAP 25 See Protein(s) SNARE complex, 177 SNPs. See Single nucleotide polymorphisms SOD. See Superoxide dismutase SPECT. See Tomography Spectroscopy 31 P magnetoresonance, 257–259, 285 optical reﬂection (ORS), 264

INDEX

Spine(s). See also Disease(s), Alzheimer’s calcium and, 11–12 dendritic, 1–4, 6, 11–12, 121, 142 density, 5 estrogen/plasticity and, 3–4 HD and, 8 HIV and, 7–8 learning/long-term potentiation and, 4–5, 34 neuropil components and, 6 plasticity, 2–3 schizophrenia and, 9–11 SR. See Serine racemase Src homology 3 (SH3) domains, 227–228, 228f, 232–233 SRE. See Serum response element SRF. See Serum response factor SSRIs. See Inhibitors Stanley Foundation Neuropathology Consortium, 213, 485 STAT. See Signal transducer and activator of transcription Stress, 112 BDNF and, 437–438 chronic, 135–136 D3 receptor and, 437–438 effects of, 126, 126f immobilization, 137 immunology and, 310–311 oxidative, 22, 285, 310–311 prenatal, 368–369 psychological, 311 related disorders, 132 Superoxide dismutase (SOD), 303 Sweden, 363 Synapses, 175, 447–448 dentate gyrus, 132 development of, 1–2 excitatory, 221–222, 225–226 glutamate, 19, 23–24 inhibitory, 82–83 markers of, 48 remodeling of, 287 Synapsins, 178, 386 Synaptic activities, 283 Synaptic pruning, 175 Synaptobrevins, 177 Synaptogenesis, 61, 131 Synaptophysins, 62f–63f, 102, 176–177, 185, 386 Synaptotagmins, 178

SynCAM. See Molecules, synaptic cell adhesion Syndromes, deletion 22q13.3, 234 SynGAP. See Protein(s) Syntaxins, 177, 178f Synthase, inducible nitric oxide (iNOS), 453–454 T TBARS. See Thiobarbituric acid-reactive substances TCF. See Ternary complex factor Ternary complex factor ( TCF), 207 Tetrahydrocannabinol, D9 (THC), 304 TF. See Transcription factor Tf. See Transferrin Thalamus, 9–10 THC. See Tetrahydrocannabinol, D9 Theta rhythms, 102 Thiobarbituric acid-reactive substances (TBARS), 285–286 3-methoxy-4-hydroxyphenylglycol (MOPEG), 138 TKRs. See Receptor(s) TMS. See Transcranial magnetic stimulation TNF-a. See Tumor necrosis factor-a Tomography positron emission, 341 single photon emission (SPECT), 285 Transcranial magnetic stimulation (TMS), 101–102 Transcription, 77–78 Transcription factor (TF ), 206–207, 208 Transcription factor, activating (ATF ), 207 Transferrin (Tf ), 396, 400–401 Tryptophan-phenylalanine-cysteine ( WFC) region, 472–473 Tryptophans, 120–122, 121f–122f 5-hydroxy (5-HTP), 139 dietary/plasma, 121f, 138–139 serotonin and, 128–130, 138–139 Tumor necrosis factor-a (TNF-a), 306 TXR. See Receptor(s) U United Kingdom, 363 UVR. See Radiation, ultraviolet

529

530 V VanderPutten, D. M., 56 Vasodilator, PGD2, 261–262 Vawter, M. P., 56 VDR. See Receptor(s) Vesicular glutamate transporter ( VGLUT1), 58 VGLUT1. See Vesicular glutamate transporter Virus, papovavirus JC, 476 Vitamin A, 360–362 Vitamin D biology of, 364 brain and, 364–367

INDEX

gene transcription and, 206, 367–368 hypothesis, 362–369 receptor (VDR), 364, 367–368 schizophrenia and, 351, 362–370 VLDL. See Receptor(s) W Waddington, Conrad H., 73–75, 85–86 WFC. See Tryptophan-phenylalanine-cysteine region X Xenopus, 473, 502

CONTENTS OF RECENT VOLUMES

Volume 37

Memory and Forgetting: Long-Term and Gradual Changes in Memory Storage Larry R. Squire

Section I: Selectionist Ideas and Neurobiology in

Implicit Knowledge: New Perspectives on Unconscious Processes Daniel L. Schacter

Population Thinking and Neuronal Selection: Metaphors or Concepts? Ernst Mayr

Section V: Psychophysics, Psychoanalysis, and Neuropsychology

Selectionist and Neuroscience Olaf Sporns

Instructionist

Ideas

Selection and the Origin of Information Manfred Eigen

Phantom Limbs, Neglect Syndromes, Repressed Memories, and Freudian Psychology V. S. Ramachandran

Section II: Populations

Neural Darwinism and a Conceptual Crisis in Psychoanalysis Arnold H. Modell

Development

and

Neuronal

Morphoregulatory Molecules and Selectional Dynamics during Development Kathryn L. Crossin

A New Vision of the Mind Oliver Sacks

Exploration and Selection in the Early Acquisition of Skill Esther Thelen and Daniela Corbetta

index

Population Activity in the Control of Movement Apostolos P. Georgopoulos

Volume 38

Section III: Functional Integration in the Brain

Segregation

and

Reentry and the Problem of Cortical Integration Giulio Tononi Coherence as an Organizing Principle of Cortical Functions Wolf Singerl

Regulation of GABAA Receptor Function and Gene Expression in the Central Nervous System A. Leslie Morrow Genetics and the Organization of the Basal Ganglia Robert Hitzemann, Yeang Olan, Stephen Kanes, Katherine Dains, and Barbara Hitzemann

Section IV: Memory and Models

Structure and Pharmacology of Vertebrate GABAA Receptor Subtypes Paul J. Whiting, Ruth M. McKernan, and Keith A. Wafford

Selection versus Instruction: Use of Computer Models to Compare Brain Theories George N. Reeke, Jr.

Neurotransmitter Transporters: Biology, Function, and Regulation Beth Borowsky and Beth J. Hoffman

Temporal Mechanisms in Perception Ernst Po¨ppel

531

Molecular

532

CONTENTS OF RECENT VOLUMES

Presynaptic Excitability Meyer B. Jackson

Volume 40

Monoamine Neurotransmitters in Invertebrates and Vertebrates: An Examination of the Diverse Enzymatic Pathways Utilized to Synthesize and Inactivate Biogenic Amines B. D. Sloley and A. V. Juorio

Mechanisms of Nerve Cell Death: Apoptosis or Necrosis after Cerebral Ischemia R. M. E. Chalmers-Redman, A. D. Fraser, W. Y. H. Ju, J. Wadia, N. A. Tatton, and W. G. Tatton

Neurotransmitter Systems in Schizophrenia Gavin P. Reynolds

Changes in Ionic Fluxes during Cerebral Ischemia Tibor Kristian and Bo K. Siesjo

Physiology of Bergmann Glial Cells Thomas Mu¨ller and Helmut Kettenmann

Techniques for Examining Neuroprotective Drugs in Vitro A. Richard Green and Alan J. Cross

index Volume 39

Techniques for Examining Neuroprotective Drugs in Vivo Mark P. Goldberg, Uta Strasser, and Laura L. Dugan

Modulation of Amino Acid-Gated Ion Channels by Protein Phosphorylation Stephen J. Moss and Trevor G. Smart

Calcium Antagonists: Their Role in Neuroprotection A. Jacqueline Hunter

Use-Dependent Regulation Receptors Eugene M. Barnes, Jr.

GABAA

Sodium and Potassium Channel Modulators: Their Role in Neuroprotection Tihomir P. Obrenovich

Synaptic Transmission and Modulation in the Neostriatum David M. Lovinger and Elizabeth Tyler

NMDA Antagonists: Their Role in Neuroprotection Danial L. Small

of

The Cytoskeleton and Neurotransmitter Receptors Valerie J. Whatley and R. Adron Harris

Development of the NMDA Ion-Channel Blocker, Aptiganel Hydrochloride, as a Neuroprotective Agent for Acute CNS Injury Robert N. McBurney

Endogenous Opioid Regulation of Hippocampal Function Michele L. Simmons and Charles Chavkin

The Pharmacology of AMPA Antagonists and Their Role in Neuroprotection Rammy Gill and David Lodge

Molecular Neurobiology of the Cannabinoid Receptor Mary E. Abood and Billy R. Martin

GABA and Neuroprotection Patrick D. Lyden

Genetic Models in the Study of Anesthetic Drug Action Victoria J. Simpson and Thomas E. Johnson Neurochemical Bases of Locomotion and Ethanol Stimulant Effects Tamara J. Phillips and Elaine H. Shen Effects of Ethanol on Ion Channels Fulton T. Crews, A. Leslie Morrow, Hugh Criswell, and George Breese index

Adenosine and Neuroprotection Bertil B. Fredholm Interleukins and Cerebral Ischemia Nancy J. Rothwell, Sarah A. Loddick, and Paul Stroemer Nitrone-Based Free Radical Traps as Neuroprotective Agents in Cerebral Ischemia and Other Pathologies Kenneth Hensley, John M. Carney, Charles A. Stewart, Tahera Tabatabaie, Quentin Pye, and Robert A. Floyd

CONTENTS OF RECENT VOLUMES

Neurotoxic and Neuroprotective Roles of Nitric Oxide in Cerebral Ischemia Turgay Dalkara and Michael A. Moskowitz

Sensory and Cognitive Functions Lawrence M. Parsons and Peter T. Fox

A Review of Earlier Clinical Studies on Neuroprotective Agents and Current Approaches Nils-Gunnar Wahlgren

Skill Learning Julien Doyon

index

533

Section V: Clinical and Neuropsychological Observations Executive Function and Motor Skill Learning Mark Hallett and Jordon Grafman

Volume 41 Section I: Historical Overview

Verbal Fluency and Agrammatism Marco Molinari, Maria G. Leggio, and Maria C. Silveri

Rediscovery of an Early Concept Jeremy D. Schmahmann

Classical Conditioning Diana S. Woodruff-Pak

Section II: Anatomic Substrates

Early Infantile Autism Margaret L. Bauman, Pauline A. Filipek, and Thomas L. Kemper

The Cerebrocerebellar System Jeremy D. Schmahmann and Deepak N. Pandya Cerebellar Output Channels Frank A. Middleton and Peter L. Strick Cerebellar-Hypothalamic Axis: Basic Circuits and Clinical Observations Duane E. Haines, Espen Dietrichs, Gregory A. Mihailoff, and E. Frank McDonald Section III. Physiological Observations Amelioration of Aggression: Response to Selective Cerebellar Lesions in the Rhesus Monkey Aaron J. Berman Autonomic and Vasomotor Regulation Donald J. Reis and Eugene V. Golanov Associative Learning Richard F. Thompson, Shaowen Bao, Lu Chen, Benjamin D. Cipriano, Jeffrey S. Grethe, Jeansok J. Kim, Judith K. Thompson, Jo Anne Tracy, Martha S. Weninger, and David J. Krupa

Olivopontocerebellar Atrophy and Friedreich’s Ataxia: Neuropsychological Consequences of Bilateral versus Unilateral Cerebellar Lesions The´re`se Botez-Marquard and Mihai I. Botez Posterior Fossa Syndrome Ian F. Pollack Cerebellar Cognitive Affective Syndrome Jeremy D. Schmahmann and Janet C. Sherman Inherited Cerebellar Diseases Claus W. Wallesch and Claudius Bartels Neuropsychological Abnormalities in Cerebellar Syndromes—Fact or Fiction? Irene Daum and Hermann Ackermann Section VI: Theoretical Considerations Cerebellar Microcomplexes Masao Ito

Visuospatial Abilities Robert Lalonde

Control of Sensory Data Acquisition James M. Bower

Spatial Event Processing Marco Molinari, Laura Petrosini, and Liliana G. Grammaldo

Neural Representations of Moving Systems Michael Paulin

Section IV: Functional Neuroimaging Studies Linguistic Processing Julie A. Fiez and Marcus E. Raichle

How Fibers Subserve Computing Capabilities: Similarities between Brains and Machines Henrietta C. Leiner and Alan L. Leiner

534

CONTENTS OF RECENT VOLUMES

Cerebellar Timing Systems Richard Ivry

Volume 43

Attention Coordination and Anticipatory Control Natacha A. Akshoomoff, Eric Courchesne, and Jeanne Townsend

Early Development of the Drosophila Neuromuscular Junction: A Model for Studying Neuronal Networks in Development Akira Chiba

Context-Response Linkage W. Thomas Thach

Development of Larval Body Wall Muscles Michael Bate, Matthias Landgraf, and Mar Ruiz Gmez Bate

Duality of Cerebellar Motor and Cognitive Functions James R. Bloedel and Vlastislav Bracha Section VII: Future Directions Therapeutic and Research Implications Jeremy D. Schmahmann

Volume 42 Alzheimer Disease Mark A. Smith Neurobiology of Stroke W. Dalton Dietrich Free Radicals, Calcium, and the Synaptic Plasticity-Cell Death Continuum: Emerging Roles of the Trascription Factor NFB Mark P. Mattson AP-I Transcription Factors: Short- and LongTerm Modulators of Gene Expression in the Brain Keith Pennypacker

Development of Electrical Properties and Synaptic Transmission at the Embryonic Neuromuscular Junction Kendal S. Broadie Ultrastructural Correlates of Neuromuscular Junction Development Mary B. Rheuben, Motojiro Yoshihara, and Yoshiaki Kidokoro Assembly and Maturation of the Drosophila Larval Neuromuscular Junction L. Sian Gramates and Vivian Budnik Second Messenger Systems Underlying Plasticity at the Neuromuscular Junction Frances Hannan and Yi Zhong Mechanisms of Neurotransmitter Release J. Troy Littleton, Leo Pallanck, and Barry Ganetzky Vesicle Recycling at the Drosophila Neuromuscular Junction Daniel T. Stimson and Mani Ramaswami Ionic Currents in Larval Muscles of Drosophila Satpal Singh and Chun-Fang Wu

Ion Channels in Epilepsy Istvan Mody

Development of the Adult Neuromuscular System Joyce J. Fernandes and Haig Keshishian

Posttranslational Regulation of Ionotropic Glutamate Receptors and Synaptic Plasticity Xiaoning Bi, Steve Standley, and Michel Baudry

Controlling the Motor Neuron James R. Trimarchi, Ping Jin, and Rodney K. Murphey

Heritable Mutations in the Glycine, GABAA, and Nicotinic Acetylcholine Receptors Provide New Insights into the Ligand-Gated Ion Channel Receptor Superfamily Behnaz Vafa and Peter R. Schofield

Volume 44

index

Human Ego-Motion Perception A. V. van den Berg Optic Flow and Eye Movements M. Lappe and K.-P. Hoffman

CONTENTS OF RECENT VOLUMES

The Role of MST Neurons during Ocular Tracking in 3D Space K. Kawano, U. Inoue, A. Takemura, Y. Kodaka, and F. A. Miles Visual Navigation in Flying Insects M. V. Srinivasan and S.-W. Zhang Neuronal Matched Filters for Optic Flow Processing in Flying Insects H. G. Krapp A Common Frame of Reference for the Analysis of Optic Flow and Vestibular Information B. J. Frost and D. R. W. Wylie Optic Flow and the Visual Guidance of Locomotion in the Cat H. Sherk and G. A. Fowler Stages of Self-Motion Processing in Primate Posterior Parietal Cortex F. Bremmer, J.-R. Duhamel, S. B. Hamed, and W. Graf Optic Flow Perception C. J. Duffy

Analysis

for

Self-Movement

Neural Mechanisms for Self-Motion Perception in Area MST R. A. Andersen, K. V. Shenoy, J. A. Crowell, and D. C. Bradley Computational Mechanisms for Optic Flow Analysis in Primate Cortex M. Lappe Human Cortical Areas Underlying the Perception of Optic Flow: Brain Imaging Studies M. W. Greenlee

535

Brain Development and Generation of Brain Pathologies Gregory L. Holmes and Bridget McCabe Maturation of Channels and Receptors: Consequences for Excitability David F. Owens and Arnold R. Kriegstein Neuronal Activity and the Establishment of Normal and Epileptic Circuits during Brain Development John W. Swann, Karen L. Smith, and Chong L. Lee The Effects of Seizures of the Hippocampus of the Immature Brain Ellen F. Sperber and Solomon L. Moshe Abnormal Development and Catastrophic Epilepsies: The Clinical Picture and Relation to Neuroimaging Harry T. Chugani and Diane C. Chugani Cortical Reorganization and Seizure Generation in Dysplastic Cortex G. Avanzini, R. Preafico, S. Franceschetti, G. Sancini, G. Battaglia, and V. Scaioli Rasmussen’s Syndrome with Particular Reference to Cerebral Plasticity: A Tribute to Frank Morrell Fredrick Andermann and Yuonne Hart Structural Reorganization of Hippocampal Networks Caused by Seizure Activity Daniel H. Lowenstein Epilepsy-Associated Plasticity in gammaAmniobutyric Acid Receptor Expression, Function and Inhibitory Synaptic Properties Douglas A. Coulter

What Neurological Patients Tell Us about the Use of Optic Flow L. M. Vaina and S. K. Rushton

Synaptic Plasticity and Secondary Epileptogenesis Timothy J. Teyler, Steven L. Morgan, Rebecca N. Russell, and Brian L. Woodside

index

Synaptic Plasticity in Epileptogenesis: Cellular Mechanisms Underlying Long-Lasting Synaptic Modifications that Require New Gene Expression Oswald Steward, Christopher S. Wallace, and Paul F. Worley

Volume 45 Mechanisms of Brain Plasticity: From Normal Brain Function to Pathology Philip. A. Schwartzkroin

Cellular Correlates of Behavior Emma R. Wood, Paul A. Dudchenko, and Howard Eichenbaum

536

CONTENTS OF RECENT VOLUMES

Mechanisms of Neuronal Conditioning David A. T. King, David J. Krupa, Michael R. Foy, and Richard F. Thompson

Biosynthesis of Neurosteroids and Regulation of Their Synthesis Synthia H. Mellon and Hubert Vaudry

Plasticity in the Aging Central Nervous System C. A. Barnes

Neurosteroid 7-Hydroxylation Products in the Brain Robert Morfin and Luboslav Sta´rka

Secondary Epileptogenesis, Kindling, and Intractable Epilepsy: A Reappraisal from the Perspective of Neuronal Plasticity Thomas P. Sutula Kindling and the Mirror Focus Dan C. McIntyre and Michael O. Poulter Partial Kindling and Behavioral Pathologies Robert E. Adamec The Mirror Epileptogenesis B. J. Wilder

Focus

and

Secondary

Hippocampal Lesions in Epilepsy: A Historical Review Robert Naquet Clinical Evidence for Secondary Epileptogensis Hans O. Luders Epilepsy as a Progressive (or Nonprogressive ‘‘Benign’’) Disorder John A. Wada Pathophysiological Aspects of Landau-Kleffner Syndrome: From the Active Epileptic Phase to Recovery Marie-Noelle Metz-Lutz, Pierre Maquet, Annd De Saint Martin, Gabrielle Rudolf, Norma Wioland, Edouard Hirsch, and Chriatian Marescaux Local Pathways of Seizure Propagation in Neocortex Barry W. Connors, David J. Pinto, and Albert E. Telefeian Multiple Subpial Assessment C. E. Polkey

Transection:

A

Clinical

The Legacy of Frank Morrell Jerome Engel, Jr. Volume 46 Neurosteroids: Beginning of the Story Etienne E. Baulieu, P. Robel, and M. Schumacher

Neurosteroid Analysis Ahmed A. Alomary, Robert L. Fitzgerald, and Robert H. Purdy Role of the Peripheral-Type Benzodiazepine Receptor in Adrenal and Brain Steroidogenesis Rachel C. Brown and Vassilios Papadopoulos Formation and Effects of Neuroactive Steroids in the Central and Peripheral Nervous System Roberto Cosimo Melcangi, Valerio Magnaghi, Mariarita Galbiati, and Luciano Martini Neurosteroid Modulation of Recombinant and Synaptic GABAA Receptors Jeremy J. Lambert, Sarah C. Harney, Delia Belelli, and John A. Peters GABAA -Receptor Plasticity during LongTerm Exposure to and Withdrawal from Progesterone Giovanni Biggio, Paolo Follesa, Enrico Sanna, Robert H. Purdy, and Alessandra Concas Stress and Neuroactive Steroids Maria Luisa Barbaccia, Mariangela Serra, Robert H. Purdy, and Giovanni Biggio Neurosteroids in Learning and Processes Monique Valle´e, Willy Mayo, George F. Koob, and Michel Le Moal

Memory

Neurosteroids and Behavior Sharon R. Engel and Kathleen A. Grant Ethanol and Neurosteroid Interactions in the Brain A. Leslie Morrow, Margaret J. VanDoren, Rebekah Fleming, and Shannon Penland Preclinical Development of Neurosteroids as Neuroprotective Agents for the Treatment of Neurodegenerative Diseases Paul A. Lapchak and Dalia M. Araujo

CONTENTS OF RECENT VOLUMES

Clinical Implications of Circulating Neurosteroids Andrea R. Genazzani, Patrizia Monteleone, Massimo Stomati, Francesca Bernardi, Luigi Cobellis, Elena Casarosa, Michele Luisi, Stefano Luisi, and Felice Petraglia Neuroactive Steroids and Central Nervous System Disorders Mingde Wang, Torbjo¨rn Ba¨ckstro¨m, Inger Sundstro¨m, Go¨ran Wahlstro¨m, Tommy Olsson, Di Zhu, Inga-Maj Johansson, Inger Bjo¨rn, and Marie Bixo Neuroactive Steroids in Neuropsychopharmacology Rainer Rupprecht and Florian Holsboer Current Perspectives on the Role of Neurosteroids in PMS and Depression Lisa D. Griffin, Susan C. Conrad, and Synthia H. Mellon

537

Processing Human Brain Tissue for in Situ Hybridization with Radiolabelled Oligonucleotides Louise F. B. Nicholson In Situ Hybridization of Astrocytes and Neurons Cultured in Vitro L. A. Arizza-McNaughton, C. De Felipe, and S. P. Hunt In Situ Hybridization on Organotypic Slice Cultures A. Gerfin-Moser and H. Monyer Quantitative Analysis of in Situ Hybridization Histochemistry Andrew L. Gundlach and Ross D. O’Shea Part II: Nonradioactive in Situ hybridization

index

Nonradioactive in Situ Hybridization Using Alkaline Phosphatase-Labelled Oligonucleotides S. J. Augood, E. M. McGowan, B. R. Finsen, B. Heppelmann, and P. C. Emson

Volume 47

Combining Nonradioactive in Situ Hybridization with Immunohistological and Anatomical Techniques Petra Wahle

Introduction: Studying Gene Expression in Neural Tissues by in Situ Hybridization W. Wisden and B. J. Morris

Nonradioactive in Situ Hybridization: Simplified Procedures for Use in Whole Mounts of Mouse and Chick Embryos Linda Ariza-McNaughton and Robb Krumlauf

Part I: In Situ Hybridization with Radiolabelled Oligonucleotides In Situ Hybridization with Oligonucleotide Probes Wl. Wisden and B. J. Morris

index

Cryostat Sectioning of Brains Victoria Revilla and Alison Jones

Volume 48

Processing Rodent Embryonic and Early Postnatal Tissue for in Situ Hybridization with Radiolabelled Oligonucleotides David J. Laurie, Petra C. U. Schrotz, Hannah Monyer, and Ulla Amtmann

Assembly and Intracellular GABAA Receptors Eugene Barnes

Trafficking

of

Processing of Retinal Tissue for in Situ Hybridization Frank Mu¨ller

Subcellular Localization and Regulation of GABAA Receptors and Associated Proteins Bernhard Lu¨scher and Jean-Marc Fritschy D1 Dopamine Receptors Richard Mailman

Processing the Spinal Cord for in Situ Hybridization with Radiolabelled Oligonucleotides A. Berthele and T. R. To¨lle

Molecular Modeling of Ligand-Gated Ion Channels: Progress and Challenges Ed Bertaccini and James R. Trudel

538

CONTENTS OF RECENT VOLUMES

Alzheimer’s Disease: Its Diagnosis and Pathogenesis Jillian J. Kril and Glenda M. Halliday DNA Arrays and Functional Genomics in Neurobiology Christelle Thibault, Long Wang, Li Zhang, and Michael F. Miles index

The Treatment of Infantile Spasms: An Evidence-Based Approach Mark Mackay, Shelly Weiss, and O. Carter Snead III ACTH Treatment of Infantile Spasms: Mechanisms of Its Effects in Modulation of Neuronal Excitability K. L. Brunson, S. Avishai-Eliner, and T. Z. Baram

Volume 49

Neurosteroids and Infantile Spasms: The Deoxycorticosterone Hypothesis Michael A. Rogawski and Doodipala S. Reddy

What Is West Syndrome? Olivier Dulac, Christine Soufflet, Catherine Chiron, and Anna Kaminski

Are there Specific Anatomical and/or Transmitter Systems (Cortical or Subcortical) That Should Be Targeted? Phillip C. Jobe

The Relationship between encephalopathy and Abnormal Neuronal Activity in the Developing Brain Frances E. Jensen

Medical versus Surgical Treatment: Which Treatment When W. Donald Shields

Hypotheses from Functional Neuroimaging Studies Csaba Juha´sz, Harry T. Chugani, Ouo Muzik, and Diane C. Chugani

Developmental Outcome with and without Successful Intervention Rochelle Caplan, Prabha Siddarth, Gary Mathern, Harry Vinters, Susan Curtiss, Jennifer Levitt, Robert Asarnow, and W. Donald Shields

Infantile Spasms: Unique Sydrome or General Age-Dependent Manifestation of a Diffuse Encephalopathy? M. A. Koehn and M. Duchowny

Infantile Spasms versus Myoclonus: Is There a Connection? Michael R. Pranzatelli

Histopathology of Brain Tissue from Patients with Infantile Spasms Harry V. Vinters

Tuberous Sclerosis as an Underlying Basis for Infantile Spasm Raymond S. Yeung

Generators of Ictal and Interictal Electroencephalograms Associated with Infantile Spasms: Intracellular Studies of Cortical and Thalamic Neurons M. Steriade and I. Timofeev

Brain Malformation, Epilepsy, and Infantile Spasms M. Elizabeth Ross

Cortical and Subcortical Generators of Normal and Abnormal Rhythmicity David A. McCormick Role of Subcortical Structures in the Pathogenesis of Infantile Spasms: What Are Possible Subcortical Mediators? F. A. Lado and S. L. Moshe´ What Must We Know to Develop Better Therapies? Jean Aicardi

Brain Maturational Aspects Relevant to Pathophysiology of Infantile Spasms G. Auanzini, F. Panzica, and S. Franceschetti Gene Expression Analysis as a Strategy to Understand the Molecular Pathogenesis of Infantile Spasms Peter B. Crino Infantile Spasms: Criteria for an Animal Model Carl E. Stafstrom and Gregory L. Holmes index

CONTENTS OF RECENT VOLUMES

Volume 50 Part I: Primary Mechanisms How Does Glucose Generate Oxidative Stress In Peripheral Nerve? Irina G. Obrosova Glycation in Diabetic Neuropathy: Characteristics, Consequences, Causes, and Therapeutic Options Paul J. Thornalley Part II: Secondary Changes Protein Kinase C Changes in Diabetes: Is the Concept Relevant to Neuropathy? Joseph Eichberg Are Mitogen-Activated Protein Kinases Glucose Transducers for Diabetic Neuropathies? Tertia D. Purves and David R. Tomlinson Neurofilaments in Diabetic Neuropathy Paul Fernyhough and Robert E. Schmidt Apoptosis in Diabetic Neuropathy Aviva Tolkovsky Nerve and Ganglion Blood Flow in Diabetes: An Appraisal Douglas W. Zochodne Part III: Manifestations Potential Mechanisms of Neuropathic Pain in Diabetes Nigel A. Calcutt Electrophysiologic Measures of Diabetic Neuropathy: Mechanism and Meaning Joseph C. Arezzo and Elena Zotova Neuropathology and Pathogenesis of Diabetic Autonomic Neuropathy Robert E. Schmidt Role of the Schwann Cell in Diabetic Neuropathy Luke Eckersley

539

Nerve Growth Factor for the Treatment of Diabetic Neuropathy: What Went Wrong, What Went Right, and What Does the Future Hold? Stuart C. Apfel Angiotensin-Converting Enzyme Inhibitors: Are there Credible Mechanisms for Beneficial Effects in Diabetic Neuropathy? Rayaz A. Malik and David R. Tomlinson Clinical Trials for Drugs Against Diabetic Neuropathy: Can We Combine Scientific Needs With Clinical Practicalities? Dan Ziegler and Dieter Luft index

Volume 51 Energy Metabolism in the Brain Leif Hertz and Gerald A. Dienel The Cerebral Glucose-Fatty Acid Cycle: Evolutionary Roots, Regulation, and (Patho) physiological Importance Kurt Heininger Expression, Regulation, and Functional Role of Glucose Transporters (GLUTs) in Brain Donard S. Dwyer, Susan J. Vannucci, and Ian A. Simpson Insulin-Like Growth Factor-1 Promotes Neuronal Glucose Utilization During Brain Development and Repair Processes Carolyn A. Bondy and Clara M. Cheng CNS Sensing and Regulation of Peripheral Glucose Levels Barry E. Levin, Ambrose A. Dunn-Meynell, and Vanessa H. Routh

Part IV: Potential Treatment

Glucose Transporter Protein Syndromes Darryl C. De Vivo, Dong Wang, Juan M. Pascual, and Yuan Yuan Ho

Polyol Pathway and Diabetic Peripheral Neuropathy Peter J. Oates

Glucose, Stress, and Hippocampal Neuronal Vulnerability Lawrence P. Reagan

540

CONTENTS OF RECENT VOLUMES

Glucose/Mitochondria in Neurological Conditions John P. Blass Energy Utilization in the Ischemic/Reperfused Brain John W. Phillis and Michael H. O’Regan Diabetes Mellitus and the Central Nervous System Anthony L. McCall Diabetes, the Brain, and Behavior: Is There a Biological Mechanism Underlying the Association between Diabetes and Depression? A. M. Jacobson, J. A. Samson, K. Weinger, and C. M. Ryan Schizophrenia and Diabetes David C. Henderson and Elissa R. Ettinger Psychoactive Drugs Affect Glucose Transport and the Regulation of Glucose Metabolism Donard S. Dwyer, Timothy D. Ardizzone, and Ronald J. Bradley index

Neural Control of Salivary S-IgA Secretion Gordon B. Proctor and Guy H. Carpenter Stress and Secretory Immunity Jos A. Bosch, Christopher Ring, Eco J. C. de Geus, Enno C. I. Veerman, and Arie V. Nieuw Amerongen Cytokines and Depression Angela Clow Immunity and Schizophrenia: Autoimmunity, Cytokines, and Immune Responses Fiona Gaughran Cerebral Lateralization and the Immune System Pierre J. Neveu Behavioral Conditioning of the Immune System Frank Hucklebridge Psychological and Neuroendocrine Correlates of Disease Progression Julie M. Turner-Cobb The Role of Psychological Intervention in Modulating Aspects of Immune Function in Relation to Health and Well-Being J. H. Gruzelier index

Volume 52

Volume 53

Neuroimmune Relationships in Perspective Frank Hucklebridge and Angela Clow Sympathetic Nervous System Interaction with the Immune System Virginia M. Sanders and Adam P. Kohm Mechanisms by Which Cytokines Signal the Brain Adrian J. Dunn Neuropeptides: Modulators of Responses in Health and Disease David S. Jessop

Immune

Brain–Immune Interactions in Sleep Lisa Marshall and Jan Born Neuroendocrinology of Autoimmunity Michael Harbuz Systemic Stress-Induced Th2 Shift and Its Clinical Implications Ibia J. Elenkov

Section I: Mitochondrial Structure and Function Mitochondrial DNA Structure and Function Carlos T. Moraes, Sarika Srivastava, Ilias Kirkinezos, Jose Oca-Cossio, Corina van Waveren, Markus Woischnick, and Francisca Diaz Oxidative Phosphorylation: Structure, Function, and Intermediary Metabolism Simon J. R. Heales, Matthew E. Gegg, and John B. Clark Import of Mitochondrial Proteins Matthias F. Bauer, Sabine Hofmann, and Walter Neupert Section II: Primary Respiratory Chain Disorders Mitochondrial Disorders of the Nervous System: Clinical, Biochemical, and Molecular Genetic Features Dominic Thyagarajan and Edward Byrne

CONTENTS OF RECENT VOLUMES

Section III: Secondary Respiratory Chain Disorders Friedreich’s Ataxia J. M. Cooper and J. L. Bradley Wilson Disease C. A. Davie and A. H. V. Schapira

541

The Mitochondrial Theory of Aging: Involvement of Mitochondrial DNA Damage and Repair Nadja C. de Souza-Pinto and Vilhelm A. Bohr index

Hereditary Spastic Paraplegia Christopher J. McDermott and Pamela J. Shaw Cytochrome c Oxidase Deficiency Giacomo P. Comi, Sandra Strazzer, Sara Galbiati, and Nereo Bresolin Section IV: Toxin Induced Mitochondrial Dysfunction Toxin-Induced Mitochondrial Dysfunction Susan E. Browne and M. Flint Beal Section V: Neurodegenerative Disorders Parkinson’s Disease L. V. P. Korlipara and A. H. V. Schapira Huntington’s Disease: The Mystery Unfolds? A˚sa Peterse´n and Patrik Brundin Mitochondria in Alzheimer’s Disease Russell H. Swerdlow and Stephen J. Kish Contributions of Mitochondrial Alterations, Resulting from Bad Genes and a Hostile Environment, to the Pathogenesis of Alzheimer’s Disease Mark P. Mattson Mitochondria and Amyotrophic Lateral Sclerosis Richard W. Orrell and Anthony H. V. Schapira

Volume 54

Unique General Anesthetic Binding Sites Within Distinct Conformational States of the Nicotinic Acetylcholine Receptor Hugo R. Ariaas, William, R. Kem, James R. Truddell, and Michael P. Blanton Signaling Molecules and Receptor Transduction Cascades That Regulate NMDA ReceptorMediated Synaptic Transmission Suhas. A. Kotecha and John F. MacDonald Behavioral Measures of Alcohol Self-Administration and Intake Control: Rodent Models Herman H. Samson and Cristine L. Czachowski Dopaminergic Mouse Mutants: Investigating the Roles of the Different Dopamine Receptor Subtypes and the Dopamine Transporter Shirlee Tan, Bettina Hermann, and Emiliana Borrelli Drosophila melanogaster, A Genetic Model System for Alcohol Research Douglas J. Guarnieri and Ulrike Heberlein index

Section VI: Models of Mitochondrial Disease Models of Mitochondrial Disease Danae Liolitsa and Michael G. Hanna

Volume 55

Section VII: Defects of Oxidation Including Carnitine Deficiency

Section I: Virsu Vectors For Use in the Nervous System

Defects of Oxidation Including Carnitine Deficiency K. Bartlett and M. Pourfarzam

Non-Neurotropic Adenovirus: a Vector for Gene Transfer to the Brain and Gene Therapy of Neurological Disorders P. R. Lowenstein, D. Suwelack, J. Hu, X. Yuan, M. Jimenez-Dalmaroni, S. Goverdhama, and M.G. Castro

Section VIII: Mitochondrial Involvement in Aging

542

CONTENTS OF RECENT VOLUMES

Adeno-Associated Virus Vectors E. Lehtonen and L. Tenenbaum Problems in the Use of Herpes Simplex Virus as a Vector L. T. Feldman Lentiviral Vectors J. Jakobsson, C. Ericson, N. Rosenquist, and C. Lundberg Retroviral Vectors for Gene Delivery to Neural Precursor Cells K. Kageyama, H. Hirata, and J. Hatakeyama

Volume 56 Behavioral Mechanisms and the Neurobiology of Conditioned Sexual Responding Mark Krause NMDA Receptors in Alcoholism Paula L. Hoffman, Processing and Representation of SpeciesSpecific Communication Calls in the Auditory System of Bats George D. Pollak, Achim Klug, and Eric E. Bauer Central Nervous System Control of Micturition Gert Holstege and Leonora J. Mouton

Section II: Gene Therapy with Virus Vectors for Specific Disease of the Nervous System

The Structure and Physiology of the Rat Auditory System: An Overview Manuel Malmierca

The Principles of Molecular Therapies for Glioblastoma G. Karpati and J. Nalbatonglu

Neurobiology of Cat and Human Sexual Behavior Gert Holstege and J. R. Georgiadis

Oncolytic Herpes Simplex Virus J. C. C. Hu and R. S. Coffin

index

Recombinant Retrovirus Vectors for Treatment of Brain Tumors N. G. Rainov and C. M. Kramm Adeno-Associated Viral Vectors for Parkinson’s Disease I. Muramatsu, L. Wang, K. Ikeguchi, K-i Fujimoto, T. Okada, H. Mizukami, Y. Hanazono, A. Kume, I. Nakano, and K. Ozawa HSV Vectors for Parkinson’s Disease D. S. Latchman Gene Therapy for Stroke K. Abe and W. R. Zhang Gene Therapy for Mucopolysaccharidosis A. Bosch and J. M. Heard index

Volume 57 Cumulative Subject Index of Volumes 1-25

Volume 58 Cumulative Subject Index of Volumes 26–50