Dompaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson's Disease (Neuroscience Intelligence Unit)

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

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Lazaros C. Triarhou, M.D., Ph.D.

Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

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

NEUROSCIENCE INTELLIGENCE UNIT 7

Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease Lazaros C. Triarhou, M.D., Ph.D. University of Macedonia Deptartment of Educational and Social Policy Thessaloniki, Greece

LANDES BIOSCIENCE GEORGETOWN, TEXAS U.S.A.

EUREKAH.COM AUSTIN, TEXAS U.S.A.

DOPAMINERGIC NEURON TRANSPLANTATION IN THE WEAVER MOUSE MODEL OF PARKINSON’S DISEASE Neuroscience Intelligence Unit EUREKAH.COM LANDES BIOSCIENCE Georgetown, Texas, U.S.A. Copyright ©2001 EUREKAH.COM All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: EUREKAH.COM / Landes Bioscience 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081 www.landesbioscience.com www.eurekah.com ISBN: 1-58706-051-5 (hard cover version) ISBN: 1-58706-087-6 (soft cover version) While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data Triarhou, Lazaros Constantinos, 1957Dopaminergic neuron transplantation in the weaver mouse model of Parkinson’s disease / Lazaros C. Triarhou p. cm. -- (Neuroscience intelligence unit) Includes bibliographical references and index. ISBN 1-58706-051-5 (alk. paper) 1. Parkinson’s disease. 2. Dopaminergic neurons--Transplantation. 3. Parkinson’s disease--Surgery. I. Title II. Series. RC382.T752000 617.4'810592--dc21 00-046207

CONTENTS 1. Introduction ........................................................................................... 1 Dopamine and Parkinson’s Disease ....................................................... 1 Experimental Models of Parkinsonism in Laboratory Animals ............... 4 Graft-Assisted Neural Reconstruction (“Brainware Engineering”?) ........ 6 2. Biology and Pathology of the Weaver Mutant Mouse .......................... 13 Introduction ........................................................................................ 13 Cellular and Molecular Genetics of the Weaver Mutation ................... 13 Alterations of the Mesotelencephalic Dopamine Projection System ..... 14 Cerebellar Phenotype of the Weaver Mutant ....................................... 23 Hippocampal Morphology .................................................................. 27 Biology of Normal ↔ Mutant Cell Associations ................................. 27 Behavioral Phenotype .......................................................................... 29 Structural Clues to the Weaver Riddle ................................................. 30 3. Histochemical Properties of Intrastriatal Mesencephalic Grafts ........... 37 Introduction ........................................................................................ 37 Methodological Considerations ........................................................... 37 Expression of Catecholaminergic Neurotransmitter-Related Molecules and Quantitative Aspects of Dopaminergic Neuron Survival ........... 39 Comparative Survival of Dopaminergic Neurons in Grafts Placed in Weaver and in 6-OHDA Lesion Hosts ........................................ 43 Expression of Neuropeptides and Structural Proteins .......................... 45 4. Structural Correlates of Process Outgrowth and Circuit Reconstruction .................................................................. 53 Axonal Reinnervation of the Host Striatum ......................................... 53 Synaptic Investment of Graft-Derived Dopamine Terminals ............... 55 Compartmental Specificity of the Striatal Reinnervation ..................... 62 Innervation of Nonstriatal Regions by the Grafts ................................ 62 Chemoaffinity and Axon-Target Recognition in Development and in Transplantation .................................................................... 64 Dendrite Extension from the Graft into the Host Striatum ................. 64 Expression of Molecules Related to Axonal and Dendritic Outgrowth ............................................................... 66 5. Neurochemical Indices of Functional Restoration ................................ 75 Dopamine Uptake Markers ................................................................. 75 Autoradiography of [3H]Dopamine Uptake ........................................ 75 Dopamine Receptors ........................................................................... 81 Neurotensin Receptors ........................................................................ 83 Excitatory and Inhibitory Amino Acid Receptors ................................ 85

6. Behavioral Recovery of Functional Responses ...................................... 89 Unilateral Grafts and Circling Behavior ............................................... 89 Correlation of Turning Bias with Structural and Biochemical Parameters ............................................................ 91 Dissociation of the Functional Contribution of Graft-Derived Axons and Dendrites in Rotational Asymmetry .... 92 Enhancement of Motor Performance after Bilateral Transplantation ... 98 7. Directions for Future Research ........................................................... 106 Introduction ...................................................................................... 106 Analysis of Early Events in Graft-Host Interactions ........................... 107 Trophic Considerations ..................................................................... 109 Neurotransmitter Mechanisms .......................................................... 112 Supplemental Restoration of the Interrupted Nigro-Striato-Nigral Loop by Striatal/Nigral Double Grafts ......... 113 Index .................................................................................................. 120

PREFACE

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arkinson’s disease, a degenerative brain disorder, affects an estimated 0.25-0.50% of the population. Symptoms result from neuronal degeneration in the nigrostriatal dopaminergic pathway and include tremor, rigidity and gradual slowness of spontaneous movement. The cause of Parkinson’s disease is only partially understood; both environmental factors and a genetic predisposition have been implicated in its etiopathogenesis. Neural transplantation is being used experimentally for providing an alternative biological source of dopamine both in animal studies and in experimental clinical trials. This book is the epitome of 15 years of research on the transplantation of dopaminergic neurons in the striatum of the weaver mouse, a neurological mutant characterized by genetically-determined degeneration of midbrain dopamine neurons, and in that respect constituting the only available laboratory model with a chronic progressive disease that mimics Parkinsonism. The other two models currently used to investigate dopaminergic mechanisms rely on the use of the neurotoxins 6-hydroxydopamine and methylphenyltetrahydropyridine for the selective removal of dopaminergic neurons from an otherwise healthy organism. Structural and functional aspects of transplantation of mesencephalic dopaminergic grafts into the striatum of weaver mice are reviewed, including histochemical correlates of graft survival and integration, numerical aspects of donor neuron survival, ultrastructural findings on synaptogenesis, neurochemical indices of dopamine uptake function and receptor binding, gene expression of several structural and neurotransmitter-receptor related molecules, the levels of striatal amino acid receptors, and the behavioral effects of unilateral and bilateral neuronal transplantations. One finding of particular interest is that weaver hosts appear to offer a suboptimal environment for dopamine cell survival compared to wild-type hosts subjected to 6-hydroxydopamine lesions. While the weaver brain permits the survival and histotypic differentiation of donor dopaminergic neurons to a degree sufficient for bringing about functional improvement, there may be microenvironmental factor(s) putatively influencing the final number of surviving dopamine cells in the grafts. It is pertinent to mention that at birth, many young neurons undergo degeneration in the weaver striatum just beneath the subependymal plate, and by one year of age an estimated 22% of medium-sized striatal neurons have been lost. The possibility that the disease process exerts an influence on graft survival has been repeatedly brought up in clinical transplantation studies in Parkinsonism. Such considerations add value to the weaver mutant as a model of nigrostriatal dopamine degeneration, particularly in studying host-graft interactions.

ACKNOWLEDGMENTS

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he author thanks all of his collaborators and co-authors of the original studies, particularly those who have devotedly shared the laboratory bench, for making this work possible; special thanks for that matter to Drs. Patrik Brundin, Guy Doucet, Walter Low, Lupe Mengod, Adamantia Mitsacos, Jay Simon, Carme Solà, Lefteri Tsoukalas and Thomas Witt. Words of deep gratitude are extended to Drs. Shirley Bayer, Anders Björklund, Manuel del Cerro, Joseph Hingtgen, Elias Kouvelas and Chema Palacios for expanding the author’s scientific horizons; to Drs. Biagio Azzarelli, Ben Boukai, Jans Muller and Sidney Ochs for inspired discussions; to the American Society for Neural Transplantation and Repair for the exciting forum it has created and maintained; to the Bodossakis Foundation, Athens, for honoring this research with the bestowal of its Science Award; to the following agencies and foundations for supporting the author’s scientific efforts over the years: the Association for the Advancement of Mental Health Research and Education, the Ataxia-Telangiectasia Children’s Project, Eli Lilly and Company, the National Ataxia Foundation, the National Institutes of Health, and the Research and Sponsored Programs of Indiana University; and to Dr. Ronald Landes personally and the staff of Landes Bioscience Publishers for their encouragement, understanding and care during the composition of this monograph.

CHAPTER 1

Introduction Dopamine and Parkinson’s Disease

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ovement control is accomplished by complex interactions among various groups of nerve cells in the central nervous system. One such important group of neurons is located in the substantia nigra in the ventral midbrain. Nigral neurons give rise to an extensive network of axonal processes that innervate the basal ganglia, establishing predominantly symmetrical synapses with dendritic spines and shafts of medium spiny projection neurons.1,2 Neurons of the substantia nigra communicate with neurons of the basal ganglia by liberating the neurotransmitter dopamine (DA). Such an interaction at the biochemical level is responsible for the fine tuning of an organism’s movements. Parkinson’s disease or paralysis agitans3 is a neurological disorder that affects movement control. In Parkinson’s disease, neurons of the substantia nigra progressively degenerate4 (Fig. 1.1); as a result, the amount of DA available for neurotransmission in the corpus striatum is lowered.5 The biochemical imbalance manifests with typical clinical symptoms that include resting tremor, rigidity, bradykinesia, i.e., a gradual slowness of spontaneous movement, and loss of postural reflexes or, in other words, poor balance and motor coordination.6-9 An estimated half-a-million people are affected with Parkinson’s disease and related disorders in the United States.10 Reductions in DA content and uptake indices have been documented in Parkinson’s disease by a variety of techniques, including [3H]mazindol binding11 or computer-aided analyses of neuromelanin pigment12 in postmortem brain tissues, as well as positron emission tomography following the administration of 6-L-[18F]-fluorodopa or [11C]nomifensine as DA uptake tracers in vivo.13 A selective increase of N-methyl-D-aspartate (NMDA)sensitive glutamate binding but not of (RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate occurs in the striatum of postmortem brain tissue from patients with Parkinson’s disease.14 An important indirect action of DA in the striatum may actually be the tuning down of the cortical excitation of striatal neurons.15 Consequently, the impairment of dopaminergic neurotransmission that occurs in Parkinsonism may lead to an increase in the physiological state of corticostriatal glutamatergic transmission, which may further contribute towards reinforcing the imbalance between subsets of striatal neuronal systems controlling the functional output of the basal ganglia, and the available evidence suggests an overactive striatal γ-aminobutyric acid (GABA) output, especially to the lateral segment of the globus pallidus.16 The commonest age of onset of idiopathic Parkinson’s disease is during the fifth and sixth decades of life.6-10 The causes of cellular death in Parkinson’s disease are only partially Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease, by Lazaros C. Triarhou. ©2001 Eurekah.com.

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

Fig. 1.1. Appearance of normal (upper) and Parkinsonian (lower) human midbrain. Depigmentation of the substantia nigra is the main macroscopic neuropathological hallmark of Parkinson’s disease. Magnification x0.7. Courtesy Dr. Jans Muller.

understood. An intracellular eosinophilic inclusion, the Lewy body, is found in neurons of the Parkinsonian substantia nigra (Fig. 1.2). The Lewy body consists of fibrillary elements that share common antigenic determinants with intermediate filaments.7 Research studies back several theories that are still being explored. It has been proposed that Parkinson’s disease is a heterogeneous entity, in the etiology of which both environmental and genetic factors could play a role. The various theories implicate endogenous chemical reactions,17 exposure to specific environmental factors and neurotoxins,18 and genetically-determined susceptibility or predisposition.19,20 In addition, there is a juvenile form of Parkinson’s disease, i.e., characterized by an early onset, which is familial

Introduction

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Fig. 1.2. Lewy bodies are circular eosinophilic hyaline structures found in neurons of the substantia nigra and other brain areas in Parkinson’s disease. The pigmented nigral neuron of the upper micrograph has an eccentric nucleus and an eosinophilic Lewy body amidst the melanin pigment granules. The nonpigmented brainstem neuron of the lower micrograph also contains a Lewy body. Magnification x450. Hematoxylin and eosin. Courtesy Dr. Biagio Azzarelli (upper), Dr. Jans Muller (lower).

and clearly due to genetic factors.21-24 Any one or a combination of these theories may eventually prove to be the cause of Parkinson’s disease. The most effective mode of treatment has been the administration of the L-isomer of 3,4-dihydroxyphenylalanine (L-DOPA), a DA precursor.25 It is thought that certain antiParkinsonian agents may exert their clinical effects via blockade of NMDA receptors.26,27 In animal models of Parkinson’s disease, NMDA and AMPA receptor antagonists were found to reverse Parkinsonian signs28 or potentiate the ability of L-DOPA to reverse akinesia and to alleviate muscular rigidity.29 Accordingly, the clinical use of NMDA antagonists

4

Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

has been considered for the symptomatic treatment of Parkinson’s disease, based also on the observation that low doses of NMDA antagonists potentiate the therapeutic effects of DA agonists and on the hypothesis that even the beneficial effects of anticholinergic drugs may be mediated in part by NMDA receptor blockade.30 Polypharmacy with L-DOPA and a glutamate antagonist as adjuvant may be a realistic prospect in the pharmacological management of Parkinsonian symptoms, based on the pathophysiological hint that Parkinson’s disease is a glutamate hyperactivity disorder.31 In addition, GABA receptor agonists have been used in clinical trials, where they are thought of having a dual action, depending on dose.32 Alternative neurosurgical procedures performed clinically to alleviate Parkinsonian symptoms include posteroventral pallidotomy33 and intrastriatal implantation of dopaminergic neurons that have the ability of releasing DA.34 The latter approach has been stimulated by studies showing that grafts of fetal mesencephalic DA neurons implanted into experimental models with DA deficiency counteract the behavioral effects caused by the lesion.35,36

Experimental Models of Parkinsonism in Laboratory Animals The DA deficiency observed in the mesostriatal system in Parkinson’s disease is the main event underlying the pathophysiology of the motoric symptomatology. Accordingly, appropriate experimental models in laboratory animals should feature the typical loss of DA neurons in the substantia nigra and an associated DA reduction in the corpus striatum in order to be useful in investigating ways of therapeutic intervention. Typically, three main experimental models have been used in the laboratory as dopaminergic phenocopies of Parkinson’s disease to address cellular mechanisms of DA deficiency and restoration. Two of those models rely on selective neurotoxins to chemically destroy dopaminergic nigral neurons. The third model is the weaver mutant mouse (wv/wv), which has a genetic mutation that leads to mesencephalic DA neuron degeneration.37-41 1. The local injection of 6-hydroxydopamine (6-OHDA) into the midbrain of rats and mice causes an acute degeneration of dopaminergic neurons42 (Fig. 1.3). The 6-OHDA molecule is recognized by nigral neurons as DA and is taken up by the cell; with its entrance in the cytoplasm, 6-OHDA expresses its toxicity and destroys monoaminergic cells selectively. Rats with unilateral 6-OHDA lesions of the substantia nigra present with a characteristic motor syndrome that includes rotation behavior ipsilaterally to the side of the lesion, either spontaneously or in response to DA-releasing agents such as amphetamine.43 The interruption of the nigrostriatal projection is associated with an increase in striatal DA receptors, a phenomenon referred to as denervation-induced supersensitivity.44 2. The N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin was accidentally found to cause a Parkinsonian syndrome in humans.45,46 MPTP-induced Parkinsonism presents with the typical clinical signs of tremor, rigidity and bradykinesia, just like idiopathic Parkinson’s disease. The MPTP molecule seems to be selectively neurotoxic for humans and nonhuman primates. For that reason, it has been used in the laboratory to induce experimental Parkinsonism in monkeys. In vitro studies have shown that, once inside the cell, MPTP becomes oxidized to 1-methyl-4-phenylpyridine (MPP + ) through the action of the enzyme monoaminooxidase B. MPP+ is the form that is toxic to dopaminergic neurons.46 3. The formation and maintenance of brain circuitry is in part regulated by an organism’s genetics. Spontaneous heritable changes–or mutations–often take place in the genes. When a certain gene undergoes a mutation, the chromosome in

Introduction

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Fig. 1.3. Unilateral destruction of the substantia nigra in the laboratory mouse through local stereotactic injection of 6-hydroxydopamine. Micrographs from upper to lower correspond to coronal levels from rostral to caudal. Immunocytochemistry with antityrosine hydroxylase antiserum. Magnification x5. From work by T.C. Witt and L.C. Triarhou.

which the gene is located may be abnormal in some functional aspect. Currently, more than 140 spontaneous mutations are known to affect the nervous system of laboratory mice. These mutant mice are valuable models for investigating various pathological conditions that modify brain function either during development or in adulthood. In the weaver mutant mouse, there is a selective decrease of neurons in the substantia nigra, resulting in a depletion of DA stores in the basal ganglia.47,48 A naturally occurring model of DA deficiency of genetic causes in rodents is particularly valuable, as it may shed new light into pathological mechanisms of degeneration related to Parkinson’s disease and the application of techniques to restore lost function. Having a relatively short lifespan, the mouse avails itself to rigorous experimental analyses. Furthermore, there is the possibility of using large samples of animals with a consistent neurological defect to obtain biological, physiological and behavioral correlates of the restoration of lost function by means of various treatments. The weaver model is a valuable complement to the chemical models; its uniqueness lies in the fact that the mesostriatal DA depletion is progressive, taking place over several months, and incomplete, in contrast with the acute degeneration typical of the toxic models. Thus, laboratory studies in the

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

weaver can address specific aspects of experimental interference with the chronic pathological central nervous system.

Graft-Assisted Neural Reconstruction (“Brainware Engineering”?) As a rule, the genesis of neuronal populations, including midbrain DA cells, is concluded during embryonic life,49 and the regenerative capacity of the adult central nervous system is largely confined to compensatory fiber sprouting and not mitotic divisions of nerve cells.50,51 Therefore, neurons that die as a result of regressive phenomena can only be replaced through implantation of cells or tissues harvested from external sources. In the past quarter of a century the field of neural transplantation has witnessed an unparalleled blooming. The publication of numerous books and periodicals attests to that effect.52-64 Neural transplantation has been used successfully to effect cell replacement in conditions characterized by focal loss of a selective group of neurons both in laboratory animals and in clinical trials. The survival and growth of embryonic substantia nigra transplants in particular has been documented in rodents and in primates with lesions of the substantia nigra.34-36, 38-41,47,65-84 Experiments in rodents have shown that it is possible to establish a terminal axonal network in the DA-denervated striatum by intracerebral grafting of fetal mesencephalic tissue.34-36,38,39,41,47,65-70 The transplant-derived innervation leads to release of DA in the striatum as determined by methods of in vivo microdialysis71 and in vivo voltammetry.72 DA fibers from grafts form synaptic connections with striatal neurons of the host.73,74 The increase in DA D2 receptor binding, which occurs after 6-OHDA lesions in rats, can be normalized by nigral transplants.75 Mesencephalic grafts contain physiologically active neurons76 and restore specific behavioral functions.77-81 The precise mechanisms by which grafts promote a functional recovery are partially understood. It appears as if a multitude of trophic, neurohumoral and synaptic mechanisms may be responsible for such a recovery.82 Synaptic formation has been considered as one of the mechanisms underlying the recovery of function in the nigrostriatal system. Normal synaptogenesis is the result of a prolonged two-way communication between presynaptic and postsynaptic neuronal elements during development. In the case of neural grafting, however, embryonic donor tissue is led to develop inside an adult recipient brain. From both a theoretical and practical viewpoint, it is important to know the extent to which grafted cells mimic normal developmental patterns or participate in aberrant patterns of synaptic interactions with the denervated striatal cells of the adult recipient organisms. In the Parkinson’s disease model, the growth of human fetal mesencephalic neurons after transplantation has been monitored in human-to-rat grafting experiments as well.83,84 Clinical trials with fetal mesencephalic grafts into the caudate nucleus or putamen have been reported in Parkinsonian patients in medical centers of several countries, including Sweden,85-100 England,101-112 Mexico,113,114 U.S.A.,115-131 Cuba,132 Russia,133 Czech Republic, 134 Slovakia,135 Canada,136 Spain,137,138 China,139 Poland140 and France.141 Such trials have been prompted by encouraging results from the extensive experimental results from studies in the rodent and primate models. Evidence for graft survival 88,96,97,100,110,112,119,126,128,141 and functional improvement of clinical signs85-113,115-141 has been presented in several of those studies. Reported variations in the outcome of the procedure might relate among other factors to the technique and site of

Introduction

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grafting, the age and method of preparation of donor tissue(s), the stage of advancement of the disease in the host, and the pharmacological scheme of patient treatment prior to the transplantation operation. Clinical neural transplantation studies are monitored in the United States by the Registry Committee of the American Society for Neural Transplantation and Repair (ASNTR), which collects basic demographic, morbidity and mortality data and carries out efficacy evaluations.142 In the European Union, a concerted effort for the development of efficient, reliable, safe and ethically acceptable transplantation therapies for neurodegenerative diseases has been carried out by the Network of European CNS Transplantation and Restoration.143,144 References 1. Pickel VM, Beckley SC, Joh TH et al. Ultrastructural immunocytochemical localization of tyrosine hydroxylase in the neostriatum. Brain Res 1981; 225:373-385. 2. Freund TF, Powell JF, Smith AD. Tyrosine hydroxylase-immunoreactive boutons in synaptic contact with identified striatonigral neurons, with particular reference to dendritic spines. Neuroscience 1984; 13:1189-1215. 3. Parkinson J. An Essay on the Shaking Palsy. London: Sherwood, Neely, and Jones, 1817. 4. Trétiakoff C. Contribution à l’étude de l’anatomie pathologique du locus niger de Sömmering. Paris: Université de Paris, 1919. 5. Ehringer H, Hornykiewicz O. Verteilung von Noradrenalin und Dopamin (3-Hydroxytyramin) in Gehirn des Menschen und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems. Klin Wochenschr 1960; 38:1236-1239. 6. Walton JN. Brain’s Diseases of the Nervous System. Oxford: Oxford University Press, 1977. 7. Wooten GF. Parkinsonism. In: Pearlman AL, Collins RC, eds. Neurobiology of Disease. New York–Oxford: Oxford University Press, 1990:454-468. 8. Rewcastle NB. Degenerative diseases of the Central Nervous System. In: Davis RL, Robertson DM, eds. Textbook of Neuropathology, 2nd edn. Baltimore: Williams & Wilkins 1991:904-961. 9. Fahn S. Parkinson’s disease and other basal ganglion disorders. In: Asbury AK, McKhann GM, McDonald WI, eds. Diseases of the Nervous System: Clinical Neurobiology, 2nd ed. Philadelphia: W.B. Saunders Co. 1992:1144-1158. 10. American Academy of Neurology. Publication “Parkinson’s Disease” (Brain Matters Series). St. Paul, MN 1997. 11. Chinaglia G, Alvarez FJ, Probst A et al. Mesostriatal and mesolimbic dopamine uptake binding sites are reduced in Parkinson’s disease and progressive supranuclear palsy: A quantitative autoradiographic study using [3H]mazindol. Neuroscience 1992; 49:317-327. 12. German DC, Manaye K, Smith WK et al. Midbrain dopaminergic cell loss in Parkinson’s disease: Computer visualization. Ann Neurol 1989; 26:507-514. 13. Leenders KL, Salmon EP, Tyrrell P et al. The nigrostriatal dopaminergic system assessed in vivo by positron emission tomography in healthy volunteer subjects and patients with Parkinson’s disease. Arch Neurol 1990; 47:1290-1298. 14. Ulas J, Weihmuller FB, Brunner LC et al. Selective increase of NMDA-sensitive glutamate binding in the striatum of Parkinson’s disease, Alzheimer’s disease, and mixed Parkinson’s disease/Alzheimer’s disease patients: An autoradiographic study. J Neurosci 1994; 14:6317-6324. 15. Nieoullon A, Kerkerian-Le Goff L. Cellular interactions in the striatum involving neuronal systems using “classical” neurotransmitters: Possible functional implications. Mvmt Disord 1992; 7:311-325. 16. Robertson RG, Clarke CA, Boyce S et al. The role of striatopallidal neurones utilizing gammaaminobutyric acid in the pathophysiology of MPTP-induced parkinsonism in the primate: Evidence from [3H]flunitrazepam autoradiography. Brain Res 1990; 531:95-104. 17. Carlsson A, Fornstedt B. Possible mechanisms underlying the special vulnerability of dopaminergic neurons. Acta Neurol Scand 1991; 84[Suppl 136]:16-18. 18. Calne S, Schoenberg B, Martin W et al. Familial Parkinson’s disease: Possible role of environmental factors. J Can Sci Neurol 1987; 14:303-305. 19. Golbe LI. The genetics of Parkinson’s disease: A reconsideration. Neurology 1990; 40[Suppl 3]:7-14.

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease 20. Golbe LI, Di Iorio G, Bonavita V et al. A large kindred with autosomal dominant Parkinson’s disease. Ann Neurol 1990; 27:276-282. 21. Martin WE, Resch JA, Baker AB. Juvenile Parkinsonism. Arch Neurol 1971; 25:494-500. 22. Yokochi M, Narabayashi H, Iizuka R et al. Juvenile Parkinsonism—Some clinical, pharmacological, and neuropathological aspects. Adv Neurol 1984; 40:407-413. 23. Narabayashi H, Yokochi M, Iizuka R et al. Juvenile Parkinsonism. In: Vinken PJ, Bruyn GW, Klawans HL, eds. Handbook of Clinical Neurology. Amsterdam: Elsevier 1986; 49:153-165. 24. Matsumine H, Saito M, Shimoda-Matsubayashi S et al. Localization of a gene for an autosomal recessive form of juvenile Parkinsonism to chromosome 6q25.2-27. Am J Hum Genet 1997; 60:588-596. 25. Cotzias GC, Papavasiliou PS, Gellene R. Modification of Parkinsonism: Chronic treatment with L-Dopa. N Engl J Med 1969; 280:337-345. 26. Lustig HS, von B Ahern K, Greenberg DA. Antiparkinsonian drugs and in vitro excitotoxicity. Brain Res 1992; 597:148-150. 27. Olney JW, Price MT, Labruyere J et al. Anti-Parkinsonian agents are phencyclidine agonists and N-methyl-aspartate antagonists. Eur J Pharmacol 1987; 142:319-320. 28. Klockgether T, Turski L, Honoré T et al. The AMPA receptor antagonist NBQX has antiparkinsonian effects in monoamine-depleted rats and MPTP-treated monkeys. Ann Neurol 1991; 30:717-723. 29. Klockgether T, Turski L. NMDA antagonists potentiate antiparkinsonian actions of L-dopa in monoamine-depleted rats. Ann Neurol 1990; 28:539-546. 30. Greenamyre JT, O’Brien CF. N-methyl-D-aspartate antagonists in the treatment of Parkinson’s disease. Arch Neurol 1991; 48:977-981. 31. Starr MS. Glutamate/dopamine D1/D2 balance in the basal ganglia and its relevance to Parkinson’s disease. Synapse 1995; 19:264-293. 32. Bartholini G, Scatton B, Zivkovic B et al. GABA receptor agonists and extrapyramidal motor function: Therapeutic implications for Parkinson’s disease. Adv Neurol 1987; 45:79-83. 33. Laitinen LV, Bergenheim AT, Hariz MI. Leksell’s posteroventral pallidotomy in the treatment of Parkinson’s disease. J Neurosurg 1992; 76:53-61. 34. Olson L. On the use of transplants to counteract the symptoms of Parkinson’s disease: Background, experimental models, and possible clinical applications. In: Cotman CW, ed. Synaptic Plasticity. New York: Guilford Press 1986:485-505. 35. Björklund A, Stenevi U. Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants. Brain Res 1979; 177:555-560. 36. Perlow MJ, Freed WJ, Hoffer BJ et al. Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system. Science 1979; 204:643-647. 37. Zigmond MJ, Stricker EM. Animal models of Parkinsonism using selective neurotoxins: Clinical and basic implications. Int Rev Neurobiol 1989; 31:1-79. 38. Brundin P, Duan W-M, Sauer H. Functional effects of mesencephalic dopamine neurons and adrenal chromaffin cells grafted to the rodent striatum. In: Dunnett SB, Björklund A, eds. Functional Neural Transplantation. New York: Raven Press, 1994:9-46. 39. Witt TC, Triarhou LC. Transplantation of mesencephalic cell suspensions from wild-type and heterozygous weaver mice into the denervated striatum: Assessing the role of graft-derived dopaminergic dendrites in the recovery of function. Cell Transpl 1995; 4:323-333. 40. Bakay RAE, Fiandaca MS, Barrow DL et al. Preliminary report on the use of fetal tissue transplantation to correct MPTP-induced Parkinson-like syndrome in primates. Appl Neurophysiol 1985; 48:358-361. 41. Triarhou LC, Low WC, Doucet G et al. The weaver mutant mouse as a model for intrastriatal grafting of fetal dopamine neurons. In: Hefti F, Weiner WJ, eds. Progress in Parkinson’s Disease Research–2. Mt. Kisco, New York: Futura Publishing Company 1992:383-393. 42. Ungerstedt U. 6-Hydroxydopamine-induced degeneration of central monoamine neurons. Eur J Pharmacol 1968; 5:107-110. 43. Ungerstedt U, Arbuthnott GW. Quantitative recording of rotational behavior in rats after 6hydroxydopamine lesions of the nigrostriatal dopamine system. Brain Res 1970; 24:485-493. 44. Marshall JF, Ungerstedt U. Supersensitivity to apomorphine following destruction of the ascending dopamine neurons: Quantification using the rotational model. Eur J Pharmacol 1977; 41:361-367.

Introduction

9

45. Burns RS, Chiueh CC, Markey SP et al. A primate model of Parkinsonism: Selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6tetrahydropyridine. Proc Natl Acad Sci USA 1983; 80:4546-4550. 46. Langston JW. MPTP and Parkinson’s disease. Trends Neurosci 1985; 8:79-83. 47. Triarhou LC, Low WC, Ghetti B. Transplantation of ventral mesencephalic anlagen to hosts with genetic nigrostriatal dopamine deficiency. Proc Natl Acad Sci USA 1986; 83:8789-8793. 48. Triarhou LC. Weaver gene expression in central nervous system. In: Conn PM, ed. Gene Expression in Neural Tissues. San Diego: Academic Press 1992:209-227. 49. Taber Pierce E. Time of origin of neurons in the brain stem of the mouse. Prog Brain Res 1973; 40:53-65. 50. Cotman CW. Synaptic Plasticity. New York: Guilford Press 1986. 51. Baudry M, Thompson RF, Davis JL. Synaptic Plasticity: Molecular, Cellular, and Functional Aspects. Cambridge, MA: MIT Press 1993. 52. Wallace RB, Das GD, eds. Neural Tissue Transplantation Research. New York-Berlin-HeidelbergTokyo: Springer-Verlag 1983. 53. Sladek JR Jr, Gash DM, eds. Neural Transplants: Development and Function. New York: Raven Press, 1984. 54. Björklund A, Stenevi U, eds. Neural Grafting in the Mammalian CNS. Amsterdam: Elsevier; 1985. 55. Azmitia EC, Björklund A, eds. Cell and Tissue Transplantation into the Adult Brain. New York: The New York Academy of Sciences 1987. 56. Gash DM, Sladek JR Jr, eds. Transplantation into the Mammalian CNS. Amsterdam-New YorkOxford: Elsevier 1988. 57. Dunnett SB, Richards S-J, eds. Neural Transplantation: From Molecular Basis to Clinical Applications. Amsterdam-New York-Oxford: Elsevier 1990. 58. Lindvall O, Björklund A, Widner H, eds. Intracerebral Transplantation in Movement Disorders: Experimental Basis and Clinical Experiences. Amsterdam-London-New York-Tokyo: Elsevier 1991. 59. Dunnett SB, Björklund A, eds. Neural Transplantation: A Practical Approach. Oxford-New YorkTokyo: Oxford University Press 1992. 60. Dunnett SB, Björklund A, eds. Functional Neural Transplantation. New York: Raven Press, 1994. 61. Sanberg PR, Wictorin K, Isacson O. Cell Transplantation for Huntington’s Disease. Austin, TX: RG Landes Co. 1994. 62. Vrbová G, Clowry G, Nógrádi A et al. Transplantation of Neural Tissue into Spinal Cord. Austin, TX: RG Landes Co. 1994. 63. Triarhou LC. Neural Transplantation in Cerebellar Ataxia. Austin, TX: RG Landes Co. 1997. 64. Freed, W.J. Neural Transplantation: An Introduction. Cambridge, MA: MIT Press 2000. 65. Stenevi U, Björklund A, Svendgaard N-A. Transplantation of central and peripheral monoamine neurons to the adult rat brain: Techniques and conditions for survival. Brain Res 1976; 114:1-20. 66. Björklund A, Dunnett SB, Stenevi U et al. Reinnervation of the denervated striatum by substantia nigra transplants: Functional consequences as revealed by pharmacological and sensorimotor testing. Brain Res 1980; 199:307-333. 67. Schmidt RH, Ingvar M, Lindvall O et al. Functional activity of substantia nigra grafts reinnervating the striatum: Neurotransmitter metabolism and [ 14C]2-deoxy- D -glucose autoradiography. J Neurochem 1982; 38:737-748. 68. Jaeger CB. Cytoarchitectonics of substantia nigra grafts: A light and electron microscopic study of immunocytochemically identified dopaminergic neurons and fibrous astrocytes. J Comp Neurol 1985; 231:121-135. 69. Strömberg I, Johnson S, Hoffer BJ et al. Reinnervation of dopamine-denervated striatum by substantia nigra transplants: Immunohistochemical and electrophysiological correlates. Neuroscience 1985; 14:981-990. 70. Brundin P, Björklund A. Survival, growth and function of dopaminergic neurons grafted to the brain. Prog Brain Res 1987; 71:293-308. 71. Zetterström T, Brundin P, Gage FH et al. Spontaneous release of dopamine from intrastriatal nigral grafts as monitored by the intracerebral dialysis technique. Brain Res 1986; 362:344-349. 72. Rose G, Gerhardt G, Strömberg I et al. Monoamine release from dopamine-depleted rat caudate nucleus reinnervated by substantia nigra transplants: An in vivo electrochemical study. Brain Res 1985; 341:92-100.

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

73. Freund TF, Bolam JP, Björklund A et al. Efferent synaptic connections of grafted dopaminergic neurons reinnervating the host neostriatum: A tyrosine hydroxylase immunocytochemical study. J Neurosci 1985; 5:603-616. 74. Mahalik TJ, Finger TE, Strömberg I et al. Substantia nigra transplants into denervated striatum of the rat: Ultrastructure of graft and host interconnections. J Comp Neurol 1985; 240:60-70. 75. Freed WJ, Ko GN, Niehoff DL et al. Normalization of spiroperidol binding in the denervated rat striatum by homologous grafts of substantia nigra. Science 1983; 222:937-939. 76. Arbuthnott G, Dunnett SB, MacLeod N. Electrophysiological properties of single units in dopamine-rich mesencephalic transplants in rat brain. Neurosci Lett 1985; 57:205-210. 77. Björklund A, Schmidt RH, Stenevi U. Functional reinnervation of the neostriatum in the adult rat by use of intraparenchymal grafting of dissociated cell suspensions from the substantia nigra. Cell Tissue Res 1980; 212:39-45. 78. Björklund A, Stenevi U, Dunnett SB et al. Functional reactivation of the deafferented neostriatum by nigral transplants. Nature (Lond) 1981; 289:497-499. 79. Dunnett SB, Björklund A, Stenevi U et al. Behavioural recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal dopamine pathway. I. Unilateral lesions. Brain Res 1981; 215:147-161. 80. Dunnett SB, Björklund A, Stenevi U et al. Behavioural recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal dopamine pathway. II. Bilateral lesions. Brain Res 1981; 229:457-470. 81. Brundin P, Isacson O, Gage FH et al. The rotating 6-hydroxydopamine-lesioned mouse as a model for assessing functional effects of neuronal grafting. Brain Res 1986; 366:346-349. 82. Björklund A, Lindvall O, Isacson O et al. Mechanisms of action of intracerebral neural implants: Studies on nigral and striatal grafts to the lesioned striatum. Trends Neurosci 1987; 10:509-516. 83. Brundin P, Nilsson OG, Strecker RE et al. Behavioural effects of human fetal dopamine neurons grafted in a rat model of Parkinson’s disease. Exp Brain Res 1986; 65:235-240. 84. Clarke DJ, Brundin P, Strecker RE et al. Human fetal dopamine neurons grafted in a rat model of Parkinson’s disease: Ultrastructural evidence for synapse formation using tyrosine hydroxylase immunocytochemistry. Exp Brain Res 1988; 73:115-126. 85. Lindvall O, Rehncrona S, Gustavii B et al. Fetal dopamine-rich mesencephalic grafts in Parkinson’s disease. Lancet 1988; ii:1483-1484. 86. Lindvall O, Rehncrona S, Brundin P et al. Human fetal dopamine neurons grafted into the striatum in two patients with severe Parkinson’s disease: A detailed account of methodology and a 6-month follow-up. Arch Neurol 1989; 46:615-631. 87. Lindvall O. Transplantation into the human brain: Present status and future possibilities. J Neurol Neurosurg Psychiat 1989; Suppl:39-54. 88. Lindvall O, Brundin P, Widner H et al. Grafts of fetal dopamine neurons survive and improve motor function in Parkinson’s disease. Science 1990; 247:574-577. 89. Brundin P, Odin P, Widner H. Promising new results with transplantation of nerve cells to the brain in Parkinson disease. Lakartidningen 1990; 87:3761-3763. 90. Brundin P, Björklund A, Lindvall O. Practical aspects of the use of human fetal brain tissue for intracerebral grafting. Prog Brain Res 1990; 82:707-714. 91. Lindvall O, Rehncrona S, Brundin P et al. Neural transplantation in Parkinson’s disease: The Swedish experience. Prog Brain Res 1990; 82:729-734. 92. Lindvall O. Prospects of transplantation in human neurodegenerative diseases. Trends Neurosci 1991; 14:376-384. 93. Lindvall O, Björklund A, Widner H, eds. Intracerebral Transplantation in Movement Disorders: Experimental Basis and Clinical Experiences. Amsterdam: Elsevier, 1991. 94. Widner H, Brundin P, Rehncrona S et al. Transplanted allogeneic fetal dopamine neurons survive and improve motor function in idiopathic Parkinson’s disease. Transpl Proc 1991; 23:793-795. 95. Lindvall O. Transplants in Parkinson’s disease. Eur Neurol 1991; 31[Suppl 1]:17-27. 96. Lindvall O, Widner H, Rehncrona S et al. Transplantation of fetal dopamine neurons in Parkinson’s disease: One-year clinical and neurophysiological observations in two patients with putaminal implants. Ann Neurol 1992; 31:155-165. 97. Widner H, Tetrud J, Rehncrona S et al. Bilateral fetal mesencephalic grafting in two patients with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). N Engl J Med 1992; 327:1556-1563.

Introduction

11

98. Widner H, Tetrud J, Rehncrona S et al. Fifteen months’ follow-up on bilateral embryonic mesencephalic grafts in two cases of severe MPTP-induced parkinsonism. Adv Neurol 1993; 60:729-733. 99. Widner H, Rehncrona S. Transplantation and surgical treatment of parkinsonian syndromes. Curr Opin Neurol Neurosurg 1993; 6:344-349. 100. Lindvall O, Sawle G, Widner H et al. Evidence for long-term survival and function of dopaminergic grafts in progressive Parkinson’s disease. Ann Neurol 1994; 35:172-180. 101. Hitchcock ER, Clough C, Hughes R et al. Embryos and Parkinson’s disease. Lancet 1988; I:1274. 102. Hitchcock ER, Kenny BG, Clough CG et al. Stereotactic implantation of fetal mesencephalon. Stereotact Funct Neurosurg 1990; 54/55:282-289. 103. Quinn NP. The clinical application of cell grafting techniques in patients with Parkinson’s disease. Prog Brain Res 1990; 82:619-625. 104. Hitchcock ER, Kenny BG, Clough CG et al. Stereotactic implantation of foetal mesencephalon (STIM): The UK experience. Prog Brain Res 1990; 82:723-728. 105. Henderson BTH, Kenny BG, Hitchcock ER et al. A comparative evaluation of clinical rating scales and quantitative measurements in assessment pre and poststriatal implantation of human foetal mesencephalon in Parkinson’s disease. Acta Neurochir 1991; Suppl 52:48-50. 106. Henderson BT, Clough CG, Hughes RC et al. Implantation of human fetal ventral mesencephalon to the right caudate nucleus in advanced Parkinson’s disease. Arch Neurol 1991; 48:822-827. 107. Hitchcock ER, Kenny BG, Henderson BTH et al. A series of experimental surgery for advanced Parkinson’s disease by foetal mesencephalic transplantation. Acta Neurochir 1991; Suppl 52:54-57. 108. Hitchcock ER. Neural implants and recovery of function: Human work. Adv Exp Med Biol 1992; 325:67-78. 109. Sinden JD, Patel SN, Hodges H. Neural transplantation: Problems and prospects for therapeutic application. Curr Opin Neurol Neurosurg 1992; 5:902-908. 110. Sawle GV, Bloomfield PM, Björklund A et al. Transplantation of fetal dopamine neurons in Parkinson’s disease: PET [18F]6-L-fluorodopa studies in two patients with putaminal implants. Ann Neurol 1992; 31:166-173. 111. Henderson B, Good PA, Hitchcock ER et al. Visual evoked cortical responses and electroretinograms following implantation of human fetal mesencephalon to the right caudate nucleus in Parkinson’s disease. J Neurol Sci 1992; 107:183-190. 112. Sawle GV, Myers R. The role of positron emission tomography in the assessment of human neurotransplantation. Trends Neurosci 1993; 16:172-176. 113. Madrazo I, León V, Torres C et al. Transplantation of fetal substantia nigra and adrenal medulla to the caudate nucleus in two patients with Parkinson’s disease. N Engl J Med 1988; 318:51. 114. Madrazo I, Franco-Bourland R, Ostrosky-Solis F et al. Neural transplantation (auto-adrenal, fetal nigral and fetal adrenal) in Parkinson’s disease: The Mexican experience. Prog Brain Res 1990; 82:593-602. 115. Freed CR, Breeze RE, Rosenberg NL et al. Transplantation of human fetal dopamine cells for Parkinson’s disease: Results at 1 year. Arch Neurol 1990; 47:505-512. 116. Freed CR, Breeze RE, Rosenberg NL et al. Therapeutic effects of human fetal dopamine cells transplanted in a patient with Parkinson’s disease. Prog Brain Res 1990; 82:715-721. 117. Fiandaca MS. Brain grafting for Parkinson’s disease. Transplantation 1991; 51:549-556. 118. Spencer DD, Robbins RJ, Naftolin F et al. Unilateral transplantation of human fetal mesencephalic tissue into the caudate nucleus of patients with Parkinson’s disease. N Engl J Med 1992; 327:1541-1548. 119. Freed CR, Breeze RE, Rosenberg NL et al. Survival of implanted fetal dopamine cells and neurologic improvement 12 to 46 months after transplantation for Parkinson’s disease. N Engl J Med 1992; 327:1549-1555. 120. Bakay RAE. Central nervous system grafting: Animal and clinical results. Stereotact Funct Neurosurg 1992; 58:67-78. 121. Thompson L. Fetal transplants show promise. Science 1992; 257:868-870. 122. Langston JW, Widner H, Goetz CG et al. Core assessment program for intracerebral transplantation (CAPIT). Movt Dis 1992; 7:2-13. 123. Goetz CG, De Long MR, Penn RD et al. Neurosurgical horizons in Parkinson’s disease. Neurology 1993; 43:1-7.

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

124. Freed CR, Breeze RE, Rosenberg NL et al. Embryonic dopamine cell implants as a treatment for the second phase of Parkinson’s disease: Replacing failed nerve terminals. Adv Neurol 1993; 60:721-728. 125. Redmond DE Jr, Robbins RJ, Naftolin F et al. Cellular replacement of dopamine deficit in Parkinson’s disease using human fetal mesencephalic tissue: Preliminary results in four patients. Res Publ Assoc Res Nerv Ment Dis 1993; 71:325-359. 126. Rauch RA, Markham CH, Rand RW et al. MR imaging findings after transplant surgery for Parkinson disease. J Magn Reson Imag 1994; 4:19-24. 127. Freeman TB, Olanow CW, Hauser RA et al. Bilateral fetal nigral transplantation into the postcommissural putamen in Parkinson’s disease. Ann Neurol 1995; 38:379-388. 128. Kordower JH, Freeman TB, Snow BJ et al. Neuropathological evidence of graft survival and striatal reinnervation after the transplantation of fetal mesencephalic tissue in a patient with Parkinson’s disease. N Engl J Med 1995; 332:1118-1124. 129. Price LH, Spencer DD, Marek KL et al. Psychiatric status after human fetal mesencephalic tissue transplantation in Parkinson’s disease. Biol Psychiat 1995; 38:498-505. 130. Olanow CW, Kordower JH, Freeman TB. Fetal nigral transplantation as a therapy for Parkinson’s disease. Trends Neurosci 1996; 19:102-109. 131. Kopyov OV, Jacques DS, Lieberman A et al. Clinical study of fetal mesencephalic intracerebral transplants for the treatment of Parkinson’s disease. Cell Transplantation 1996; 5:327-337. 132. Molina H, Quiñones R, Alvarez L et al. Transplantation of human fetal mesencephalic tissue in caudate nucleus as treatment for Parkinson’s disease: The Cuban experience. Restor Neurol 1991; 4:99-110. 133. Bekhtereva NP, Gilerovich EG, Gurchin FA et al. Transplantation of embryonal nerve tissues in the treatment of Parkinson disease. Z Nevropatol Psikhiat SS Korsakova 1990; 90:10-13. 134. Subrt O, Tichy M, Vladyka V et al. Grafting of fetal dopamine neurons in Parkinson’s disease: The Czech experience with severe akinetic patients. Acta Neurochir 1991; Suppl 52:51-53. 135. Marsala J, Zigova T, Badonic T et al. Neurotransplantation, critical analysis and perspectives. Bratislav Lekar List 1992; 93:111-122. 136. Jones D. Halifax hospital first in Canada to proceed with controversial fetal-tissue transplant. Can Med Assoc J 1992; 146:389-391. 137. Lopez-Lozano JJ, Brera B, Bravo G et al. Neural transplants in Parkinson’s disease. Transpl Proc 1993; 25:1005-1011. 138. Lopez-Lozano JJ, Bravo G, Brera B et al. Long-term follow-up in 10 Parkinson’s disease patients subjected to fetal brain grafting into a cavity in the caudate nucleus: The Clinica Puerta de Hierro experience. Transpl Proc 1995; 27:1395-1400. 139. Iacono RP, Tang ZS, Mazziotta JC et al. Bilateral fetal grafts for Parkinson’s disease: 22 months’ results. Stereotact Funct Neurosurg 1992; 58:84-87. 140. Zabek M, Mazurowski W, Dymecki J et al. Transplantation of fetal dopaminergic cells in Parkinson disease. Neurol Neurochir Polsk 1992; Suppl 1:13-19. 141. Remy P, Samson Y, Hantraye P et al. Clinical correlates of [18F]fluorodopa uptake in five grafted parkinsonian patients. Ann Neurol 1995; 38:580-588. 142. Goetz CG, Bakay RAE, Fine A et al. American Society for Neural Transplantation Registry for fetal mesencephalic implants: Demographic and baseline data. Abstr Am Soc Neural Transpl 1996; 3:25. 143. Boer GJ. Ethical guidelines for the use of human embryonic or fetal tissue for experimental and clinical neurotransplantation and research. Network of European CNS Transplantation and Restoration (NECTAR). J Neurol 1994; 242:1-13. 144. Wolfslast G. Legal aspects of neurotransplantation. Zbl Neurochir 1995; 56:210-214.

CHAPTER 2

Biology and Pathology of the Weaver Mutant Mouse Introduction

T

he weaver mutant mouse (wv/wv ) is characterized by a genetically induced degeneration of mesostriatal dopamine (DA) neurons. In that sense, it can be viewed as a pathophysiological phenocopy of Parkinsonism and, therefore, an invaluable experimental model for investigating mechanisms of progressive DA neuron degeneration, as well as issues of the survival and growth of intrastriatally grafted fetal DA neurons in the chronically denervated striatum.1-3 The weaver (wv) mutation also interferes with the survival and organization of neurons in the cerebellar cortex and the hippocampus. The anatomical systems affected by the wv gene are of particular interest regarding development and degeneration. In particular, the cerebellar lesion of the weaver cerebellum has presented a forum for extensive studies on mechanisms of neurite extension, neuronal migration, and the remodeling of synaptic circuitry. The weaver model has particular merit if one considers that in humans, geneticallydetermined loss of mesencephalic DA neurons occurs in familial Parkinson’s disease and in cases of multiple systems atrophies that include olivopontocerebellar degeneration (Menzel type) and primary degeneration of the granular layer of the cerebellum of Norman type.4-12 Such diseases provide a link between cerebellar atrophies and nigrostriatal/striatonigral degenerations.

Cellular and Molecular Genetics of the Weaver Mutation The weaver mutation occurred spontaneously in 1961 in a C57BL/6J mouse genetic stock.13 Since only homozygotes (wv/wv) displayed neurological locomotor signs, the mutation was initially considered to be autosomal recessive.14 However, aberrations in cerebellar cytoarchitectonics have been observed in heterozygotes (wv/+), to a milder degree than homozygotes, which indicates that the mutation is incomplete dominant or semidominant.15-17 The wv allele has been mapped by means of translocation-carrying animals to the distal end of mouse chromosome (Mmu ) 16 at a distance of 60 centimorgans (cM) away from the centromere.18-19 In the same vicinity there have been assigned the regional positions of the loci for soluble superoxide dismutase (Sod-1 ),20 mammalian homolog of the 3´ domain of avian leukemia protooncogene (Ets-2),21 and β-amyloid protein precursor (βAPP);22,23 these three genes occupy a 3.2 cM region proximal to wv. The same three Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease by Lazaros C. Triarhou. ©2001 Eurekah.com.

,

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson's Disease

genes have been mapped to human chromosome (Hsa) 21, the telemetric region of which is highly homologous to Mmu 16 and phylogenetically conserved.24,25 The wv mutation has been identified as a missense mutation, with a G→A substitution in nucleotide 953 of the inward-rectifier K+ channel gene Girk2, and an ensuing Gly→Ser replacement at residue 156 of the GIRK2 protein.26 There is a human equivalent ATP-sensitive K+ channel gene that maps to Hsa 21q22.1.27 Electrophysiological experiments to assess the functional implications of the Gly→Ser substitution have not demonstrated inward rectifying K+ currents in cultured granule cells from either wild-type or weaver mice at postnatal day eight.28 Subsequent analyses of GIRK2 weaver and GIRK1 channels in Xenopus oocytes found that GIRK2 weaver homomultimeric channels lose their selectivity for K+ ions, giving rise to inappropriate receptor-activated and basally active Na+ currents, while heteromultimers of GIRK2 weaver and GIRK1 appear to have reduced current.29 Cultured weaver granule cells are proteolytically overactive and secrete excessive amounts of tissue plasminogen activator, which is likely to interfere with neurite outgrowth potential on a laminin substratum.30 Electrophysiology shows that weaver granule cells have poor resting membrane potentials (-38 mV); aprotinin, a protease inhibitor, can restore the resting membrane potential to near normal (-59 mV), rescue weaver granule cells from death on the laminin substratum and promote neurite outgrowth.30

Alterations of the Mesotelencephalic Dopamine Projection System Midbrain Dopamine Cells Neuron loss occurs in all three areas of the mesencephalic DA cell system, i.e., substantia nigra or area A9, ventral tegmental area or area A10, and retrorubral nucleus or area A8 (Fig. 2.1). The substantia nigra is the main source of dopaminergic innervation of the caudate-putamen complex, while the ventral tegmental area and the retrorubral nucleus provide DA innervation to limbic and cortical projection fields.31 The atrophy of the substantia nigra was first seen in sections conventionally stained for histology32 (Fig. 2.2). Tyrosine hydroxylase (TH, tyrosine monooxygenase) immunocytochemical studies verified the involvement of nigral DA neurons33-35 (Fig. 2.3). Direct evidence of neuronal cell death in the weaver substantia nigra and ventral tegmental area has been obtained by electron microscopy36 (Fig. 2.4). Neurons with osmiophilic cytoplasm, disrupted organelles, and pyknotic nuclei are seen in both areas of weaver homozygotes 16-45 days old; the cellular debris is surrounded by reactive astroglia. A 31% decrease in DA levels has been described in the weaver midbrain,37,38 which can readily be explained by the loss of DA cell bodies. Quantitative immunocytochemical studies in serial paraffin sections of the midbrain disclosed that the weaver substantia nigra has 42% fewer DA cells than the wild-type on postnatal day 20 and 69% fewer DA cells at three months of age.34,35 A second wave of DA neuron degeneration takes place during the second year of life, which brings total DA cell loss at the end of two years of age to 85% in the substantia nigra.39 The weaver ventral tegmental area and retrorubral nucleus are normal at 20 days of age. However, at three months, there is a 26% loss of DA neurons in the ventral tegmental area and a 56% loss in the retrorubral nucleus.34,35 By the end of the second year of life, DA neuron losses amount to 35% in A10 and to 60% in A8.39

Biology and Pathology of the Weaver Mutant Mouse

15

Fig. 2.1. Schematic drawing of tyrosine hydroxylase immunoreactivity in coronal sections of the midbrain in wildtype mouse (left) and homozygous weaver mutant (right). Abbreviations: A8, retrorubral nucleus; A9, substantia nigra; A9L substantia nigra pars lateralis; A9V, substantia nigra pars ventralis; A10, ventral tegmental area; A10C ventral tegmental area pars caudalis; FR, fasciculus retroflexus of Meynert; LM, lemniscus medialis; IP, nucleus interpeduncularis. Numbers on the left indicate distance from the rostral beginning of the substantia nigra pars compacta in mm. Reprinted with permission from: Triarhou LC, Norton J, Ghetti B. Exp Brain Res 1988; 70:256-265. © Springer-Verlag.

Nigral DA cell numbers in the weaver model from birth to senescence were regressed upon age to obtain the best mathematical function in the weaver model (Fig. 2.5).40 Regression fits show that DA neuron fallout combines two independent components, an initial exponential decay, superceded by a linear regression, with a threshold at around 100 days. The first wave of (exponential) cell loss follows the general form Yt = α + Y0 e -λt, where Yt is a dependent variable representing DA cell count with respect to age, Y0 is the initial

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson's Disease

Fig. 2.2. Histological sections of the pars compacta of the substantia nigra in a normal mouse (upper) and in a weaver mutant mouse (lower). The substantia nigra of the mutant contains far fewer neurons that that of the control. Gallocyanin Nissl. Magnification x200. Reprinted with permission from: Triarhou LC, Ghetti B. Exp Brain Res 1987; 68:434-435. © Springer-Verlag.

number of cells, λ is the constant of proportionality known as the decay constant, age t is an independent variable, and constant term α represents a horizontal asymptote. The halflife T1/2 of neurons degenerating in this phase is around 58 days. The exponential pattern implies that the probability per unit time that a neuron will die is a constant (λ); that probability is an inherent characteristic and can be calculated at 1.2% per day. Such a property is natural, cell-specific, and independent of time. In the second (linear) phase of degeneration, the probability of a neuron dying is a function of time and decreases with age, i.e., the longer a cell survives, the less likely it becomes to degenerate.

Biology and Pathology of the Weaver Mutant Mouse

17

Fig. 2.3. Tyrosine hydroxylase immunoreactivity in the substantia nigra of a wild-type (upper) and a weaver mutant (lower) mouse. There is a substantial cellular deficit in the mutant. Dark-field illumination. Magnification x200. Reprinted with permission from: Triarhou LC, Norton J, Ghetti B. Exp Brain Res 1988; 70:256-265. © SpringerVerlag.

Immunocytochemical studies have associated the selective vulnerability of wv DA neurons with differences in their histochemical signatures. Specifically, DA neurons coexpressing the 28 kDa Ca++-binding protein appear to be more resistant to degeneration.41 Further, analyses of the timetables of neurogenesis by means of combined [3H]thymidine dating and TH immunocytochemistry (Fig. 2.6) indicate that DA neurons generated later in embryonic life are preferentially targeted by the weaver mutation.42,43 The mathematical findings described above prove the existence of two independent DA neuron subsets with regards to degeneration, which might relate to structural and developmental neuronal idiosyncrasies.

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson's Disease

Fig. 2.4. Dendrites of the weaver substantia nigra at advanced stages of degeneration. Magnification x27000. Reprinted with permission from: Ghetti B, Triarhou LC (ref. 36) © 1992 Futura Publishing Co., Inc.

Fig. 2.5. Numbers of tyrosine hydroxylase immunoreactive neurons in the substantia nigra of weaver mutants expressed as percent of the wild-type values over the life-span of the animals. Note that values in the abscissa are not linear, but rather represent the ages for which actual experimental data have been collected. Numbers combined from several previously published articles.

Nigral Dopaminergic Dendrites Heterozygous weaver mice have normal DA neuron numbers in all three areas at 20 and 90 days of age.34,35 Using TH immunocytochemistry (Fig. 2.7), a subcellular site was found to be defective in the substantia nigra of mice both heterozygous and homozygous for the wv gene, involving the dendritic DA projection that extends from the substantia nigra pars compacta into the substantia nigra pars reticulata.34,35,44 Measurements of total

Biology and Pathology of the Weaver Mutant Mouse

19

Fig. 2.6. Dating of dopaminergic nigral neurons by a double-labeling technique combining tyrosine hydroxylase immunocytochemistry with [3H]thymidine autoradiography. Magnification x370. Unpublished micrograph by S.A. Bayer and L.C. Triarhou.

Fig. 2.7. Representative fields of the pars reticulata of the substantia nigra in wild-type (a), weaver heterozygous (b) and weaver homozygous (c) mice. Immunocytochemistry with antityrosine hydroxylase antiserum. Note the high density and formation of bundles in the wild-type, markedly in contrast with the sparsity and atrophic appearance of immunopositive dendrites in the heterozygotes, despite the presence of normal numbers of dopamine neuron somata in the pars compacta of the latter. The number of immunopositive dendrites in the weaver homozygote is reduced even further, being extremely small to match the number of surviving dopamine neurons in the pars compacta. Magnification x400. Reprinted with permission from: Triarhou LC, Ghetti B. Brain Res 1989; 501:373-381. © Elsevier Science Publishers B.V.

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson's Disease

dendritic lengths in camera lucida drawings of immunocytochemical preparations44 indicate that in the substantia nigra pars reticulata of weaver heterozygotes, DA dendrites are reduced by 60% at 20 days of age, when nigral DA neuron number, striatal DA content, and pattern of synaptic connectivity of nigrostriatal axon terminals are all normal. At that same age, the deficit of DA dendrites in the substantia nigra pars reticulata of weaver homozygotes is 76% and disproportionate to the 42% loss of nigral DA cell bodies. The structural defect of nigral dendrites can also be seen following immunocytochemical labeling with antibodies against microtubule-associated protein 2 (MAP2), a marker for dendritic processes in nervous tissue;45 an overall reduction of MAP2 immunoreactive dendrites is observed in the substantia nigra pars reticulata of heterozygous and homozygous weaver mutants (Fig. 2.8). The state of surviving dopaminergic dendrites was also studied by Golgi and by ultrastructural immunocytochemical methods.46 In Golgi preparations (Fig. 2.9), dendrites of the substantia nigra pars reticulata normally display typical fusiform varicosities.47,48 The diameter of such varicosities is reduced in the surviving dendrites of weaver homozygotes by almost 41% compared to controls, and is also accompanied by an elongation of the intervaricose segment (denoting fewer varicosities per unit length). In heterozygotes, the diameter of fusiform varicosities is reduced by about 33% relative to the wild-type, thus being intermediate in magnitude between homozygous mutants and normal. Using pre-embedding electron microscopic immunocytochemistry (Fig. 2.10), one finds deficiencies in both weaver genotypes. The percentage of TH-labeled dendrites that receive synaptic input from unlabeled (to a large extent striatonigral) afferent axon terminals is 50% in wild-type animals, 24% in wv/+, and 9% in wv/wv.46 These findings strengthen the view that the dendritic DA projection of the substantia nigra represents a subcellular target of wv gene action in both the heterozygous and homozygous states. Furthermore, such data indicate that in addition to being fewer, the remaining DA dendrites of wv/+ and wv/wv are aberrant in structural and probably functional aspects of synaptic connectivity. One thus gains insight into how the wv mutation may hinder process outgrowth and maintenance, and possibly explain the generalized convulsions intermittently manifested by weaver heterozygotes, as the substantia nigra has been implicated in the pathophysiology of experimental seizures.49,50 There are two theoretical possibilities in explaining the dendritic defect in homozygotes: first, that the dendritic defect is simply an aftereffect of the loss of DA somata; secondly, that a deficit in dendritogenesis might precede the death of DA cell bodies, an idea that is also compatible with the notion that in heterozygosity the dendritic defect is seen early, in the absence of DA cell body loss, and that in homozygosity, the extent of the dendritic deficit (76%) is disproportionate to the extent of cell body loss (42%). Considering that cerebellar granule cells have a defect in process maintenance, it is meaningful to consider subcellular pathways interfering with similar functions in formulating a unifying mechanism of wv gene action.

Telencephalic Terminal Projection Fields A 52% reduction in DA levels was first described in the cerebrum of four-to-six months old weaver homozygotes.51 It was then proposed that DA pathways of the substantia nigra and caudate nucleus would be altered to account for the depletion of one-half of cerebral DA, whether such a mechanism involved changes in storage, synthesis, or degeneration of pathways.

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Fig. 2.8. Immunocytochemistry with a monoclonal antibody against microtubule-associated protein 2 (MAP2), a molecule selectively localized in dendrites, to show dendritic extension into the substantia nigra pars reticulata in wild-type (a), heterozygous weaver (b) and homozygous weaver midbrain (c). At 20 days of age, heterozygotes have a normal number of dopamine cell bodies in pars compacta, but lack 60% of dendrites in pars reticulata. The dendritic deficit in weaver homozygotes is 76% and disproportionate to the 42% cell loss at that age. Magnification x100. Reprinted with permission from: Triarhou LC. Meth Neurosci 1992; 9:209-227. © Academic Press, Inc.

Fig. 2.9. Golgi-Cox preparations showing varicose dendrites in the substantia nigra pars reticulata of +/+ (a), wv/ + (b) and wv/wv (c) mutant mouse. Note the substantial reduction of varicose size and the elongation of the intervaricose segment in wv/wv. Magnification x150. Unpublished micrographs by the author.

Neuron losses in the mesencephalic DA cell groups lead to DA deficiency in several telencephalic areas to which A8, A9, and A10 normally supply DA innervation. DA levels in the caudate-putamen complex, which normally receives innervation from all three groups,31 are decreased by 75%.32 DA levels in the olfactory tubercle, which normally receives innervation mainly from area A10,31 are decreased by 27%.32 DA levels in the frontal cortex, which normally receives input from areas A9 and A10,31 are decreased by 77%.32 DA levels in the lateral septal nucleus, which normally receives innervation from A10,31 are decreased by 37%.38 TH enzyme activity in the weaver striatum is reduced by 43% at two weeks and by 77% at four months of age.32

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson's Disease

Fig. 2.10. Electron microscopic immunocytochemistry for tyrosine hydroxylase showing the formation of synapses between unlabeled axon terminals and immunopositive dendrites in the substantia nigra of normal (a), heterozygous weaver (b) and homozygous weaver (c). Magnification x22200 (a), x24200 (b), x22300 (c). Unpublished micrographs by L. Graham and L.C. Triarhou.

The striatal DA deficiency has been documented anatomically by catecholamine histofluorescence, by TH immunocytochemistry, and by DA immunocytochemistry.34,37,52,53 Using such techniques, one sees a substantial reduction of fiber innervation in the dorsal striatum but not in the nucleus accumbens. A reduction of DA fiber innervation has been also documented in the frontal cortex of weaver mutants by means of TH immunocytochemistry.54 The use of a quantitative autoradiography technique of [3H]DA uptake by striatal slices reveals an almost complete absence of DA axons in the dorsal aspect of the weaver neostriatum and an increasing density of DA innervation toward ventral areas; the remaining neostriatum is overlaid by diffuse silver grains, suggesting a deficient DA uptake and storage mechanism in residual DA fibers.53 Further, DA axons of the nucleus accumbens,

Biology and Pathology of the Weaver Mutant Mouse

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where DA content does not differ from normal, also display features of deficient uptake and storage of DA.53 In agreement with those findings, autoradiographic studies of mazindol binding (Fig. 2.11) and biochemical studies on the high-affinity uptake of [3H]DA by synaptosomal preparations of striatum have shown defective DA uptake in both the dorsal striatum and the nucleus accumbens.55,56 Both in normal and weaver striatum, junctional synaptic contacts formed by TH immunoreactive nerve terminals are predominantly of the symmetrical type. In addition to DA axon terminals being fewer in number in the weaver striatum, the remaining striatal DA afferents establish an inadequate synaptic connectivity on resident striatal neurons.34,52 It has been estimated that 90% of the contacts display a junctional membrane specialization in the striatum of the normal mouse (Fig. 2.12), whereas only 50% of the surviving axons establish junctional synaptic relationships with their target neurons in the weaver striatum at 20 days of age,52 that proportion further declining to one-fourth of normal by eight months.57 Moreover, there is an increase in the incidence of axosomatic contacts formed by incoming DA fibers, a phenomenon that may be indicative of synaptic immaturity, as such contacts are commonly seen during the early ontogeny of the nigrostriatal anlage. The DA denervation of the weaver striatum is associated with a slight increase in DA D2 receptor binding. One set of studies reports a 21% increase in DA D2 receptors in the dorsolateral striatal quadrant of six months old mutants, as measured by specific [3H]spiperone binding.58 An independent study59 reports 11% and 12% increases in DA D2 receptors in the dorsomedial and dorsolateral quadrants, respectively, on the basis of [3H]spiroperidol binding. DA D1 binding sites, as revealed by the specific antagonist [3H]SCH 23390, are reported to be either normal60 or reduced by 15-20%59 in the weaver caudate-putamen complex. DA receptor changes could be induced in the weaver neostriatum either as a result of DA denervation or be simply related to a geometrical rearrangement associated with the decrease in striatal volume, as the weaver brain is smaller than normal. Since DA D1 receptors are produced by striatal but not intrinsic nigral neurons,61 changes in DA D1 binding sites would reflect alterations of receptors belonging to intrinsic striatal neurons. On the other hand, since both striatal and nigral cells synthesize DA D2 receptors,62 it remains unknown to what extent the changes in DA D2 binding reflect alterations of receptors belonging to striatal cells or to nigrostriatal axons. Finally, one neuropathological observation in the weaver striatum that may prove to be of particular significance is that at birth, many young neurons undergo degeneration in the area located just beneath the subependymal plate.63 Quantitative studies further show that by the end of the first year of life, an estimated 22% of medium-sized striatal neurons have been lost.64

Cerebellar Phenotype of the Weaver Mutant Granule Cells

The cerebellum of homozygous weaver mutants is grossly atrophic.65,66 Despite normal mitotic activity,15 the majority of postmitotic granule cell precursors in the external germinal layer (EGL) do not emit axons, fail to migrate inward to the internal granular layer (IGL), and die massively at the interface of the EGL and the molecular layer during the first two weeks of postnatal life.66 The residual EGL persists in weaver mutants for

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson's Disease

Fig. 2.11. Autoradiography of [3H]mazindol binding to +/+ (upper) and wv/wv (lower) forebrain. Binding signal in the wild-type is dense throughout the entire striatum. In the weaver striatum, binding is severely reduced, especially in the dorsal striatal complex. Magnification x12. Reprinted with permission from: Stotz EH, Palacios JM, Landwehrmeyer B et al. J Neural Transm [Gen Sect] 1994; 97:51-64. © Springer-Verlag.

about a week longer than in control mice.14 The adult cerebellum of weaver mutants appears agranular with the exception of the lateralmost portions of the hemispheres and the paraflocculus, where granule cell loss is less pronounced.14 Heterozygous weaver mice (wv/+) are associated with a reduced rate of granule cell migration and an intermediate degree of dying granule cell count;15,66 arrested granule cells are seen at the interface of the molecular and Purkinje cell layers of adult animals. Evidence of cell death in the postmitotic zone of the EGL is already apparent at birth, suggesting that an earlier event in granule cell development, such as the exit of neuroblasts from the cell cycle or axonogenesis, might be affected by the wv gene.67 Delaying EGL

Biology and Pathology of the Weaver Mutant Mouse

25 Fig. 2.12. Ultrastructural appearance of tyrosine hydroxylase immunoreactive nerve terminals in the dorsolateral striatum of normal and heterozygous weaver mice forming axodendritic synapses with immunonegative targets. The synapse in the upper micrograph is symmetrical (type II), whereas the one in the lower micrograph is asymmetrical (type I), being characterized by the presence of subsynaptic dense particles. Magnification x59100 (upper), x75100 (lower). Original unpublished micrograph by the author (upper). Lower micrograph reprinted with permission from: Triarhou LC, Norton J, Ghetti B. J Neurocytol 1988; 17:221-232. ©Kluwer Academic Publishers

cells from exiting the cell cycle by hormonal manipulation68 reduces granule cell death in both wv/wv and wv/+, thus supporting the notion that granule cell degeneration occurs after cell division has been completed. In parallel with granule cell loss, the levels of several molecules associated with granule cells in cerebellum decline postnatally. Such molecules include the neuronal form of the c-src protooncogene-encoded protein-tyrosine kinase pp60c-src(+);69,70 the ATP-dependent glutamate uptake system in synaptic vesicles;71 sulfonylurea receptors associated with ATP-regulated K+ channels,72 saxitoxin-sensitive Na+ channels,73 and receptors for ω-conotoxin GVIA, an irreversible blocker of N-type Ca++ channel,74 which are all thought

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson's Disease

to be positioned presynaptically on parallel fibers; nerve growth factor;75 and a developmentally regulated, neural-specific protein recognized by monoclonal antibody OZ42 and possibly involved in the early stages of granule cell axonal elongation.76 Decreases in the granule cell-specific protein β2 chimerin and in calcicludine high-affinity binding sites, as well as in the Purkinje cell-specific protein Wnt-3, have been reported in the weaver cerebellum.77-79 The cellular localization of growth-associated phosphoprotein GAP-43, microtubule-associated protein MAP2 and βAPP mRNAs correlates with the corresponding anatomical deficits.80,81 On the other hand, the expression of D1.1 ganglioside, which is normally restricted to the EGL and later disappears as developing granule cells cease mitosis and begin migrating toward the IGL, is prolonged in the weaver cerebellar cortex and fails to disappear at later ages;82 D1.1 ganglioside could be involved in adhesive interactions that regulate the timing of granule cell migration.82 Cell agglutination assays of cerebellar microcultures in the presence of plant lectins to assess defects in cell surface moieties have shown that concanavalin A and wheat germ agglutinin [specific for binding carbohydrates α-methyl-D-mannoside and (D-GlcNAc)3, respectively] induce agglutination rates 20- and 10-fold higher in wv/wv cells than in +/+ cells;83 this suggests the postnatal persistence of embryonic cell surface elements on weaver cells. There has been a debate as to whether Bergmann radial glial fibers, which granule cells normally use as a guide in their migratory pathway, represent a target of the wv mutation. Bergmann glial fibers exhibit some atypical morphological features in the weaver cerebellum.66 Nonetheless, they are numerous, highly hypertrophic, and contain bundles of intermediate filaments characteristic of reactive astroglia.84,85 Based on the observation that some granule cells do succeed in migrating to the IGL and then degenerate, it was suggested that granule cells are likely to be the primary target of the wv mutation.85 The astroglial abnormalities could then secondarily result from the granule cell deficit through an impairment of specific neuronal-astrocytic associations.83

Purkinje Cells Purkinje cell counts in midsagittal sections of the weaver cerebellum have disclosed a reduction of Purkinje cell number, evident as early as on the fifth postnatal day, both in heterozygosity and in homozygosity, with little change occurring thereafter.15 The average reduction in vermal Purkinje cell number is 48% in wv/wv and 20% in wv/+.15 Similar counts have been confirmed independently in three-to-nine month old mice, in which the vermal Purkinje cell deficit is estimated at 50% and 21% in genotypes wv/wv and wv/+, respectively.86 The overall reduction in Purkinje cell number is 28% in wv/wv and 14% in wv/+,86 indicating that Purkinje cell loss is more severe in the vermis than in the cerebellar hemispheres. It remains unclear whether weaver Purkinje cells are generated normally during ontogeny and then degenerate, or whether there is a deficit in their initial formation. The remaining Purkinje cells of the wv/wv cerebellar cortex do not form a single layer and are found in ectopic positions.66,87 Purkinje cell arrangement in more than one row is also seen in the wv/+ cerebellar cortex, albeit to a lesser extent than in weaver homozygotes. The apical dendrites of Purkinje cells in the weaver cerebellum are oriented randomly, are often inversed, and occasionally display a “weeping willow” shape of arborization.87-89 Results from network analyses of Golgi preparations show that weaver Purkinje cells possess only 12% of the total number of segments present in a normal dendritic tree and that the mean distance between soma and tree periphery is reduced by 28%.90 Further, Purkinje

Biology and Pathology of the Weaver Mutant Mouse

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cell dendrites do not develop spiny branchlets, while their primary and secondary branches have an irregular rough surface owing to the presence of numerous spines.91 Aberrant dendritic trees of a lesser severity are seen in wv/+ Purkinje cells.87 At the ultrastructural level, one sees unattached Purkinje dendritic spines, often complete with postsynaptic specialization densities, and usually devoid of a presynaptic input from parallel fibers. Structurally, the dendritic spines are indistinguishable from those of normal cerebellar cortex with a presynaptic element.87,91-93 The synaptic remodeling of the neuronal circuitry in the weaver cerebellum involves formation of heterologous synapses on free spines by axon terminals of mossy fiber rosettes and by climbing fiber varicosities, which never reach such targets under normal circumstances, and the formation of attachment plate-like junctions by free postsynaptic sites of opposing Purkinje dendritic spines.91 One of the major afferents to Purkinje cells are the olivocerebellar climbing fibers. In normal development, there is a transient multiple innervation of Purkinje cells by climbing fibers, which later regresses, reaching a monoinnervation in adulthood. Parallel fiber input to Purkinje cells is thought to be involved in the regression of supernumerary climbing fiber collaterals.94 Since granule cells degenerate in the weaver cerebellum, the multiple innervation of Purkinje cells by climbing fiber collaterals persists in adult mutants at a mean ratio of 3.5 between the two neuronal elements.94-96

Hippocampal Morphology By studying the anatomy and histology of the hippocampus in one-to-two month old weaver homozygotes, Sekiguchi et al97 have documented pyramidal cell ectopia and alterations of the mossy fiber trajectories. The pyramidal cell layer of area CA3 frequently appears to be thicker than normal, with an apparent increase in cell-free spaces corresponding to neuropil; furthermore, the pyramidal cell layer seems to be subdivided into two or three layers. Small clusters of ectopic pyramidal cells are seen throughout the dorsoventral extent of the hippocampus, including the stratum radiatum and the stratum oriens. Mossy fibers of the weaver hippocampus appear to emerge diffusely from a region between the suprapyramidal and the infrapyramidal mossy fiber layers, and travel obliquely down through the pyramidal cell layer. In other cases, short discontinuous bundles of mossy fibers diverge from the infrapyramidal mossy fiber layer and invade the thickened pyramidal cell layer. The presence of ectopic neurons most likely reflects a disturbance of neuronal migration.97 The fact that the abnormalities of the weaver hippocampus appear only in area CA3, coupled with the finding that the number of ectopic cells is fairly small compared to the total number of hippocampal pyramidal cells, would indicate that the observed defect may not be consistent with a massive and general abnormality in neuronal migration, but rather, with a normal production of most of the pyramidal cells in the ventricular zone; nonetheless, a smaller number of late-generated pyramidal neurons, destined for the pyramidal cell layer of CA3, may terminate their migratory route prematurely.

Biology of Normal ↔ Mutant Cell Associations Once neuron types that are affected by a mutation are identified, it is important to determine how gene action brings about its effects on the involved lineage; appropriate confrontations between wild-type and mutant cells can be designed through the use of mosaic chimeras, tissue culture systems, and neural grafts.98 All of these approaches have been used insofar as the weaver cerebellum is concerned.

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson's Disease

Chimeras Studies employing normal ↔ heterozygous chimeras (+/+ ↔ wv/+) indicate that the wv gene intrinsically affects granule cells in causing them to be ectopic, since granule cell ectopia in the molecular layer is exclusively associated with the wv/+ component of the chimeric organism.99 Studies with intraspecific and interspecies normal ↔ homozygous chimeras lead to the conclusion that granule cell death is most likely due to an intrinsic action of the wv allele on granule cells, the genotype of Purkinje cells and Bergmann glia being apparently irrelevant to the survival of granule cells.100 Studies with chimeric mice, produced by fusing homozygous or heterozygous weaver embryos with wild-type embryos and using comparative measures of chimerism, indicate that the decrease in Purkinje cell number seen in the mutant cerebellum is a direct effect of the wv gene.17 However, the disorganized positioning of Purkinje cells and the aborted orientation of their dendritic arbor are due to extrinsic factors, thus being indirect effects of the mutation.17,99 Similar effects on Purkinje cell form are seen in experimentally induced agranularity of the cerebellum after X-ray irradiation,101 which can be viewed as a phenocopy of the weaver granuloprival cortex. The dissociation of genetic and epigenetic factors in Purkinje cell formation is compatible with the proposition that the development of different subcellular properties of Purkinje cells is controlled by different genes.88,91 Results from experiments with tetraparental normal ↔ weaver mouse chimeras indicate that only nigral DA cells of weaver lineage are lost, pointing to the idea that the wv gene acts intrinsically to nigral DA cells in causing their loss as well.102

Cell and Tissue Cultures Granule cell survival and neurite outgrowth were studied in tissue culture preparations.103-105 Neurons were obtained from seven days old cerebella of +/+, wv/+, and wv/wv animals. After six days in culture, more than 80% of homozygous weaver neurons die, whereas heterozygous neurons show an intermediate level of survival (about 40%). By prelabeling dividing neurons in vivo with [3H]thymidine six hours prior to their harvesting for culture, it was determined that weaver cells which die are the ones generated more recently. This parallels the in vivo situation, where weaver granule cells die soon after their genesis.15 Homozygous weaver neurons in culture possess abnormally shaped growth cones, neuritic shafts, and cell somata.104 Neurite outgrowth was followed in cerebellar cultures by measuring the mean total length of processes generated per neuron; after six days in vitro, that parameter is reduced by 88% in wv/wv and by 53% in wv/+, compared to +/+ cerebellum. Time-lapse microcinematography shows that the defect in homozygous weaver cells is not in the initiation of neurites, but in neurite maintenance and elongation.98,104 In vitro studies in microcultures of dissociated neuronal and astroglial cells have shown that +/+ granule cells can successfully attach to and migrate along both +/+ and wv/wv Bergmann glia, while wv/wv granule cells have an impaired association with either +/+ or wv/wv radial glia.106 Further, wv/wv granule cells were found to create a dysmorphic appearance in +/+ Golgi epithelial cells; such an effect could be due to an impaired trophic interaction between weaver granule cells and normal astrocytes, possibly mediated by astrotactin, a 100 kDa protein localized to cerebellar granule cells and reduced by more than 95% in the weaver cerebellum.107

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Cerebellar Grafts Solid grafts of gestational day 15 (E15) wild-type cerebellar tissue were transplanted into the cerebellomedullary cistern of weaver mutant hosts, between the uvula vermis and the dorsal surface of the brainstem, to study survival, growth, and synaptic properties within the microenvironment provided by the cerebrospinal fluid of the mutants.108-110 The grafts displayed a layered cellular organization reminiscent of the normal cerebellar cortex, with identifiable molecular, Purkinje cell and granule cell layers. Parallel fiber axon terminals presynaptic to Purkinje cell dendritic spines were identified in the molecular layer of the grafts. However, the number of parallel fibers was reduced compared to the normal cerebellar cortex, a phenomenon commonly seen in cerebellum in tissue culture or in cerebellar transplants into normal hosts. It was concluded that the weaver environment does not pose any apparent limitations beyond those inherent in the process of cerebellar growth and differentiation outside the normal anatomical context. In another study, pieces of E15 wild-type cerebellar tissue were transplanted into the cerebellum of four week old weaver mutants.111,112 Six weeks after transplantation, donor tissue developed a trilaminar organization, which contrasted with the granuloprival cerebellar cortex of the hosts. Evidence for the migration of implanted granule cells into the host cerebellum was presented. Positive immunoreactivity for synapsin I, a synaptic vesicle membrane-specific phosphoprotein, was taken as an index of synapse formation by donor granule and Purkinje cells, possibly on host cerebellar neurons. Cerebellar grafts from weaver into normal animals have also been performed.113 Mutant granule cell precursors were prepared from the external germinal layer of the cerebellum of postnatal day 5-6 weaver animals and implanted into the cerebellum of five day old wildtype hosts. Three to six days after transplantation, some wv/wv transplanted cells displayed features of differentiated granule cells, e.g., parallel fiber extension, migration through the molecular and Purkinje cell layers, and extension of dendrites. The conclusion of that study was that the wv gene acts nonautonomously in vivo, and that local cell interactions required for granule cell migration may induce early steps in neuronal differentiation.113 However, a number of surviving granule cells is observed in the weaver cerebellum anyway, and the holding view is that the wv gene acts intrinsically within mutant neurons in causing their cellular death.100

Behavioral Phenotype Homozygous weaver mutants (wv/wv) display a behavioral syndrome that includes locomotor, spatial orientation, and memory deficits. In particular, they manifest instability of gait, poor limb coordination, and resting and intention tremors.14 In addition, they display navigational deficits by not acquiring a forced swimming-induced immobility response, reduced activity in open-field tests, delayed spontaneous alternation, and a hind paw clasping reflex when lifted by the tail.114-118 Weaver homozygotes show a nonlinear course during the slow phase of horizontal optokinetic nystagmus evoked by a random dot pattern moving at a constant speed, and a reduction in nystagmic beat frequency for most of the stimulus velocities spectrum;119 since the cerebellum is known to participate in optokinetic nystagmus functions, the most likely explanation for such differences between weaver and normal mice could be attributed to the cerebellar lesions. Some of the behavioral deficits observed in weaver homozygotes, such as the exploration of a hole-board matrix, are not seen in mutants that have cerebellar lesions only, such

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson's Disease

as the nervous (nr/nr).120 On the other hand, forced swimming-induced immobility, which is defective in weaver mutants, is known to be altered by drugs that interfere with central dopaminergic activity.121 Furthermore, hind paw clasping responses can be induced in rodents after subjection to 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal DA projection.122 Pharmacological experiments indicate that direct-acting DA agonists, such as apomorphine and pergolide, induce an increase in the number of photocell crossings by weaver mice in a locomotor activity chamber, whereas amphetamine, a presynaptic DA releasing agent, leads to a decrease in activity.32 Taking into account all of these considerations, it is most likely that the weaver behavioral syndrome is probably underlain by both the cerebellar and nigrostriatal pathologies.118 In that sense, the weaver mouse is a model combining ataxia and Parkinsonism. It is difficult to precisely define the relative contribution of the cerebellar and the mesostriatal pathology to the behavioral phenotype. Granule cells and Purkinje cells have different roles in the local cerebellar circuitry, functioning respectively as cortical interneurons and as output projection neurons. It is thought that loss of up to 90% of Purkinje cells produces only minor effects on selected motor capabilities.123 Based on such reasoning, the extent of Purkinje cell loss observed in weaver mice would not suffice to induce ataxic signs on its own. Most of the other neurologically mutant mice, which are characterized by heredodegenerative ataxia, display severe Purkinje cell defects and often secondary granule cell loss.124,125 One might then speculate that the Purkinje cell deficit of the weaver mouse may conceivably not contribute too much to the neurological syndrome; the granule cell deficit may have a certain role in compromising movement control; and the DA deficiency may be responsible for the manifestation of specific catecholaminemediated behavioral signs. Heterozygous weaver mice (wv/+) do not present locomotor abnormalities or tremor; however, they intermittently manifest generalized tonic/clonic convulsions that are usually lethal.126,127 One logical explanation for such an activity could pertain to the dopaminergic dendrite deficit of the substantia nigra pars reticulata,44 as the substantia nigra has been implicated in the pathophysiology of experimental seizures.49,50 Homozygous weaver mutants seldom manifest seizures. One additional form of behavior not encountered in other cerebellar mutants is a remarkable leaping upward following agitation, preceded by spreading of the limbs, arching of the back, and hyperextension of the neck, and frequently exceeding a quarter of a meter into the air.14 Incapacitating seizure activity may be induced in wv/wv by attempts at radial arm-maze task testing.128 It has been theorized44 that the lower incidence of seizures in weaver homozygotes compared to heterozygotes could be associated with a compensatory effect of the loss of DA cell bodies in addition to dendrites, as bilateral lesions of the substantia nigra involving nigral efferent cells are anticonvulsant.

Structural Clues to the Weaver Riddle

The identification of the wv gene as a base substitution in the DNA encoding a K+ channel26 has provided an answer to the important question as to the molecular nature of the mutation; at the same time, it has opened new queries regarding the subcellular pathways leading to the associated neuroanatomical phenotype. By identifying the genetic basis of the mutation, the epicenter of the riddle is transposed from a DA neuron problem, a granule cell problem, a Purkinje cell problem and a hippocampal pyramidal neuron

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problem to a K+ channel problem, neuron loss being the final expression of a molecular cascade of events. Certain developmental and physiological possibilities relating the Girk2 mutation to the cerebellum and substantia nigra have been discoursed upon.129 Presumably, the mutated GIRK2 subunits lead to increased cell death as a result of basal nonselective channel opening ensuing from a wv mediated loss of K+ selectivity, whether channels are expressed either as wv GIRK2 homomultimers or as GIRK1-wv GIRK2 heteromultimers, and a concomitant loss of sensitivity to the heterotrimeric guanine nucleotide-binding protein (G protein) βγ dimers.130 Simple morphological observations through the microscope can be of a complementary nature to the information provided by the powerful molecular genetic techniques in one’s attempt at understanding how the wv mutation causes its diverse neuropathological aberrations. Structural and behavioral clues from the nigrostriatal, cerebellar, and hippocampal systems, such as those reviewed in the present Chapter, may provide useful pieces of information in the quest for a solution to the complex equation of weaver gene action. References 1. Triarhou LC, Low WC, Doucet G et al. The weaver mutant mouse as a model for intrastriatal grafting of fetal dopamine neurons. In: Hefti F, Weiner WJ, eds. Progress in Parkinson’s Disease Research - 2. Mount Kisco, NY: Futura Publishing, 1992:389-400. 2. Bankiewicz K, Mandel RJ, Sofroniew M. Trophism, transplantation, and animal models of Parkinson’s disease. Exp Neurol 1993; 124:140-149. 3. Brundin P, Duan W-M, Sauer H. Functional effects of mesencephalic dopamine neurons and adrenal chromaffin cells grafted to the rodent striatum. In: Dunnett SB, Björklund A, eds. Functional Neural Transplantation. New York: Raven Press, 1994:9-46. 4. Norman RM. Primary degeneration of the granular layer of the cerebellum: An unusual form of familial cerebellar atrophy occurring in early life. Brain 1940; 63:365-379. 5. Jervis GA. Early familial cerebellar degeneration (Report of three cases in one family). J Nerv Ment Dis 1950; 111:398-407. 6. Jervis GA. Concordant primary atrophy of the cerebellar granules in monozygotic twins. Acta Genet Med Gemellol 1954; 3:153-162. 7. Norman RM, Urich H. Cerebellar hypoplasia associated with systemic degeneration in early life. J Neurol Neurosurg Psychiat 1958; 21:159-166. 8. Hirano A, Dembitzer HM, Ghatak NR et al. On the relationship between human and experimental granule cell type cerebellar degeneration. J Neuropathol Exp Neurol 1973; 32:493-502. 9. Ferrer I, Sirvent J, Manresa JM et al. Primary degeneration of the granular layer of the cerebellum (Norman type): A Golgi study. Acta Neuropathol (Berl) 1987; 75:203-208. 10. Chou SM, Mizuno Y, Rothner AD. Congenital granuloprival hypoplasia of cerebellar and hippocampal cortex. J Child Neurol 1987; 2:279-286. 11. Mathews KD, Afifi AK, Hanson JW. Autosomal recessive cerebellar hypoplasia. J Child Neurol 1989; 4:189-193. 12. Wilhelmsen KC, Weeks DE, Nygaard TG et al. Genetic mapping of “Lubag” (X-linked dystoniaParkinsonism) in a Filipino kindred to the pericentromeric region of the X chromosome. Ann Neurol 1991; 29:124-131. 13. Lane PW. Mouse News Lett 1964; 30:32. 14. Sidman RL, Green MC, Appel SH. Catalog of the Neurological Mutants of the Mouse. Cambridge, MA: Harvard University Press, 1965:66-67. 15. Rezai Z, Yoon CH. Abnormal rate of granule cell migration in the cerebellum of ‘weaver’ mutant mice. Dev Biol 1972; 29:17-26. 16. Sotelo C. Mutant mice and the formation of cerebellar circuitry. Trends Neurosci 1980; 3:33-36. 17. Smeyne RJ, Goldowitz D. Purkinje cell loss is due to a direct action of the weaver gene in Purkinje cells: Evidence from chimeric mice. Dev Brain Res 1990; 52:211-218. 18. Lane PW, Sweet HO. Mouse News Lett. 1979; 60:46,50.

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19. Davisson MT, Roderick TH. Linkage map. In: Lyon MF, Searle AG, eds. Genetic Variants and Strains of the Laboratory Mouse. 2nd ed. Oxford–Stuttgart: Oxford University Press–Gustav Fischer Verlag, 1989:416-427. 20. Reeves RH, Gallahan D, O’Hara BF et al. Genetic mapping of Prm-1, Igl-1, Smst, Mtv-6, Sod-1 , and Ets-2 and localization of the Down syndrome region on mouse chromosome 16. Cytogenet Cell Genet 1987; 44:76-81. 21. Watson DK, McWilliams-Smith MJ, Kozak C. et al. Conserved chromosomal positions of dual domains of the ets protooncogene in cats, mice, and humans. Proc Natl Acad Sci USA 1986; 83:1792-1796. 22. Reeves RH, Robakis NK, Oster-Granite ML et al. Genetic linkage in the mouse of genes involved in Down syndrome and Alzheimer’s disease in man. Mol Brain Res 1987; 2:215-221. 23. Lovett M, Goldgaber D, Ashley P et al. The mouse homolog of the human amyloid β protein (AD-AP) gene is located on the distal end of mouse chromosome 16: Further extension of the homology between human chromosome 21 and mouse chromosome 16. Biochem Biophys Res Commun 1987; 144:1069-1075. 24. Reeves RH, Crowley MR, Lorenzon N et al. The mouse neurological mutant weaver maps within the region of chromosome 16 that is homologous to human chromosome 21. Genomics 1989; 5:522-526. 25. Mjaatvedt AE, Citron MP, Reeves RH. High-resolution mapping of D16Led-1, Gart, Gas-4, Cbr, Pcp-4 , and Erg on distal mouse chromosome 16. Genomics 1993; 17:382-386. 26. Patil N, Cox DR, Bhat D et al. A potassium channel mutation in weaver mice implicates membrane excitability in granule cell differentiation. Nature Genet 1995; 11:126-129. 27. Tsaur M-L, Menzel S, Lai F-P et al. Isolation of a cDNA clone encoding a KATP channel-like protein expressed in insulin-secreting cells, localization of the human gene to chromosome band 21q22.1, and linkage studies with NIDDM. Diabetes 1995; 44:592-596. 28. Mjaatvedt AE, Cabin DE, Cole SE et al. Assessment of a mutation in the H5 domain of Girk2 as a candidate for the weaver mutation. Genome Res 1995; 5:453-463. 29. Slesinger PA, Patil N, Liao J et al. Functional effects of the mouse weaver mutation on G proteingated inwardly rectifying K+ channels. Neuron 1996; 16:321-331. 30. Murtomaki S, Trenkner E, Wright JM et al. Increased proteolytic activity of the granule neurons may contribute to neuronal death in the weaver mouse cerebellum. Dev Biol 1995; 168:635-648. 31. Björklund A, Lindvall O. Dopamine-containing systems in the CNS. In: Björklund A, Hökfelt T, eds. Handbook of Chemical Neuroanatomy. Amsterdam: Elsevier, 1984; 2:55-122. 32. Schmidt MJ, Sawyer BD, Perry KW et al. Dopamine deficiency in the weaver mutant mouse. J Neurosci 1982; 2:376-380. 33. Triarhou LC, Low WC, Ghetti B. Transplantation of ventral mesencephalic anlagen to hosts with genetic nigrostriatal dopamine deficiency. Proc Natl Acad Sci USA 1986; 83:8789-8793. 34. Triarhou LC. Definition of the Mesostriatal Dopamine Deficit in the Weaver Mutant Mouse and Reconstruction of the Damaged Pathway by Means of Neural Transplantation. Ann Arbor, MI: University Microfilms International, 1987. 35. Triarhou LC, Norton J, Ghetti B. Mesencephalic dopamine cell deficit involves areas A8, A9 and A10 in weaver mutant mice. Exp Brain Res 1988; 70:256-265. 36. Ghetti B, Triarhou LC. Combined degeneration of cerebellar granule cells and of midbrain dopamine neurons in the weaver mutant mouse. In: Hefti F, Weiner WJ, eds. Progress in Parkinson’s Disease Research-2. Mt. Kisco, NY: Futura Publishing, 1992:369-382. 37. Roffler-Tarlov S, Graybiel AM. Weaver mutation has differential effects on the dopamine-containing innervation of the limbic and nonlimbic striatum. Nature (Lond) 1984; 307:62-66. 38. Roffler-Tarlov S, Graybiel AM. Expression of the weaver gene in dopamine-containing neural systems is dose-dependent and affects both striatal and nonstriatal regions. J Neurosci 1986; 6:3319-3330. 39. Ghetti B, Triarhou LC. Profile of mesencephalic dopamine neuron loss in weaver mutant mice during life-span. Soc Neurosci Abstr 1990; 16:1138. 40. Triarhou LC, Tsoukalas LH. Clues to the pathogenesis of dopaminergic neuron degeneration in the weaver mouse midbrain. Exp Neurol 1999; 159:615. 41. Gaspar P, Ben Jelloun N, Febvret A. Sparing of the dopaminergic neurons containing calbindinD28k and of the dopaminergic mesocortical projections in weaver mutant mice. Neuroscience 1994; 61:293-305.

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42. Bayer SA, Wills KV, Triarhou LC et al. Selective vulnerability of late-generated dopaminergic neurons of the substantia nigra in weaver mutant mice. Proc Natl Acad Sci USA 1995; 92:9137-9140. 43. Bayer SA, Wills KV, Triarhou LC et al. Systematic differences in time of dopaminergic neuron origin between normal mice and homozygous weaver mutants. Exp Brain Res 1995; 105:200-208. 44. Triarhou LC, Ghetti B. The dendritic dopamine projection of the substantia nigra: Phenotypic denominator of weaver gene action in hetero– and homozygosity. Brain Res 1989; 501:373-381. 45. Triarhou LC. Weaver gene expression in central nervous system. In: Conn PM, ed. Gene Expression in Neural Tissues. San Diego: Academic Press, 1992:209-227. 46. Triarhou LC, Ghetti B. Further characterization of the dopaminergic dendrite deficit in substantia nigra pars reticulata of heterozygous and homozygous weaver mutant mice: Golgi, MAP2 and synaptic connectivity studies. Soc Neurosci Abstr 1991; 17:159. 47. Ramón y Cajal S. Histologie du système nerveux de l’homme et des vertébrés, tome II. Paris: Maloine, 1911:275-278. 48. Björklund A, Lindvall O. Dopamine in dendrites of substantia nigra neurons: Suggestions for a role in dendritic terminals. Brain Res 1975; 83:531-537. 49. Lindvall O, Ingvar M, Gage FH. Short term status epilepticus in rats causes specific behavioral impairments related to substantia nigra nectosis. Exp Brain Res 1986; 64:143-148. 50. La Grutta V, Sabatino M. Substantia nigra-mediated anticonvulsant action: A possible role of a dopaminergic component. Brain Res 1990; 515:87-93. 51. Lane JD, Nadi NS, McBride WJ et al. Contents of serotonin, norepinephrine and dopamine in the cerebrum of the ‘staggerer’, ‘weaver’ and ‘nervous’ neurologically mutant mice. J Neurochem 1977; 29:349-350. 52. Triarhou LC, Norton J, Ghetti B. Synaptic connectivity of tyrosine hydroxylase immunoreactive nerve terminals in the striatum of normal, heterozygous and homozygous weaver mutant mice. J Neurocytol 1988; 17:221-232. 53. Doucet G, Brundin P, Seth S et al. Degeneration and graft-induced restoration of dopamine innervation in the weaver mouse neostriatum: A quantitative radioautographic study of [3H]dopamine uptake. Exp Brain Res 1989; 77:552-568. 54. Triarhou LC, Low WC, Ghetti B. Layer-specific innervation of the dopamine-deficient frontal cortex in weaver mutant mice by grafted mesencephalic dopaminergic neurons. Cell Tissue Res 1988; 254:11-15. 55. Triarhou LC, Stotz EH, Low WC et al. Studies on the striatal dopamine uptake system of weaver mutant mice and effects of ventral mesencephalic grafts. Neurochem Res 1994; 19:1349-1358. 56. Roffler-Tarlov S, Pugatch D, Graybiel AM. Patterns of cell and fiber vulnerability in the mesostriatal system of the mutant mouse weaver. II. High affinity uptake sites for dopamine. J Neurosci 1990; 10:734-740. 57. Triarhou LC, Low WC, Norton J et al. Reinstatement of synaptic connectivity in the striatum of weaver mutant mice following transplantation of ventral mesencephalic anlagen. J Neurocytol 1988; 17:233-243. 58. Kaseda Y, Ghetti B, Low WC et al. Age-related changes in striatal dopamine D2 receptor binding in weaver mice and effects of ventral mesencephalic grafts. Exp Brain Res 1990; 83:1-8. 59. Pullara JM, Marshall JF. Striatal dopamine innervation and receptor density: Regional effects of the weaver mutation. Brain Res 1989; 480:225-233. 60. Ohta K, Graybiel AM, Roffler-Tarlov S. Dopamine D1 binding sites in the striatum of the mutant mouse weaver. Neuroscience 1989; 28:69-82. 61. Mengod G, Vilaró MT, Niznik HB et al. Visualization of a dopamine D1 receptor mRNA in human and rat brain. Mol Brain Res 1991; 10:185-191. 62. Mengod G, Martinez-Mir MI, Vilaró MT et al. Localization of the mRNA for dopamine D2 receptor in the rat brain by in situ hybridization histochemistry. Proc Natl Acad Sci USA 1989; 86:8560-8564. 63. Ghetti B, Triarhou LC. Nigrostriatal aberrations induced by weaver gene are present at birth. Soc Neurosci Abstr 1992; 18:156. 64. Bayer SA, Triarhou LC, Thomas JD et al. Correlated quantitative studies of the neostriatum, nucleus accumbens, substantia nigra, and ventral tegmental area in normal and weaver mutant mice. J Neurosci 1994; 14:6901-6910.

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65. Sidman RL. Development of interneuronal connections in brains of mutant mice. In: Carlson FD, ed. Physiological and Biochemical Aspects of Nervous Integration. Englewood Cliffs, NJ: Prentice Hall, 1968:163-193. 66. Rakic P, Sidman RL. Sequence of developmental abnormalities leading to granule cell deficit in cerebellar cortex of weaver mutant mice. J Comp Neurol 1973; 152:103-132. 67. Smeyne RJ, Goldowitz D. Development and death of external granular layer cells in the weaver mouse cerebellum: A quantitative study. J Neurosci 1989; 9:1608-1620. 68. Smeyne RJ, Goldowitz D. Postnatal development of the wild-type and weaver cerebellum after embryonic administration of propylthiouracil (PTU). Dev Brain Res 1990; 54:282-286. 69. Wiestler OD, Trenkner E, Walter G. Progressive loss of neuronal src protein in postnatal weaver and staggerer cerebellum. Exp Cell Biol 1988; 56:190-195. 70. Brugge JS, Lustig A, Messer A. Changes in the pattern of expression of pp60c-src in cerebellar mutants of mice. J Neurosci Res 1987; 18:532-538. 71. Fischer-Bovenkerk C, Kish PE, Ueda T. ATP-dependent glutamate uptake into synaptic vesicles from cerebellar mutant mice. J Neurochem 1988; 51:1054-1059. 72. Mourre C, Widmann C, Lazdunski M. Sulfonylurea binding sites associated with ATP-regulated K+ channels in the central nervous system: Autoradiographic analysis of their distribution and ontogenesis, and of their localization in mutant mice cerebellum. Brain Res 1990; 519:29-43. 73. Mourre C, Widmann C, Lazdunski M. Saxitoxin-sensitive Na+ channels: Presynaptic localization in cerebellum and hippocampus of neurological mutant mice. Brain Res 1990; 533:196-202. 74. Maeda N, Wada K, Yuzaki M et al. Autoradiographic visualization of a calcium channel antagonist, [125I]ω-conotoxin GVIA, binding site in the brains of normal and cerebellar mutant mice (pcd and weaver). Brain Res 1989; 489:21-30. 75. Matsui K, Furukawa S, Shibasaki H et al. Reduction of nerve growth factor level in the brain of genetically ataxic mice (weaver, reeler). Fed Eur Biochem Soc Lett 1990; 276:78-80. 76. Pickford LB, Mayer DN, Bolin LM et al. Transiently expressed, neural-specific molecule associated with premigratory granule cells in postnatal mouse cerebellum. J Neurocytol 1989; 18:465-478. 77. Leung T, How BE, Manser E et al. Cerebellar β2-chimærin, a GTPase-activating protein for p21 Ras-related Rac is specifically expressed in granule cells and has a unique N-terminal SH2 domain. J Biol Chem 1994; 269:12888-12892. 78. Schweitz H, Heurteaux C, Bois P et al. Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons. Proc Natl Acad Sci USA 1994; 91:878-882. 79. Salinas PC, Copeland NG, Jenkins NA et al. Maintenance of Wnt-3 expression in Purkinje cells of the mouse cerebellum depends on interactions with granule cells. Development 1994; 120:1277-1286. 80. Triarhou LC, Solà C, Palacios JM et al. MAP2 and GAP-43 expression in normal and weaver mouse cerebellum: Correlative immunohistochemical and in situ hybridization studies. Arch Histol Cytol 1998; 61:233-242. 81. Solà C, Mengod G, Ghetti B et al. Regional distribution of the alternatively spliced isoforms of βAPP RNA transcript in the brain of normal, heterozygous and homozygous weaver mutant mice as revealed by in situ hybridization histochemistry. Mol Brain Res 1993; 17:340-346. 82. Johnstone SR, Stallcup WB. Altered expression of the D1.1 ganglioside in the cerebellum of the weaver mouse. J Neurochem 1988; 51:1655-1657. 83. Hatten ME, Liem RKH, Mason CA. Defects in specific associations between astroglia and neurons occur in microcultures of weaver mouse cerebellar cells. J Neurosci 1984; 4:1163-1172. 84. Bignami A, Dahl D. The development of Bergmann glia in mutant mice with cerebellar malformation: reeler, staggerer and weaver. Immunofluorescence study with antibodies to the glial fibrillary acidic protein. J Comp Neurol 1974; 155: 219-230. 85. Sotelo C, Changeux J-P. Bergmann fibers and granular cell migration in the cerebellum of homozygous weaver mutant mouse. Brain Res 1974; 77:484-491. 86. Blatt GJ, Eisenman LM. A qualitative and quantitative light microscopic study of the inferior olivary complex of normal, reeler, and weaver mutant mice. J Comp Neurol 1985; 232:117-128. 87. Rakic P, Sidman RL. Organization of cerebellar cortex secondary to deficit of granule cells in weaver mutant mice. J Comp Neurol 1973; 152:133-162. 88. Sotelo C. Dendritic abnormalities of Purkinje cells in the cerebellum of neurologic mutant mice (weaver and staggerer). Adv Neurol 1975; 12:335-351.

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89. Sotelo C. Purkinje cell ontogeny: Formation and maintenance of spines. Prog Brain Res 1978; 48:149-170. 90. Bradley P, Berry M. The Purkinje cell dendritic tree in mutant mouse cerebellum. A quantitative Golgi study of weaver and staggerer mice. Brain Res 1978; 142:135-141. 91. Sotelo C. Anatomical, physiological and biochemical studies of the cerebellum from mutant mice. II. Morphological study of cerebellar cortical neurons and circuits in the weaver mouse. Brain Res 1975; 94:19-44. 92. Hirano A, Dembitzer HM. Cerebellar alterations in the weaver mouse. J Cell Biol 1973; 56:478-486. 93. Hanna RB, Hirano A, Pappas GD. Membrane specializations of dendritic spines and glia in the weaver mouse cerebellum: A freeze-fracture study. J Cell Biol 1976; 68:403-410. 94. Crepel F, Mariani J. Multiple innervation of Purkinje cells by climbing fibers in the cerebellum of the weaver mutant mouse. J Neurobiol 1976; 7:579-582. 95. Puro DG, Woodward DJ. The climbing fiber system in the weaver mutant. Brain Res 1977; 129:141-146. 96. Mariani J. Extent of multiple innervation of Purkinje cells by climbing fibers in the olivocerebellar system of weaver, reeler, and staggerer mutant mice. J Neurobiol 1982; 13:119-126. 97. Sekiguchi M, Nowakowski RS, Nagato Y, Tanaka O, Guo H, Madoka M, Abe H. Morphological abnormalities in the hippocampus of the weaver mutant mouse. Brain Res 1995; 696:262-267 98. Sidman RL. Mutations affecting the central nervous system in the mouse. In: Schmitt FO, Bird SJ, Bloom FE, eds. Molecular Genetic Neuroscience. New York: Raven Press, 1982:389-400. 99. Goldowitz D, Mullen RJ. Granule cell as a site of gene action in the weaver mouse cerebellum: Evidence from heterozygous mutant chimeras. J Neurosci 1982; 2:1474-1485. 100. Goldowitz D. The weaver granuloprival phenotype is due to intrinsic action of the mutant locus in granule cells: Evidence from homozygous weaver chimeras. Neuron 1989; 2:1565-1575. 101. Altman J. Morphological development of the rat cerebellum and some of its mechanisms. Exp Brain Res [Suppl] 1982; 6:8-49. 102. Goldowitz D. Genetic studies search for answer to substantia nigra dopamine cell death. Parkinson Rep 1991; 12:6. 103. Willinger M, Margolis DM, Sidman RL. Neuronal differentiation in cultures of weaver mutant mouse cerebellum. J Supramol Struct Cell Biochem 1981; 17:79-86. 104. Willinger M, Margolis DM. Effect of the weaver (wv) mutation on cerebellar neuron differentiation. I. Qualitative observations of neuron behavior in culture. Dev Biol 1985; 107:156-172. 105. Willinger M, Margolis DM. Effect of the weaver (wv) mutation on cerebellar neuron differentiation. II. Quantitation of neuron behavior in culture. Dev Biol 1985; 107:173-179. 106. Hatten ME, Liem RKH, Mason CA. Weaver mouse cerebellar granule neurons fail to migrate on wild-type astroglial processes in vitro. J Neurosci 1986; 6:2676-2683. 107. Edmondson JC, Liem RKH, Kuster JE et al. Astrotactin: A novel neuronal cell surface antigen that mediates neuron-astroglial interactions in cerebellar microcultures. J Cell Biol 1988; 106:505-517. 108. Triarhou LC, Ghetti B, Low WC. Purkinje and granule cells survive in cerebellar grafts implanted into hosts with genetically-determined Purkinje or granule cell degeneration. Ann Neurol 1986; 20:138. 109. Low WC, Triarhou LC, Ghetti B. Cerebellar transplants into mutant mice with Purkinje and granule cell degeneration. Ann NY Acad Sci 1987; 495:740-744. 110. Triarhou LC, Low WC, Ghetti B. Transplantation of cerebellar anlagen to hosts with genetic cerebellocortical atrophy. Anat Embryol (Berl) 1987; 176:145-154. 111. Takayama H, Kohsaka S, Shinozaki T et al. Immunohistochemical studies on synapse formation by embryonic cerebellar tissue transplanted into the cerebellum of the weaver mutant mouse. Neurosci Lett 1987; 79:246-250. 112. Takayama H, Toya S, Shinozaki T et al. Possible synapse formation by embryonic cerebellar tissue grafted into the cerebellum of the weaver mutant mouse. Acta Neurochir [Suppl] 1988; 43:154-158. 113. Gao W-Q, Hatten ME. Neuronal differentiation rescued by implantation of weaver granule cell precursors into wild-type cerebellar cortex. Science 1993; 260:367-370. 114. Lalonde R. Acquired immobility response in weaver mutant mice. Exp Neurol 1986; 94:808-811. 115. Lalonde R. Motor abnormalities in weaver mutant mice. Exp Brain Res 1987; 65:479-481. 116. Lalonde R, Botez MI. Navigational deficits in weaver mutant mice. Brain Res 1986; 398:175-177. 117. Lalonde R. Delayed spontaneous alternation in weaver mutant mice. Brain Res 1986; 398:178-180.

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118. Triarhou LC, Ghetti B. Neuroanatomical substrate of behavioural impairment in weaver mutant mice. Exp Brain Res 1987; 68:434-435. 119. Grüsser-Cornehls U, Böhm P. Horizontal optokinetic ocular nystagmus in wild-type (B6CBA+/+) and weaver mutant mice. Exp Brain Res 1988; 72:29-36. 120. Lalonde R, Botez MI. Exploration of a hole-board matrix in nervous mutant mice. Brain Res 1985; 343:356-359. 121. Porsolt RD, Bertin A, Blavet N et al. Immobility induced by forced swimming in rats: Effects of agents which modify central catecholamine and serotonin activity. Eur J Pharmacol 1979; 57:201-210. 122. Whishaw IQ, Dunnett SB. Dopamine depletion, stimulation or blockade in the rat disrupts spatial navigation and locomotion dependent upon beacon or distal cues. Behav Brain Res 1985; 18:11-29. 123. Wetts R, Moran T, Oster-Granite M et al. Effect of Purkinje cell loss on complex motor behavior. Soc Neurosci Abstr 1985; 11:1037. 124. Sotelo C, Triller A. Fate of presynaptic afferents to Purkinje cells in the adult nervous mutant mouse: A model to study presynaptic stabilization. Brain Res 1979; 175:11-36. 125. Triarhou LC. Rate of neuronal fallout in a transsynaptic cerebellar model. Brain Res Bulletin 1998; 47:219-222. 126. Seyfried TN. Convulsive disorders. In: Foster HL, Small JD, Fox JG, eds. The Mouse in Biomedical Research. New York: Academic Press, 1982; 4:97-124. 127. Eisenberg B, Messer A. Tonic/clonic seizures in a mouse mutant carrying the weaver gene. Neurosci Lett 1989; 96:168-172. 128. Goldowitz D, Koch J. Performance of normal and neurological mutant mice on radial arm maze and active avoidance tasks. Behav Neural Biol 1986; 46:216-226. 129. Goldowitz D, Smeyne RJ. Tune into the weaver channel. Nature Genet 1995; 11:107-109. 130. Navarro B, Kennedy ME, Velimirovic B et al. Nonselective and Gby-insensitive weaver K+ channels. Science 1996; 272:1950-1953.

CHAPTER 3

Histochemical Properties of Intrastriatal Mesencephalic Grafts Introduction

T

he rationale behind neural transplantation studies using the weaver mouse model has been to replace degenerated neurons that are lost in the neurogenetic disease by intracerebrally grafted fetal mesencephalic cells.1 The cellular properties of dopaminergic grafts had been studied extensively in the neurotoxic models of dopamine (DA) deficiency induced by 6-hydroxydopamine (6-OHDA) and N-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP).2-5 A natural model of DA neuron degeneration such as the weaver is a valuable complement to the chemical models. The uniqueness of the weaver model lies in the fact that the mesostriatal DA depletion is progressive, taking place over several months, and incomplete, in contrast with the acute degeneration characteristic of the neurotoxic models. Thus, neural transplantation studies in the weaver can address specific aspects of graft integration with the chronic pathological nervous system.

Methodological Considerations It is generally thought that the optimal developmental stage for harvesting embryonic neurons for neural grafting is around the time of the cessation of mitotic divisions of the corresponding neuroblasts. Neurons of the mouse substantia nigra originate by embryonic day (E) 12.6 To prepare mesencephalic grafts of dissociated cell suspensions we have used E12 donor tissue. For grafting solid pieces of tissue we have used slightly elder animals (E14-E15); in the latter case, the embryonic brain yields firmer chunks of tissue, which can be grafted intact. Donor mesencephalic tissue can be implanted into the striatum of recipient animals in three different ways (Fig. 3.1):1,7 1. Solid grafts can be inserted stereotactically into the lateral cerebral ventricle, adjacent to the caudate-putamen complex of the host (Fig. 3.2). The stereotactic coordinates are: 0.8 mm mediolaterally, 0.0 mm anteroposteriorly with respect to bregma, and 2.0 mm dorsoventrally with respect to the dural surface.8 2. Solid grafts can be placed into a neocortical cavity, prepared in advance by aspiration of the cortex overlying the dorsal neostriatum, according to a “delayed-cavity” transplantation protocol.9 The aspiration cavity occupies a space bounded by the coordinates: 1.0-2.0 mm mediolaterally, 0.5-1.5 mm anteroposteriorly with respect to bregma, 0.0-1.0 mm dorsoventrally with respect to the dural surface.10

Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease, by Lazaros C. Triarhou. ©2001 Eurekah.com.

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

Fig. 3.1. Schematic drawing of the methods for graft placement into the striatum. Upper: Graft into the lateral cerebral ventricle. Middle: Graft into a preformed cortical cavity. Lower: Intrastriatal cell suspension. Abbreviations: G, graft; CP, caudate-putamen; FR, frontal cortex; LS, lateral septum; NA, nucleus accumbens; TU, olfactory tubercle. Reprinted with permission from: Triarhou LC, Low WC, Doucet G et al. 1992 © Futura Publishing Co., Inc.

3. Donor tissue can be made into a cell suspension by gentle enzymatic and mechanical dissociation (Fig. 3.3) and grafted by stereotactic surgery directly into the striatal parenchyma.11 The stereotactic coordinates are: 1.9 mm mediolaterally, 1.0 mm anteriorly with respect to bregma, and 2.7-3.0 mm dorsoventrally with respect to the dural surface (Fig. 3.4).12 In our studies we have used adult weaver mutant mice (two to four months old at the time of grafting) as recipients of fetal mesencephalic tissue. We have applied all of the aforementioned surgical approaches, i.e., solid intraventricular grafts,8 solid grafts into a neocortical cavity,10,13-17 and intrastriatal cell suspensions.12,18-24 The choice of a particular technique depends on the specific queries related to a particular experiment. Generally, cell suspension grafts yield better survival rates than solid grafts (i.e., more than 90% compared to about 50%).

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Fig. 3.2. Solid mesencephalic grafts into the lateral ventricle of weaver hosts, lying between the lateral septum and the caudate-putamen complex. DeMyer silver-impregnated section (left) for the demonstration of nerve fibers; tyrosine hydroxylase immunocytochemistry (right) showing immunopositive neurons and processes. Magnification x95 (left), x105 (right). Reprinted with permission from: Triarhou LC, Low WC, Ghetti B. Proc Natl Acad Sci USA 1986; 83:8789-8793.

Fig. 3.3. A portion of an embryonic cell suspension laid on a hemocytometer prior to grafting for a viability count, based on the Trypan blue (0.2%) exclusion principle. Unstained cells are viable; stained cells are nonviable. Magnification x110. Original unpublished micrograph by the author.

Expression of Catecholaminergic Neurotransmitter-Related Molecules and Quantitative Aspects of Dopaminergic Neuron Survival Histological evidence for the survival of transplanted DA neurons inside the weaver mutant’s brain has been obtained from experiments with both solid and cell suspension grafts. Immunopositivity for tyrosine hydroxylase (TH) and immunonegativity for DA

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

Fig. 3.4. A mesencephalic cell suspension graft placed into the weaver striatum (left), as seen with tyrosine hydroxylase immunocytochemistry three months after transplantation. Higher-power view of individual grafted neurons with dendritic and axonal processes in a solid transplant (right). Magnification x25 (left) x285 (right). Reprinted with permission from: Solà C, Mengod G, Low WC et al. Eur J Neurosci 1993; 5:1442-1454. © Blackwell Science Ltd. (left); Triarhou LC, Low WC, Norton J et al. J Neurocytol 1988; 17:233-243. © Kluwer Academic Publishers (right).

β-hydroxylase (DBH),8 or immunopositivity directly for DA,18 have been taken as evidence for the dopaminergic “signature” of grafted cells. Surviving transplants usually contain between 100 and 1200 DA neurons. Grafted DA neurons and the resulting fiber outgrowth are sustained over periods of time when the intrinsic nigrostriatal system of the weaver mutant has considerably declined. Observations on long-term weaver mice at nine months after transplantation suggest that, in spite of good survival of grafted DA neurons, graft-derived DA fiber outgrowth may be reduced in the affected striatum.18

Solid Grafts

In the case of intracerebroventricular grafts,8 donor tissue has been found within the lateral ventricle, attached to the caudate-putamen complex of the recipient animals (Fig. 3.2). The ependymal lining was not present at the levels of tissue adhesion. Individual pieces of grafted tissue reached dimensions of 0.2-0.9 mm anteroposteriorly, 0.2-0.6 mm mediolaterally, and 0.3-1.1 mm dorsoventrally. Nerve cell bodies were identified in preparations stained with Nissl stain or hematoxylin–eosin. A dense network of neuronal processes was seen in sections of tissue impregnated with DeMyer silver. Immunocytochemical labeling with antiserum against TH revealed the presence of positively immunoreactive neurons inside the grafts. The same grafts did not immunoreact with DBH antiserum. The fact that grafted nerve cells express TH and not DBH immunoreactivity indicates that these neurons are dopaminergic. Nonetheless, due to the physical proximity of the A8 (retrorubral), A9 (nigral), or A10 (ventral tegmental) anatomical fields25 in the primordial mesencephalon, it is difficult to determine the exact field of origin of the transplanted DA neurons.

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In the immunocytochemical specimens, there was a clustering of TH-immunoreactive cell bodies and islands of TH fibers. The diameters of TH-immunoreactive somata ranged from 8-18 µm, with the majority of neurons being in the 10-16 µm range. In the weaver mouse experiments, grafts contained an estimated 130-830 TH-positive neurons. In experiments conducted in rats, the number of surviving grafted DA neurons has ranged from 100-4000 cells.26-28 It has been estimated in the 6-OHDA rat model that 320 grafted DA cells may be sufficient for exerting a behavioral effect.26 Considering that the mesencephalic DA system of the mouse contains 6000-8000 cells,29,30 whereas that of the rat contains 30000-40000 cells,31 it would be meaningful to expect that the number of surviving DA neurons in the mouse model would also exert detectable behavioral effects (cf. Chapter 6). In studies with mesencephalic grafts into a preformed surgical cavity and survival times of four to five months after transplantation,10,13,14 donor tissue was situated in the forebrain cavity, lying over the head of the caudate-putamen complex and contained an estimated 100-1000 TH immunoreactive neurons. Grafted TH immunoreactive neurons reached 10-20 µm in diameter. Dendrites and axons emanated from the neuronal somata, making the neuropil of the grafts appear rich with a dense network of TH-immunolabeled fibers. When examined under the electron microscope, TH-immunoreactive cell bodies were readily recognized in the transplants; electron-dense reaction product was present in their cytoplasm but absent from the nucleus. The somata of grafted TH immunoreactive neurons received synaptic input from unlabeled axon terminals. TH-immunolabeled dendrites in the grafts were also invested by unlabeled axon terminals, which contained spheroid or flattened synaptic vesicles. Such axodendritic synapses were predominantly asymmetrical, characterized by the presence of a thick postsynaptic membrane density.

Cell Suspension Grafts Compared to the implantation of solid DA-containing grafts, the use of mesencephalic cell suspensions for grafting purposes is generally associated with better intraparenchymal graft survival, different dynamics of neuronal growth and spatial interaction with the host parenchyma, less surgical trauma, and the possibility for precise direction of graft placement into deep host brain sites.11,32 The potential of cell suspension grafts for better survival is one of the reasons why that approach was adopted in the use of human fetal mesencephalic tissue for transplantation into immunosuppressed laboratory rats33,34 and in clinical trials in the striatum of patients with Parkinson’s disease.35,36 When dissociated cell suspensions, prepared from the ventral midbrain of normal mouse fetuses (Fig. 3.3), were stereotactically implanted into the neostriatum of weaver mice,12 grafts were found to contain an estimated 100-700 TH immunoreactive neurons (Fig. 3.4). In electron microscopy, the reaction product in TH immunolabeled somata of the graft appeared granular and was localized in a dispersed manner within the cytoplasm (Fig. 3.5). TH immunoreactive cell somata were found to receive synaptic input from unlabeled axon terminals. In certain instances they were surrounded by electron-lucent astroglial sheaths or by thin myelinated axons. The cell nucleus was free of immunoreactive precipitate. Immunocytochemical labeling with monoclonal antibodies against a DA-glutaraldehyde-ovalbumin complex revealed the presence of a large number of DA-positive cell bodies in cell suspension grafts at nine months after transplantation.18 On postnatal day 90 the substantia nigra of wild-type mice contains a total of 3400 DA cells in both sides; the weaver substantia nigra contains 70% fewer DA neurons, i.e.,

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

Fig. 3.5. Ultrastructural appearance of tyrosine hydroxylase immunoreactive neuronal somata from a cell suspension graft. Magnification x6500. Reprinted with permission from: Triarhou LC, Brundin P, Doucet G et al. Exp Brain Res 1990; 79:3-17. © Springer-Verlag.

approximately 1000 cells on both sides.30 The dopaminergic supply of the dorsolateral striatum is provided by neurons of the substantia nigra proper.31 Over 99% of the striatal DA input from the substantia nigra is ipsilateral.37 One may therefore estimate that each side of the normal mouse striatum derives its innervation from approximately 1700 nigral DA cells. In the weaver condition that innervation would originate from the 500 DA cells that remain in the ipsilateral substantia nigra. Nonetheless, although present, those wv/wv neurons provide a biochemically deficient and synaptically inadequate striatal innervation.

Histochemical Properties of Intrastriatal Mesencephalic Grafts

43

The true number of DA cells in the grafts studied could presumably be higher, as the antibody may not penetrate through the entire thickness of a tissue section in the absence of Triton X-100.38 This implies that the innervation derived from the graft is from a cell population which is probably more than one-half of that normally arriving from the weaver substantia nigra. However, two essential points worthy of consideration are that 1. grafted DA cells are located within the target area and can therefore provide a more concentrated innervation to the dorsal neostriatum as compared to the host mesostriatal projection and 2. the wild-type genotype of donor DA cells could be responsible for a physiologically more efficient innervation of striatal domains when compared to the remnant innervation provided by the genotypically mutant intrinsic DA neurons. As a matter of fact, by applying a quantitative technique of [3H]DA uptake autoradiography to the weaver condition, Doucet et al18 found that, while the intrinsic weaver DA axons have a strong defect in DA uptake and storage mechanisms, graft-derived DA fibers display normal features of DA uptake and storage (cf. Chapter 5). A DA recurrent collateral axonal plexus is not found in normal substantia nigra pars reticulata.39 Inside solid grafts of fetal mesencephalon into rats with 6-OHDA lesions of the mesostriatal DA projection, Bolam et al40 found that approximately 50% of the TH immunoreactive boutons were in contact with TH immunoreactive dendrites or somata, an observation markedly differing from the intact substantia nigra. As these investigators noted, there is a possibility that some of the TH terminals may belong to host catecholaminergic axons; however, the most likely explanation is that such connections between pre- and postsynaptic DA neuronal elements represent an anomaly in the local establishment of synapses within the graft. Our own observations in the weaver model did not reveal recurrent DA axon collaterals synapsing on TH immunoreactive dendrites in the recipient striatum or within cell suspension grafts.12 In that respect, the absence of a DA recurrent axonal plexus mimics the situation found normally in substantia nigra pars reticulata. The dissimilarity of our observation to that of Bolam et al40 might be related to different techniques of transplantation or to the biology of the recipient organisms or to a combination of these parameters.

Comparative Survival of Dopaminergic Neurons in Grafts Placed in Weaver and in 6-OHDA Lesion Hosts Heterozygous weaver mice (wv/+) have normal numbers of dopaminergic neurons in the midbrain and a normal innervation of the striatum by DA axon terminals.30,41 However, the substantia nigra of weaver heterozygotes contains 60% fewer DA dendrites than that of wild-type mice.42,43 In one study we transplanted two types of grafts (+/+ and wv/+) unilaterally into the right striatum of two types of hosts, i.e., in wv/wv mutants, which have the bilateral genetically-induced lesions of the mesostriatal DA pathway, and in +/+ mice with unilateral right lesions induced by 6-OHDA.22 Among the aims of the study were to determine whether DA cells survive in comparable numbers in grafts of +/+ and wv/+ origin, and whether the host type plays any role in determining surviving graft cell number. Wild-type mice received a stereotactic unilateral injection of 6-OHDA (2 µl of a solution containing 4 µg/µl 6-OHDA and 0.2 µg/µl ascorbic acid) into the right substantia nigra three weeks prior to grafting. The stereotactic coordinates had been determined

44

Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

in advance by using Cresyl violet injection in a test animal; they were 4.0 mm posterior to bregma, 1.5 mm lateral to the midline, and 5.0 mm dorsoventrally with respect to the dural surface. Pairs of wild-type mice (to yield +/+ embryos) or pairs of wv/wv females and +/+ males (to yield wv/+ embryos) were placed in the same cage for a 48 hour mating period. Ventral mesencephalic primordia were dissected out of +/+ and wv/+ fetuses and used in the transplantation surgery sessions. Average cell viability, determined in 5 µl aliquots using a hemocytometer and Trypan blue exclusion, was about 80% in the more dissociated portions of the suspensions. Counts of TH-immunoreactive cells were obtained in coronal sections of the brains that contained the matured grafts. TH-immunopositive cells were seen in the transplanted side of the striatum of both the 6-OHDA and wv/wv recipient mice, and grafts of both +/+ and wv/+ origin exhibited a robust development with numerous TH-immunoreactive cell bodies. In some cases, TH immunopositive cells were also found in areas outside the caudate-putamen, such as the overlying neocortex and corpus callosum, lateral ventricle and globus pallidus. To compare the number of surviving DA cells in +/+ and wv/+ grafts and to determine whether host caudate-putamen factors play any role in determining surviving graft cell number, a two-way general linear model analysis of variance was carried out, with host type and graft genotype as the fixed main effects.44 The results showed a significant host type effect and nonsignificant graft genotype and host x graft interaction effects. This indicates that the host types differ in influencing graft survival, regardless of the genotype of donor tissue. The mean TH cell numbers in the two types of hosts (averaged over graft genotypes) were 764 TH cells in the case of 6-OHDA hosts and 95 TH cells in the case of wv/wv hosts. On the other hand, +/+ and wv/+ grafts did not differ significantly from each other with respect to surviving TH cell number; neuronal count means, averaged over host types, were 215 cells for +/+ grafts and 337 for wv/+ grafts. The results of these experiments showed that DA cells obtained from +/+ or wv/+ fetal midbrain survive in comparable numbers after intrastriatal transplantation into either type of host.22 Such a finding is in line with cell count studies in the midbrain of adult +/+ and wv/+ mice, which have shown no virtual difference in DA neuron number between these two genotypes in any of the mesencephalic DA cell groups.30 What is of particular interest is that weaver homozygous hosts seem to offer a less good environment for DA cell survival than wild-type hosts with 6-OHDA lesions. This was the first study that compared the effects of the two host environments on the graft in the same inbred mouse stock. In our previous studies, in which weaver homozygotes had been used as a host model of mesostriatal DA deficiency,8,10,12,13-21 grafted neurons had survived in satisfactory numbers to induce behavioral improvements. The findings reviewed in this segment indicated that, while the wv/wv brain permits the survival and histotypic differentiation of donor DA neurons, there may be microenvironmental factor(s) putatively influencing the final number of surviving DA cells in the grafts. To adequately test this hypothesis further, aliquots of cells from the same cell suspension should be transplanted into both wv/wv and +/+ hosts at the same time. In the reported study, the cell suspensions used for transplantation into the two groups of hosts had been adjusted such that each animal received 120,000 viable cells. One should keep in mind that the Trypan blue exclusion method, used to determine viability, effectively assesses membrane integrity; it is quite possible that many cells that exclude Trypan blue are undergoing developmental cell death which may be the case for cell suspensions with

Histochemical Properties of Intrastriatal Mesencephalic Grafts

45

lower viability. Still, the observed 80% cell viability in the suspensions may not be sufficient to justify a 4:1 ratio in the average DA cell survival in caudate-putamen between the two types of hosts.22 The suggestion of a differential effect of wild-type and mutant host environments on the graft needs to be looked at carefully. Along a similar line of reasoning, it should be mentioned that at birth, many young neurons undergo dark degeneration in the wv/wv striatum just beneath the subependymal plate,45 and by one year of age, an estimated 22% of medium-sized striatal neurons have been lost.46 The possibility that the disease process may exert an influence on graft survival has been repeatedly brought up in the clinical studies on DA neuron grafting in patients with ongoing Parkinson’s disease.47-49 These considerations add an extra value to the weaver mutant as a natural disease model of nigrostriatal DA degeneration in studying host-graft interactions.

Expression of Neuropeptides and Structural Proteins Cholecystokinin and Neurotensin Following transplantation to the mutant striatum, embryonic mesencephalic neurons retain, at least in qualitative terms, features of their normal histochemical phenotypes. Grafts have been tested for immunopositivity to cholecystokinin and neurotensin,15 two endogenous neuropeptides normally colocalized with DA in neurons of the ventral midbrain. Normally, about 40% of DA cell bodies in ventral tegmental area and virtually all DA cell bodies in pars compacta of the anterior substantia nigra and in pars lateralis contain cholecystokinin-like-immunoreactivity.50 Neurotensin-like immunoreactivity is seen in a small population of DA cell bodies in the ventral tegmental area and retrorubral nucleus.51 These neuropeptides are thought to modulate DA-mediated neurotransmission.52 Grafts contained relatively large numbers of cholecystokinin-immunoreactive cell bodies, about 400 cells on average.15 By analyzing adjacent serial sections alternately labeled with TH and cholecystokinin antisera, cholecystokinin immunoreactivity was found to be expressed either in combination with TH immunoreactivity or alone. Neurotensin immunoreactive cells were also seen, but their number was very small.

Calcium-Binding Protein A subset of DA neurons in substantia nigra, ventral tegmental area and retrorubral nucleus are immunoreactive for 28 kDa calcium-binding protein (CaBP). 53,54 DA/CaBP-containing neurons of the substantia nigra are believed to innervate the striatal compartment referred to as “matrix” (defined on the basis of acetylcholinesterase histochemical positivity55) that contains CaBP-immunoreactive neurons as well, whereas striatal “patches” (characterized by Met-enkephalin immunopositivity and µ-opiate receptor binding56), which are devoid of CaBP-immunoreactive neurons, are also avoided by nigrostriatal DA/CaBP fibers.53 Calcium-binding proteins in the nervous system are thought to provide a finer level of regulation for calcium-dependent intracellular processes.57 CaBP-immunoreactive neurons were abundant in mesencephalic grafts.15 A proportion of CaBP immunopositive neurons also contained TH, as gathered from the analysis of serial sections. CaBP immunoreactivity was localized in several telencephalic areas of the host, including cerebral cortex, lateral septum, nucleus accumbens and caudate-putamen complex, as previously reported in the rat.57,58

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

Amyloid Beta-Protein Precursor

Using in situ hybridization histochemistry with [32P]oligonucleotide probes, we studied the cellular localization of RNA transcripts for amyloid β-protein precursor (βAPP) in the mesostriatal system of normal and weaver mutant mice and in ventral mesencephalic cell suspensions transplanted to the weaver striatum.59,60 The reasoning was that the properties of cells that have matured after transplantation inside a pathological system need to be compared with those of cells that have grown in the normal organism and in the recipient. The alternative splicing of the βAPP transcript was studied by using oligonucleotide probes recognizing the four different βAPP mRNAs. The DNA oligonucleotide probe sequences were complementary to mouse βAPP mRNAs. Immunocytochemical labeling for TH was used to verify DA neuron survival in the grafts; further, anti-βAPP antibodies were used to detect the respective antigens in grafted tissue. The βAPP molecule is a transmembrane protein possibly mediating cell-cell adhesion and is abundantly expressed in many brain areas.61-64 The gene for βAPP has been localized on human chromosome 21 and on mouse chromosome 16, in the vicinity of the weaver locus.65,66 The βAPP includes in its sequence the β-amyloid peptide, the main component of amyloid deposits found in dementia of Alzheimer type, in late Down syndrome, and in the aged human brain.67 Four species of βAPP mRNAs have been described in human,61,68-73 rat,74,75 and mouse brain,76,77 which are thought to result from the alternative splicing of the βAPP pre-mRNA transcript: βAPP695, βAPP714, βAPP751 and βAPP770 mRNAs, where numbers correspond to the number of amino acids encoded by the respective open-reading frames. The distribution of βAPP695, βAPP714 and βAPP751 mRNAs in the mouse brain is wide and comparable; on the other hand, the distribution of βAPP770 mRNA is more limited than that of the other three isoforms and has a different pattern.78 Transcripts encoding isoforms βAPP695, βAPP714 and βAPP751 were present in normal substantia nigra and progressively reduced in weaver;59 such a reduction was correlated with DA neuron loss. High levels of hybridization signal were found in the grafts for RNA transcripts encoding isoforms βAPP695, βAPP714 and βAPP751 (Fig. 3.6).60 The βAPP770 species–normally seen in striatum and not substantia nigra–was not expressed in the grafts, but it was present in the recipient striatum, thus giving an image of a “negative” graft inside a “positive” striatum.21,60 In the autoradiographic emulsions, the high hybridization signal corresponded to the area of graft location. By using antibodies for immunocytochemistry, βAPP immunoreactivity was mainly found in cell bodies in the grafts. Quantitative results were obtained on the density of hybridization signal for the various mRNAs studied. All of the mRNAs (isoforms βAPP695, βAPP714, βAPP751) that are important, in the sense that they constitute molecules normally expressed by nigral neurons, were very close and did not show any statistically significant differences between the cell suspensions and the normal substantia nigra. Messenger RNAs for βAPP695, βAPP714 and βAPP751 are localized in the substantia nigra pars compacta and ventral tegmental area of normal mouse midbrain. Signal levels are much reduced in homozygous weaver mice; such a reduction is correlated with the previously documented loss of mesencephalic DA neurons. The appearance of RNA message for βAPP in ventral mesencephalic grafts suggests that grafted cells mature in a similar manner to those in the normal substantia nigra in spite of their development in a foreign environment. Since the grafts contain large numbers of TH-positive cells, it is likely that the transcripts studied are expressed by DA cells, although one cannot exclude the possibility that other cells of mesencephalic origin in the grafts may also express these transcripts.

Histochemical Properties of Intrastriatal Mesencephalic Grafts

47

Fig. 3.6. Autoradiographic films showing detection by in situ hybridization histochemistry of mRNA for βAPP695 in a mesencephalic cell suspension graft. Note the high level of hybridization signal in the graft. Magnification x15. Reprinted with permission from: Solà C, Mengod G, Low WC et al. Eur J Neurosci 1993; 5:1442-1454. ©Blackwell Science Ltd.

Several possible roles have been postulated for βAPP in the central nervous system, including growth-promoting action,79 neurotrophic effects either as a sprouting promoter80,81 or inhibitor,82,83 and surface receptors mediating cell–cell and cell–matrix recognition.68,74,84-86 In lesion studies, it was found that in specific anatomical systems βAPP expression is modified subsequent to axotomy or denervation.87-90 Following axotomy of the facial and hypoglossal motor nerves in the rat, mRNAs coding for the different βAPP forms are increased in motor neurons of the corresponding cranial nuclei; after regeneration, βAPP mRNA levels return to normal.87 Similarly, βAPP695, βAPP751 and βAPP770 mRNA levels are increased in sensory neurons of the rat dorsal root ganglia after crushing of the sciatic nerve;88 after reinnervation, βAPP695 mRNA levels return to normal, but Kunitz protease inhibitor-containing βAPP mRNAs remain elevated. On the other hand, anterior horn motor neurons express reduced levels of βAPP695 following axotomy.88 An increased amount of βAPPs is seen in the rat cerebral cortex following N-methyl-D-aspartate lesions of the nucleus basalis of Meynert, which supplies a major cholinergic innervation to the cortex, indicating that denervation from an afferent input may transsynaptically regulate the expression of βAPP in specific brain areas.89 It is worth noting the lack of response of the Kunitz domain-containing βAPP mRNA isoforms in the areas with neuronal loss in the weaver, i.e., the substantia nigra and ventral tegmental area, because an increased expression of these isoforms has repeatedly been described in Alzheimer’s disease brain73,91-93 and in the brain of experimental animals in response to different kinds of experimentally induced neuronal injury.88,90,94 Around the areas of cellular damage after intracerebroventricular injection of kainic acid into rats, an increase was detected in the levels of Kunitz protease inhibitor-containing βAPP mRNAs in association with reactive astrogliosis.90 Electron microscopic studies in substantia nigra and ventral tegmental area of 20-45-day-old weaver mutant mice have disclosed the presence

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

of neuronal debris surrounded by astroglial processes.95 Using glial fibrillary acidic protein immunocytochemistry, one detects a mild reaction early on, but in elder mice the difference in glial fibrillary acidic protein immunoreactivity between wild-type and weaver mutants is not strong. The lack of persistence of a pronounced glial reaction in areas where cells are dying could be one reason why there is no increased expression of βAPP mRNAs. On the other hand, loss of the nigrostriatal DA input to the striatum does not appear to affect βAPP expression in that denervated area of the weaver brain, either as an increase in the expression of βAPP770 or a novel expression of the other three forms following denervation. It is possible that the chronic progressive evolution of the weaver lesion may account for such a lack of changes. In a separate report, PC12 cells transfected with retroviral recombinants expressing the carboxyl-terminal 104 amino acids of βAPP were transplanted into the brain of newborn mice.96 Four months after transplantation, recipient mice exhibited a significant cortical atrophy; some mice showed Alz-50 immunoreactivity in the somatodendritic domain of neurons in the cortex surrounding the transplants, and a disorganization of the neuropil in the CA2 and CA3 regions of the hippocampus, suggesting that the C-terminal fragment of βAPP may cause specific neuropathological changes and neurodegeneration in vivo. References 1. Triarhou LC, Low WC, Doucet G et al. The weaver mutant mouse as a model for intrastriatal grafting of fetal dopamine neurons. In: Hefti F, Weiner WJ (eds). Progress in Parkinson’s Disease Research–2. Mt. Kisco, New York: Futura Publishing Company, 1992:383-393. 2. Björklund A, Lindvall O, Isacson O et al. Mechanisms of action of intracerebral neural implants: Studies on nigral and striatal grafts to the lesioned striatum. Trends Neurosci 1987; 10:509-516. 3. Brundin P, Björklund A. Survival, growth and function of dopaminergic neurons grafted to the brain. Prog Brain Res 1987; 71:293-308. 4. Redmond DE, Sladek JR Jr, Roth RH et al. Fetal neuronal grafts in monkeys given methylphenyltetrahydropyridine. Lancet 1986; i:1125-1127. 5. Bohn MC, Cupit L, Marciano F et al. Adrenal medulla grafts enhance recovery of striatal dopaminergic fibers. Science 1987; 237:913-916. 6. Taber Pierce E. Time of origin of neurons in the brain stem of the mouse. Prog Brain Res 1973; 40:53-65. 7. Olson L. On the use of transplants to counteract the symptoms of Parkinson’s disease: Background, experimental models, and possible clinical applications. In: Cotman CW, ed. Synaptic Plasticity. New York: Guilford, 1985:485-505. 8. Triarhou LC, Low WC, Ghetti B. Transplantation of ventral mesencephalic anlagen to hosts with genetic nigrostriatal dopamine deficiency. Proc Natl Acad Sci USA 1986; 83:8789-8793. 9. Stenevi U, Björklund A, Svendgaard N-A. Transplantation of central and peripheral monoamine neurons to the adult rat brain: Techniques and conditions for survival. Brain Res 1976; 114:1-20. 10. Low WC, Triarhou LC, Kaseda Y et al. Functional innervation of the striatum by ventral mesencephalic grafts in mice with inherited nigrostriatal dopamine deficiency. Brain Res 1987; 435:315-321. 11. Björklund A, Schmidt RH, Stenevi U. Functional reinnervation of the neostriatum in the adult rat by use of intraparenchymal grafting of dissociated cell suspensions from the substantia nigra. Cell Tissue Res 1980; 212:39-45. 12. Triarhou LC, Brundin P, Doucet G et al. Intrastriatal implants of mesencephalic cell suspensions in weaver mutant mice: Ultrastructural relationships of dopaminergic dendrites and axons issued from the graft. Exp Brain Res 1990; 79:3-17. 13. Triarhou LC, Low WC, Ghetti B. Synaptic investment of striatal cellular domains by grafted dopamine neurons in weaver mutant mice. Naturwissenschaften 1987; 74:591-593. 14. Triarhou LC, Low WC, Norton J et al. Reinstatement of synaptic connectivity in the striatum of weaver mutant mice following transplantation of ventral mesencephalic anlagen. J Neurocytol 1988; 17:233-243.

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15. Triarhou LC, Low WC, Ghetti B. Dopamine neurone grafting to the weaver mouse neostriatum. Prog Brain Res 1990; 82:187-195. 16. Kaseda Y, Ghetti B, Low WC et al. Age-related changes in striatal dopamine D2 receptor binding in weaver mice and effects of ventral mesencephalic grafts. Exp Brain Res 1990; 83:1-8. 17. Triarhou LC, Low WC, Ghetti B. Genetic mesotelencephalic dopamine deficiency in weaver mutant mice: Reinstatement of neuronal connectivity by solid grafts of fœtal mesencephalon. Fidia Res Series 1988; 15:183-192. 18. Doucet G, Brundin P, Seth S et al. Degeneration and graft-induced restoration of dopamine innervation in the weaver mouse neostriatum: A quantitative radioautographic study of [3H]dopamine uptake. Exp Brain Res 1989; 77:552-568. 19. Solà C, Mengod G, Low WC et al. GAP-43, MAP2 and βAPP gene expression in the nigrostriatal system of normal and weaver mutant mice and in intrastriatal mesencephalic grafts. J Neuropathol Exp Neurol 1992; 51:351. 20. Triarhou LC, Stotz EH, Low WC et al. Studies on the striatal dopamine uptake system of weaver mutant mice and effects of ventral mesencephalic grafts. Neurochem Res 1994; 19:1349-1358. 21. Triarhou LC, Solà C, Mengod G et al. Ventral mesencephalic grafts in the neostriatum of the weaver mutant mouse: Structural molecule and receptor studies. Cell Transpl 1995; 4:39-48. 22. Witt TC, Triarhou LC. Transplantation of mesencephalic cell suspensions from wild-type and heterozygous weaver mice into the denervated striatum: Assessing the role of graft-derived dopaminergic dendrites in the recovery of function. Cell Transpl 1995; 4:323-333. 23. Triarhou LC, Norton J, Hingtgen JN. Amelioration of the behavioral phenotype in weaver mutant mice through bilateral intrastriatal grafting of fetal dopamine cells. Exp Brain Res 1995; 104:191-198. 24. Stasi K, Mitsacos A, Giompres P et al. Autoradiographic study of amino acid receptors in the striatum of weaver mice receiving nigral transplants. Soc Neurosci Abstr 1997; 23:2000. 25. Dahlström A, Fuxe K. Evidence of the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta Physiol Scand [Suppl] 1964; 232:1-55. 26. Björklund A, Stenevi U. Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants. Brain Res 1979; 177:555-560. 27. Björklund A, Dunnett SB, Stenevi U et al. Reinnervation of the denervated striatum by substantia nigra transplants: Functional consequences as revealed by pharmacological and sensorimotor testing. Brain Res 1980; 199:307-333. 28. Jaeger CB. Cytoarchitectonics of substantia nigra grafts: A light and electron microscopic study of immunocytochemically identified dopaminergic neurons and fibrous astrocytes. J Comp Neurol 1985; 231:121-135. 29. Baker H, Joh TH, Reis DJ. Genetic control of number of midbrain dopaminergic neurons in inbred strains of mice: Relationship to size and neuronal density of the striatum. Proc Natl Acad Sci USA 1980; 77:4369-4373. 30. Triarhou LC, Norton J, Ghetti B. Mesencephalic dopamine cell deficit involves areas A8, A9 and A10 in weaver mutant mice. Exp Brain Res 1988; 70:256-265. 31. Björklund A, Lindvall O. Dopamine-containing systems in the CNS. In: Björklund A, Hökfelt T, eds. Handbook of Chemical Neuroanatomy, Vol. 2. Amsterdam: Elsevier, 1984:55-122. 32. Brundin P, Isacson O, Björklund A. Monitoring of cell viability in suspensions of embryonic CNS tissue and its use as a criterion for intracerebral graft survival. Brain Res 1985; 331:251-259. 33. Brundin P, Nilsson OG, Strecker RE et al. Behavioural effects of human fetal dopamine neurons grafted in a rat model of Parkinson’s disease. Exp Brain Res 1986; 65:235-240. 34. Clarke DJ, Brundin P, Strecker RE et al. Human fetal dopamine neurons grafted in a rat model of Parkinson’s disease: Ultrastructural evidence for synapse formation using tyrosine hydroxylase immunocytochemistry. Exp Brain Res 1988; 73:115-126. 35. Hitchcock ER, Clough C, Hughes R et al. Embryos and Parkinson’s disease. Lancet 1988; i:1274. 36. Lindvall O, Rehncrona S, Gustavii B et al. Fetal dopamine-rich mesencephalic grafts in Parkinson’s disease. Lancet 1988; ii:1483-1484. 37. Morgan S, Steiner H, Rosenkranz C et al. Dissociation of crossed and uncrossed nigrostriatal projections with respect to site of origin in the rat. Neuroscience 1986; 17:609-614. 38. Sternberger LA. Immunocytochemistry. New York: Wiley, 1986. 39. Wassef M, Berod A, Sotelo C. Dopaminergic dendrites in the pars reticulata of the rat substantia nigra and their striatal input: Combined immunocytochemical localization of tyrosine hydroxylase and anterograde degeneration. Neuroscience 1981; 6:2125-2139.

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40. Bolam JP, Freund TF, Björklund A et al. Synaptic input and local output of dopaminergic neurons in grafts that functionally reinnervate the host neostriatum. Exp Brain Res 1987; 68:131-146. 41. Triarhou LC, Norton J, Ghetti B. Synaptic connectivity of tyrosine hydroxylase immunoreactive nerve terminals in the striatum of normal, heterozygous and homozygous weaver mutant mice. J Neurocytol 1988; 17:221-232. 42. Triarhou LC. Definition of the Mesostriatal Dopamine Deficit in the Weaver Mutant Mouse and Reconstruction of the Damaged Pathway by Means of Neural Transplantation. Ann Arbor: University Microfilms International, 1987. 43. Triarhou LC, Ghetti B. The dendritic dopamine projection of the substantia nigra: Phenotypic denominator of weaver gene action in hetero- and homozygosity. Brain Res 1989; 501:373-381. 44. Sokal RR, Rohlf FJ. Biometry, 2nd edn. New York: W. H. Freeman and Company, 1981. 45. Ghetti B, Triarhou LC. Nigrostriatal aberrations induced by weaver gene are present at birth. Soc Neurosci Abstr 1992; 18:156. 46. Bayer SA, Triarhou LC, Thomas JD et al. Correlated quantitative studies of the neostriatum, nucleus accumbens, substantia nigra, and ventral tegmental area in normal and weaver mutant mice. J Neurosci 1994; 14:6901-6910. 47. Lindvall O. Transplants in Parkinson’s disease. Eur Neurol 1991; 31[Suppl 1]:17-27. 48. Lindvall O, Brundin P, Widner H et al. Grafts of fetal dopamine neurons survive and improve motor function in Parkinson’s disease. Science 1990; 247:574-577. 49. Lindvall O, Sawle G, Widner H et al. Evidence for long-term survival and function of dopaminergic grafts in progressive Parkinson’s disease. Ann Neurol 1994; 35:172-180. 50. Hökfelt T, Skirboll L, Everitt B et al. Distribution of cholecystokinin-like immunoreactivity in the nervous system: Coexistence with classical neurotransmitters and other neuropeptides. Ann NY Acad Sci 1985; 448:255-274. 51. Hökfelt T, Everitt BJ, Theodorsson-Norheim E et al. Occurrence of neurotensin-like immunoreactivity in subpopulations of hypothalamic, mesencephalic, and medullary catecholamine neurons. J Comp Neurol 1984; 222:543-559. 52. Kalivas PW. Interactions between neuropeptides and dopamine neurons in the ventro-medial mesencephalon. Neurosci Biobehav Rev 1985; 9:573-587. 53. Gerfen CR, Baimbridge KG, Miller JJ. The neostriatal mosaic: Compartmental distribution of calcium-binding protein and parvalbumin in the basal ganglia of the rat and monkey. Proc Natl Acad Sci USA 1985; 82:8780-8784. 54. Gerfen CR, Baimbridge KG, Thibault J. The neostriatal mosaic: III. Biochemical and developmental dissociation of patch-matrix mesostriatal systems. J Neurosci 1987; 7:3935-3944. 55. Graybiel AM, Ragsdale CW. Histochemically distinct compartments in the striatum of human, monkey, and cat demonstrated by acetylthiocholinesterase staining. Proc Natl Acad Sci USA 1978; 75:5723-5726. 56. Pert CB, Kuhar MJ, Snyder SH. Opiate receptor: Autoradiographic localization in rat brain. Proc Natl Acad Sci USA 1976; 73:3729-3733. 57. Enderlin S, Norman AW, Celio MR. Ontogeny of the calcium binding protein calbindin D-28k in the rat nervous system. Anat Embryol (Berl) 1987; 177:15-28. 58. García-Segura LM, Baetens D, Roth J et al. Immunohistochemical mapping of calcium-binding protein immunoreactivity in the rat central nervous system. Brain Res 1984; 296:75-86. 59. Solà C, Mengod G, Ghetti B et al. Regional distribution of the alternatively spliced isoforms of βAPP RNA transcript in the brain of normal, heterozygous and homozygous weaver mutant mice as revealed by in situ hybridization histochemistry. Mol Brain Res 1993; 17:340-346. 60. Solà C, Mengod G, Low WC et al. Regional distribution of amyloid β-protein precursor, growthassociated phosphoprotein-43 and microtubule-associated protein 2 mRNAs in the nigrostriatal system of normal and weaver mutant mice and effects of ventral mesencephalic grafts. Eur J Neurosci 1993; 5:1442-1454. 61. Tanzi RE, Gusella JF, Watkins PC et al. Amyloid β protein gene: cDNA, mRNA distribution, and genetic linkage near the Alzheimer locus. Science 1987; 235:880-884. 62. Bendotti C, Forloni GL, Morgan RA et al. Neuroanatomical localization and quantification of amyloid precursor protein mRNA by in situ hybridization in the brains of normal, aneuploid, and lesioned mice. Proc Natl Acad Sci USA 1988; 85:3628-3632. 63. Manning RW, Reid CM, Lampe RA et al. Identification in rodents and other species of a mRNA homologous to the human β-amyloid precursor. Mol Brain Res 1988; 3:293-298.

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64. Mita S, Schon EA, Herbert J. Widespread expression of amyloid beta-protein precursor gene in rat brain. Am J Pathol 1989; 134:1253-1261. 65. Reeves RH, Robakis NK, Oster-Granite ML et al. Genetic linkage in the mouse of genes involved in Down syndrome and Alzheimer’s disease in man. Mol Brain Res 1987; 2:215-221. 66. Lovett M, Goldgaber D, Ashley P et al. The mouse homolog of the human amyloid β protein (AD-AP ) gene is located on the distal end of mouse chromosome 16: Further extension of the homology between human chromosome 21 and mouse chromosome 16. Biochem Biophys Res Commun 1987; 144:1069-1075. 67. Selkoe DJ (1989) Biochemistry of altered proteins in Alzheimer’s disease. Ann Rev Neurosci 1989; 12:463-490. 68. Kang J, Lemaire H-G, Unterbeck A et al. The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature (Lond) 1987: 325:733-736. 69. Goldgaber D, Lerman MI, McBride OW et al. Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer’s disease. Science 1987; 235:877-880. 70. Tanzi RE, McClatchey AI, Lamperti ED et al. Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer’s disease. Nature (Lond) 1988; 331:528-530. 71. Kitaguchi N, Takahashi Y, Takushima Y et al. Novel precursor of Alzheimer’s disease amyloid protein shows protease inhibitory activity. Nature (Lond) 1988; 331:530-532. 72. Ponte P, Gonzalez-De Whitt P, Schilling J et al. A new A4 amyloid mRNA contains a domain homologous to serine protease inhibitors. Nature (Lond) 1988; 331:525-527. 73. Golde TE, Estus S, Usiak M et al. Expression of β amyloid protein precursor mRNAs: Recognition of a novel alternatively spliced form and quantitation in Alzheimer’s disease using PCR. Neuron 1990; 4:253-267. 74. Shivers BD, Hilbich C, Multahaup G et al. Alzheimer’s disease amyloidogenic glycoprotein: Expression pattern in rat brain suggests a role in cell contact. EMBO J 1988; 7:1365-1370. 75. Kang J, Müller-Hill B. Differential splicing of Alzheimer’s disease amyloid A4 precursor RNA in rat tissue. PreA4(695) mRNA is predominantly produced in rat and human brain. Biochem Biophys Res Commun 1990; 166:1192-1200. 76. Yamada T, Sasaki H, Furuya H et al. Complementary DNA for the mouse homolog of the human amyloid beta protein precursor. Biochem Biophys Res Commun 1987; 149:665-671. 77. Yamada T, Sasaki H, Dohura K et al. Structure and expression of the alternatively-spliced forms of mRNA for the mouse homolog of Alzheimer’s disease amyloid beta protein precursor. Biochem Biophys Res Commun 1989; 158:906-912. 78. Solà C, Mengod G, Probst A et al. Differential regional and cellular distribution of the β-amyloid precursor protein messenger RNAs containing and lacking the Kunitz protease inhibitor domain in the brain of human, rat and mouse. Neuroscience 1993; 53:267-295. 79. Saitoh T, Sundsmo M, Roch J-M et al. Secreted form of amyloid β protein precursor is involved in the growth regulation of fibroblasts. Cell 1989; 58:615-622. 80. Whitson JS, Selkoe DJ, Cotman CW. Amyloid β protein enhances the survival of hippocampal neurons in vitro. Science 1989; 243:1488-1490. 81. Whitson JS, Glabe CG, Shintani E et al. β-Amyloid protein promotes neuritic branching in hippocampal cultures. Neurosci Lett 1990; 110: 319-324. 82. Yankner BA, Duffy LK, Kirschner DA. Neurotrophic and neurotoxic effects of amyloid β protein: Reversal by tachykinin neuropeptides. Science 1990; 250:279-282. 83. Roher AE, Ball MJ, Bhave SV et al. β-Amyloid from Alzheimer disease brain inhibits sprouting and survival of sympathetic neurons. Biochem. Biophys. Res. Commun 1991; 174:572-579. 84. Schubert D, Jin LW, Saitoh T et al. The regulation of amyloid β protein precursor secretion and its modulatory role in cell adhesion. Neuron 1989; 3:689-694. 85. Klier FG, Cole G, Stallcup W et al. Amyloid beta-protein precursor is associated with extracellular matrix. Brain Res 1990; 515:336 -342. 86. Breen KC, Bruce M, Anderton BH. Beta amylold precursor protein mediates neuronal cell–cell and cell–surface adhesion. J Neurosci Res 1991; 28:90-100. 87. Mengod G, Solà C, García-Ladona FJ et al. β-Amyloid precursor protein expression in rat brain: Increased levels of Kunitz domain containing forms after neuronal lesions. Soc Neurosci Abstr 1991; 17:1105. 88. Scott JN, Parhad IM, Clark AW. β-Amyloid precursor protein gene is differentially expressed in axotomized sensory and motor systems. Mol Brain Res 1991; 10:315-325.

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89. Wallace WC, Bragin V, Robakis NK et al. Increased biosynthesis of Alzheimer amyloid precursor protein in the cerebral cortex of rats with lesions of the nucleus basalis of Meynert. Mol Brain Res 1991; 10:173-178. 90. Solà C, García-Ladona FJ, Mengod G et al. Increased levels of the Kunitz protease inhibitorcontaining βAPP mRNAs in rat brain following neurotoxic damage. Mol Brain Res 1993; 17:41-52. 91. Tanaka S, Nakamura S, Ueda K et al. Three types of amyloid protein precursor mRNA in human brain: Their differential expression in Alzheimer’s disease. Biochem Biophys Res Commun 1988; 157:472-479. 92. Tanaka S, Shiojiri S, Takahashi Y et al. Tissue-specific expression of three types of β-protein precursor mRNA: Enhancement of protease inhibitor-harboring types in Alzheimer’s disease brains. Biochem Biophys Res Commun 1989; 165:1406-1414. 93. Palmert MR, Golde TE, Cohen ML et al. Amyloid protein precursor messenger RNAs: Differential expression in Alzheimer’s disease. Science 1988; 241:1080-1084. 94. Abe K, Tanzi RE, Kogure K. Selective induction of Kunitz-type protease inhibitor domain-containing amyloid precursor protein mRNA after persistent focal ischemia in rat cerebral cortex. Neurosci Lett 1991; 125:172-174. 95. Ghetti B, Triarhou LC. Degeneration of mesencephalic dopamine neurons in weaver mutant mice. Neurochem Int [Suppl] 1992; 20:305-307. 96. Neve RL, Kammersheidt A, Hohmann CF. Brain transplants of cells expressing the carboxyl-terminal fragment of the Alzheimer amyloid protein precursor cause specific neuropathology in vivo. Proc Natl Acad Sci USA 1992; 89:3448-3452.

CHAPTER 4

Structural Correlates of Process Outgrowth and Circuit Reconstruction Axonal Reinnervation of the Host Striatum

C

ertain cellular mechanisms by which grafts promote recovery in experimental animals have been deciphered.1,2 It has been suggested that a multitude of trophic, neurohumoral and synaptic mechanisms could be involved in bringing about functional recovery in the nigrostriatal models.3 Grafts of embryonic ventral mesencephalon taken from genetically normal mice and transplanted into the lateral ventricle of adult weaver mutants were shown to survive and grow in the mutant host environment and to reinnervate target regions of the recipient brain.4 A dense network of neuronal processes was seen in sections of tissue impregnated with silver. Fine tyrosine hydroxylase (TH)-immunoreactive fibers were detectable within the host caudate-putamen complex, in the vicinity of the mesencephalic grafts; they were much scarcer in the contralateral nongrafted caudate. The depth of reinnervation of the host caudate at one month after grafting was about 250 µm. At longer survival times of 4.5 months, the reinnervation of the recipient caudate-putamen by fine TH immunoreactive fibers displaying the characteristic varicosities of central catecholaminergic axons extended to a depth of 1000 µm in the dorsoventral plane.5-8 Reinnervation occurred to a similar extent in the rostrocaudal direction. The density of TH immunoreactive fibers in the grafted striatum was remarkably rich in comparison with the contralateral, nongrafted side, which displayed a minimal amount of TH immunoreactivity (Figs. 4.1 and 4.2). The density of TH immunoreactive fibers in the grafted neostriata was somewhat lower than the density of TH immunoreactive fibers in the dorsal neostriatum of wild-type animals. When compared to the nongrafted striatum of 8.5 months old weaver mutants, TH fiber density is much higher in the grafted side. Increased fiber density following transplantation is a consequence of the innervation of the recipient striatum by grafted mesencephalic DA neurons. At 4.5 months after grafting, reinnervation extends to 1000 µm into the host striatum, a distance that covers the dorsal striatal aspect fully and that is a little longer than one-half of the entire dorsoventral diameter of the striatum. The idea that the depth of reinnervation can increase with longer survival times has also been described in the case of DA neuron grafting to the striatum of rats with 6-OHDA lesions of the substantia nigra.9-13 The depth of reinnervation of the recipient weaver caudate is comparable to that reported in the 6-OHDA rat model;9-14 in the latter studies, DA fibers derived from the

Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease, by Lazaros C. Triarhou. ©2001 Eurekah.com.

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Fig. 4.1. TH immunoreactivity in the nongrafted (left) and in the grafted (right) striatum of a weaver mutant that received a solid mesencephalic graft in a preformed cortical cavity over the neostriatum. Immunolabeled fibers are sparse in the nongrafted side; fiber density is substantially higher in the transplanted side, shown four months postoperatively. Dark-field illumination. Magnification x370. Reprinted with permission from: Triarhou LC, Low WC, Norton J et al. J Neurocytol 1988; 17:233-243. © Kluwer Academic Publishers

Fig. 4.2. A unilateral mesencephalic cell suspension graft placed into the right weaver striatum, as seen with tyrosine hydroxylase immunocytochemistry three months after transplantation. The density of tyrosine hydroxylase positive fibers is much higher in the transplanted side compared to the contralateral, nongrafted left side. Magnification x18. Unpublished micrograph.

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grafts innervate the host caudate-putamen of rats to a depth of 500 µm with a near normal density10 and more extensive depths of penetration are observed with increasing survival time after transplantation.12,13

Synaptic Investment of Graft-Derived Dopamine Terminals Solid Grafts DA fibers originating in the substantia nigra terminate in the striatum and establish predominantly symmetrical synapses with dendritic spines and dendritic shafts of the medium spiny projection neurons.15,16 Normal synaptogenesis is the result of a prolonged two-way communication between presynaptic and postsynaptic neuronal elements during development.17 In the case of neural grafting, however, embryonic donor tissue is led to develop inside an adult recipient brain. From both a theoretical and practical viewpoint, it is important to know whether grafted DA cells are capable of re-establishing synaptic connections with the progressively denervated striatal neurons in the weaver model; this question has been addressed. Solid grafts were obtained from normal embryos at a gestational age of 14-15 days and implanted into a surgical cavity overlying the dorsal striatum of adult weaver recipients.5,6 Both in normal and weaver striatum, junctional contacts formed by TH immunoreactive nerve terminals are predominantly of the symmetrical type. By applying appropriate stereological corrections,18 it has been estimated that about 90% of the contacts are junctional in normal striatum, whereas only 50% of the few remaining axons in the weaver striatum display a junctional membrane specialization at 20 days of age,19 the latter proportion declining further to about one-fourth of normal by 8.5 months of age.6 Following DA neuron transplantation, the incidence of junctional contacts is reinstated to normal values (91%);5,6 further, the profile of the newly established synaptic connectivity resembles the normal situation in regard with subcellular domains of host striatal neurons that are synaptically invested by graft-derived DA axons. Thus, the majority of contacts in the reinnervated striatum (84%) are made with dendritic shafts and spines, which is the case in the normal situation (92% in wild-type animals). A total of 114 TH immunoreactive nerve terminals were examined in the nongrafted dorsal striata of weaver mutants.5,6 These terminals contacted in total 324 spines, dendrites, axons and somata. In addition, they were apposed to 97 unlabeled terminal boutons without an indication of a synaptic connection. At 8.5 months of age, the mean proportion of ajunctional contacts was 90%, and that of junctional contacts was 8%. Among junctional synapses, the majority was of the symmetrical type, and less than 1% was of the asymmetrical type. The percentages corresponding to ajunctional (90%) and junctional (8%) contacts markedly contrasted with those in 20-day-old weaver homozygotes, in which the respective values were 83% and 17% and with those in wild-type mice, in which the respective values were 73% and 27%.19 Although the majority of total contacts was with dendritic spines and shafts (89%), the proportions of junctional synapses with spines and dendrites were extremely low (2% and 5% respectively). TH immunoreactive nerve terminals were relatively rare to find in the nongrafted striatum of weaver animals. TH immunoreactive nerve terminals were found much more frequently in the grafted striatum of weaver recipients. A total of 205 TH immunoreactive nerve terminals were examined, which contacted 379 spines, dendrites, axons and somata. In addition, they were apposed to 129 unlabeled terminal boutons without an indication of synaptic

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connection. The proportion of ajunctional contacts in the grafted neostriata was 70% and that of junctional synapses was 29%. Among junctional synapses, the majority was of the symmetrical type, and less than 3% were of the asymmetrical type (Fig. 4.3). The percentages corresponding to ajunctional (70%) and junctional (27%) contacts contrasted markedly with the respective percentages in the nongrafted side (90% and 8%), whereas they approximated the corresponding percentages in normal animals (73% and 27% respectively19). The majority of contacts (84%) were with dendritic spines and shafts. The proportion of contacts with axons (9%) and with perikarya (6%) appeared increased in relation to normal (5% and 3% respectively19). TH immunoreactive nerve terminals were found in symmetrical synaptic contact with dendritic spines which were also receiving asymmetrical synapses from unlabeled axon terminals. The axodendritic synapses of TH immunoreactive nerve terminals were predominantly of the symmetrical type and only rarely of the asymmetrical type. On several occasions, multiple TH immunoreactive nerve terminals were found to contact the soma of the same striatal neuron. Those findings show that transplantation of ventral mesencephalic grafts to the striatum of adult weaver mutants leads to 1. an increase in TH fiber density in the grafted striatum, 2. an increase in the proportion of junctional contacts and 3. a pattern of overall synaptic connectivity closely resembling that found in the normal mouse neostriatum. In the dorsal striatum of normal mice, 27% of the relations of TH immunoreactive nerve terminals are characterized by the presence of junctional membrane specializations in random single sections; this implies that 85% of the contacts might be truly junctional,19 if one applies the stereological correction formula of Beaudet and Sotelo.18 In 20-day-old weaver mutants, 17% of the relations of TH immunoreactive nerve terminals are characterized by the presence of junctional membrane specializations in random single sections, which may imply that 53% of the contacts may be truly junctional.19 The percentage of junctional contacts in weaver mutant mice is reduced with age such that by 8.5 months only 8% of the relations of TH immunoreactive nerve terminals display junctional membrane specializations in random single sections (nongrafted side); by applying the stereological formula, we calculate that 26% of the contacts might be truly junctional in the weaver striatum at that age. Following transplantation, the percentage of junctional contacts in random single sections becomes 29%, which may correspond to a 91% value of true junctional contacts after applying the stereological correction. The percentage of junctional contacts in the region of the grafted striatum analyzed thus approximates the normal situation. These data indicate that the graft-derived innervation forms a new DA axonal plexus and reinstates synaptic connectivity in the recipient mutant striatum. The majority of the synaptic contacts of TH immunoreactive boutons in the reinnervated striatum (84%) were with dendritic spines and dendritic shafts. This is the case in the normal rat15,16 and mouse.19 The fact that over 80% of the new synapses are formed with the appropriate cellular targets (i.e., dendrites and spines) supports the notion that the reafferentation of the host striatum by graft-derived DA afferents preserves its synaptic specificity. In this context, it is possible that in repopulating the denervated weaver striatum, DA afferents originating in the grafts invest postsynaptic sites of the host brain that had either been vacated from their intrinsic presynaptic DA input or had never received such an input. Other studies have also documented formation of synaptic connections in rats with 6-OHDA lesions after transplantation of DA-rich grafts.11

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Fig. 4.3. An asymmetrical synapse (type I) formed by a TH immunoreactive nerve terminal and characterized by the presence of a pronounced postsynaptic membrane density. x126000. Reprinted with permission from: Triarhou LC, Low WC, Norton J et al. J Neurocytol 1988; 17:233-243. © Kluwer Academic Publishers

The proportion of total contacts of TH immunoreactive boutons with neuronal perikarya following transplantation (6%) is double than that seen in normal animals (3%). Furthermore, the proportion of axosomatic junctional synapses following transplantation (2.4%) is three times the corresponding value in normal mice (0.8%). Such an increase may indicate a state of anatomical immaturity, as prevalence of axosomatic contacts is a morphological feature of the primordial mesostriatal DA projection.20 Such an immaturity might result from a temporal mismatch in the molecular interactions between the grafted mesencephalic anlage and the adult, denervated weaver striatum; in other words, growing axons of grafted DA neurons may not receive the appropriate target-derived molecular signals from the adult recipient striatum in the same manner as they would have done during normal development in situ. The persistence of axosomatic contacts may accordingly be associated with a lack of regulatory molecules that would normally induce DA axon terminals to translocate from somatic to dendritic synaptic sites. The presence of axosomatic contacts between terminals of grafted DA cells and giant striatal interneurons, which has been found in rats subjected to 6-OHDA lesions following grafting of DA-containing transplants, has been indicated to represent an aberrant form of synaptic connectivity not seen under normal circumstances.11 In particular, Freund et al11 had reported an increased proportion of axosomatic contacts following grafting and pointed out that a second class of cell received input in the grafted rats in contrast to the normal striatum. In another study we used a double-labeling immunocytochemical technique to analyze the innervation of chemically-identified neurons of the host by graft-derived fibers.21 After graft survival periods of three months, sections were sequentially incubated with TH and choline acetyltransferase (ChAT) antisera. TH immunoreactivity was shown by Ni(NH 4)2(SO4)2 intensification (blue/gray product), and ChAT by conventional 3,3´-diaminobenzidine hydrochloride reaction (red/brown product). Grafts exhibited a

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robust development, with numerous DA cells surviving and a rich TH fiber outgrowth into the host. The somata of large cholinergic striatal interneurons were innervated by a dense TH fiber plexus, a phenomenon not seen normally. This aberrant type of connectivity may in part explain how a relatively small number of grafted DA cells (compared to normal substantia nigra) exerts a strong functional effect, as measured by the rotational bias scores after methamphetamine challenge.11,21 The findings on long-term survival and synapse formation lend credence to the idea that normal embryonic DA neurons are capable of integration with a pathological nervous system that results from a naturally occurring degenerative process. The increased TH fiber density, as determined by light microscopy, and the different pattern of connectivity of TH immunoreactive nerve terminals following transplantation, as determined by quantitative electron microscopy, indirectly demonstrate that synapses are formed between graft-derived DA axon terminals and host striatal neurons. A concomitant paracrine action of DA released from nonsynaptic axon terminals cannot be precluded solely on the basis of morphological grounds, as it has been postulated that catecholamine axons exert such an activity in specific regions of the nervous system.22,23 The existence of newly formed synapses in the weaver studies indicates that grafted DA cells may exert their functional effects,24 at least in part, through synaptic activity. In turn, grafted DA neurons may be synaptically influenced by other neurons inside, or perhaps outside, the transplant, as their somata and dendrites receive input from immunocytochemically unlabeled terminal boutons. This latter point has also been made in studies where DA-containing grafts were implanted to rats with 6-OHDA lesions.12,25

Cell Suspension Grafts Cell suspensions were prepared from the ventral midbrain of normal mouse fetuses and stereotaxically implanted into the neostriatum of two to three months old homozygous weaver mutant mice.26 Prior to perfusion, which was carried out at 80 days after transplantation surgery, the grafted striata of the weaver recipients were deprived of their intrinsic mesostriatal DA input through local injections of 6-OHDA into the remnant ipsilateral substantia nigra, so that we could selectively study the innervation derived from the grafts. Such lesions resulted in a 96-100% depletion of TH immunoreactive cell bodies in areas A9 and A8, whereas area A10 was left relatively unaffected. Considering that the substantia nigra is the sole source of endogenous DA supply to the dorsolateral neostriatal region, reinnervating fibers in that striatal quadrant would have their origin in the graft. Grafts contained an estimated 100-700 TH immunoreactive neurons. An ultrastructural analysis demonstrated that graft-derived TH immunoreactive axons were in apposition primarily with unlabeled dendrites or spines of the recipient striatum in more than 90% of the cases (Fig. 4.4.a). Graft-derived dopaminergic dendrites received synaptic input from unlabeled axon terminals (Fig. 4.4.b) and, in a few instances, were apposed to the unlabeled somata of striatal neurons. The reinnervated area of the dorsolateral striatum was divided into two parts: one proximal to the graft (i.e., at a distance of 0-500 µm from it) and one distal (i.e., 500-1000 µm). A total of 490,150 µm2 from the proximal area of four blocks from two animals and a total of 498,000 µm2 from the distal area of the same blocks (after trimming and re-embedding) were scanned at a primary magnification of x 20,000. Overall, the area proximal to the graft contained 111 TH immunoreactive profiles, and the area distal to the graft contained 68 TH immunoreactive profiles.

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Fig. 4.4. Electron micrograph (a) showing a graft-derived TH immunoreactive axon terminal in symmetrical (type II) synaptic contact with an unlabeled dendrite of the host striatum. A graft-derived TH immunopositive dendrite (b) is shown, receiving an immunonegative presynaptic axon terminal. Magnification x46000 (left), x44800 (right). Reprinted with permission from: Triarhou LC, Brundin P, Doucet G et al. Exp Brain Res 1990; 79:3-17. © Springer-Verlag.

In one subset of TH immunoreactive neuronal processes the peroxidase reaction product was localized within the cytoplasm in a dispersed granular manner. The pattern of immunoprecipitation in those neuronal processes and the fact that they were postsynaptic to unlabeled axon terminals strongly suggested that they were dendrites. The diameter of those profiles varied from 0.5-2.5 µm. Such TH immunoreactive dendritic processes were found in both the proximal and distal parts of the reinnervated dorsolateral neostriatum, suggesting that they occasionally extended 500 µm or more from the cell somata. Unlabeled axon terminals that were in synaptic contact with TH immunoreactive dendrites belonged to morphologically heterogeneous populations; they contained either small spheroid or flattened clear synaptic vesicles, and occasionally large granular vesicles. Synapses formed between such unlabeled axons and TH immunoreactive dendrites were predominantly asymmetrical. TH immunolabeled dendrites were also seen apposed to the perikarya of unlabeled cells in the host striatum, but without clear-cut evidence for the presence of specialized synaptic membranes. The incidence of such “dendrosomatic” appositions was 13.2%, i.e., 5 instances out of a total of 38 dendrites examined in the area proximal to the graft. Similar appositions were not seen in the area distal to the graft. In the second subset of neuronal processes, the peroxidase reaction product was localized in the cytoplasm and was organized around spherical vesicles whose core appeared clear. These processes were interpreted as axons. The diameter of the clear-core synaptic vesicles was approximately 50 nm. In contrast to previous observations on solid mesencephalic grafts implanted into a cortical cavity in rats,25 TH immunolabeled axon terminals were

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not found in synaptic contact with TH immunolabeled dendrites or somata either in the reinnervated striatum or inside the grafts, suggesting the absence of recurrent axon collaterals in grafted DA neurons. The overall ratio of TH immunoreactive axons over that of TH immunoreactive dendrites in the reinnervated striatum was three-to-one: 26 from a total of 179 TH immunopositive profiles, 70.9% were identified as axons and 22.9% as dendrites; in 6.1% of the profiles an identification was not clear-cut and these were classified as “indiscernible”. TH immunoreactive dendrites were found at a much higher frequency in the area proximal to the graft (34.2% of the total number of TH immunoreactive profiles) than in the area distal to the graft (where the corresponding value was 4.4%). TH immunoreactive nerve terminals were found in contact with unlabeled dendritic spines, dendritic shafts, axons and perikarya in the grafted striatum. Postsynaptic densities were seen occasionally. The average axon diameters in the three conditions, i.e., wild-type striatum, weaver striatum, and graft-derived innervation of the weaver striatum, were 425 nm, 315 nm and 269 nm respectively.26 The appropriate statistical tests showed that all three groups differed significantly from each other: the mean diameter of TH immunoreactive axons was significantly smaller in weaver than in wild-type mice, and the diameter of graft-derived axons was significantly smaller than both the wild-type and the weaver. The finding that graft-derived DA axons are smaller in diameter than even the endogenous DA axons in the homozygous weaver striatum merits attention. Such a reduction in diameter could theoretically be either due to some biological parameter(s) present in the mutant host striatum or be a property inherent in the process of DA neuron transplantation. That question could be answered by studying axon diameters of DA neurons grafted to the striatum of wild-type mice previously subjected to 6-OHDA lesions of the mesostriatal DA pathway. Alternatively, this finding could be related to the number of varicosities per unit length of axon. It has been documented in rats with neurotoxic lesions of the mesostriatal DA projection that the graft-derived innervation is associated with a formation of new synaptic connections with host striatal neurons.11,12 In weaver mice the evidence for new synapse formation following transplantation of solid mesencephalic grafts had been based on a comparison of the pattern of synaptic connectivity in the grafted and nongrafted striatum6 (Fig. 4.5.a). In the cell suspension study26 the remaining mesostriatal projection of the weaver hosts was removed by 6-OHDA lesions of the substantia nigra shortly before perfusing the animals, and after the grafts had developed (Fig. 4.5.b). Such an experimental manipulation provides direct evidence for innervation of the host striatum by graft-derived afferents rather than by fibers belonging to the host and being in some way stimulated by the graft to sprout, a mechanism which has been suggested in the case of adrenal medullary grafts to the striatum of methylphenyltetrahydropyridine (MPTP)-treated mice.27 Striatal DA fibers are very fine and have abundant varicosities.28 As sites of neurotransmitter uptake, storage, and presumably release, axonal varicosities constitute more than 80% of the elements that are labeled by [3H]DA-uptake autoradiography.29 Catecholamine varicosities are also referred to as “terminals”, “endings” or “boutons” irrespective of whether they actually constitute the physical end of an axon or not.30 Criteria used to characterize an axonal profile as a varicosity in electron microscopy include diameter, the establishment of synaptic contacts, and number of synaptic vesicles.30 However, those criteria may not always be present in immunocytochemical preparations used for two-dimensional electron microscopy, as single sections through DA axonal varicosities:

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Fig. 4.5. Schematic drawing of two approaches used to study the graft-derived innervation: (a) in one study the transplanted side, consisting of the graft (G)-derived plus the right (R) endogenous innervation is compared to the nongrafted left (L) side; (b) in another study, the graft-derived innervation is studied after the right endogenous innervation is subtracted through a 6-hydroxydopamine lesion of the ipsilateral substantia nigra.

1. may be distant from the equatorial plane, 2. may not be inclusive of the macular density, and 3. synaptic vesicles may be confounded by the peroxidase immunoprecipitate. An additional source of uncertainty is the lack of sufficient information on whether these parameters remain unaltered in the weaver condition and in the transplantation setting. Using analysis of serial sections, Freund et al16 classified TH immunopositive axonal profiles in the normal rat striatum into the following categories: small presynaptic axons (diameter 0.1-0.4 µm) and thin en passant synapse-forming fibers (sometimes < 0.2 µm in diameter), larger varicosities (diameter 0.3-0.5 µm) containing relatively few synaptic vesicles considering their size, and very thick main axons (diameter 0.5-1.0 µm) rarely seen and never found to make synaptic contact. In the striatum of the mouse the proportion of contacts formed by TH immunoreactive nerve endings and displaying junctional membrane specializations in single sections is 27%, implying, after using appropriate stereological corrections,18 that at least 85% of the contacts could be truly junctional in the three dimensions.19 Of the contacts formed by TH immunoreactive nerve terminals in the striatum of normal rats or mice, 92%-93% are with dendritic spines or shafts.16,19 In previous studies of rat-to-rat,11 human-to-rat31 and normal-to-mutant mouse6 grafting experiments, respectively 89%, 83% and 84% of the contacts of TH immunoreactive nerve terminals were found to be axospinous and axodendritic. However, the proportion of axosomatic contacts was approximately double than that normally seen in all three situations studied, a phenomenon which may be indicative of either an aberrant form of connectivity or of the persistence of a developmental profile during the synaptic ontogeny of the graft-derived innervation.6,11,20

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Compartmental Specificity of the Striatal Reinnervation Following transplantation to the weaver mutant striatum, embryonic mesencephalic neurons retain features of their normal histochemical phenotypes. Under the appropriate circumstances, it appears that the innervations displayed by TH, 28 kDa calcium-binding protein (CaBP) and cholecystokinin (CCK)-immunoreactive fibers following mesencephalic tissue transplantation recapitulate specific patterns of nigral-striatal neuron interactions occurring normally.32 TH-immunoreactive fibers are scanty in the nongrafted striatum of weaver mutants, whereas a remnant TH innervation is observed in the nucleus accumbens and in the olfactory tubercle. In the side of the transplant, innervation of the dorsal striatum by graft-derived TH-immunoreactive fibers is rich and homogeneous; such fibers cover a distance of 500-1000 µm away from the graft into the host striatum in both the dorsoventral and rostrocaudal planes. In the adult mouse striatal DA innervation is homogeneous, following the disappearance of developmental DA-rich islands.33 In that regard, the graft-derived DA innervation of the host striatum in weaver mutants appears to reach maturity, as the pattern of innervation appears homogeneous. The innervation of the host striatum by 28 kDa calcium-binding protein (CaBP)-immunoreactive fibers is rich, as deduced from comparing grafted and nongrafted sides of the weaver striatum, but confined to the striatal matrix; the striatal patches appear to be avoided by graft-derived CaBP-immunoreactive fibers and thus stay immunonegative. Therefore, based on the expression of immunoreactive CaBP, the patch–matrix dissociation that is characteristic of the normal striatal innervation appears to be recapitulated in the graft-induced anatomical situation. Graft-derived CaBP-immunoreactive fibers show a preference for the striatal matrix of the host and avoid the striatal patches.32 Grafted CaBP neurons that provide an innervation to the striatum could originate either in the substantia nigra of the primordial donor mesencephalic tissue or in other areas included in the dissected pieces, such as nucleus interpeduncularis, supramammillaris, mammillaris lateralis and posterior, zona incerta and substantia grisea centralis.34,35 As it would be difficult to identify the embryological origin of CaBP-immunoreactive neurons in the grafts, one cannot claim with certainty that the innervation observed in the weaver striatum after grafting originates specifically from “nigral” CaBP neurons. In the event that this is the case, however, the patch–matrix dissociation characteristic of the normal nigrostriatal innervation would be recapitulated in the transplantation situation as well. The innervation of the dorsal striatum by cholecystokinin (CCK)-immunoreactive fibers from the transplant is rather limited.32 Such a finding extends observations from studies in rats with 6-OHDA lesions of the mesotelencephalic DA projection and ventral mesencephalic transplants.36 Schultzberg et al36 have suggested that such a limited fiber ingrowth might indicate the presence of selective growth-regulating mechanisms in the denervated striatum that would favor innervation by fibers from the appropriate sources, i.e., subsets of DA neurons lacking CCK-immunoreactivity.

Innervation of Nonstriatal Regions by the Grafts Lateral Septum Grafts implanted into the lateral ventricle may occasionally become attached to the septum of the host.4 In that case, fibers emanating from TH-immunoreactive neurons can

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invade the septum and innervate large septal neurons by forming perisomatic basket-like arrays around them. Neurons of the substantia nigra normally send collateral branches to the lateral septum, as has been shown in multiple-labeling studies.37 The pattern of reinnervation of the weaver septum is comparable to the normal DA innervation of the lateral septal nucleus, which is mainly composed of perisomatic basket formations.38

Frontal Cortex The dopaminergic innervation of the frontal cortex has its origin in cell bodies of the A9 and A10 mesencephalic DA cell groups.39-45 The mesocortical DA projection is involved in affective and cognitive behavior,46,47 normal exploratory behavior48 and the response to physical stress.49 An association between loss of mesolimbocortical DA and cognitive impairment has been made in humans with Parkinson’s disease.50 Although the dementia seen in some patients with Parkinson’s disease may also be related to the severe degeneration of the nucleus basalis of Meynert and its cholinergic projection to the neocortex,51,52 regional depletion of DA from the prefrontal cortex has been associated with the pathophysiology of cognitive impairment in primates subjected to experimental lesions.53 DA deficiency in the frontal cortex is a component of the phenotype of the weaver mutant;54 DA levels in the frontal cortex of weaver homozygotes are 77% lower than control values. Decreased DA levels in the telencephalon are associated with losses of DA cell bodies in the A9 and A10 mesencephalic DA cell groups.55 We have examined the pattern of anatomical innervation of the weaver frontal cortex by grafted mesencephalic DA neurons.56 TH-immunoreactive fibers originating in the grafts that had been placed into a cortical cavity above the striatal complex of the hosts spread both mediolaterally and rostrocaudally into the cortical layers of the recipients to an extent of 1000 µm. The contralateral, nongrafted anteromedial frontal and anterior cingulate cortices displayed a sparsity of TH-immunoreactive fibers compared to the wildtype. In the anteromedial frontal lobe of the hosts, the graft-induced reinnervation was confined to the basal cortical layers. TH-immunoreactive fibers were abundant in layers IV-VI. In the anterior cingulate cortex, TH-immunoreactive fibers ascended to superficial layers, including the molecular layer, in addition to the innervation of basal cortical layers. Such a dichotomy in the anatomical pattern of innervation of the two different cortical areas was consistent: the animal-to-animal variation regarding reinnervation of the frontal cortex was limited to the amount of TH innervation distributed preferentially to layers IV, V or VI; in the cingulate cortex, TH-immunoreactive fibers ascended to the molecular layer in all cases examined. Under normal circumstances, the DA innervation of the anteromedial frontal lobe is distributed among the basal cortical layers (IV-VI), whereas the DA innervation of the cingulate cortex extends to superficial layers both in the rat44 and in the mouse.56 The pattern of innervation seen after transplantation of embryonic DA neurons into the frontal cortex of weaver mutant mice resembles the normal DA innervation and indicates that, in repopulating DA-deficient cortical areas of the recipients, the graft-derived DA fibers selectively exhibit a preference for those cortical layers that are normally invested by DA afferents. Thus, mechanisms for selective axon-target recognition may be operating at the cortical level in the central nervous system. Moreover, the genetic process responsible for the degeneration of the mesotelencephalic DA system in weaver mutant mice does not seem

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to abolish the ability of target cortical domains for selective recognition by ingrowing axons from normal grafted DA neurons.

Chemoaffinity and Axon-Target Recognition in Development and in Transplantation The concept of chemotactic affinity during neural development and regeneration has been known for a long period of time.57-59 Experimental evidence supporting the existence of mechanisms responsible for the selective establishment of connections during development and regeneration has been presented for the neuromuscular junction,60-62 the sympathetic 63 and parasympathetic 64 nervous systems, and the retinotectal projection.65-67 Concerning grafts of mesencephalic DA cells, Björklund et al68 have pointed out that there is a selectivity of DA fiber outgrowth from the graft into surrounding host tissue, depending largely on whether the latter is a normal DA target area or not. For example, mesencephalic DA cell suspensions grafted to the neostriatum or nucleus accumbens– which normally receive a substantial innervation from the midbrain DA cell system–give rise to extensive DA fiber outgrowth into the host tissue; by contrast, grafts placed in brain sites that are non-DA terminal areas, such as the parietal cortex and the lateral hypothalamus, exhibit very poor radiation of DA fibers into the surrounding host tissue.68 Furthermore, Herman et al69 have described normal patterns of reinnervation after grafting of DA cell suspensions to the nucleus accumbens, the lateral septum and the anteromedial frontal cortex of rats subjected to 6-OHDA lesions. In other studies dealing with transplantation of central neural tissues, layer-specific connectional selectivity has been observed during reinnervation of the hippocampus by grafts of locus coeruleus70 and of septal nucleus,71,72 and during reinnervation of the occipital cortex by tectal grafts.73 This selectivity occurs in spite of the abnormal route taken by fibers from the graft in establishing an appropriate laminar pattern of innervation. The findings in the weaver model point to the notion that graft-derived DA afferents are capable of distinguishing, or being distinguished by or both, cellular and subcellular domains of the host brain that normally receive DA innervation. Such a selective recognition may be related to an inherent property of fibers of grafted cells to locate their original sites of termination. It is also possible that host cellular subsets are able to attract ingrowing fibers through molecular properties that bias microenvironmental influences on incoming afferents. The end result of such interactions is a stereotyped pattern of connectivity that parallels connectional patterns encountered normally.

Dendrite Extension from the Graft into the Host Striatum Normally, the striatum receives only axons of DA neurons from the pars compacta of the substantia nigra, while DA dendrites are confined to the pars reticulata of the substantia nigra.44 When mesencephalic cell suspension grafts were placed intrastriatally, grafted DA neurons displayed an extensive arborization with both fine varicose and with thicker processes, corresponding to the typical morphologies of axons and dendrites, respectively (Fig. 4.6). The ratio of the incidence of TH immunoreactive axons over that of TH immunoreactive dendrites in the reinnervated striatum is two-to-one in an area proximal to the graft (i.e., at distances less than 500 µm) and twenty-to-one in areas 500-1000 µm distally from the graft.26 This effect brings up a difference between the graft-derived innervation and the normal striatal DA supply. DA dendrites of the substantia nigra pars reticulata

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Fig. 4.6. TH-immunopositive axon (upper) and dendrite (lower) extended from a cell suspension graft into the striatum of the host weaver mutant. Magnification x285. Reprinted with permission from: Solà C, Mengod G, Low WC et al. Eur J Neurosci 1993; 5:1442-1454. © Blackwell Science Ltd.

represent a brain area where dendritic release of neurotransmitter has been demonstrated.74,75 It has been theorized that the dendritic release of DA may contribute, in conjunction with the synaptic input of DA nerve terminals, to the functional effects of the transplants8,26 (cf. also Chapter 6). As Björklund and Lindvall74 had pointed out, the release and uptake of DA from nigral neurons may not be an exclusive property of axon terminals, and intradendritic DA could serve neurotransmitter secretory functions as well; this contention has been supported

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by experimental evidence in rats76,77 and cats.75 It still remains unclear to what extent nigral DA dendrites normally form presynaptic specializations.78 Although synapses between DA presynaptic dendrites and non-DA postsynaptic dendrites were not seen in our ultrastructural studies,26 “appositions” between DA dendrites and unlabeled neuronal elements have been found, and one cannot exclude the possibility that nonsynaptic release of DA from dendrites may contribute to the functional effects of the DA cell suspensions. It is possible that after intrastriatal grafting of nigral cell suspensions, graft-derived DA dendrites extending into the recipient striatum may be receiving synaptic input from the host. The existence of synapses between unlabeled axon terminals containing flattened synaptic vesicles (possibly GABAergic) or large granular vesicles (possibly peptidergic) and DA dendrites emerging from the graft into the recipient striatum might be indicative of such a phenomenon.26 TH dendrites receiving synaptic input from unlabeled axon terminals have previously been found inside solid grafts implanted to a cortical cavity in rats with 6-OHDA lesions,25 in the reinnervated striatum of rats with 6-OHDA lesions and solid grafts in preformed cavities12 or intrastriatal cell suspension grafts,31 and within solid grafts implanted to a cortical cavity in weaver mutants.6 Dendrites emanating from the intrastriatal nigral grafts can thus be viewed as part of a heterotopic pars reticulata. In the ectopic position, DA dendrites of the graft may participate in “homologous” physiological functions through nonsynaptic release of DA,75 including regulation of the activity of grafted DA neurons, of the release of neurotransmitter from afferent axons to the graft, and of the activity of non-DA grafted nigral neurons. In addition, DA dendrites of the graft may serve “heterologous” or novel functions, such as modulation of the activity of corticostriatal afferents or of the physiological activity of striatal cells. In turn, the activity of grafted DA neurons might be regulated by afferents from the host striatum to the dopaminergic dendrites of the grafts.

Expression of Molecules Related to Axonal and Dendritic Outgrowth A battery of immunocytochemical markers was used to monitor aspects of process outgrowth in the grafts.79 Furthermore, in situ hybridization histochemistry with [32P]oligonucleotide probes has been employed to investigate the cellular localization of RNA transcripts encoding axon and dendrite-related molecules.79,80 Weaver hosts, three to four months old at the time of grafting, received cell suspension grafts into the right striatum. Survival times of three months after transplantation were allowed. Axon growth was studied by using 1. a rabbit antiserum against rat brain neural cell adhesion molecule (N-CAM), an integral membrane glycoprotein that modulates the aggregation and fasciculation of individual neuronal processes into fiber bundles;81,82 2. monoclonal antibodies against growth-associated phosphoprotein-43 (GAP-43), which is involved in axonal outgrowth during ontogeny, regeneration and plasticity,83-87 3. monoclonal antibodies against phosphorylated and nonphosphorylated antigenic determinants in neurofilaments (clones SMI-31 and SMI-32);88,89 4. a monoclonal antibody against synaptophysin, a glycosylated membrane polypeptide of presynaptic vesicles (Mr = 38 kDa, also designated protein P-38).90,91

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N-CAM immunoreactivity was seen at high levels in layer I of cerebral cortex and in moderate levels in the remaining cortical layers and striatum.79 Cell suspension grafts exhibited strong N-CAM immunoreactivity in the form of fiber streams that also extended into dorsolateral and ventrolateral striatum. GAP-43, a growth cone-associated, neuron-specific protein, has been implicated in axonal outgrowth in ontogeny, regeneration and synaptic plasticity.86,92 The GAP-43 gene was localized to mouse chromosome 16.93 GAP-43 expression is strong during development. However, GAP-43 is also abundant in many adult brain structures, including DA neurons of the substantia nigra and ventral tegmental area.83 With immunocytochemistry, one could see streams of GAP-43 immunoreactive fibers in the area of the substantia nigra. GAP-43 immunoreactivity in the grafts had a similar pattern to that of N-CAM and was seen forming streams of fibers in the areas that corresponded to the implanted cell suspensions.79,80 SMI-31 immunolabeling was found in axon bundles in the grafts and striatum, but not in neuronal somata.79 SMI-32 immunolabeling was seen in some neuronal bodies in cortical layers II, IV and globus pallidus, and in very few cells of the striatum and of the grafts. This indicated a normal compartmentation of the functional states of neurofilaments dependent upon phosphorylation in individual nerve cells. A similar, normal phosphorylation pattern of neurofilaments has been reported in intraparenchymal and intraventricular cerebellar grafts into the rat brain where, in contrast to the situation in trauma or disease, phosphorylated neurofilament epitopes never appeared in the perikarya of transplanted neurons as well.88 High levels of synaptophysin immunoreactivity were seen in cortical layers I, II and IV and striatum; intermediate-to-high levels were seen in cortical layers III, V, VI and globus pallidus, as well as in the grafts, attesting to normal synaptogenesis and the formation of neurosecretory processes by transplanted mesencephalic cells.79 Histochemical correlates of dendrite outgrowth were studies using antibodies against microtubule-associated protein 2 (MAP2), a major MAP of brain tissue selectively localized in dendritic trees.94 MAP2 is known to promote microtubule assembly, and MAP2 molecules form side-arms in microtubules. MAP2 also interacts with neurofilaments, actin and other cytoskeletal elements. MAP2 is expressed at higher levels in some types of neurons than others. With immunocytochemistry, a dense network of MAP2-immunoreactive dendrites was seen in substantia nigra pars reticulata of normal midbrain. MAP2 immunoreactivity was seen in the form of crowded cell processes in the grafts and in their vicinity. Using in situ hybridization histochemistry with [32P]oligonucleotide probes, we studied the cellular localization of RNA transcripts for GAP-43 and MAP2 in the mesostriatal system of normal and weaver mutant mice.80 In addition, expression of the same messages was studied in ventral mesencephalic cell suspensions transplanted to the weaver striatum. Brains were quickly removed from the cranium after decapitation and frozen on dry ice. Coronal sections were cut at 20 µm in a cryostat. Synthetic oligonucleotide probes were labeled with [32P]. The DNA oligonucleotide probe sequences were complementary to rat GAP-43 mRNA and mouse MAP2 mRNA. Transcripts encoding GAP-43 were seen in abundance in normal mouse substantia nigra, in agreement with previous findings in the rat.83 In the autoradiographic emulsions, individual cells with a strong hybridization signal could be distinguished in the substantia nigra pars compacta. MAP2 mRNA hybridization signal was seen in the substantia nigra

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pars compacta of normal mice. The intensity of the autoradiographic signal was less strong than that for GAP-43. Transcripts encoding GAP-43 and MAP2 were progressively reduced in the weaver midbrain. Hybridization signal for GAP-43 mRNA was seen in a very small number of substantia nigra cells in weaver homozygotes. The hybridization signal obtained for MAP2 mRNA in the weaver substantia nigra was extremely low. Quantitative results on the density of hybridization signal for the mRNAs studied were collected in the substantia nigra pars compacta and in the caudate-putamen complex. The average signal density in caudate-putamen was about the same in both genotypes for both mRNAs. On the other hand, there were obvious differences in the density of hybridization signal in the substantia nigra of weaver mutant mice and that of wild-type animals, with a trend for lower values in the mutants for both mRNAs. Values for both mRNAs were consistently lower in the weaver substantia nigra (GAP-43 by 36%; MAP2 by 19%). GAP-43 mRNA was seen in a localized area of the host striatum in the transplanted side that corresponded to the graft, while the contralateral, nongrafted side had a low hybridization signal (Fig. 4.7.a). Similar results were seen for MAP2 mRNA, i.e., an area of high hybridization signal in the transplanted side, and low signal in the contralateral side (Fig. 4.7.b). In quantitative terms, GAP-43 and MAP2 mRNA were very close and did not show any statistically significant differences between the cell suspensions and the normal substantia nigra. The appearance of RNA message for GAP-43 and MAP2 in ventral mesencephalic grafts suggests that grafted cells mature in a similar manner to those in the normal substantia nigra in spite of their development in a foreign environment. The data offer a molecular correlate of axonal and dendritic protein gene transcription by transplanted mesencephalic cells. GAP-43, a protein kinase C substrate, is a rapidly transported axonal protein most prominently expressed in regenerating and developing nerves. GAP-43 mRNA is seen in many regions of the adult central nervous system, including entorhinal cortex, hippocampal pyramidal cells, olfactory bulb mitral cells, lateral habenula, substantia nigra pars compacta, ventral tegmental area, nucleus raphé dorsalis, nucleus centralis superior, locus coeruleus and cerebellar granule cells.83,95,96 Interestingly, the neuropil of the caudate-putamen complex is among the brain areas that retain a high content of GAP-43 throughout life.97 Considering that detectable levels persist in several adult neuronal groups, it has been suggested that GAP-43 may be engaged in accelerating process outgrowth and in structural remodeling.95,96 Further evidence supporting the involvement of GAP-43 in functional plasticity comes from the finding that mature mammalian central nervous system neurons regenerating into a peripheral nervous system graft display a marked increase in their content of GAP-43.98 Following selective lesions of the substantia nigra by 6-OHDA, hybridization signal for GAP-43 mRNA disappeared, indicating that high levels of GAP-43 are synthesized in the catecholamine neurons of the substantia nigra.83 The finding that GAP-43 mRNA is greatly reduced in the weaver substantia nigra, from which DA neurons are known to undergo degeneration and disappear, extend that observation. Previous transplantation studies in rats with 6-OHDA lesions of the nigrostriatal bundle showed GAP-43 immunoreactivity in solid ventral mesencephalic grafts;99 levels of immunoreactivity appeared to be higher during the first two weeks of graft development. Using a double-labeling technique of GAP-43 mRNA in situ hybridization and TH immunocytochemistry, Clayton

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Fig. 4.7. Autoradiographic films showing detection by in situ hybridization histochemistry of mRNAs for (a) GAP43 and (b) MAP2 in mesencephalic cell suspension grafts. Note the high levels of hybridization signal in the cell suspension grafts in the right hemisphere. Magnification x15. Reprinted with permission from: Solà C, Mengod G, Low WC et al. Eur J Neurosci 1993; 5:1442-1454. © Blackwell Science Ltd.

and Mahalik100 showed expression of GAP-43 mRNA mainly within grafted ventral mesencephalic neurons chemically identified as dopaminergic; this finding indicated that, although small non-DA cells may occasionally express GAP-43 mRNA as well, dopaminergic cells show high levels of signal and a vigorous outgrowth that may be linked to their ability to access a normal target cell population in vivo. The cytoskeletal protein MAP2, a high molecular weight MAP in the central nervous system, is selectively found in dendrites of developing and mature neurons. The dephosphorylation of MAP2 by a phosphatase, possibly the Ca++/calmodulin-dependent protein

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phosphatase calcineurin, may account for some of the effects of specific neurotransmitters on postsynaptic neurons.101 In addition to normal localization in neuronal dendrites, MAP2 immunoreactivity is seen in spheroid bodies in dystrophic dendrites of nigrostriatal DA neurons and in Lewy bodies in the substantia nigra pars compacta of Parkinsonian brains, whereas spheroid bodies in the pars reticulata and in the distal nigrostriatal axons do not react with MAP2 antibodies, in a pattern typical of axonal as opposed to dendritic spheroid bodies.102 Immunocytochemistry with anti-MAP2 antibodies in normal mouse substantia nigra reveals a dense network of dendrites extending from the pars compacta into the pars reticulata; an overall reduction of MAP2 immunoreactive dendrites is observed in the substantia nigra pars reticulata of weaver mutants in association with the loss of nigral DA neurons.103,104 In a transplantation study, embryonic ventral mesencephalon was grafted into the sciatic nerve of rats to examine the expression of neuronal cytoskeletal proteins in the grafts.105 Dense dendritic arbors were immunostained with MAP2 antibody; however, in one year old grafts, prominent cytoskeletal changes were observed in the somata, axons and dendrites, with an apparent regression of the dendritic trees as evidenced by MAP2 immunocytochemistry. Those grafts had been isolated in the peripheral nervous system, and such aberrant cytoskeletal modifications might have been associated with the lack of trophic interactions with appropriate efferent and afferent connections to and from the host tissue. The data on GAP-43 and MAP2 mRNA gene expression in the cell suspension grafts offer molecular correlates, respectively, of axonal and dendritic protein gene transcription following transplantation. These findings also indicate that grafted mesencephalic cells mature similarly to those in the wild-type substantia nigra, presenting normal levels of mRNAs expressed by mesencephalic cells in control mouse midbrain. Grafted mesencephalic cells mature similarly to those in the normal substantia nigra with respect to the expression of N-CAM, GAP-43, MAP2, neurofilament epitopes and synaptophysin. Some of these substances may form the biochemical substrate that underlies the cellular interactions between growing processes of donor neurons and their targets in the host caudate-putamen, and mediate selective processes of recognition, whereby inherent properties of fibers of grafted cells may locate their original sites of termination. References 1. Isacson O, Deacon T. Neural transplantation studies reveal the brain’s capacity for continuous reconstruction. Trends Neurosci 1997; 20:477-482. 2. Olson L. Regeneration in the adult central nervous system: Experimental repair strategies. Nature Med 1997; 3:1329-1335. 3. Björklund A, Lindvall O, Isacson O et al. Mechanisms of action of intracerebral neural implants: Studies on nigral and striatal grafts to the lesioned striatum. Trends Neurosci. 1098; 10:509-516. 4. Triarhou LC, Low WC, Ghetti B. Transplantation of ventral mesencephalic anlagen to hosts with genetic nigrostriatal dopamine deficiency. Proc Natl Acad Sci USA 1986; 83:8789-8793. 5. Triarhou LC, Low WC, Ghetti B. Synaptic investment of striatal cellular domains by grafted dopamine neurons in weaver mutant mice. Naturwissenschaften 1987; 74:591-593. 6. Triarhou LC, Low WC, Norton J et al. Reinstatement of synaptic connectivity in the striatum of weaver mutant mice following transplantation of ventral mesencephalic anlagen. J Neurocytol 1988; 17:233-243. 7. Triarhou LC, Low WC, Ghetti B. Genetic mesotelencephalic dopamine deficiency in weaver mutant mice: Reinstatement of neuronal connectivity by solid grafts of foetal mesencephalon. Fidia Res Series 1988; 15:183-192.

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8. Triarhou LC, Low WC, Doucet G et al. The weaver mutant mouse as a model for intrastriatal grafting of fetal DA neurons. In: Hefti F, Weiner WJ, eds. Progress in Parkinson’s Disease Research– 2. Mount Kisco, New York: Futura Publishing Co., 1992:389-400. 9. Björklund A, Stenevi U. Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants. Brain Res 1979; 177:555-560. 10. Björklund A, Dunnett SB, Stenevi U et al. Reinnervation of the denervated striatum by substantia nigra transplants: Functional consequences as revealed by pharmacological and sensorimotor testing. Brain Res 1980; 199:307-333. 11. Freund TF, Bolam JP, Björklund A et al. Efferent synaptic connections of grafted dopaminergic neurons reinnervating the host neostriatum: A tyrosine hydroxylase immunocytochemical study. J Neurosci 1985; 5:603-616. 12. Mahalik TJ, Finger TE, Strömberg I et al. Substantia nigra transplants into denervated striatum of the rat: Ultrastructure of graft and host interconnections. J Comp Neurol 1985; 240:60-70. 13. Strömberg I, Johnson S, Hoffer B et al. Reinnervation of dopamine-denervated striatum by substantia nigra transplants: Immunohistochemical and electrophysiological correlates. Neuroscience 1985; 14:981-990. 14. Aguayo A, Björklund A, Stenevi U et al. Fetal mesencephalic neurons survive and extend long axons across peripheral nervous system grafts inserted into the adult striatum. Neurosci Lett 1984; 45:53-58. 15. Pickel VM, Beckley SC, Joh TH et al. Ultrastructural immunocytochemical localization of tyrosine hydroxylase in the neostriatum. Brain Res 1981; 225:373-385. 16. Freund TF, Powell TF, Smith AD. TH immunoreactive boutons in synaptic contact with identified striatonigral neurons, with particular reference to dendritic spines. Neuroscience 1984; 13:1189-1215. 17. Purves D, Lichtman JW. Principles of Neural Development. Sunderland: Sinauer, 1985. 18. Beaudet A, Sotelo C. Synaptic remodelling of serotonin axon terminals in rat agranular cerebellum. Brain Res 1981; 206:305-329. 19. Triarhou LC, Norton J, Ghetti B. Synaptic connectivity of tyrosine hydroxylase immunoreactive nerve terminals in the striatum of normal, heterozygous and homozygous weaver mutant mice. J Neurocytol 1988; 17:221-232. 20. Specht LA, Pickel VM, Joh TH et al. Fine structure of the nigrostriatal anlage in fetal rat brain by immunocytochemical localization of tyrosine hydroxylase. Brain Res 1981; 218:49-65. 21. Triarhou LC, Low WC, Ghetti B. Dopaminergic–cholinergic interactions following transplantation of ventral mesencephalic grafts to the weaver mouse neostriatum. Neurology 1991; 41[Suppl 1]:398. 22. Nestler EJ, Greengard P. Distribution of protein I and regulation of its state of phosphorylation in the rabbit superior cervical ganglion. J Neurosci 1982; 2:1011-1023. 23. Mobley P, Greengard P. Evidence for widespread effects of noradrenaline on axon terminals in the rat frontal cortex. Proc Natl Acad Sci USA 1985; 82:945-947. 24. Low WC, Triarhou LC, Kaseda Y et al. Functional innervation of the striatum by ventral mesencephalic grafts in mice with inherited nigrostriatal dopamine deficiency. Brain Res 1987; 435:315-321. 25. Bolam JP, Freund TF, Björklund A et al. Synaptic input and local output of dopaminergic neurons in grafts that functionally reinnervate the host neostriatum. Exp Brain Res 1987; 68:131-146. 26. Triarhou LC, Brundin P, Doucet G et al. Intrastriatal implants of mesencephalic cell suspensions in weaver mutant mice: Ultrastructural relationships of dopaminergic dendrites and axons issued from the graft. Exp Brain Res 1990; 79:3-17. 27. Bohn MC, Cupit L, Marciano F et al. Adrenal medulla grafts enhance recovery of striatal dopaminergic fibers. Science 1987; 237:913-916. 28. Fuxe K, Hökfelt T, Nilsson O. Observations on the cellular localization of dopamine in the caudate nucleus of the rat. Z Zellforsch 1964; 63:701-706. 29. Doucet G, Descarries L, Garcia S. Quantification of the dopamine innervation in adult rat neostriatum. Neuroscience 1986; 19:427-445. 30. Hökfelt T. In vitro studies on central and peripheral monoamine neurons at the ultrastructural level. Z Zellforsch 1968; 91:1-74. 31. Clarke DJ, Brundin P, Strecker RE et al. Human fetal dopamine neurons grafted in a rat model of Parkinson’s disease: Ultrastructural evidence for synapse formation using tyrosine hydroxylase immunocytochemistry. Exp Brain Res 1988; 73:115-126.

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32. Triarhou LC, Low WC, Ghetti B. Dopamine neuron grafting to the weaver mouse neostriatum. Prog Brain Res 1990; 82:187-195. 33. Roffler-Tarlov S, Graybiel AM. The postnatal development of the dopamine-containing innervation of dorsal and ventral striatum: Effects of the weaver gene. J Neurosci 1987; 7:2364-2372. 34. Garcia-Segura LM, Baetens D, Roth J et al. Immunohistochemical mapping of calcium-binding protein immunoreactivity in the rat central nervous system. Brain Res 1984; 296:75-86. 35. Enderlin S, Norman AW, Celio MR. Ontogeny of the calcium binding protein calbindin D-28k in the rat nervous system. Anat Embryol (Berl) 1987; 177:15-28. 36. Schultzberg M, Dunnett SB, Björklund A et al. Dopamine and cholecystokinin immunoreactive neurons in mesencephalic grafts reinnervating the neostriatum: Evidence for selective growth regulation. Neuroscience 1984; 12:17-32. 37. Fallon JH. Collateralization of monoamine neurons: Mesotelencephalic dopamine projections to caudate, septum, and frontal cortex. J Neurosci 1981; 1:1361-1368. 38. Lindvall O. Mesencephalic dopaminergic afferents to the lateral septal nucleus of the rat. Brain Res 1975; 87:89-95. 39. Thierry AM, Blanc G, Sobel A et al. Dopaminergic terminals in the rat cortex. Science 1973; 182:499-501. 40. Fuxe K, Hökfelt T, Johansson O et al. The origin of the dopamine nerve terminals in limbic and frontal cortex. Evidence for meso-cortico dopamine neurons. Brain Res 1974: 82:349-355. 41. Lindvall O, Björklund A, Moore RY et al. Mesencephalic dopamine neurons projecting to neocortex. Brain Res 1974; 81:325-331. 42. Lindvall O, Björklund A, Divac I. Organization of catecholamine neurons projecting to the frontal cortex in rat. Brain Res 1978; 142:1-24. 43. Emson PC, Koob GF. The origin and distribution of dopamine-containing afferents to the rat frontal cortex. Brain Res 1978; 142:249-267. 44. Björklund A, Lindvall O. Dopamine-containing systems in the CNS. In: Björklund A, Hökfelt T (eds). Handbook of Chemical Neuroanatomy, vol 2. Amsterdam–New York–Oxford: Elsevier, 1984:55-122. 45. Van Eden CG, Hoorneman EMD, Buijs RM et al. Immunocytochemical localization of dopamine in the prefrontal cortex of the rat at the light and electron microscopic level. Neuroscience 1987; 22:849-862. 46. Routtenberg A. The reward system of the brain. Sci Am 1978; 239:154-164. 47. Scarnati E, Forchetti C, Ruggieri S et al. Dopamine and dementia. An animal model with destruction of the mesocortical dopaminergic pathway: A preliminary study. In: Amaducci L, Davison AN, Antuono P, eds. Aging of the Brain and Dementia. New York: Raven Press, 1980:139-145. 48. Fink JS, Smith GP. Mesolimbic and mesocortical dopaminergic neurons are necessary for normal exploratory behavior in rats. Neurosci Lett 1980; 17:61-65. 49. Thierry AM, Tassin JP, Blanc G et al. Selective activation of the mesocortical DA system by stress. Nature (Lond) 1976; 263:242-244. 50. Scatton B, Rouquier L, Javoy-Agid F et al. Dopamine deficiency in the cerebral cortex in Parkinson disease. Neurology 1982; 32:1039-1040. 51. Candy JM, Perry RH, Perry EK et al. Pathological changes in the nucleus of Meynert in Alzheimer’s and Parkinson’s diseases. J Neurol Sci 1983; 59:277-289. 52. Whitehouse PJ, Hedreen JC, White CL III et al. Basal forebrain neurons in the dementia of Parkinson disease. Ann Neurol 1983; 13:243-248. 53. Brozoski TJ, Brown RM, Rosvold HE et al. Cognitive deficit caused by regional depletion of dopamine in prefrontal cortex of rhesus monkey. Science 1979; 205:929-932. 54. Schmidt MJ, Sawyer BD, Perry KW et al. Dopamine deficiency in the weaver mutant mouse. J Neurosci 1982; 2:376-380. 55. Triarhou LC, Norton J, Ghetti B (1988) Mesencephalic dopamine cell deficit involves areas A8, A9 and A10 in weaver mutant mice. Exp Brain Res 1988; 70:256-265. 56. Triarhou LC, Low WC, Ghetti B. Layer-specific innervation of the dopamine-deficient frontal cortex in weaver mutant mice by grafted mesencephalic dopaminergic neurons. Cell Tissue Res 1988; 254:11-15. 57. Langley JN. Note on regeneration of preganglionic fibres of the sympathetic. J Physiol 1895; 18:280-284. 58. Cajal SR (1929) Studies on Vertebrate Neurogenesis (Guth L, transl). Springfield: Charles C Thomas, 1960.

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59. Sperry RW. Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc Natl Acad Sci USA 1963; 50:703-710. 60. Sperry RW, Arora HL. Selectivity in regeneration of the oculomotor nerve in the cichlid fish, Astronatus ocellatus. J Embryol Exp Morphol 1965; 14:307-317. 61. Hoh JFY. Selective and nonselective reinnervation of fast-twitch and slow-twitch rat skeletal muscle. J Physiol 1975; 251:791-801. 62. Van Essen DC, Jansen JKS. The specificity of reinnervation by identified sensory and motor neurons in the leech. J Comp Neurol 1977; 171:433-454. 63. Purves D, Thompson W, Yip JW. Re-innervation of ganglia transplanted to the neck from different levels of the guinea-pig sympathetic chain. J Physiol 1981; 313:49-63. 64. Landmesser L, Pilar G. Selective reinnervation of two cell populations in the adult pigeon ciliary ganglion. J Physiol 1970; 211:203-216. 65. Sperry RW (1943) Effect of 180 degree rotation of the retinal field on visuomotor coordination. J Exp Zool 1943; 92:263-279. 66. Stone LS. Functional polarization in the retinal development and its reestablishment in regenerating retinae of rotated grafted eyes. Proc Soc Exp Biol Med 1944; 57:13-14. 67. Attardi DG, Sperry RW. Preferential selection of central pathways by regenerating optic fibers. Exp Neurol 1963; 7:46-64. 68. Björklund A, Stenevi U, Schmidt RH et al. Survival and growth of nigral cell suspensions implanted in different brain sites. Acta Physiol Scand 1983; [Suppl] 522:9-18. 69. Herman JP, Choulli K, Geffard M et al. Reinnervation of the nucleus accumbens and frontal cortex of the rat by dopaminergic grafts and effects on hoarding behavior. Brain Res 1986; 372:210-216. 70. Björklund A, Stenevi U, Svendgaard N-A. Growth of transplanted monoaminergic neurones into the adult hippocampus along the perforant path. Nature (Lond) 1976; 262:787-790. 71. Björklund A, Stenevi U. Reformation of the severed septohippocampal cholinergic pathway in the adult rat by transplanted septal neurons. Cell Tissue Res 1977; 185:289-302. 72. Dunnett SB, Low WC, Iversen SD et al. Septal transplants restore maze learning in rats with fornix-fimbria lesions. Brain Res 1982; 251:335-348. 73. Sharkey MA, Steedman JG, Lund RD et al. Tectal transplants into the occipital cortex of the newborn rat. Dev Brain Res 1987; 31:119-123. 74. Björklund A, Lindvall O. Dopamine in dendrites of substantia nigra neurons: Suggestions for a role in dendritic terminals. Brain Res 1975; 83:531-537. 75. Chéramy A, Leviel V, Glowinski J. Dendritic release of dopamine in the substantia nigra. Nature (Lond) 1981; 289:537-542. 76. Korf J, Zieleman M, Westerink BHC. Dopamine release in substantia nigra? Nature (Lond) 1976; 260:257-258. 77. Geffen LB, Jessell TM, Cuello AC et al. Release of dopamine from dendrites in rat substantia nigra. Nature 1976; 260:258-260. 78. Wassef M, Berod A, Sotelo C. Dopaminergic dendrites in the pars reticulata of the rat substantia nigra and their striatal input: Combined immunocytochemical localization of tyrosine hydroxylase and anterograde degeneration. Neuroscience 1981; 6:2125-2139. 79. Triarhou LC, Solà C, Mengod G et al. Ventral mesencephalic grafts in the neostriatum of the weaver mutant mouse: Structural molecule and receptor studies. Cell Transpl 1995; 4:39-48. 80. Solà C, Mengod G, Low WC et al. Regional distribution of amyloid β-protein precursor, growthassociated phosphoprotein-43 and microtubule-associated protein 2 mRNAs in the nigrostriatal system of normal and weaver mutant mice and effects of ventral mesencephalic grafts. Eur J Neurosci 1993; 5:1442-1454. 81. Edelman GM, Crossin KL. Cell adhesion molecules: Implications for a molecular histology. Ann Rev Biochem 1991; 60:155-190. 82. Sanes JR. Extracellular matrix molecules that influence neural development. Ann Rev Neurosci 1989; 12:491-516. 83. Bendotti C, Servadio A, Samanin R. Distribution of GAP-43 mRNA in the brain stem of adult rats as evidenced by in situ hybridization: Localization within monoaminergic neurons. J Neurosci 1991; 11:600-607. 84. Benowitz LI, Apostolides PJ, Perrone-Bizzozero N et al. Anatomical distribution of the growthassociated protein GAP-43/B-50 in the adult rat brain. J Neurosci 1988; 8:339-352.

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85. Meiri K, Bickerstaff LE, Schwob JE. Monoclonal antibodies show that kinase C phosphorylation of GAP-43 during axonogenesis is both spatially and temporally restricted in vivo. J Cell Biol 1991; 112:991-1005. 86. Meiri KF, Pfenninger KH, Willard MB. Growth-associated protein, GAP-43, a polypeptide that is induced when neurons extend axons, is a component of growth cones and corresponds to pp46, a major polypeptide of a subcellular fraction enriched in growth cones. Proc Natl Acad Sci USA 1986; 83:3537-3541. 87. Palacios G, Mengod G, Sarasa M et al. De novo synthesis of GAP-43: In situ hybridization histochemistry and light and electron microscopy immunocytochemical studies in regenerating motor neurons of cranial nerve nuclei in the rat brain. Mol Brain Res 1994; 24:107-117. 88. Poltorak M, Freed WJ, Sternberger LA et al. A comparison of intraventricular and intraparenchymal cerebellar allografts in rat brain: evidence for normal phosphorylation of neurofilaments. J Neuroimmunol 1988; 20:63-72. 89. Sternberger LA, Sternberger NH. Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. Proc Natl Acad Sci USA 1983; 80:6126-6130. 90. Jahn R, Schiebler W, Ouimet C et al. A 38,000-dalton membrane protein (p38) present in synaptic vesicles. Proc Natl Acad Sci USA 1985; 82:4137-4141. 91. Wiedenmann B, Franke WW. Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell 1985; 41:1017-1028. 92. Goslin K, Schreyer DJ, Skene JH et al. Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones. Nature (Lond) 1988; 336:672-674. 93. Kosik KS, Orecchio LD, Bruns GA et al. Human GAP-43: its deduced amino acid sequence and chromosomal localization in mouse and human. Neuron 1988; 1:127-132. 94. Matus A. Microtubule-associated proteins: their potential role in determining neuronal morphology. Ann Rev Neurosci 1988; 11:29-44. 95. De la Monte SM, Federoff HJ, Ng S et al. GAP-43 gene expression during development: Persistence in a distinctive set of neurons in the mature central nervous system. Dev Brain Res 1989; 46:161-168. 96. Meberg PJ, Routtenberg A. Selective expression of protein F1/(GAP-43) mRNA in pyramidal but not granule cells of the hippocampus. Neuroscience 1991; 45:721-733. 97. DiFiglia M, Roberts RC, Benowitz LI. Immunoreactive GAP-43 in the neuropil of adult rat neostriatum: Localization in unmyelinated fibers, axon terminals and dendritic spines. J Comp Neurol 1990; 302:992-1001. 98. Campbell G, Anderson PN, Turmaine M et al. GAP-43 in the axons of mammalian CNS neurons regenerating into peripheral nerve grafts. Exp Brain Res 1991; 87:67-74. 99. Clayton GH, Mahalik TJ, Finger TE. GAP-43 and 5B4-CAM immunoreactivity during the development of transplanted fetal mesencephalic neurons. Exp Neurol 1991; 114:1-10. 100. Clayton GH, Mahalik TJ. GAP-43 expression in neurochemically identified subpopulations of neurons within fetal ventral mesencephalic transplants. Restor Neurol Neurosci 1992; 4:160. 101. Halpain S, Greengard P. Activation of NMDA receptors induces rapid dephosphorylation of the cytoskeletal protein MAP2. Neuron 1990; 5:237-246. 102. Yamada T, Akiyama H, McGeer PL. Two types of spheroid bodies in the nigral neurons in Parkinson’s disease. Can J Neurol Sci 1991; 18:287-294. 103. Triarhou LC, Ghetti B. Further characterization of the dopaminergic dendrite deficit in substantia nigra pars reticulata of heterozygous and homozygous weaver mutant mice: Golgi, MAP2 and synaptic connectivity studies. Soc Neurosci Abstr 1991; 17:159. 104. Triarhou LC. Weaver gene expression in central nervous system. In: Conn PM, ed. Methods in Neurosciences, vol 9: Gene Expression in Neural Tissues. San Diego: Academic Press, 1992:209-227. 105. Doering LC. Transplantation of fetal CNS tissue into the peripheral nervous system: A model to study aberrant changes in the neuronal cytoskeleton. J Neural Transpl Plast 1991; 2:193-205.

CHAPTER 5

Neurochemical Indices of Functional Restoration Dopamine Uptake Markers

A

general asset of the weaver model is that one can study graft development at the same time as the animal’s own dopamine (DA) system continues to undergo a progressive degeneration, which is reflected in the relatively slow loss of cells in the mutant, compared with the rapid and traumatic loss in neurotoxic lesion experiments. Adaptive changes to the striatal DA denervation also differ in many ways between the weaver mutant and the 6-hydroxydopamine (6-OHDA) model.1 In that respect, studies with lesion models2 and with genetic dissection through mutations3 may provide a complementary profile of graft effects and of graft-recipient interactions at structural and functional levels, pertinent to understanding cellular mechanisms of graft action both in the basic neuroscience and in the clinical setting, where 6-L-[18F]fluorodopa uptake is used to monitor the survival of human mesencephalic grafts in Parkinsonian patients.4,5

Autoradiography of [3H]Dopamine Uptake Mesencephalic grafts into the denervated rat striatum can restore striatal DA tissue content and spontaneous DA release up to normal levels.6-9 The ability of grafted DA neurons to reinnervate the weaver mouse neostriatum is of particular interest since the mesencephalic neuronal degeneration in the host is spontaneous, due to the genetic defect, and striatal denervation is incomplete. Previous nigral transplantation studies have, with a few exceptions,10-12 been conducted in rats with near complete neurotoxic lesions of the mesostriatal DA system, rendering the host striatum virtually devoid of DA innervation. In the clinical context, fetal DA neurons grafted to Parkinsonian patients who do not totally lack striatal DA innervation will have to compete with remaining host mesostriatal DA fibers in establishing a novel DA terminal network. In that regard, the weaver striatum represents an interesting model of the human disease. A quantitative autoradiographic method of in vitro [3H]DA uptake by brain slides developed earlier14 was used to evaluate the distribution and extent of striatal DA reinnervation resulting from cell suspension grafts of fetal ventral mesencephalic tissue in weaver mutant mice.15 Brain slices from normal mice and unilaterally grafted weaver mice were incubated in [3H]DA, in the presence of desipramine and pargyline, three to nine months after graft surgery. Semithin sections from the fixed and re-embedded slices were subsequently exposed on tritium sensitive film and afterwards dipped in nuclear emulsion for light microscopic autoradiography. Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease, by Lazaros C. Triarhou. ©2001 Eurekah.com.

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The results revealed a virtually complete absence of DA axons in the dorsomedial quadrant of the weaver neostriatum and an increasing density of DA innervation toward ventrolateral areas. In nonoperated mutants, two different labeling patterns were observed in distinct regions of the striatum. The first pattern consisted of small silver grain clusters standing out against a relatively low background of diffuse silver grains. These silver grain clusters were found essentially in the neuropil, avoided neuronal somata and myelinated fascicles, and had the typical autoradiographic appearance of DA varicosities.13 Regionally, they were found in very high numbers throughout the striatum of normal mice. In weaver mutant mice, silver grain clusters were largely restricted to a narrow ventral and periventricular zone of the caudate-putamen complex and to the ventral striatal complex (i.e., nucleus accumbens and olfactory tubercle). Nevertheless, the number of such labeled varicosities was reduced in the lateral portion of both the nucleus accumbens and the olfactory tubercle. The second labeling pattern was characteristic of the weaver neostriatum: being barely above background in the dorsomedial quadrant and covering with high density the lateral half of the neostriatum in some mice, it was restricted to the ventrolateral quadrant of the rostral levels in other mice. Such labeling consisted of a low to moderately high density of diffuse silver grains with very little clustering. Similarly to silver grain clusters, it was confined to the neuropil and essentially avoided myelinated fascicles. The diffuse silver grain autoradiographic pattern suggested a dysfunctioning uptake-storage mechanism in the residual dopaminergic fibers of this region. The average density values were approximately 10-fold higher in the ventral and ventrolateral parts of the caudate-putamen than in dorsal and dorsomedial areas. In comparison, the DA innervation of wild-type striatum was much more uniform, and the largest difference between the dorsolateral and ventromedial areas was 2-fold. Consistent with previous observations in the cell body regions of the normal midbrain processed for DA uptake in vitro, transplanted dopaminergic somata were not visualized in the autoradiographs. Figure 5.1 illustrates examples of graft-derived DA reinnervation in the weaver neostriatum. DA varicosities of graft origin clearly corresponded to the normal type of silver grain clusters in adjacent autoradiographs. This made them easily distinguishable against the diffusely labeled residual host DA innervation in dorsal striatal aspects. There was considerable variation in the extent of reinnervation among mice, but graft-derived fibers were mostly confined to the dorsal one-third to one-half of the neostriatum. In contrast to the intrinsic weaver neostriatal DA innervation, DA fibers of graft origin exhibited the normal, clustered type of varicosity labeling. The computerized image analysis of silver grain density in film autoradiographs was calibrated by counting these labeled varicosities in selected areas of light microscopic autoradiographs from the same sections. Comparisons between grafted and nongrafted sides for each striatal subdivision showed significant differences in most regions of the dorsal neostriatum at three coronal levels examined. In grafted weaver mice, the average DA reinnervation of neostriatal tissue surrounding the grafts was about 20% of the density seen in wild-type mice in the area closest to the graft. In the most extensively reinnervated animal, the graft-derived DA varicosities reached 80% of normal density in the immediate vicinity of the graft. In the poorest graft, the corresponding value was 3%. There was a gradual decline in DA reinnervation with increasing distance up to 1.0-1.4 mm away from the graft. Observations on long-term mice, at nine months after transplantation, suggest that the graft-derived DA fiber outgrowth may become reduced in the affected striatum with

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Fig. 5.1. Film autoradiographs of [3H]DA uptake and storage by brain slices, showing the distribution of DA innervation in the nongrafted striatum of a weaver mutant mouse (left hemisphere), and the superimposed dopaminergic “neoinnervation” from a mesencephalic cell suspension graft (right hemisphere). Upper micrograph is from a more rostral level, and lower micrograph is from a more caudal level. The outline of the graft is evident in the lower micrograph, as well as a gradient of the reinnervation away from the graft. Magnification: x12. Reprinted with permission from: Doucet G, Brundin P, Seth S et al. Exp Brain Res 1989; 77:552-568. © SpringerVerlag.

time, in spite of good survival of grafted neurons. Ventral parts of the neostriatum, which contained higher numbers of residual intrinsic DA fibers, were much more sparsely reinnervated than dorsal and dorsomedial areas. The poor reinnervation of the ventral neostriatum is compatible with the view that the residual intrinsic afferent input may prevent a robust graft-derived fiber ingrowth in that area. The adopted autoradiographic method offers several advantages for the quantification of DA innervation in neostriatum. DA storage sites visualized as silver grain clusters

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are presumably equivalent to the terminal release sites and they are probably the most meaningful morphological unit for a quantitative description of the DA terminal network. The specificity of silver grain clusters as labels for DA axonal varicosities in normal rat striatum under similar experimental conditions has been discussed at length elsewhere.13 However, in the study on weaver mutants, the [3H]DA uptake and autoradiography were performed on mouse striata containing heterotopically grafted DA neurons. Under those in vitro conditions, DA cell bodies were not labeled; the eventual contribution of graft-derived DA dendritic labeling in the host striatum remains unclear. In a parallel electron microscopic study, the overall ratio of tyrosine hydroxylase-immunolabeled axons to dendrites of graft origin was found to be three-to-one: about two-to-one in an area 0.5 mm proximal to the graft, and about twenty-to-one in an area 0.5-1.0 mm distal to it.15 Thus, even if DA dendrites were labeled as strongly as DA axonal varicosities, the latter would still represent the vast majority of silver grain clusters in the host striatum. The DA varicosities that originate in the graft produce silver grain clusters of the normal type of appearance. They are thus readily distinguishable from the diffuse labeling of the intrinsic weaver neostriatal innervation. The DA reinnervation by the graft was largely restricted to the dorsal region of the neostriatum. In part, this could be explained by the fact that the bulk of the grafted tissue was located in that region. However, even in cases when the graft extended into more ventral regions of neostriatum there was little innervation ventrally. In fact, the density of DA varicosities just ventral to the graft was markedly lower in cases when the graft reached more ventral levels. In some of these cases, there were no DA terminals at all ventral to the graft. This is in marked contrast to previous observations in 6-OHDA denervated rat striata where DA fiber outgrowth from a graft was found to be symmetrical in all directions.16 A plausible explanation may relate to the fact that the weaver striatum is not denervated to the same extent ventrally as it is dorsally. Transplantation studies in the hippocampal formation17-19 have indicated that vacated synaptic sites are critical for the growth of graft axons into an adult host brain region, and this may apply for ingrowing DA axons as well.11 The normal appearance of silver grain clusters produced by DA varicosities originating in the graft suggests that they have functional DA uptake-storage mechanisms. In ultrastructural studies, it has been estimated that virtually all DA terminals from ventral mesencephalic grafts display synaptic differentiations in weaver hosts.20 Further, the pattern of connectivity with target profiles was comparable to that previously reported for mesencephalic grafts in the 6-OHDA denervated neostriatum of rats21,22 and to the normal DA innervation in mouse23 or rat neostriatum.24 Thus, after survival periods of three to six months, it appears that grafted DA neurons establish a more functional innervation of the host caudate-putamen than the intrinsic weaver nigrostriatal DA neurons, both with respect to uptake-storage mechanisms and synaptic connectivity. Developmental studies in weaver mice have indicated that the dorsal neostriatum never receives much DA innervation.25 Therefore, with figures of 20% and up to 80% reinnervation in this region, it seems likely that numerous postsynaptic sites remain receptive to connection by grafted DA neurons even though they had never received any DA contacts during development. Since the number of DA D1 and D2 receptors is normal26 or even increased27 in adult weaver mutants, such receptivity may also be functional. In summary, transplantation of wild-type fetal mesencephalic tissue into the weaver neostriatum, an environment where intrinsic DA axons are strongly deficient, provides a quantitatively significant DA reinnervation of the host tissue that is functional with regards

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to DA uptake-storage mechanisms. Such a graft-derived DA innervation is densest in the tissue surrounding the graft and becomes gradually diminished with distance.

Autoradiography of [ 3H]mazindol and [ 3H]GBR 12935 Binding The reductions in DA uptake indices in the weaver mouse resemble those seen in human neurological conditions characterized by striatal DA deficiency, such as Parkinson’s disease and progressive supranuclear palsy, where [3H]mazindol binding to postmortem brain tissue is reduced as well.28 The DA uptake system was analyzed in the striatal complex of wild-type (+/+) and homozygous weaver mutant (wv/wv) mice and in weaver mutants that had received intrastriatal grafts of ventral mesencephalic cell suspensions in order to determine the degree of functional reinnervation.29 Whole brains were frozen on dry ice for autoradiography using [3H]mazindol as a ligand for DA uptake sites. Brains were cut into 10 µm thick coronal sections in a cryostat. Sections were then thaw-mounted on acid-cleaned, gelatin-coated slides. Autoradiographic studies were carried out using [3H]mazindol as a ligand for DA uptake sites. The incubation medium also contained 0.3 µM desipramine to block binding to noradrenaline uptake sites. [3H]Mazindol binding was studied in serial coronal sections of wild-type mice, nonoperated weaver mice, and of weaver mutants with grafts to the right hemisphere. [3H]Mazindol binding was dense in the +/+ striatum, throughout both the dorsal and ventral aspects. [3H]mazindol binding in the nongrafted wv/wv striatum was clearly reduced compared to the wild-type. Such a depletion was most prevalent in the dorsal caudateputamen (86-87% reduction). [3H]Mazindol binding in the ventral caudate-putamen complex was reduced by 67% relative to the control. In the [3H]mazindol binding autoradiographic films, a much higher silver grain density was evident in the dorsal part of the grafted striata. The animals that had received transplants had on the average 40-64% higher [3H]mazindol binding values in the right side than in the left, depending on dorsoventral topography. Such a graft-induced partial restoration of [3H]mazindol binding in the right side reached 21% and 46% of the wildtype values in the dorsal and ventral regions of the striatum, respectively. The increase in [3H]mazindol binding to DA uptake sites is 40-64% in the various striatal quadrants of transplanted weaver mice compared to nongrafted mutants; the restoration of [3H]mazindol binding is partial as well and represents 21-46% of the levels measured in the striatum of wild-type mice. Biochemical and pharmacological studies suggest that in rodent striatum, [3H]mazindol is functionally related to the DA uptake carrier complex, to which it binds with high affinity; as a matter of fact, mazindol has the ability to inhibit the reuptake process of DA.30,31 There are other substances that inhibit DA reuptake and are used to radiolabel DA uptake sites for autoradiography. These include cocaine, CFI, GBR 12909 and GBR 12935, and the results of several studies are in agreement with a one-site model in which all of these markers share a common binding site.31-35 The functional integration of fetal mesencephalic grafts into the striatum of weaver mutant mice has been studied by investigating the topographical levels of the DA transporter by means of autoradiography for [3H]GBR 12935, another ligand that labels DA uptake sites. Recipient weaver mice showed a 55-65% increase in [3H]GBR 12935 binding in the grafted dorsal striatum over the opposite side.36

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The dopaminergic reinnervation of the striatum by mesencephalic cell suspensions has been also studied in rats subjected to 6-OHDA denervation, using autoradiography with [3H]GBR 12935.37 In that study, the depth of reinnervation reached several hundred µm in the striatum of the host, and it was estimated that the reinnervated area represented on the average 83% of the striatal surface at specific levels in the coronal plane. Tests of rotational responses using the DA D1 receptor agonist SKF 38393 and the DA D2 receptor agonist LY 171555 showed that, in graft-bearing animals, the lesion-induced contralateral turning bias was completely abolished. The same ligand, [3H]GBR 12935, was used to quantify the graft-induced innervation of the host in a further study in rats.38 Deposits of grafted tissue were surrounded by a halo of dense [3H]GBR 12935 binding, which showed a pronounced gradient from the graft-host border to the periphery of the host caudate-putamen complex. A clear graftderived DA reinnervation extended for several hundred µm into the striatum of the host. The 6-OHDA lesion-induced ipsiversive turning response evoked by the administration of amphetamine was markedly attenuated or reversed by the grafts. In addition, the grafts affected the rate of contraversive turning elicited by the administration of apomorphine.

Synaptosomal Uptake of [ 3H]Dopamine

Biochemical studies on the high-affinity uptake of [3H]DA by synaptosomal preparations of striatum have shown defective DA uptake in both the dorsal striatum and nucleus accumbens.39-43 Assays of [3H]DA uptake were carried out in vitro in striatal synaptosomal fractions from wild-type mice (+/+) and from the two hemispheres of weaver mutant mice (wv/wv) that had received unilateral grafts of mesencephalic cell suspensions to the right side. Striata were homogenized for the preparation of synaptosomal fractions by differential ultracentrifugation.29 Assays of [3H]DA uptake were carried out in vitro in striatal synaptosomal fractions from wild-type mice (+/+) and from the two hemispheres of homozygous weaver mutants (wv/wv) that had received unilateral grafts of mesencephalic cell suspensions to the right side. The unbiased estimators of parametric group means for net [3H]DA uptake were 49.6, 7.8 and 10.0 pmol/mg-protein/2-min for wild-type, nongrafted weaver and grafted weaver groups, respectively. [3H]DA uptake in wild-type mice differed significantly from both nongrafted and grafted weaver striata. In comparing [3H]DA uptake between right and left sides of the recipient weaver mice, there was a significant side effect between nongrafted and grafted sides. The right side was higher than the left by 28% (computed as average right over average left, or by 38% [computed as the mean of all individual (R-L)/L values, which may be a more appropriate estimator, as each observation for graft treatment is paired with the observation for no treatment in the same animal]. The summarized findings indicate that grafts of fetal mesencephalic cell suspensions placed into the striatum of weaver mutants lead to increases in DA uptake parameters. The synaptosomal uptake of [3H]DA into preparations of entire striatum is increased by 28-38% over the nongrafted side, which means that the extent of DA effects in areas immediately surrounding the graft and associated strictly with motoric activity may be underestimated, and regional values could be higher. The restoration in overall DA uptake function is partial and represents 20% of normal. By conducting measurements of synaptosomal DA uptake in vitro and of the binding characteristics of mazindol in brain slices by autoradiography, one has the advantage of combining the anatomical resolution of uptake site visualization with a dynamic indicator of function for DA uptake in the nerve terminal. In other words, the two approaches are

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complementary: the binding of [3H]mazindol to the DA uptake carrier is an indicator of the number of DA uptake sites, and the in vitro synaptosomal uptake of [3H]DA is considered a functional marker, as it indicates the effectiveness of the DA uptake carrier system in transporting DA into the nerve terminals.

Dopamine Receptors Tissue Binding of [ 3H]Spiperone DA D2 receptor binding is increased in the striatum of 5-6 month old weaver mutant mice27 in response to the loss of DA neurons in the midbrain and the decrease in DA content. Specific [3H]spiperone binding was measured in the dorsolateral, dorsomedial and ventrolateral striatum and in the nucleus accumbens of wild-type and homozygous weaver mutants with or without mesencephalic grafts to determine if such grafts would reverse the increase in DA D2 receptor binding in the striatum. Aspiration cavities were prepared in the cortex of weaver mice, and ventral mesencephalic tissue from E14-E15 fetuses was placed on the surface of the right dorsal striatum when the recipients were 3 months old. Compared with the nongrafted side, the decreases in [3H]spiperone binding on the right (grafted) side in the dorsolateral and dorsomedial striatum of the transplanted group were significantly greater than the differences in the other two groups. Since the grafts were introduced at three months of age, at which no significant difference in binding is seen between wv/wv and +/+, it appears that the grafts may prevent the increase in DA D2 receptor binding which is seen in nongrafted weaver mice at six months of age. Specific [3H]spiperone binding was determined in groups grafted, cavity-only and unoperated. The following results were obtained: in the dorsolateral striatum the L-R difference in [3H]spiperone binding for the transplanted group was significantly greater than that for the cavity-only group, but not significantly different from that for the unoperated group; in the dorsomedial striatum the L-R difference in binding for the transplanted group was significantly greater than those for both the cavity-only group and the unoperated group; in the nucleus accumbens none of the L-R differences were significantly different. Since DA D2 receptor binding is increased in wv/wv compared to +/+ at six months of age, we tested the effect of grafting at about three months of age on D2 receptor binding measured at six months. In mice which received ventral mesencephalic grafts unilaterally, specific [3H]spiperone binding was decreased in the dorsomedial and dorsolateral striatum, but not in the nucleus accumbens on the transplanted side. Such a decrease may reflect prevention of the upregulation of DA D2 receptors, which otherwise occurs by six months in the dorsolateral striatum. This may in turn suggest that synapses formed by graft-derived DA axon terminals20 may influence the neurochemistry of host striatal neurons. Our present results are analogous to those found in rats with 6-OHDA lesions with fetal nigral transplants, which normalize the increase in DA D2 receptors of the striatum.44,45 A factor to consider is the status of DA D1 receptors in the weaver mouse. DA D1 receptors are in fact more abundant than DA D2 receptors in striatum, nucleus accumbens and substantia nigra, and in the ventral tegmental area DA D2 receptors are undetectable.46 DA D1 and D2 receptors seem to interact in the striatum and the interaction is different, depending on the presence of a lesion.47 While DA D1 receptor binding is unchanged in the weaver,26 it is possible that changes in DA D1 receptors or their interaction with DA D2 receptors in the nucleus accumbens after transplantation might account for a correlation of turning behavior and [3H]spiperone binding there after transplantation.

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This could occur without any overall change in [3H]spiperone binding which occurs to DA D1 sites at much lower affinity than to DA D2 sites.

Autoradiography of [ 3H]CV 205-502 Binding and Distribution of Dopamine Receptor RNA Transcripts

In situ hybridization histochemistry with [32P]oligonucleotide probes was employed to investigate the cellular localization of DA receptor RNA transcripts; ligand binding autoradiography was carried out to compare receptor distribution with receptor gene expression.48 DNA oligonucleotide probe sequences were complementary to the coding region of human DA D1 receptor mRNA, and rat DA D2 and D3 receptor mRNAs.49-51 DA receptor autoradiography, using the D2/D3 agonist [3H]CV 205-502 as a ligand, gave strong binding in the caudate-putamen complex of wild-type mice, as well as in nucleus accumbens, isles of Calleja, olfactory tubercle, substantia nigra and ventral tegmental area; intermediate levels were seen in lateral septal nucleus.48 Comparative analyses in the rat brain combining binding autoradiography of D2/D3 ligands with in situ hybridization histochemistry for DA D2 and D3 receptor RNA messages suggest that binding sites are of different pharmacology, consistent with the presence of D3 sites in the isles of Calleja, a predominance of D2 in caudate-putamen, and a coexistence of D2 and D3 in nucleus accumbens.49 Homozygous weaver mice showed a reduction in [3H]CV 205-502 binding in substantia nigra and ventral tegmental area. Interestingly, there was no binding in the nigral transplants (Fig. 5.2), despite the presence of tyrosine hydroxylase-immunopositive cells in the grafts in adjacent serial sections, indicating an apparent abnormality of grafted cells. DA D1 receptor mRNA in +/+ mice was detected at high levels in caudate-putamen, nucleus accumbens, olfactory tubercle, isles of Calleja, and at intermediate-to-low levels in cerebral cortex, particularly entorhinal cortex. In the substantia nigra and ventral tegmental area, signal was not detectable. Thus, the anatomical distribution of DA D1 receptor mRNA was comparable to that seen in the rat brain.51 In wv/wv mice, a reduction of about 15% in signal strength was seen in the caudate-putamen nucleus. DA D2 receptor mRNA was seen at high levels in the caudate-putamen, nucleus accumbens, olfactory tubercle, substantia nigra, ventral tegmental area and intermediate lobe of the pituitary of +/+ mice; intermediate-to-low levels were seen in lateral septal nucleus. In wv/wv mice, there were reductions in signal in the substantia nigra and ventral tegmental area of the orders of 43% and 30%, respectively. The striatum was essentially unchanged. No hybridization signal was detected in the grafts for DA D2 receptor mRNA, which is normally expressed by nigral DA neurons. DA D3 receptor mRNA signal was strong in the isles of Calleja in both +/+ and wv/wv animals, and a slight increase in message was seen in the nucleus accumbens of weaver mutants. Concerning DA receptors in the striatum, it is known that the D2 receptor supersensitivity induced by 6-OHDA treatment is normalized by a nigral graft.37,44,52 In the wv/wv striatum, there is only a slight increase in DA D2 receptor binding in the dorsolateral quadrant in the order of 21%, which can be reversed by intrastriatal ventral mesencephalic grafts.27 The findings reported in the present set of studies suggest that specific structural properties of grafted nigral cells are maintained after transplantation, while other aspects of their cellular biology may be compromised. Of interest are the differences between the grafts and the control SN in regard to the expression of DA D2 receptors; such perturbations

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Fig. 5.2. DA receptor autoradiography using the agonist [3H]CV 205-502 as a ligand shows no binding in the transplant, indicating an apparent abnormality of grafted cells. Although [3H]CV 205-502 binds to both D2 and D3 sites, the binding in caudate-putamen and substantia nigra is primarily due to the D2 type.49 Magnification x10. Reprinted with permission from: Triarhou LC, Solà C, Mengod G et al. Cell Transpl 1995; 4:39-48. © Cognizant Communication Corp.

could be hypothetically linked to the spatial and temporal mismatch in the molecular interactions between the grafted mesencephalic anlage and the adult, denervated striatum. Nevertheless, under different circumstances, many receptor types have been reported to be present in transplanted tissues regardless of graft location and surrounding environment.53

Neurotensin Receptors Neurotensin receptor binding was visualized by autoradiography with 0.1 nM monoiodo([125I]-Tyr3)neurotensin.48 The technique of receptor autoradiography was used to examine the distribution of neurotensin receptor binding sites in the striatum and related regions of normal and weaver mutant mice. In normal mice, neurotensin receptors were localized on DA cells and their processes. Compared to the wild-type, weaver mutants showed an 86% reduction in grain density in the dorsal striatum, associated with the DA axon loss. A similar decrease of neurotensin receptors is seen in the dorsal part of the caudate head in postmortem brain tissues from patients affected with Parkinson’s disease.54 In the transplantation experiments, neurotensin receptor binding was intense in the grafts (Fig. 5.3) and correlated with the distribution of tyrosine hydroxylase immunoreactivity. Autoradiographic grain density in the reinnervated weaver striatum reached on the average 63%, and occasionally 77%, of wild-type values, resulting in all likelihood from the graft-induced innervation of the recipient. The cellular localization of neurotensin receptor RNA transcript was studied by in situ hybridization.48 The mRNA was present in normal substantia nigra and ventral tegmental area, but the signal was substantially reduced from both areas in wv/wv mutants, as expected after the loss of midbrain DA cells. Neurotensin receptor mRNA, which is normally expressed by nigral DA neurons, was not detected in the grafts, despite the positive

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Fig. 5.3. Autoradiography of neurotensin binding sites labeled with [125I]neurotensin. Binding signal is observed in the cell suspension in the right hemisphere. The increase in [125I]neurotensin binding signal seen in the transplanted side of the striatum corresponds to reinnervation by graft-derived dopaminergic fibers. Upper micrograph is from a more rostral level, lower micrograph is from a more caudal level. Magnification: x14. Reprinted with permission from: Triarhou LC, Solà C, Mengod G et al. Cell Transpl 1995; 4:39-48. © Cognizant Communication Corp.

receptor binding, indicating the possibility that receptors could be present but sustaining RNA message levels and receptor turnover might be too low to allow detection in the in situ hybridization studies. No hybridization signal was detected in the grafts for neurotensin receptor mRNAs, which is normally expressed by nigral DA neurons. Neurotensin binding sites, labeled with [125I]neurotensin, were visualized in the suspensions, indicating the possibility that

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receptors could be present but that RNA message levels might be too low to allow detection. These findings have suggested that specific functional properties of grafted nigral cells are maintained after transplantation, while other aspects of their cellular biology may be compromised.

Excitatory and Inhibitory Amino Acid Receptors The effect of dopaminergic denervation on N-methyl-D-aspartate (NMDA) receptors has been investigated in the rat striatum. It was found that three weeks after 6-OHDA lesions of the medial forebrain bundle, there is a selective 13% increase in the level of NR2A receptor subunit mRNA, as determined by in situ hybridization histochemistry with oligonucleotide probes.55 At the same time, NMDA receptor binding, as determined by autoradiography with L-[3H]glutamate, was unchanged, thus indicating that dopaminergic denervation may exert differential effects on NMDA gene expression. In another study, the 6-OHDA lesion rat model was used to study the response of non-NMDA receptors to striatal DA denervation by ligand binding autoradiography.56 One to four weeks after the lesions, the maximum number of [3H]AMPA [(RS)-α-amino3-hydroxy-5-methylisoxazole-4-propionate] and [3H]kainate binding sites was found decreased by 17-26%, suggesting that about one-fourth to one-fifth of the AMPA and kainate receptors in the rat striatum are localized on presynaptic terminals of dopaminergic axons and pointing to a possible role of non-NMDA glutamate receptors in the presynaptic regulation of DA. The functional integration of fetal mesencephalic grafts into the striatum of weaver mutant mice was studied by investigating the levels of excitatory and inhibitory amino acid receptors.36,57,58 Cell suspensions were prepared from +/+ mice at E12 and implanted unilaterally into the striatum of wv/wv mutants. Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM [3H]GBR 12935. The average increase in [3H]GBR 12935 binding in grafted dorsal striatum compared to nongrafted wv/wv striatum was 60% three months after grafting. Levels of excitatory amino acid receptors were studied by means of autoradiography of 80 nM [3H]CNQX (6-cyano-7-nitro-quinoxaline-2,3-dione) and 100 nM NMDAsensitive [3H]glutamate binding to visualize the topography of non-NMDA and NMDA receptors, respectively.57 Increases of 30% or more were found for [3H]CNQX binding in the dorsal nongrafted weaver striatum compared to +/+, and a further 6-9% increase in grafted weaver compared to nongrafted side. The added increase of non-NMDA receptors in the transplanted striatum might be explained by a presence of such receptors on DA presynaptic endings of graft origin. A 20% increase in NMDA-sensitive [3H]glutamate binding was measured in the dorsal nongrafted weaver striatum compared to +/+. NMDAsensitive [3H]glutamate binding in the transplanted side of weaver mutants tended to be slightly higher in all areas of the striatal complex compared to the nongrafted side, without reaching, however, conventional levels of statistical significance. Using in situ hybridization histochemistry with synthetic [32P]labeled oligonucleotide probes, we investigated RNA transcripts encoding the four AMPA receptor subunits. RNA transcripts in the striatum are seen with a decreasing signal intensity in the following order: GluRB > GluRA > GluRC > GluRD. The weaver caudate-putamen shows a 12% increase in GluRA subunit mRNA compared to +/+, while mesencephalic neuron

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transplantation leads to slight increases (3%) in the levels of GluRB mRNA in nucleus accumbens. The levels of inhibitory amino acid receptors in transplanted wv/wv mice were studied by means of autoradiography of 8 nM [3H]flunitrazepam and 12 nM [3H]muscimol binding,58 in order to visualize the distribution of GABAA receptors. A 17% increase in [3H]flunitrazepam binding and a 20% increase in [3H]muscimol binding was found in the nongrafted dorsal striatum of weaver homozygotes compared to wild-type (+/+) control levels. The functional mesencephalic grafts had a partial normalizing effect on both [3H]flunitrazepam and [3H]muscimol binding in the dorsal striatum of the weaver recipients. The normalization brought about by the grafts was around 20% for [3H]flunitrazepam binding and more than 40% for [3H]muscimol binding. The results of the amino acid receptor binding studies relate to the important interaction between the converging glutamatergic corticostriatal and dopaminergic nigrostriatal pathways in controlling the functional output of the basal ganglia in Parkinson’s disease and in experimental models of DA deficiency.57,58 References 1. Triarhou LC, Stotz EH, Mengod G et al. The weaver mutant mouse as a neurogenetic model of nigrostriatal dopamine deficiency: Adaptive changes and differences from the 6-OHDA model. Exp Neurol 1994; 129:13. 2. Björklund A. Dopaminergic transplants in experimental parkinsonism: Cellular mechanisms of graftinduced functional recovery. Curr Opin Neurobiol 1992; 2:683-689. 3. Triarhou LC, Low WC, Doucet G et al. The weaver mutant mouse as a model for intrastriatal grafting of fetal dopamine neurons. In: Hefti F, Weiner WJ, eds. Progress in Parkinson’s Disease Research–2. Mt. Kisco, NY: Futura Publishing Co., 1992:389-400. 4. Lindvall O, Brundin P, Widner H et al. Grafts of fetal dopamine neurons survive and improve motor function in Parkinson’s disease. Science 1990; 247:574-577. 5. Sawle GV, Myers R. The role of positron emission tomography in the assessment of human neurotransplantation. Trends Neurosci 1993; 16:172-176. 6. Schmidt RH, Ingvar M, Lindvall O et al. Functional activity of substantia nigra grafts reinnervating the striatum: Neurotransmitter metabolism and [ 14C]2-deoxy- D -glucose autoradiography. J Neurochem 1982; 38:737-748. 7. Schmidt RH, Björklund A, Stenevi U et al. Intracerebral grafting of neuronal cell suspensions. III. Activity of intrastriatal nigral suspension implants as assessed by measurements of dopamine synthesis and metabolism. Acta Physiol Scand [Suppl] 1983; 522:19-28. 8. Zetterström T, Brundin P, Gage FH et al. Spontaneous release of dopamine from intrastriatal nigral grafts as monitored by the intracerebral dialysis technique. Brain Res 1986; 362:344-349. 9. Strecker RE, Sharp T, Brundin P et al. Autoregulation of dopamine release and metabolism by intrastriatal nigral grafts as revealed by intracerebral dialysis. Neuroscience 1987; 22:169-178. 10. Schmidt RH, Björklund A, Stenevi U. Intracerebral grafting of dissociated CNS tissue suspensions: A new approach for neuronal transplantation to deep brain sites. Brain Res 1981; 218:347-356. 11. Gage FH, Björklund A, Stenevi U et al. Intracerebral grafting of neuronal cell suspensions. VIII. Survival and growth of implants of nigral and septal cell suspensions in intact brains of aged rats. Acta Physiol Scand [Suppl] 1983; 522:67-75. 12. Dunnett SB, Hernandez TD, Summerfield A et al. Graft-derived recovery from 6-OHDA lesions: Specificity of ventral mesencephalic graft tissues. Exp Brain Res 1988; 71:411-424. 13. Doucet G, Descarries L, Garcia S. Quantification of the dopamine innervation in adult rat neostriatum. Neuroscience 1986; 19:427-445. 14. Doucet G, Brundin P, Seth S et al. Degeneration and graft-induced restoration of dopamine innervation in the weaver mouse neostriatum: A quantitative radioautographic study of [3H]dopamine uptake. Exp Brain Res 1989; 77:552-568. 15. Triarhou LC, Brundin P, Doucet G et al. Intrastriatal implants of mesencephalic cell suspensions in weaver mutant mice: Ultrastructural relationships of dopaminergic dendrites and axon terminals issued from the graft. Exp Brain Res 1990; 79:3-17.

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16. Björklund A, Stenevi U, Schmidt RH et al. Intracerebral grafting of neuronal cell suspensions. I. Introduction and general methods of preparation. Acta Physiol Scand [Suppl] 1983; 522:9-18. 17. Zhou CF, Raisman G, Morris RJ. Specific patterns of outgrowth from transplants to host mice hippocampi, shown immunohistochemically by the use of allelic forms of THY1. Neuroscience 1985; 16:819-833. 18. Gage FH, Björklund A. Enhanced graft survival in the hippocampus following selective denervation. Neuroscience 1986; 17:89-98. 19. Zhou FC, Auerbach SB, Azmitia EC. Stimulation of serotonergic neuronal maturation after fetal mesencephalic raphe transplantation into the 5,7-DHT-lesioned hippocampus of the adult rat. Ann NY Acad Sci 1987; 495:138-152. 20. Triarhou LC, Low WC, Norton J et al. Reinstatement of synaptic connectivity in the striatum of weaver mutant mice following transplantation of ventral mesencephalic anlagen. J Neurocytol 1988; 17:233-243. 21. Freund TF, Bolam JP, Björklund A et al. Efferent synaptic connections of grafted dopaminergic neurons reinnervating the host neostriatum: A tyrosine hydroxylase immunocytochemical study. J Neurosci 1985; 5:603-616. 22. Clarke DJ, Brundin P, Strecker RE et al. Human fetal dopamine neurons grafted in a rat model of Parkinson’s disease: Ultrastructural evidence for synapse formation using tyrosine hydroxylase immunocytochemistry. Exp Brain Res 1988; 73:115-126. 23. Triarhou LC, Norton J, Ghetti B. Synaptic connectivity of tyrosine hydroxylase immunoreactive nerve terminals in the striatum of normal, heterozygous and homozygous weaver mutant mice. J Neurocytol 1988; 17:221-232. 24. Freund TF, Powell JF, Smith AD. Tyrosine hydroxylase-immunoreactive boutons in synaptic contact with identified striatonigral neurons, with particular reference to dendritic spines. Neuroscience 1984; 13:1189-1215. 25. Roffler-Tarlov S, Graybiel AM. The postnatal development of the dopamine-containing innervation of dorsal and ventral striaturn: Effects of the weaver gene. J Neurosci 1987; 7:2364-2372. 26. Ohta K, Graybiel AM, Roffler-Tarlov S. Dopamine D1 binding sites in the striatum of the mutant mouse weaver. Neuroscience 1988; 28:69-82. 27. Kaseda Y, Ghetti B, Low WC et al. Age-related changes in striatal dopamine D2 receptor binding in weaver mutant mice and effects of ventral mesencephalic grafts. Exp Brain Res 1990; 83:1-8. 28. Chinaglia G, Alvarez FJ, Probst A et al. Mesostriatal and mesolimbic dopamine uptake binding sites are reduced in Parkinson’s disease and progressive supranuclear palsy: A quantitative autoradiographic study using 3H]mazindol. Neuroscience 1992; 49:317-327. 29. Triarhou LC, Stotz EH, Low WC et al. Studies on the striatal dopamine uptake system of weaver mutant mice and effects of ventral mesencephalic grafts. Neurochem Res 1994; 19:1229-1238. 30. Javitch JA, Blaustein RO, Snyder SH. [3HMazindol binding associated with neuronal dopamine and norepinephrine uptake sites. Mol Pharmacol 1984; 26:35-44. 31. Zimányi I, Lajtha A, Reith MEA. Comparison of characteristics of dopamine uptake and mazindol binding in mouse striatum. Naunyn-Schmiedeberg’s Arch Pharmacol 1989; 340:626-632. 32. Allard PO, Eriksson K, Ross SB et al. [3H]GBR-12935 binding to dopamine uptake sites in rat striatum. Neuropsychobiology 1990/91; 23:177-181. 33. Nissbrandt H, Engberg G, Pileblad E. The effects of GBR 12909, a dopamine re-uptake inhibitor, on monoaminergic neurotransmission in rat striatum, limbic forebrain, cortical hemispheres and substantia nigra. Naunyn-Schmiedeberg’s Arch Pharmacol 1991; 344:16-28. 34. Mennicken F, Savasta M, Peretti-Renucci R et al. Autoradiographic localization of dopamine uptake sites in the rat brain with 3H-GBR 12935. J Neural Transm [Gen Sect] 1992; 87:1-14. 35. Reith ME, Selmeci G. Radiolabeling of dopamine uptake sites in mouse striatum: comparison of binding sites for cocaine, mazindol, and GBR 12935. Naunyn-Schmiedeberg’s Arch Pharmacol 1992; 345:309-318. 36. Stasi K, Mitsacos A, Giompres P et al. Autoradiographic study of amino acid receptors in the striatum of weaver mice receiving nigral transplants. Soc Neurosci Abstr 1997; 23:2000. 37. Savasta M, Mennicken F, Chritin M et al. Intrastriatal dopamine-rich implants reverse the changes in dopamine D2 receptor densities caused by 6-hydroxydopamine lesion of the nigrostriatal pathway in rats: An autoradiographic study. Neuroscience 1991; 46:729-738. 38. Cenci MA, Campbell K, Björklund A. Neuropeptide messenger RNA expression in the 6-hydroxydopamine-lesioned rat striatum reinnervated by fetal dopaminergic transplants: Differen-

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tial effects of the grafts on preproenkephalin, preprotachykinin and prodynorphin messenger RNA levels. Neuroscience 1993; 57:275-296. 39. Roffler-Tarlov S, Pugatch D, Graybiel AM. Patterns of cell and fiber vulnerability in mesostriatal system of the mutant mouse weaver. II. High affinity uptake sites for dopamine. J Neurosci 1990; 10:734-740. 40. Roffler-Tarlov S, Graybiel AM. Genetic effects on dopamine uptake sites in the striatum. Soc Neurosci Abstr 1991; 17:1289. 41. Simon JR, Ghetti B. Topographic distribution of dopamine uptake, choline uptake, choline acetyltransferase, and GABA uptake in the striata of weaver mutant mice. Neurochem Res 1992; 17:431-436. 42. Richter JA, Stotz EH, Ghetti B et al. Comparison of alterations in tyrosine hydroxylase, dopamine levels, and dopamine uptake in the striatum of the weaver mutant mouse. Neurochem Res 1992; 17:437-441. 43. Stotz EH, Palacios JM, Landwehrmeyer B et al. Alterations in dopamine and serotonin uptake systems in the striatum of the weaver mutant mouse. J Neural Transm [Gen Sect] 1994; 97:51-64. 44. Freed WJ, Ko GN, Niehoff DL et al. Normalization of spiroperidol binding in the denervated rat striatum by homologous grafts of substantia nigra. Science 1983; 222:937-939. 45. Freed WJ, Olson L, Ko GN et al. Intraventricular substantia nigra and adrenal medulla grafts: mechanisms of action and 3H]spiroperidol autoradiography. In: Björklund A, Stenevi U, eds. Neural Grafting in the Mammalian CNS. Amsterdam: Elsevier Science Publishers BV, 1985:471-488. 46. Boyson SJ, McGonigle P, Molinoff PB. Quantitative autoradiographic localization of the D1 and D2 subtypes of dopamine receptors in rat brain. J Neurosci 1986; 6:3177-3188. 47. Sonsalla PK, Manzino L, Heikkila RE. Interactions of D1 and D2 dopamine receptors on the ipsilateral vs contralateral side in rats with unilateral lesions of the dopaminergic nigrostriatal pathway. J Pharmacol Exp Ther 1988; 247:180-185. 48. Triarhou LC, Solà C, Mengod G et al. Ventral mesencephalic grafts in the neostriatum of the weaver mutant mouse: Structural molecule and receptor studies. Cell Transpl 1995; 4:39-48. 49. Landwehrmeyer B, Mengod G, Palacios JM. Differential visualization of dopamine D2 and D3 receptor sites in rat brain: A comparative study using in situ hybridization histochemistry and ligand binding autoradiography. Eur J Neurosci 1993; 5:145-153. 50. Mengod G, Martínez-Mir MI, Vilaró MT et al. Localization of the mRNA for the dopamine D2 receptor in the rat brain by in situ hybridization histochemistry. Proc Natl Acad Sci USA 1989; 86:8560-8564. 51. Mengod G, Vilaró MT, Niznik HB et al. Visualization of a dopamine D1 receptor mRNA in human and rat brain. Mol Brain Res 1991; 10:185-191. 52. Dawson TM, Dawson VL, Gage FH et al. Functional recovery of supersensitive dopamine receptors after intrastriatal grafts of fetal substantia nigra. Exp Neurol 1991; 111:282-292. 53. Lu SY, Norman AB. Neurotransmitter receptors in fetal tissue transplants: Expression and functional significance. J Neural Transpl Plastic 1993; 4:215-226. 54. Chinaglia G, Probst A, Palacios JM. Neurotensin receptors in Parkinson’s disease and progressive supranuclear palsy: An autoradiographic study in basal ganglia. Neuroscience 1990; 39:351-360. 55. Ulas J, Cotman CW. Dopaminergic denervation of striatum results in elevated expression of NR2A subunit. Neuroreport 1996; 7:1789-1793. 56. Zavitsanou K, Mitsacos A, Giompres P et al. Changes in [3H]AMPA and [3H]kainate binding in rat caudate-putamen and nucleus accumbens after 6-OHDA lesions of the medial forebrain bundle: An autoradiographic study. Brain Res 1996; 731:132-140. 57. Mitsacos A, Tomiyama M, Stasi K et al. [3H]CNQX and NMDA-sensitive [3H]glutamate binding sites and AMPA receptor subunit RNA transcripts in the striatum of normal and weaver mutant mice and effects of ventral mesencephalic grafts. Cell Transpl 1999; 8:11-23. 58. Stasi K, Mitsacos A, Giompres P et al. Partial restoration of striatal GABAA receptor balance by functional mesencephalic dopaminergic grafts in mice with hereditary Parkinsonism. Exp Neurol 1999; 157:259-267.

CHAPTER 6

Behavioral Recovery of Functional Responses Unilateral Grafts and Circling Behavior

S

tudies in rats have shown that unilateral destruction of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) results in a spontaneous rotational bias to the side ipsilateral to the lesion.1 With time, spontaneous rotational behavior subsides, but it can still be induced pharmacologically; amphetamine, an agent that releases dopamine (DA) from presynaptic terminals, causes a rotational bias to the side ipsilateral to the lesion;2 apomorphine, a DA receptor agonist, causes rotation to the side contralateral to the lesion.3 The effect of apomorphine is presumably a result of activating a greater number of DA receptors on the side of the lesion, due to the increase in receptors as a function of denervation supersensitivity.1,4 Reinnervation of the chemically denervated striatum by grafted DA neurons in those rodents has been shown to rectify the rotational bias displayed by animals with unilateral lesions.5-11 Unilateral transplantation of DA cells into the striatum of rats with unilateral 6-OHDA lesions of the nigrostriatal pathway reverses the amphetamine-induced turning bias according to the rotational asymmetry model of Ungerstedt and Arbuthnott.2 Unilateral grafts can also improve spontaneous (i.e., not drug-induced) behaviors resulting from striatal DA denervation, such as simple sensorimotor orientation, forelimb stepping functions, and response latency in disengage behavior.12,13 Bilateral grafts into the striatum of rats with bilateral lesions of the ascending DA pathway are efficient in reversing sensorimotor and akinetic impairments induced by 6-OHDA.14 Since both sides of the substantia nigra degenerate in the weaver, nongrafted mutants do not display any rotational bias to either side. On the other hand, in weaver mutants with unilateral DA-containing mesencephalic grafts into the dorsal striatum, in the form of either solid tissue or cell suspensions, methamphetamine elicits a significant circling bias toward the contralateral, nongrafted side15-21 (Fig. 6.1a,b). Such an effect seems to be specific for DA-containing cells, as vehicle-sham grafts, nonsurviving grafts, cortical cavities only, cerebellar grafts or grafts of noradrenaline-containing neurons from the locus ceruleus do not affect rotational behavior.15,16,20 Generally, turning behavior is either monitored manually by individual investigators or measured electronically in rotometers. Intraperitoneal methamphetamine doses of 1.0-2.5 mg/kg body weight are used. Following administration of amphetamine, sham-operated and graft-receiving weaver mice become motorically activated.16 Over the 30-60 min test periods usually observed, sham-operated mice show no significant side bias in motor

Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease, by Lazaros C. Triarhou. ©2001 Eurekah.com.

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Fig. 6.1. Behavioral effects of unilateral and bilateral mesencephalic grafts into the striatum of weaver mutant mice on motor performance. Unilateral grafts into the right striatum result in a rotational bias towards the left side after methamphetamine administration (a,b). Bilateral grafts lead to fewer topplings (d) and improved balance rod scores (f ) compared to nongrafted mutants (c,e). Unpublished micrographs made from videotape.

behavior. In contrast, transplanted weaver mutants show a marked motor asymmetry with preferential turning away from the grafted side. Weaver animals from several behavioral, anatomical, ultrastructural, biochemical and pharmacological studies, with unilateral mesencephalic grafts into the right striatum, display an average circling bias [expressed as the mean of all individual L/(L+R) values] of around 70-90% in the direction opposite to graft placement.15-21 Rotating mice still manifest signs of the superimposed cerebellar ataxia. The finding that unilateral reinstatement of the nigral DA component of the nigrostriatal pathway in weaver mutant mice results in a rotational bias toward the contralateral side indicates that the critical neuronal circuitry of the striatum, necessary for responding to presynaptic DA release, must be present. Rats with bilateral 6-OHDA lesions of the nigrostriatal pathway become akinetic, aphagic, and adipsic, but do not display a rotational bias;22 unilateral transplants of DA neurons produce an amphetamine-induced rotation complementary to that seen in animals with unilateral lesions of the nigrostriatal pathway. However, DA grafts do not reverse

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the akinetic, aphagic, and adipsic behaviors.22 The neurotoxic depletion of striatal DA bilaterally resembles that of the weaver mutant with respect to the dorsal striatum. However, it has been indicated that DA innervation in the limbic striatum of weaver mutants is not affected as severely as that of the nonlimbic (dorsal) striatum.23 Such a situation may be the reason why weaver mutants do not exhibit akinetic, aphagic, and adipsic behaviors.24 The effect of unilateral DA grafts on amphetamine-induced rotations in weaver mice parallels in a way that of rats with bilateral 6-OHDA lesions and unilateral DA grafts; in both situations a circling bias is displayed toward the side contralateral to the graft, which, relative to the grafted replenished side, appears as the side of a lesion.

Correlation of Turning Bias with Structural and Biochemical Parameters Several studies have demonstrated that terminal fields innervated by fibers from mesencephalic grafts contain DA and its metabolites.25 Electrovoltammetric studies have indicated that in the rat, the graft-derived fiber innervation releases DA when stimulated by K+ ions.26 Additional evidence for graft-derived DA release is the decrease in DA receptor sensitivity in the denervated striatum after grafting; Murrin et al4 have shown that DA denervation of the striatum results in an upregulation of DA receptors, which is most likely a consequence of the synthesis of additional receptors. Freed et al27 have demonstrated that in rats with 6-OHDA lesions the reinnervation of the denervated striatum by grafts of fetal substantia nigra normalizes DA receptor sensitivity. DA receptor binding is elevated in the dorsolateral striatum of weaver mutants,28 and a normalizing effect of mesencephalic grafts on DA receptor sensitivity in these animals has been measured.18 In all, grafted fetal DA neurons from normal mice are capable of functionally restoring a DA innervation in the striatum of weaver mutant mice. Grafted DA neurons and the resulting fiber outgrowth are sustained over periods of time when the intrinsic nigrostriatal system of the weaver mutant has considerably declined. The association between rotational bias and nerve cell count has been investigated in weaver mice with unilateral cell suspension grafts.17 In that study, the Pearson product moment correlation coefficient was statistically significant when a logarithmic transformation was applied to both rotational bias [Y=log10(L-R)] and nerve cell count [X=log10Nc]. In another study, a positive correlation between rotational bias and number of surviving tyrosine hydroxylase (TH) immunoreactive cells in the graft was found in weaver hosts in linear scale.29 Studies in rats and mice with 6-OHDA lesions of the mesostriatal DA pathway and intrastriatal DA neuron grafts show that the degree of behavioral recovery correlates with the extent of DA fiber ingrowth from the graft into the host striatum and with the number of surviving DA neurons in the graft.11,30,31 Both the amount of graft-derived DA innervation and the number of transplanted DA neurons show a logarithmic relationship to the graft effect on rotational behavior.30,31 Such a relationship points to the existence of a saturation-point, beyond which additional DA fiber ingrowth or additional grafted DA cells may not add to the magnitude of the behavioral effect. In line with those reports, the studies in the weaver mutant17,29 indicate correlations between DA cell number in the grafts and rotational behavior scores. The fact that in one study the logarithmic transformation on the two variables yields the strongest relationship indicates that the effect of neuron number on rotational asymmetry is stronger at lower ranges of both variables and that it levels off at higher numerical values for both variables. That would point to the existence of a saturation-point regarding turning behavior as well, a notion which is

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meaningful if one considers that the number of rotations that a mouse can physically perform during a certain period of time has a limit and, once that limit is reached, additional DA cells may do little to surpass it. In the autoradiographic study of [3H]DA uptake by Doucet et al,16 there was no clearcut correlation between degree of reinnervation and behavioral motor asymmetry scores, whereas in other reports, the correlation between rotational asymmetry and DA reinnervation,30 number of DA neurons in the graft17,31 or striatal DA content32 has been reported to be nonlinear. It is possible that an existing correlation may have gone unnoticed with the sample size used in the [3H]DA uptake study. Biochemical findings on synaptosomal [3H]DA uptake have been compared to the results from amphetamine-induced turning behavior tests.20 A plot of L-R rotations against R-L [3H]DA uptake gave a positive correlation between the two parameters, indicating that animals with a strong rotational bias to the left tended to have higher [3H]DA uptake in the right side of the striatum. Parallel measurements of motor performance and DA uptake in the same animals offer an index of behavioral recovery as a function of transmitter-related activity. Correlations between rotational bias and changes in striatal DA D2 receptors, measured by [3H]spiperone binding, have been calculated.18 The only region for which a significant correlation was observed was the nucleus accumbens. Such a result is less readily explained than e.g., a significant correlation between behavior and binding changes in dorsal striatum. Brundin et al33 studied rats with unilateral mesostriatal and bilateral accumbens 6-OHDA lesions. Unilateral transplants into the nucleus accumbens amplified the locomotor response and ipsilateral turning in response to amphetamine. Those authors emphasized the amplifier as opposed to the directional role played by the nucleus accumbens in motor responses, in agreement with earlier suggestions by Kelly and Moore.34 It remains to be determined how differences in [3H]spiperone binding in the nucleus accumbens may be induced by a transplant in the dorsal striatal aspect. Brundin et al33 have argued against a diffusion effect of DA to regions away from the graft; in that context, there was indeed a significant change in [3H]spiperone binding in the dorsolateral and dorsomedial striatal quadrants and not in the nucleus accumbens as a result of transplantation.

Dissociation of the Functional Contribution of Graft-Derived Axons and Dendrites in Rotational Asymmetry The weaver mutation leads to loss of mesencephalic DA cells and nigrostriatal DA axons in homozygosity (wv/wv) and to a deficiency of nigral dopaminergic dendrites without concomitant loss of DA cell somata or axons in heterozygosity (wv/+).35-38 The use of the laboratory mouse for transplantation purposes is particularly valuable, as it allows grafting from or to mutant strains and takes advantage of genetic mutations known to affect the murine nervous system.11,39 After transplantation of wild-type (+/+) fetal mouse mesencephalon into the weaver striatum, a functional reinnervation occurs as evidenced behaviorally by rotational asymmetry tests.15,16 Histologically, both axons and dendrites derived from the grafts innervate the host tissue and establish synaptic contacts with striatal neurons.17,40 In considering the functional significance of the dendritic innervation supplied by graft DA neurons to the host striatum, it was theorized that dendritic release of DA may contribute, in conjunction with the synaptic input of DA axon terminals, to the functional effects of the grafts17 (Fig. 6.2).

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Fig. 6.2. Neuronal connections between the substantia nigra (SN) and the caudate-putamen complex in the normal condition (left) and in a weaver mutant mouse with an intrastriatal graft of embryonic substantia nigra (right). Circuits have been simplified and interneuron circuits are not shown. Left panel: Normally, DA neurons of the substantia nigra pars compacta (SNc) project their axons to the caudate-putamen complex (CP), where they establish synaptic contact with spiny projection neurons. In turn, striatal projection neurons send their axons to the substantia nigra pars reticulata (SNr), where they impinge on DA dendrites of nigral neurons. DA dendrites in SNr are thought to regulate, through the release of DA, the activity of mesostriatal DA neurons (1), the release of neurotransmitter from incoming axons to the substantia nigra (2), and the activity of non-DA nigral neurons (3) projecting to postnigral nuclei such as the thalamus and the tectum. Right panel: In weaver mutant mice, approximately two-thirds of the DA neurons in SNc degenerate (1´), leading to an interruption of the mesostriatal DA projection (2´), a deprivation of incoming striatonigral axons of their dendritic cellular targets (3´), and a disturbance in the regulation of the activity of non-DA nigral neurons (4´). Dendrites emanating from the intrastriatal graft of substantia nigra (SNg) can be viewed as part of a heterotopic pars reticulata. In the ectopic position, DA dendrites in the graft may, through the release of DA, still participate in homologous physiological functions, such as regulation of the activity of grafted DA neurons (1), of the release of neurotransmitter from afferent axons to the graft (2), and of the activity of non-DA grafted nigral neurons (3) projecting to undetermined targets (?). In addition, DA dendrites from the graft might serve heterologous (or novel) functions, such as modulation of the activity of cortical afferents to the striatum (4), and influence of the physiological activity of striatal cells by DA released from dendrites (5). Reprinted with permission from: Triarhou LC, Brundin P, Doucet G et al. Exp Brain Res 1990; 79:3-17. © Springer-Verlag.

To address that issue, we transplanted two types of grafts (+/+ and wv/+) unilaterally into the right striatum of two types of hosts, i.e., wv/wv mutants, and +/+ mice with unilateral right 6-OHDA lesions.29 The aims of the study were to examine whether wv/+ DA cells maintain a “dendrite-poor” phenotype after transplantation to the denervated striatum, and to compare their functional effects with those of wild-type (+/+) grafts in reversing amphetamine-induced turning behavior. Such an approach would “subtractively” offer an index of the contribution of the dendritic component to functional recovery (Fig. 6.3). The viability and morphology of grafted neurons were assessed by TH immunocytochemistry on serial sections of the host forebrains. The morphology of surviving TH

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Fig. 6.3. The rationale behind using grafts of +/+ and wv/+ origin to dissociate the functional significance of the relative contribution of the axonal and dendritic innervation of the host striatum in behavioral recovery by subtraction. The anatomical phenotype of wild-type dopaminergic neurons would comprise both axonal and dendritic arbors, whereas wv/+ DA neurons would offer a relatively “clean” axonal preparation, due to their characteristic defect in dendritic outgrowth.

immunoreactive neurons depended on the genotype of donor tissue (Fig. 6.4). Cells derived from +/+ fetuses showed an extensive arborization with both fine varicose and with thicker processes, that corresponded to the typical morphology of axons and dendrites respectively. Process arborization of weaver heterozygous graft neurons was less extensive compared to that of +/+ graft neurons in both types of hosts. The vast majority of TH immunoreactive processes in wv/+ grafts were fine in thickness and had the characteristic axonal varicosities of catecholamine axons. The dendritic arborization of wv/+ cells was strikingly poorer than that of +/+ cells in grafts placed into both host types, most likely reflecting their in situ phenotypic abnormality.35,38 Mice with 6-OHDA lesions of the right substantia nigra are expected to rotate ipsilateral to the lesion; 6-OHDA mice with right lesions and right grafts are expected either to display no bias or to rotate to the left (because of denervation-induced receptor supersensitivity). This information can be summarized by predicting rotational directions of Llesion < Rlesion and Lgraft ≥ Rgraft (Fig. 6.5, upper). Following methamphetamine challenge, animals with 6-OHDA lesions registered on the average 14 left and 75 right turns before transplantation. After transplantation of +/+ tissue, 6-OHDA recipients registered on the average 117 left and 2 right turns, thus showing an average 178% reversal of asymmetry from ipsilateral turning after lesion to contralateral turning after grafting. The 6-OHDA hosts with transplants of wv/+ tissue registered on the average 42 left and 11 right turns, thus showing an average 165% reversal of asymmetry. The effects of the two types of grafts were compared by means of an ordinary student’s t-test using the average [(Lgraft-Rgraft )-(Llesion-Rlesion )] scores, which were 142 for animals with +/+ grafts and 119 for animals with wv/+ grafts. The analysis showed that the difference in turning bias between the group with +/+ grafts and the group with wv/+ grafts was not statistically significant.

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Fig. 6.4. TH immunocytochemistry of grafts of wild-type (upper) and heterozygous weaver origin (lower), placed into wv/wv hosts. In the case of +/+ grafts, the arborization of processes from TH immunoreactive neurons is extensive and comprises both axons and dendrites. In the case of wv/+ grafts, presence of dendrites is strikingly smaller. Magnification x123. Reprinted with permission from Witt TC, Triarhou LC. Cell Transpl 1995; 4:323-333. © Cognizant Communication Corp.

Nonoperated wv/wv mutants do not display any rotational bias to either side, since both sides of the substantia nigra degenerate; on the other hand, unilateral mesencephalic grafts induce a contralateral circling bias after methamphetamine administration,15,16 which can be expressed as a predicted rotational direction of Lgraft > Rgraft (Fig. 6.5, lower). In a previous study15 methamphetamine-induced rotations toward the nongrafted side were found to be statistically significant only when mice with surviving transplants–as defined by TH immunopositivity–were analyzed as a separate group from those with nonsurviving grafts; therefore, for the sake of the present analysis, we only used the mice with successful

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Fig. 6.5. Schematic cartoon of rotational biases in the case of placing a right dopaminergic neuron graft into a host with a right unilateral 6-OHDA lesion (upper), and in the case of placing a right dopaminergic neuron graft into a wv/wv host (lower), which has a bilateral genetic DA deficiency. Left drawings in each case represent the host state before transplantation, right drawings represent the situation after transplantation. A possible stronger bias to the left after grafting could be attributed to a developing DA receptor supersensitivity after denervation.

grafts, arbitrarily defining a graft as successful if it had 50 or more surviving TH immunopositive cells in the caudate-putamen. Recipient wv/wv mice with +/+ grafts displayed a circling bias [expressed as the mean of all the individual L/(L+R) values] of 88% toward the side contralateral to the graft. Recipient wv/wv mice with wv/+ grafts displayed an average circling bias of 83% in the direction opposite to graft placement. The effects of the two types of grafts were compared using the average (Lgraft-Rgraft ) scores, which were 60 for weaver mutants with +/+ grafts and 103 for weaver mutants with wv/+ grafts. The analysis showed that the difference in turning bias between the group with +/+ grafts and that with wv/+ grafts was not statistically significant. Functional recovery has been reported after DA neuron grafting in animal models with lesions of the mesostriatal DA projection system and in humans with Parkinson’s disease.7,11,15,16,41,42 One fundamental issue pertaining to the mechanisms of graft action

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is whether the host brain can integrate the donor cells into a functional neuronal circuit. Such a circuit may utilize a variety of connections between host neurons and the axons, somata, and dendrites of graft neurons. In the normal substantia nigra, neurons of the pars compacta extend dendrites into the pars reticulata. By releasing DA, dendrites may modulate the activity of dopaminergic and nondopaminergic cells in the substantia nigra and may presynaptically regulate the afferent input to nigral neurons.43,44 It was suggested that when wild-type substantia nigra is used for transplantation, grafted nigral dendrites may serve similar functions and in addition mediate the transmission of impulses from the host striatum to the graft and influence the activity of host striatal neurons.17 In the substantia nigra of heterozygous weaver mice, nigral dendrites are reduced by about 60%.38 Assuming that a similar deficiency is expressed by the wv/+ substantia nigra when it is used as a graft, one would have a source of “dendrite-poor” DA neurons for studying how such cells perform after heterotopic transplantation into the denervated striatum. Grafted wv/+ DA cells indeed express an anatomical phenotype consistent with that seen in the wv/+ substantia nigra in situ, i.e., poor in dendritic arbors. The wv/+ and the +/+ grafts to the denervated striatum induce similar functional recoveries as assessed by rotational asymmetry tests. Qualitatively, DA neurons of wv/+ grafts appear to have fewer processes than those of +/+ grafts. Such a difference is most likely due to a smaller dendritic contribution in the heterozygous weaver group. Nonetheless, the neurons of wv/+ grafts reverse the rotational behavior, despite the apparent deficiency in dendritic outgrowth. In a study with +/+ grafts into wv/wv hosts,17 the proportion of dendrites to axons was found to be 1:2 within a distance of 0.5 mm from the graft, dropping down to 1:20 in the area located 0.5-1.0 mm away from the graft. Grafted wv/+ cells display a hindered dendritic form compared to +/+ cells; in that respect, cells that are genotypically wv/+ recapitulate the structural phenotype seen in the intact substantia nigra, and do so regardless of host type environment. That finding strengthens the view that the dendritic DA projection of the substantia nigra may represent a subcellular target of the wv gene.38,45 The ability of +/+ and wv/+ grafts to influence turning behavior appears comparable. When amphetamine induces DA release from the graft, a dopaminergic asymmetry with an ispilateral excess of DA is created in the bilaterally denervated striatum of wv/wv hosts, and animals turn contralateral to the graft. In 6-OHDA hosts, an asymmetrical supply of dopaminergic nerve terminals in the striatum is created by the unilateral destruction of the substantia nigra. When animals are injected with amphetamine, the unilateral deficiency of DA causes them to turn ipsilateral to the side of the lesion.2 When a nigral graft is placed into the denervated striatum and DA release is stimulated by amphetamine, the dopaminergic asymmetry is reversed.11 Because receptor supersensitivity may develop unilaterally in the denervated striatum,46 DA release on the side of the graft may induce a greater striatal response than on the side of the intact striatum. Such a greater response to DA release could explain why the 6-OHDA host mice not only reversed the direction of their turning behavior, but also exhibited asymmetrical turning behavior toward the opposite side of the 6-OHDA lesion and the graft. In the case of +/+ cell suspensions, both axons and dendrites immunoreactive for TH extend from the graft into the recipient striatum.17,29 In their ectopic position, dopaminergic dendrites of the graft might underscore several functions through the release of DA, e.g., regulation of the activity of grafted DA neurons, of neurotransmitter release by afferent

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axon terminals to the graft, and of the activity of non-DA grafted nigral neurons in the suspensions; moreover, DA released from the heterotopic dopaminergic dendrites could presumably modulate the activity of corticostriatal afferents and influence the physiological activity of host striatal cells.17,44 The hypothesis that dendritic release of DA may contribute, in conjunction with the synaptic input of DA axon terminals, to the functional effects of the grafts17,39 was tested in the present experiments. Amphetamine-induced rotations were used as an assay for that purpose. It is known that amphetamine facilitates dopaminergic neurotransmission of nigral DA neurons by comparably releasing DA from both axon terminals and dendrites.44,47 Our results indicate that the amphetamine-induced DA release from graft-derived axons and dendrites (in the case of +/+ transplants) and from axons primarily (in the case of wv/+ transplants) bring about comparable degrees of recovery in the rotational asymmetry model, suggesting that the axonal innervation issued from the graft suffices for improving that particular behavioral function. In other words, the axonal innervation supplied by wv/+ grafts to the denervated striatum induces a functional recovery comparable to that brought about by +/+ cells, which in addition supply a substantial dendritic innervation to the host.

Enhancement of Motor Performance after Bilateral Transplantation Bilateral Solid Grafts Weaver mice display instability of gait, poor coordination of the limbs, and a fine rapid tremor of the trunk and extremities.24 Hind-limbs assume an abducted and extended position and the body is lowered to the surface of the ground. Affected animals topple over to the sides after every few steps (Fig. 6.1c) and move their limbs rapidly in an attempt to right themselves. The tremor of the extremities is particularly noticeable when animals fall and try to right themselves. The behavioral syndrome is in all likelihood underlain by both the cerebellar and mesotelencephalic perturbations.48 One may assume, for example, that intention tremor has a cerebellar basis, while resting tremor is associated with the pathology of the basal ganglia. Behavioral studies in the weaver mutant mouse have further disclosed a number of locomotor, spatial orientation and memory deficits;49-52 some of those deficits are not seen in other mutants with cerebellar lesions only,53 whereas others are known to be altered by drugs that interfere with catecholamine function54 or even to be induced by 6-OHDA lesions of the mesostriatal DA projection in normal animals.48,50,55 One would expect that behaviors of weaver mutants that are catecholamine-mediated may be improved following bilateral intrastriatal grafting of ventral mesencephalic grafts. Thus, the weaver mouse presents a model in which one may dissect out the ataxic from the Parkinsonian components of the motoric phenotype by means of neural transplantation. Spontaneous behavior in an open-field was observed in wild-type mice and in transplanted weaver mutants.56,57 After bilateral transplantation of solid mesencephalic grafts to the striatum (Fig. 6.6), weaver mutants are able to sustain the abdomen in a raised relief from the ground and to move about for relatively long periods of time without toppling over (Fig. 6.1d). The hindlimbs appear less abducted and hyperextended. Grafted animals are able to sustain the body in an upright posture, markedly contrasting to the lowered, widened stance of nongrafted mutants. Despite this apparent neurological improvement, tremor and moderate gait instability are still present.

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Fig. 6.6. Bilateral transplantation of solid mesencephalic grafts into preformed cortical cavities overlying the weaver striatum. Immunocytochemistry with anti-TH antiserum. Abbreviations: G, graft; CP, nucleus caudatus-putamen; V, ventriculus lateralis; CC, corpus callosum. Dark-field illumination. Reprinted with permission from: Triarhou LC, Low WC, Ghetti B. Prog Brain Res 1990; 82:187-195.

Toppling activity was quantified over a five-minute observation period. During that time, wild-type mice topple over zero times; nongrafted weaver mice fall on the average 14 times; weaver mice with bilateral cortical cavities fall 17 times; and weaver mice with bilateral cortical cavities and bilateral DA-containing grafts fall four times. An analysis using a t-test for paired comparisons between weaver mice with bilateral cortical cavities before and after bilateral transplantation of DA-containing neurons showed that the difference between the two groups was statistically significant, and that weaver mice with bilateral grafts topple over to the sides about 70% fewer times than nonoperated mutants.57 In equilibrium tests, animals are placed on a horizontal wooden bar suspended above the floor.58 The ability of normal mice to stay on the bar exceeds three minutes. Unoperated weaver mice fall off within a few seconds after placement on the bar (Fig. 6.1e). Weaver mice with bilateral nigral transplants have stayed on the bar for an average 2.5 times longer period compared to nongrafted mutants.57 Occasionally, a grafted mouse can make a 180˚ turn on the bar without falling off; in such an instance, the animal secures its position by wrapping the tail around the bar (Fig. 6.1f ). Thus, a balance deficit mediated at least in part by nigrostriatal DA can be improved after transplantation. The cerebellar component of the behavioral phenotype is a common denominator in the various experimental groups. In a sense, bilateral replacement of striatal DA renders weaver mutants from combined ataxic/Parkinsonian to ataxic. That problem is counterbalanced by the benefits of using the mutant striatum as a vector for analyzing donor tissue growth inside the pathological brain. The motor behavior findings indicate that a critical neuronal machinery of the striatum needed to respond appropriately to DA release must be present in weaver and that neural grafting is a viable approach for studying dopaminergic function in spontaneous neurodegenerative disorders of the mesostriatal DA projection system. The enhancement of motor behaviors after bilateral transplantation could be related to reinstatement of synaptic connectivity in the striatum.17,40,59 While mere ingrowth of DA fibers is usually sufficient for eliciting drug-induced behaviors, it is thought that to correct spontaneous behaviors that are partly driven by the host’s brain, a precise reconstruction of neuronal circuitry may be crucial.57,60,61 In essence, the findings in the weaver mutant mouse indicate the potentials of the neuron grafting technique for correcting certain catecholamine-mediated behaviors in a multiple systems atrophy of genetic etiology.

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Bilateral Cell Suspension Grafts An asset of the weaver model is that one can study graft development at the same time as the animal’s own DA system is undergoing a progressive degeneration. In carrying out studies of intrastriatal DA cell transplantation in weaver mutant mice, the cerebellar component of the behavioral phenotype is regarded as a common denominator among the various groups involved. Recovery of functional responses was also determined in a battery of behavioral tests in weaver mutants after bilateral grafting of mesencephalic cell suspensions to the striatum62 (Fig. 6.7). Equilibrium was tested on the basis of the time that mice were able to stay on a suspended balance rod before falling off. Locomotor coordination was measured by the number of times mice toppled over to the sides as they moved about in an open-field matrix, and locomotor activity was quantified by the number of square crossings in the open-field. Grafted weaver mutants perform significantly better than nongrafted mutant mice in all of these three tasks. The findings clearly demonstrate that bilateral cell suspension grafts of fetal DA cells enhance motor performance in the weaver model. Behavioral tests were carried out three to four months after grafting. As an index of equilibrium, the time interval was measured between placement on and falling off a balance rod suspended over the ground.58 The rod was wooden and had a circular cross-section 2.5 cm in diameter. Three successive trials were carried out. Since wild-type mice do not fall off the rod and generally remain on it for a long period of time, a cut-off time of 60 sec was set. In the actual experiment, all the wild-type mice invariably got 60 sec scores. Nonoperated weaver mutants stayed on the rod for an average 9.7 sec before failing off, while mutants with bilateral transplants stayed on it for an average 25 sec. Locomotor coordination and activity were evaluated in an open-field matrix.62 The apparatus was made of formica; its walls were 40 cm high; the base measured 100 x 100 cm and was equally divided into 25 squares measuring 20 x 20 cm each. After preliminary adaptation to the matrix animals were tested for five-minute sessions on three consecutive trials. On each test, the mouse was placed in a corner square facing into the corner. Behavior was monitored by an investigator sitting at one side of the open-field matrix. Locomotor coordination was quantified by counting the number of topplings in the open-field. Wild-type mice did not topple over to the sides at all as they moved about. Nonoperated weaver mutants toppled over 15.5 times, while weaver mice with bilateral grafts toppled an average 7 times during the five-minute observation period. Locomotor activity was quantified by counting the number of square crossings (defined as at least two paws entering a square) in the open-field. In the 25-square matrix, the means for the three successive trials were as follows: wild-type mice, 63, 72.2 and 77.4 (n=10); nongrafted weaver mutants, 1.6, 1.3 and 2.4 (n=9); grafted weaver mice, 9.7, 11.6 and 11.4 (n=9). The graft effect in weaver mutants was significant in all trials. The findings show that motor activities that are defective in weaver mutants can be enhanced after bilateral transplantation of mesencephalic cell suspensions to the striatum. The improvement of specific clinical signs following transplantation again suggests that those components of the behavioral phenotype are mediated, at least in part, through central catecholamine mechanisms. There is premise for testing specific motoric, sensorimotor, and memory-involving functions that are thought to be mediated, at least in part, by the nigrostriatal network.63-66 Along that line, we tested weaver mutant mice in a battery of tasks to determine graft-induced recovery of behavioral responses. With this approach, one may correlate the reinstatement of specific motoric, sensorimotor and learning behaviors to the reinstatement

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Fig. 6.7. Bilateral intrastriatal transplantation of ventral mesencephalic cell suspensions in a weaver mouse (a) and morphological appearance of grafted DA neurons at a higher power (b). TH immunocytochemistry in coronal sections of the forebrain. Magnification x18 (a), x370 (b). Reprinted with permission from: Triarhou LC, Norton J, Hingtgen JN. Exp Brain Res 1995; 104:191-198. © Springer-Verlag.

of striatal DA following bilateral intrastriatal grafting, and eventually dissociate the mesostriatal from the cerebellar components of the weaver behavioral phenotype. In 4 x 4 hole-board tests, weaver mutants display fewer visits to central and peripheral holes than normal mice.50 We have observed a similar behavior of weaver mice when placed in a 5 x 5 open-field matrix. These deficits are indicative of a locomotor abnormality. Since similar deficits are not seen in another cerebellar mutant, the nervous,53 which has normal DA levels in the brain,67 one could claim that the weaver abnormality is associated with the mesotelencephalic DA deficiency, and therefore, bilateral replenishment of striatal DA after grafting improves this behavior.

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Additional behaviors have been studied in weaver mutant mice. In delayed spontaneous alternation tests in a T-maze, weaver mutants do not alternate choices of maze arms at intertrial intervals of four or five minutes, contrary to normal mice,52 indicating a deficit in spatial working memory. In tests of navigational performance in a water-maze, weaver mutants commit more errors and take a longer time to reach the platform than control mice, indicating a possible deficit in spatial orientation.51 In a forced swimming task test, weaver mutants do not acquire the immobility response as normal mice do,49 indicating a subtle deficit in swimming behavior. Since in normal animals the immobility response can be altered by drugs that modify central catecholamine activity,54 future studies with bilateral DA grafts might show an improvement in the acquisition of the immobility response. Although the demonstration of behavioral reversal of drug-induced rotational asymmetry is an invaluable quantitative index of graft function in the currently utilized experimental models of DA deficiency, its relevance to the clinical improvement of Parkinsonian signs is unclear, whereas parameters such as spontaneous rotations, sensory neglect, tremor, rigidity and hypokinesia bear a probable or definite clinical relevance to the functional capacity of the grafts.68 The reported findings on significant behavioral effects of bilateral transplants in weaver mice in a number of motor performance tests are important, as they provide convincing evidence for the improvement of clinically-relevant signs in an experimental model with chronic progressive degeneration of the nigrostriatal DA pathway. References 1. Ungerstedt U. Postsynaptic supersensitivity after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine system. Acta Physiol Scand [Suppl] 1971; 367:69-93. 2. Ungerstedt U, Arbuthnott GW. Quantitative recording of rotational behavior in rats after 6-hydroxydopamine lesions of the nigrostriatal dopamine system. Brain Res 1970; 24:485-493. 3. Marshall JF, Ungerstedt U. Supersensitivity to apomorphine following destruction of the ascending dopamine neurons: Quantification using the rotational model. Eur J Pharmacol 1977; 41:361-367. 4. Murrin LC, Gale K, Kuhar MJ. Autoradiographic localization of neuroleptic and dopamine receptors in the caudate-putamen and substantia nigra: Effects of lesions. Eur J Pharmacol 1979; 60:229-235. 5. Björklund A, Stenevi U. Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants. Brain Res 1979; 177:555-560. 6. Perlow MJ, Freed WJ, Hoffer BJ et al. Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system. Science 1979; 204:643-647. 7. Björklund A, Schmidt RH, Stenevi U. Functional reinnervation of the neostriatum in the adult rat by use of intraparenchymal grafting of dissociated cell suspensions from the substantia nigra. Cell Tissue Res 1980; 212:39-45. 8. Björklund A, Stenevi U, Dunnett SB et al. Functional reactivation of the deafferented neostriatum by nigral transplants. Nature (Lond) 1981; 289:497-499. 9. Dunnett SB, Björklund A, Stenevi U et al. Behavioural recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal dopamine pathway. I. Unilateral lesions. Brain Res 1981; 215:147-161. 10. Björklund A, Stenevi U, Dunnett SB et al. Cross-species neural grafting in a rat model of Parkinson’s disease. Nature (Lond) 1982; 298:652-654. 11. Brundin P, Isacson O, Gage FH et al. The rotating 6-hydroxydopamine-lesioned mouse as a model for assessing functional effects of neuronal grafting. Brain Res 1986; 366:346-349. 12. Nikkhah G, Duan W-M, Knappe U et al. Restoration of complex sensorimotor behavior and skilled forelimb use by a modified nigral cell suspension transplantation approach in the rat Parkinson model. Neuroscience 1993; 56:33-43. 13. Olsson M, Nikkhah G, Bentlage C et al. Evaluation of a new stepping test to monitor limb function in the rat Parkinson model: Lesion, drug, and transplant effects. Soc Neurosci Abstr 1993; 19:1052.

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14. Dunnett SB, Björklund A, Schmidt RH et al. Behavioural recovery in rats with bilateral 6-OHDA lesions following implantation of nigral cell suspensions. Acta Physiol Scand [Suppl] 1983; 522:39-47. 15. Low WC, Triarhou LC, Kaseda Y et al. Functional innervation of the striatum by ventral mesencephalic grafts in mice with inherited nigrostriatal dopamine deficiency. Brain Res 1987; 435:315-321. 16. Doucet G, Brundin P, Seth S et al. Degeneration and graft-induced restoration of dopamine innervation in the weaver mouse neostriatum: A quantitative radioautographic study of [3H]dopamine uptake. Exp Brain Res 1989; 77:552-568. 17. Triarhou LC, Brundin P, Doucet G et al. Intrastriatal implants of mesencephalic cell suspensions in weaver mutant mice: Ultrastructural relationships of dopaminergic dendrites and axons issued from the graft. Exp Brain Res 1990; 79:3-17. 18. Kaseda Y, Ghetti B, Low WC et al. Age-related changes in striatal dopamine D2 receptor binding in weaver mutant mice and effects of ventral mesencephalic grafts. Exp Brain Res 1990; 83:1-8. 19. Solà C, Mengod G, Low WC et al. Regional distribution of amyloid β-protein precursor, growthassociated phosphoprotein-43 and microtubule-associated protein 2 mRNAs in the nigrostriatal system of normal and weaver mutant mice and effects of ventral mesencephalic grafts. Eur J Neurosci 1993; 5:1442-1454. 20. Triarhou LC, Stotz EH, Low WC et al. Studies on the striatal dopamine uptake system of weaver mutant mice and effects of ventral mesencephalic grafts. Neurochem Res 1994; 19:1229-1238. 21. Stasi K, Mitsacos A, Giompres P et al. Autoradiographic study of amino acid receptors in the striatum of weaver mice receiving nigral transplants. Soc Neurosci Abstr 1997; 23:2000. 22. Dunnett SB, Björklund A, Stenevi U et al. Behavioural recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal dopamine pathway. II. Bilateral lesions. Brain Res 1981; 229:457-470. 23. Roffler-Tarlov S, Graybiel AM. Weaver mutation has differential effects on the dopamine-containing innervation of the limbic and nonlimbic striatum. Nature (Lond) 1984; 307:62-66. 24. Sidman RL, Green MC, Appel SH. Catalog of the Neurological Mutants of the Mouse. Cambridge, MA: Harvard University Press, 1965. 25. Schmidt RH, Ingvar M, Lindvall O et al. Functional activity of substantia nigra grafts reinnervating the striatum: Neurotransmitter metabolism and [ 14C]2-deoxy- D -glucose autoradiography. J Neurochem 1982; 38:737-748. 26. Rose G, Gerhardt G, Strömberg I et al. Monoamine release from dopamine-depleted rat caudate nucleus reinnervated by substantia nigra transplants: An in vivo electrochemical study. Brain Res 1985; 341:92-100. 27. Freed WJ, Ko GN, Spoor HE et al. Normalization of spiroperidol binding in the denervated rat striatum by homologous substantia nigra transplants. Science 1983; 222:937-939. 28. Kaseda Y, Ghetti B, Low WC et al. Dopamine D2 receptors increase in the dorsolateral striatum of weaver mutant mice. Brain Res 1987; 422:178-181. 29. Witt TC, Triarhou LC. Transplantation of mesencephalic cell suspensions from wild-type and heterozygous weaver mice into the denervated striatum: Assessing the role of graft-derived dopaminergic dendrites in the recovery of function. Cell Transpl 1995; 4:323-333. 30. Björklund A, Dunnett SB, Stenevi U et al. Reinnervation of the denervated striatum by substantia nigra transplants: Functional consequences as revealed by pharmacological and sensorimotor testing. Brain Res 1980; 199:307-333. 31. Brundin P, Barbin P, Strecker RE et al. Survival and function of dissociated rat dopamine neurones grafted at different developmental stages or after being cultured in vitro. Dev Brain Res 1988; 39:233-243. 32. Schmidt RH, Björklund A, Stenevi U et al. Intracerebral grafting of neuronal cell suspensions. III. Activity of intrastriatal nigral suspension implants as assessed by measurements of dopamine synthesis and metabolism. Acta Physiol Scand [Suppl] 1983; 522:19-28. 33. Brundin P, Strecker RE, Londos E et al. Dopamine neurons grafted unilaterally to the nucleus accumbens affect drug-induced circling and locomotion. Exp Brain Res 1987; 69:183-194. 34. Kelly PH, Moore KE. Mesolimbic dopaminergic neurons in the rotational model of nigrostriatal function. Nature (Lond) 1976; 263:695-696. 35. Triarhou LC. Definition of the Mesostriatal Dopamine Deficit in the Weaver Mutant Mouse and Reconstruction of the Damaged Pathway by Means of Neural Transplantation. Ann Arbor, MI: University Microfilms International, 1987.

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36. Triarhou LC, Norton J, Ghetti B. Mesencephalic dopamine cell deficit involves areas A8, A9 and A10 in weaver mutant mice. Exp Brain Res 1988; 70:256-265. 37. Triarhou LC, Norton J, Ghetti B. Synaptic connectivity of tyrosine hydroxylase immunoreactive nerve terminals in the striatum of normal, heterozygous and homozygous weaver mutant mice. J Neurocytol 1988; 17:221-232. 38. Triarhou LC, Ghetti B. The dendritic dopamine projection of the substantia nigra: Phenotypic denominator of weaver gene action in hetero- and homozygosity. Brain Res 1989; 501:373-381. 39. Triarhou LC, Low WC, Doucet G et al. The weaver mutant mouse as a model for intrastriatal grafting of fetal dopamine neurons. In: Hefti F, Weiner WJ, eds. Progress in Parkinson’s Disease Research–2. Mt. Kisco, NY: Futura Publishing Co., 1992:389-400. 40. Triarhou LC, Low WC, Ghetti B. Synaptic investment of striatal cellular domains by grafted dopamine neurons in weaver mutant mice. Naturwissenschaften 1987; 74:591-593. 41. Lindvall O, Brundin P, Widner H et al. Grafts of fetal dopamine neurons survive and improve function in Parkinson’s disease. Science 1990; 247:574-577. 42. Lindvall O, Sawle G, Widner H et al. Evidence for long-term survival and function of dopaminergic grafts in progressive Parkinson’s disease. Ann Neurol 1994; 35:172-180. 43. Björklund A, Lindvall O. Dopamine in dendrites of substantia nigra neurons: Suggestions for a role in dendritic terminals. Brain Res 1975; 83:531-537. 44. Chéramy A, Leviel V, Glowinski J. Dendritic release of dopamine in the substantia nigra. Nature (Lond) 1981; 289:537-542. 45. Triarhou LC. Weaver gene expression in central nervous system. In: Conn PM, ed. Gene Expression in Neural Tissues. San Diego, CA: Academic Press, 1992:209-227. 46. Creese I, Snyder SH. Nigrostriatal lesions enhance striatal 3H-apomorphine and 3H-spiroperidol binding. Eur J Pharmacol 1979; 56:277-281. 47. Bernardini GL, Gu X, Viscardi E et al. Amphetamine-induced and spontaneous release of dopamine from A9 and A10 cell dendrites: An in vitro electrophysiological study in the mouse. J Neural Transm [Gen Sect] 1991; 84:183-193. 48. Triarhou LC, Ghetti B. Neuroanatomical substrate of behavioural impairment in weaver mutant mice. Exp Brain Res 1987; 68:434-435. 49. Lalonde R. Acquired immobility response in weaver mutant mice. Exp Neurol 1986; 94:808-811. 50. Lalonde R. Motor abnormalities in weaver mutant mice. Exp Brain Res 1987; 65:479-481. 51. Lalonde R, Botez MI. Navigational deficits in weaver mutant mice. Brain Res 1986; 398:175-177. 52. Lalonde R. Delayed spontaneous alternation in weaver mutant mice. Brain Res 1986; 398:178-180. 53. Lalonde R, Botez MI. Exploration of a hole-board matrix in nervous mice. Brain Res 1985; 343:356-359. 54. Porsolt RD, Bertin A, Blavet N et al. Immobility induced by forced swimming in rats: Effects of agents which modify central catecholamine and serotonin activity. Eur J Pharmacol 1979; 57:201-210. 55. Whishaw IQ, Dunnett SB. Dopamine depletion, stimulation or blockade in the rat disrupts spatial navigational and locomotion dependent upon beacon or distal cues. Behav Brain Res 1985; 18:11-29. 56. Low WC, Triarhou LC, Kaseda Y et al. Bilateral nigral grafts to the striatum of weaver mutant mice enhance locomotor coordination. Soc Neurosci Abstr 1989; 15:1355. 57. Triarhou LC, Low WC, Ghetti B. Dopamine neurone grafting to the weaver mouse neostriatum. Prog Brain Res 1990; 82:187-195. 58. Bure J, Bure_ová O, Huston JP. Techniques and Basic Experiments for the Study of Brain and Behaviour. Amsterdam–New York–Oxford: Elsevier/North-Holland Biomedical Press, 1976. 59. Triarhou LC, Low WC, Norton J et al. Reinstatement of synaptic connectivity in the striatum of weaver mutant mice following transplantation of ventral mesencephalic anlagen. J Neurocytol 1988; 17:233-243. 60. Arbuthnott G, Dunnett SB, MacLeod N. Electrophysiological properties of single units in dopamine-rich mesencephalic transplants in rat brain. Neurosci Lett 1985; 57:205-210. 61. Doucet G, Murata Y, Brundin P et al. Host afferents into intrastriatal transplants of fetal ventral mesencephalon. Exp Neurol 1989; 106:1-19. 62. Triarhou LC, Norton J, Hingtgen JN. Amelioration of the behavioral phenotype in weaver mutant mice through bilateral intrastriatal grafting of fetal dopamine cells. Exp Brain Res 1995; 104:191-198. 63. Sabol KE, Neill DB, Wages SA et al. Dopamine depletion in a striatal subregion disrupts performance of a skilled motor task in the rat. Brain Res 1985; 335:33-43.

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64. Isacson O, Dunnett SB, Björklund A. Behavioural recovery in an animal model of Huntington’s disease. Proc Natl Acad Sci USA 1986; 83:2728-2732. 65. Whishaw IQ, O’Connor WT, Dunnett SB. The contributions of motor cortex, nigrostriatal dopamine and caudate-putamen to skilled forelimb use in the rat. Brain 1986; 109:805-843. 66. White NM. Effect of nigrostriatal dopamine depletion on the posttraining, memory-improving action of amphetamine. Life Sci 1988; 43:7-12. 67. Lane JD, Nadi NS, McBride WJ et al. Contents of serotonin, norepinephrine and dopamine in the cerebrum of the ‘staggerer’, ‘weaver’ and ‘nervous’ neurologically mutant mice. J Neurochem 1977; 29:343-350. 68. Lindvall O. Neural transplantation in Parkinson’s disease. Brain Pathol 1994; 4:304.

CHAPTER 7

Directions for Future Research Introduction

O

ur groundwork, first, introduced and characterized in detail the weaver mouse as a model of spontaneous progressive dopamine (DA) deficiency similar to Parkinson’s disease, and secondly, laid the foundations for the intracerebral transplantation of catecholamine-producing neurons in this model of degeneration. Weaver mice represent the only known model of genetically-determined degeneration of the endogenous DA system and offer a unique opportunity to study the effects of a chronically ill environment on the fate of grafted DA neurons. Summarizing the previous Chapters, one can emphasize the following essential points. The weaver mutation leads to a progressive degeneration of nigrostriatal DA neurons over the animal’s life-span: 40% of nigral DA cells are lost during the first three postnatal weeks, an additional 30% by three months of age, and another 15% during the second year of life.1,2 Moreover, there is a 22% loss of striatal medium-sized neurons in one-year old animals.3 Neural transplantation studies show that fetal DA-rich grafts prepared from wildtype mouse donors survive after implantation into the weaver striatum and express many normal histochemical properties; supply an axonal and a dendritic innervation to host tissue and establish synaptic connections with host striatal neurons; lead to increases in DA uptake parameters; bring about a functional recovery, evidenced by induction of a contralateral rotational asymmetry after unilateral grafts and by an enhancement of locomotor performance after bilateral transplantation. However, grafts seem to do better in the surviving DA neuron number when implanted into wild-type (+/+) host mice with nigral 6-hydroxydopamine (6-OHDA) lesions, compared to weaver hosts. In Parkinson’s disease, the progressive degeneration of nigral neurons occurs over many years. It is conceivable that in Parkinsonian patients receiving neural grafts, the ongoing disease process might destroy donor DA neurons.4 The question as to whether one example of a disease process interferes with the survival of grafted neurons can be addressed in transplantation studies in weaver mice. Further studies could look into factors that may be responsible for the vulnerability of grafted DA cells in the chronically ill weaver environment, and determine whether or not experimental manipulations can optimize graft performance. When one uses a recipient model with chronic degeneration, one is confronted with an added degree of complexity. The pathological system inside which graft development is monitored evolves dynamically, and special care must be taken in designing experiments. The following general guidelines are meaningful:

Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease, by Lazaros C. Triarhou. ©2001 Eurekah.com.

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• Standardizing the ages of host animals at which transplantation is performed. Our past experience has shown that by proper choice of host age at around three months, we minimize the volatile effect of the dynamic degenerative process and manage to achieve greater degree of homogeneity of the experimental subjects. The major changes in DA cell number in the weaver substantia nigra occur in two waves, one between birth and three months (70% loss), and one during the second year of life (additional 15%); between those two periods there is a time of a relative steady state, which is the best temporal window to use for transplantation. • Standardizing the biological age of donor tissue and the number of injected viable donor cells and monitoring aliquots of the cell suspensions histochemically by obtaining cell counts in smears of left-over suspension using standard protocols.5,6 • Assigning animals from the same transplantation session to the various experimental groups evenly, such that noncontrolled technical parameters are equally divided among the various treatments. • Applying the appropriate designs in statistically analyzing the results and meeting the assumptions required in the validity of the various tests of significance.7 Some ideas of potential interest for the weaver model are discussed next, pertinent to the following topics: 1. immediate early events after transplantation; 2. potentially “poisonous” factors in the weaver brain and the use of “survival” factors to optimize graft growth; 3. neural transmission mechanisms of graft-induced restoration of function; 4. the supplemental reconstruction of the interrupted nigro-striato-nigral loop by double grafts. Issues of more general interest, such as the use of alternative sources of donor tissue, including progenitor cells, adult neural stem cells, immortal cell lines, and genetically modified cells,8-11 as well as the use of neuroprotective compounds to improve graft survival12-15 are covered in the appropriate bibliographical sources.

Analysis of Early Events in Graft-Host Interactions Apoptosis The limited availability of target-derived growth factors may enhance cell death in the grafts compared with normal development. Apoptotic cell death is a regressive modality altogether different from the cellular death observed during necrotic events.16 Necrosis occurs after major intoxications, hypoxia or tissue trauma. Apoptosis, on the other hand, is a more “physiological” mode of cell death observed during processes such as tissue organization and turnover. Characteristically, apoptotic cells are efficiently removed by neighboring cells or macrophages. Although this mode of cell death may comprise the majority of total cell death in nature, it is less conspicuous than necrosis and was first described comprehensively only in 1972.17 A fundamental biochemical mechanism of apoptosis is a change in chromatin structure, often associated with fragmentation of DNA into oligonucleosomal fragments of about n x 180 base pairs due to the activation of an endogenous endonuclease. Such a fragmentation occurs early in the time-course of apoptosis and precedes the disintegration of the plasma membrane. Apoptosis is known to be triggered in several cell types by inflammatory mediators such as tumor necrosis factor-α (TNF) or nitric oxide, and tissues can be damaged as the ultimate consequence of the

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release of such endogenous mediators. Histological techniques are available that allow the analysis of DNA fragmentation directly on the slide. Developmental cell death occurs in most areas of the central nervous system.18 Programmed (apoptotic) cell death is an active process that may require gene transcription and protein synthesis.19-21 Although the direct evidence for its role in nigral ontogeny is scanty, it is plausible that neuronal cell death plays an important part there as well, both in normality and in the transplantation setting. Absence of a target is one of the major causes of developmental cell death.18 It is known that nigral neurons in vitro may depend on growth factors such as brain-derived neurotrophic factor (BDNF), basic fibroblast-growth factor (bFGF) and interleukin-6 (IL-6) for their survival.22-24 The limited availability of such target-derived growth factors may enhance cell death in the transplants compared with normal development; this may be especially true for grafts placed into the weaver striatum, which seem to contain fewer surviving DA neurons than grafts placed in the striatum of wild-type mice with 6-OHDA lesions. In the past, it was generally assumed that the observed cell death in transplants was necrotic and associated with poor vascularization and tissue trauma, until a study in rats showed numerous apoptotic cells with fragmented DNA in mesencephalic grafts at 5-15 days after grafting and suggested that developmental cell death may function to eliminate cells that fail to compete successfully in connecting with appropriate postsynaptic targets during the period of vigorous axonal outgrowth and synaptogenesis.25 The current thought is that the first week after transplantation is a mostly critical period, when most implanted neurons die by apoptosis.26-29 Accordingly, antiapoptotic agents such as caspase inhibitors are helpful in improving graft survival experimentally.30

Inflammatory Cytokines Cytokines are considered as constitutive factors of the brain, involved in the regulation of nervous system development by mediating growth, lineage commitment and cellular differentiation. The analysis of the expression of interleukin-1α, IL-2 and its receptor, IL-6, macrophage-colony stimulating factor-1 (MCSF) and its receptor c-fms , and TNF and its receptor in the weaver brain might yield interesting results. Experimental studies on neural transplantation have led to the appreciation of the complex interactions between neurotrophic factors, inflammatory cytokines, the grafted tissue, and the host brain’s response.31 In that sense, neural grafting contributes to a better understanding of plasticity and the common features it shares with ontogenetic and regenerative events in the central nervous system. In transplantation experiments, one may place special emphasis on the expression of inflammatory cytokines in the early stages of graft development, as well as on the possible tissue treatment with antiinflammatory compounds such as lipopolysaccharide and indomethacin32 to enhance cellular repair. In the central nervous system, cytokines are found either in the context of normal developmental processes or in response to injury.33-35 Advances in the understanding of the structure of the receptors for neurotrophic factors reveal them to be much like the receptors used by cytokines and “traditional” growth factors operating elsewhere in the body; the distinct actions of neurotrophic factors may be linked not to the utilization of novel receptor systems, but rather to the fact that they activate these receptors in neurons.36 Further, brain-derived IL-1 has been proposed as an early contributor to a cascade of regressive events that lead to the pathophysiological changes of human neurodegenerative diseases.37

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Activated glial cells in the substantia nigra in Parkinson’s disease but not in control brains can produce toxic cytokines, such as the proinflammatory cytokine TNF, which may provoke cell death;38 furthermore, the cell bodies and processes of most dopaminergic neurons show positive immunoreactivity for TNF receptor (TNF-R), suggesting that nigral neurons may be sensitive to TNF produced in Parkinson’s disease. On the other hand, TNF may lead to an increased permissiveness of the optic nerve to neuronal adhesion and neurite extension after injury, thus promoting macrophage-mediated regeneration.39 MCSF and its receptor (c-fms ) are produced constitutively in normal mouse brain during development from E13 through adulthood.35 IL-6 and monocyte chemoattractant protein-1 (MCP1) present distinct patterns of expression during pre- and postnatal development of the rat cerebral cortex and hippocampus.40 Phagocytic microglia produce both neurotoxic factors that extend tissue damage during acute inflammatory responses in the nervous system and growth-promoting factors that support astroglia and indirectly neuronal survival.41 The neurobiological interest in interleukin-1α has increased dramatically over the last years, as it was found to be synthesized within the brain and act directly to modify local and systemic neural functions, by participating in neurotrophic, neural plasticity, and neuronal degeneration.42 Stimulated microglia produce IL-1, a potent mitogen for astroglia that indirectly promotes neuron survival through its action on astrocytes in vitro.43 IL-2, a protein secreted by T lymphocytes, is also found in mammalian brain, and its topographic distribution encompasses the dorsal neostriatum of the mouse.44 Receptor sites for IL-2, similar to the type present on the surface of T cell subsets, have been localized in brain as well, where they can mediate a variety of biological effects including neuromodulation of transmitter release.44 IL-2 activity has been described in injured rat brain.45 Immunocytochemical localization of cytokines46 in grafted and nongrafted weaver brain may lead to an improved understanding of their role in the graft-induced inflammatory reaction of the host, as well as the graft-host neurotrophic interactions. One could for example study the expression of IL-1, IL-2 and its low-affinity receptor subunit Tac, IL-6, MCSF and c-fms , TNF and TNF-R in the grafted and nongrafted striatum of animals with unilateral transplants (at early time-points, i.e., two days to two weeks after grafting), as well as in wild-type control mice, using a battery of available antibodies. In particular, the following questions could be asked: Which of those cytokines and receptors are normally produced by the mouse striatum and substantia nigra? How is the expression influenced by the ongoing degenerative process in the weaver brain? What is the response of the host brain to the transplantation intervention? Do the grafts recapitulate the normal expression in the substantia nigra in situ or are there departures from normality?

Trophic Considerations The Weaver Environment The two more readily explanations for the finding that grafts show a poorer survival inside the weaver brain compared to +/+ hosts with 6-OHDA lesions are either that the ongoing degenerative process of the weaver produces “poisonous” products or that the weaver striatum lacks “survival” factors necessary for optimal graft growth and maintenance. The differences in graft survival between weaver and wild-type 6-OHDA hosts have been observed five months after transplantation.47 Taking into account previous studies

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in weaver mutants that show continued fiber outgrowth between one and four months after transplantation, with no much change thereafter for up to nine months,48,49 one is led to think that the most crucial period for the survival of donor dopaminergic cells may be during the first one or two weeks after the operation. One way to test whether the weaver striatum lacks “survival” factors is to graft healthy striatal cells to weaver mutants ahead of time, prior to the transplantation of DA neurons in a second phase, and to determine whether such a manipulation ameliorates graft performance. An experimental design could consist of three different conditions: mesencephalic grafts placed into the weaver striatum (MES→WV); striatal/mesencephalic cografts placed in the weaver striatum (MES+STR→WV); mesencephalic grafts placed into the striatum of 6-OHDA lesion wild-type mice (MES→WT). The theoretical outcomes are: 1. (MES→WV) < (MES+STR→WV) ≤ (MES→WT). This outcome would support the idea of missing striatal “survival” factors, and suggest that supplementing missing chemical/cellular cues through striatal cell transplantation improves mesencephalic cell survival. 2. (MES→WV) = (MES+STR→WV) < (MES→WT). This outcome would imply that the wild-type host brain environment contains cues leading to better graft survival than the weaver striatal environment, regardless of supplementing or not the latter with striatal tissue grafts.

Growth Factor Treatment Factors known to have trophic effects on mesencephalic DA cells can be used to optimize graft survival in weaver hosts. For a specific growth factor treatment to be meaningful, the corresponding growth factor receptor must be present in transplanted DA cells, so that they would be able to receive an intercellular trophic signal. Contrary to the case of neurotransmission, where neurotransmitters released from presynaptic terminals act on postsynaptic receptors, in the case of growth factor-receptor interactions, the growth factor may be released by the postsynaptic neuron, internalized by the presynaptic terminal via its receptor, and retrogradely transported to the soma. There are growth factors with demonstrated trophic effects on mesencephalic DA neurons, whose genes, as well those of their receptors, have been cloned and sequenced. Basic FGF (bFGF) is a member of a family of structurally and functionally related pluripotent polypeptides with mitogenic and trophic action on cells of mesodermal and neuroectodermal origin.50 The cDNA clone encoding rat bFGF has been cloned and sequenced and corresponds to a molecule consisting of 154 amino acid residues.51 In cultures of rat mesencephalic DA neurons, bFGF was found to promote neuronal survival and to have mitogenic activity.23,52 Two groups have independently described enhancing in vivo effects of bFGF on the survival and sprouting of mesencephalic DA neuron grafts implanted to the denervated caudate-putamen of the rat.53,54 The effects of bFGF are mediated through binding to a high affinity surface receptor of 130 kDa, the complementary DNA of which has been cloned and sequenced.55,56 The bFGF receptor (FGF-R) contains three extracellular immunoglobulin-like domains, an unusual acidic region, and an intracellular tyrosine kinase domain, arranged in a pattern different from any growth factor receptor described.55 The FGF-R sequence lies between the nucleotide sequences of two highly conserved amino acid motifs from the catalytic domain of protein-tyrosine kinases.56

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To localize the FGF-R gene product, an antisense oligonucleotide complementary to positions 1447-1470 of the mouse FGF-R mRNA sequence56 can be used. A monoclonal antibody that recognizes the mouse FGF-R is available commercially from Chemicon (clone MAB125). To test the trophic influence of bFGF treatment on graft growth parameters, grafts can be treated with 10 ng/5 µl bFGF prior to transplantation. One may assess the survival rate of grafted tyrosine hydroxylase (TH) positive cell bodies in immunohistochemical preparations, as well as the dopaminergic axonal outgrowth with [3H]mazindol binding autoradiography, which gives a precise quantitative measure of the number of uptake carrier molecules and thus an indicator of the number of DA axon terminals. In previous reports with DA-rich grafts to the caudate of 6-OHDA lesioned rats and intrastriatal infusions of bFGF, it was found that TH immunopositive neuron survival was enhanced.53,54 It will be important to determine whether bFGF treatment of grafts placed into weaver hosts can bring the number of surviving DA neurons to the levels observed in grafts placed into +/+ mice with 6-OHDA lesions. BDNF is a dimeric protein of 13.5 kDa subunits, produced mainly in the central nervous system.57 It has been shown to promote the survival of dopaminergic neurons from the substantia nigra in cell cultures.22 Treatment of cultures derived from embryonic rat ventral mesencephalon with BDNF results in dose-dependent increases in the number of TH positive neurons, DA uptake activity, and DA content.58 Moreover, when BDNF was infused into the striatum of adult rats, it was found to be retrogradely transported and colocalized with DA in nigral neurons, indicating that these cells may be responsive to the trophic influences of BDNF.59 BDNF was shown to stimulate autophosphorylation of and transduce signals through the trkB member of the tyrosine kinase receptor family: the trk proto-oncogene is transcribed only in neural crest-derived components of the nervous system;60 trkB, a gene structurally related to trk, is expressed in embryonic and adult nervous systems and has been identified as the receptor for BDNF.61 To follow the temporal sequence of trkB gene expression in mesencephalic grafts at various times after transplantation, one may localize the trkB gene product by using antisense oligonucleotides complementary to bases 1879-1920 for the full-length trkB, to bases 723-767 for truncated trkB,62,63 and oligonucleotides complementary to bases 219-266 and 777-824 of the mouse BDNF mRNA sequence.64 To test the trophic influence of BDNF treatment on graft growth parameters, one could treat the cell suspensions prior to grafting with 50 ng/ml of recombinant BDNF (product # G1491, Promega, Madison, WI) and assess the survival rate of grafted TH positive cells and the dopaminergic axonal outgrowth as outlined above in the case of bFGF treatment. In a previous report with DA-rich grafts to the caudate of 6-OHDA lesioned rats and intrastriatal infusions of BDNF, it was found that functional graft effects were enhanced but TH immunopositive numbers did not change in comparison with vehicle injections.65 However, there are several ways in which the weaver model might differ from the 6-OHDA lesion model in graft responses. GDNF (glial cell line-derived neurotrophic factor) was identified and cloned; it belongs to the transforming growth factor-β superfamily, with strong evidence for trophic effects on midbrain dopaminergic neurons.66 GDNF treatment of intraocular ventral mesencephalic grafts led to doubling or quadrupling of surviving DA neuron number depending on dosage.67 GDNF was shown to exert protective and reparative effects on the nigrostriatal DA system in C57BL mice against MPTP-induced neurotoxicity68 and to prevent axotomy-induced retrograde degeneration of nigral DA neurons in rats in vivo.69

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Trophic effects of GDNF have been described in adult rat facial motor neurons and in developing avian and murine spinal motor neurons.70,71

Neurotransmitter Mechanisms The ventral striatum, specifically the nucleus accumbens and the isles of Calleja, is an area of increased interest because of the discovery of the DA D3 receptor. Investigating the accumbens is also significant, as that structure behaves differently from the dorsal striatum with regards to DA D2 receptors, nerve cell counts, and transplantation effects. DA D3 receptor gene expression is seen predominantly in the nucleus accumbens and the isles of Calleja.72 In previous studies, DA D3 receptor mRNA signal was strong in the isles of Calleja in both +/+ mice and in wv/wv mutants, and a slight increase in message was seen in the weaver nucleus accumbens.73 The ventral striatum can be studied in more detail, with particular emphasis on the effects of ventrally placed grafts. The nucleus accumbens presents particular interest in the weaver mutant for several reasons: 1. On the presynaptic end, DA content is unchanged in the wv/wv nucleus accumbens;74 however, the uptake and storage of [3H]DA inside the nerve terminals are below normal.49,75 [3H]Mazindol binding is decreased by 88% compared to +/+,76 which is disproportionate to the 36% decrease in synaptosomal [3H]DA uptake.75 This would suggest that there are large decreases in the number of DA uptake carriers, but [3H]DA uptake may be affected less severely, possibly due to compensatory changes occurring in the nucleus accumbens to actually increase the activity of the DA transporter. 2. The total number of medium-sized neurons in the wv/wv nucleus accumbens is reduced by approximately 27% from the total +/+ number.3 3. DA D2 receptors as measured by [3H]spiperone binding in tissue homogenates are reduced in the wv/wv nucleus accumbens.77 4. After DA neuron transplantation to the dorsal striatum, the only significant correlation between rotational bias and [3H]spiperone binding was observed in the nucleus accumbens region.77 This result was not anticipated and is less readily explained than e.g., in dorsal striatum. Brundin et al78 have studied rats with 6-OHDA lesions unilaterally in the mesostriatal pathway and bilaterally in the accumbens. Unilateral transplants in the accumbens amplified the locomotor response and the ipsilateral turning in response to amphetamine. These authors emphasized the “amplifier” as opposed to the “directional” role player by the accumbens in motor responses in agreement with the suggestions of Kelly and Moore.79 5. While 6-OHDA lesions in rats lead to decreases in DA D3 receptor gene expression in the nucleus accumbens,80 in the weaver accumbens we have observed a slight increase in DA D3 mRNA signal.73 This reiterates the biological differences between the acute 6-OHDA neurotoxic model of mesostriatal DA deficiency and the chronic-progressive lesion induced by the weaver gene,81 which may be related to the developmental failure of DA axon terminals to establish an adequate synaptic connectivity, to differences in receptor responses and adaptive mechanisms and to the functional complexity of monoamine interactions in different states of central DA deficiency depending on the acuteness or chronicity of the deafferentation process.

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Rotational asymmetry tests are generally conducted after methamphetamine administration. However, it is known that bilateral local depletion of DA from the nucleus accumbens with 6-OHDA causes an attenuation of the rotational behavior observed in rats with unilateral mesostriatal 6-OHDA lesions.78 Unilateral DA-rich grafts to the accumbens elevate the number of turns opposite to the direction of circling produced when a graft is placed into the ipsilateral caudate-putamen, suggesting that the accumbens plays only an amplifying and not a directional role. Based on that reasoning, one might predict that DA-rich grafts implanted into the weaver nucleus accumbens could lead to an increase in ipsilateral to the graft rotations as opposed to the contralateral turning bias induced by grafts to the dorsal striatum. One additional behavior relevant to ventrally placed grafts is the reversal of lesion-induced hyperactivity or the attenuation of supersensitive locomotor responses after apomorphine.78,82

Supplemental Restoration of the Interrupted Nigro-StriatoNigral Loop by Striatal/Nigral Double Grafts One obvious supposition is that a grafting operation in an adult degenerating brain cannot lead to a sequence of events identical to those during normal fetal ontogeny. Therefore, the reconstruction of defective neuronal circuits by means of neural transplantation is limited, owing in part to the temporal mismatch between donor and host tissues and the ensuing discrepancies in developmental patterns that involve migratory pathways, chemical communication cues, and synaptogenetic events. Double-grafting experiments can be an aggressive approach of transplantation engineering, aimed at reconstructing multiple aspects of a defective neuronal network. Double transplantation studies have been carried out intracerebrally83-88 and in oculo89-95 to address issues of survival and trophic effects. To reconstruct the interrupted nigro-striato-nigral loop, one might implant: 1. DA cells orthotopically into the substantia nigra—in addition to DA cells in the striatum—to provide for missing postsynaptic DA target cells to striatonigral, pallidosubthalamonigral, and pallidohabenulonigral projections, as well as supply the local dendritic DA effects in the substantia nigra pars reticulata; 2. striatal cells into the nigra or the dorsal pallidum in addition to DA cells in the striatum, to supplement missing striatal projection neurons to the striatonigral and striatopallidonigral pathways. DA-containing grafts lead to functional recovery in DA deficiencies. Usually, the recovery is incomplete. Although the behavioral reversal of amphetamine-induced rotational asymmetry is an invaluable index of graft function in models of DA deficiency, its relevance to the clinical improvement of Parkinsonian signs is unclear, whereas parameters such as spontaneous rotations, sensory neglect, tremor, rigidity and hypokinesia bear definite clinical relevance to the functional capacity of the grafts.4 Simple but reliable quantitative motor performance tests of spontaneous (i.e., not drug-elicited) behavioral improvement have merit in that they involve clinically relevant signs, where a precise reconstruction of local connectivity is thought necessary, as opposed to the pure release of DA from the axon terminal that is involved in drug-induced turning behavior.96,97 In replacing dopaminergic neurotransmission in the caudate-putamen by means of intrastriatal nigral transplantation, only one aspect of the anatomical pathway is restored. Several other components in the nigro-striato-nigral loop and its connections remain disrupted. For example, the nerve endings of striatonigral, pallidosubthalamonigral, and

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pallidohabenulonigral pathways still remain without a target in the substantia nigra, and the GABA (γ-aminobutyric acid) output from the substantia nigra to the mesencephalic reticular formation is interrupted in multiple ways in terms of presynaptic afferents. It is important to determine whether restructuring those components of the striatonigral network in addition to providing a source of DA neurotransmission in the striatum can lead to further improved function compared to intrastriatal DA grafts alone. As a rule, in the various models of nigrostriatal DA deficiency studied, mesencephalic DA cells are grafted into the host striatum. The reasoning is to re-establish DA neurotransmission in the terminal field of the caudate-putamen complex. Nonetheless, the nigrostriatal and striatonigral projections form a neuronal loop, where nigral DA neurons do more than just “pump” DA into the striatum: they receive synaptic input from striatonigral axon terminals, whose physiological function they are thought to modulate, and they provide DA through somatodendritic release mechanisms to nondopaminergic nigral neurons projecting to the thalamus and the contralateral substantia nigra. To achieve a greater restoration of function, those components of nigrostriatal DA neuron activity may have to be corrected as well. One approach that has been used to achieve an optimized nigrostriatal reconstruction after 6-OHDA lesions in rats was to combine DA-rich nigral grafts in the vicinity of the host substantia nigra with injections of striatal tissue grafts along a single oblique needle penetration through the frontal pole and neostriatum towards the nigra to form a nigrostriatal “bridge”.83 Such embryonic striatal “bridges” provided a continuous column of tissue from the nigral DA grafts to the host striatum. In certain ways, that approach provides a restoration of the damaged pathway that resembles more closely the normal situation. In addition to losing nigrostriatal DA cells, weaver mutant mice also manifest a loss of medium-sized striatal neurons.3 The striatal cell loss is smaller than the nigral neuron loss in magnitude (22% as opposed to >70%), and could be secondary (transsynaptic), resulting from the striatal deafferentation. Regardless of cause, the fact remains that there is a deficit in the striatal neuronal complement as well. A similar phenomenon has been seen in postmortem brain tissues from patients with Parkinson’s disease.98 The striatal primordium develops as two elevations in the rostral floor of the lateral ventricles and striatal neuron genesis occurs about two days later than the substantia nigra.99,100 Therefore, to obtain striatal tissue for grafting in the mouse, one selects embryos at E14 (CRL=12 mm). After the fetal brain is removed from the cranium, a longitudinal cut is made through the medial cortex to expose the striatal primordium and the striatal eminence is snipped off by a superficial horizontal cut.101 Regarding the temporal relationships of double transplants, although technically it is possible to place two different grafts in one animal in the same surgery session, it may be preferable to carry out the procedure in two steps. In one experiment, the “conventional” intrastriatal DA neuron grafts can be implanted first, and after one month (which allows for growth and functional maturation), the second operation can be implemented, placing nigra-to-nigra or striatum-to-nigra/pallidum grafts, as appropriate. In that way, it is possible to perform functional tests before and after surgery in the same mice, which is also the optimal design for purposes of statistical analyses, as “repeated measures” are used, where each mouse is compared to itself pre- and postoperatively, and thus animal-to-animal variation is minimized. The order of tissue transplantation can be reversed, such that striatal innervation of the substantia nigra would be replaced first, followed by DA neuron

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transplantation into the striatum. Accordingly, there are three main factors to consider in such experiments: graft site (intrastriatal and intranigral), type of donor material (mesencephalic suspensions nested within intrastriatal site, striatal suspensions nested within intranigral site, as well as sham injections nested within the two sites), and temporal order or sequence of the operation (intrastriatal followed by intranigral, and intranigral followed by intrastriatal). Multivariate statistical techniques, such as multiple regression analysis, can be used for example to correlate the number of surviving cells in the two different grafts with behavioral scores. The conventional equation for such analyses is Y = α0 + α 1X 1 + α 2X 2 where the estimate of the dependent variable Y (in this case a behavioral score) is a function of two independent variables, intrastriatal graft X 1 and intranigral graft X 2.102 A correlation coefficient such as α j denotes the regression coefficient of Y on variable X j that one would expect if all of the other variables in a regression equation had been held constant experimentally at their means. In a diagrammatic representation of a multiple regression of Y on two independent variables X 1 and X 2 the regression line becomes a plane, the slope or tilt of which is determined by the partial regression coefficients that correspond to the slope of the plane in the X 1 and in the X 2 directions. With such double-grafting experiments one can aim at a segmental reconstruction of damaged neuronal pathways through multiple placement of individual grafts at the appropriate anatomical sites, in order to provide in each case the missing input, which is important for reinstating physiological activity. References 1. Triarhou LC, Norton J, Ghetti B. Mesencephalic dopamine cell deficit involves areas A8, A9 and A10 in weaver mutant mice. Exp Brain Res 1988; 70:256-265. 2. Triarhou LC. Weaver gene expression in central nervous system. In: Conn PM, ed. Gene Expression in Neural Tissues. San Diego: Academic Press, 1992:209-227. 3. Bayer SA, Triarhou LC, Thomas JD et al. Correlated quantitative studies of the neostriatum, nucleus accumbens, substantia nigra, and ventral tegmental area in normal and weaver mutant mice. J Neurosci 1994; 14:6901-6910. 4. Lindvall O. Neural transplantation in Parkinson’s disease. In: Dunnett SB, Björklund A, eds. Functional Neural Transplantation. New York: Raven Press, 1994:103-137. 5. Brundin P, Isacson O, Björklund A. Monitoring of cell viability in suspensions of embryonic CNS tissue and its use as a criterion for intracerebral graft survival. Brain Res 1985; 331:251-259. 6. Brundin P, Strecker RE. Preparation and intracerebral grafting of dissociated fetal brain tissue in rats. In: Conn PM, ed. Lesions and Transplantation. San Diego: Academic Press, 1991:305-326. 7. Winer BJ. Statistical Principles in Experimental Design, 2nd edn. New York: McGraw-Hill, 1971. 8. Svendsen CN, Caldwell MA, Shen J et al. Long-term survival of human central nervous system progenitor cells transplanted into a rat model of Parkinson’s disease. Exp Neurol 1997; 148:135-146. 9. Svendsen CN, Caldwell MA, Ostenfeld T. Human neural stem cells: Isolation, expansion and transplantation. Brain Pathol 1999; 9:499-513. 10. Kuhn HG, Svendsen CN. Origins, functions, and potential of adult neural stem cells. Bioessays 1999; 21:625-630. 11. Freed WJ. Neural Transplantation: An Introduction. Cambridge, MA: MIT Press, 2000. 12. Nakao N, Frodl EM, Duan WM et al. Lazaroids improve the survival of grafted rat embryonic dopamine neurons. Proc Natl Acad Sci USA 1994; 91:12408-12412. 13. Othberg A, Keep M, Brundin P et al. Tirilazad mesylate improves survival of rat and human embryonic mesencephalic neurons in vitro. Exp Neurol 1997; 147:498-502. 14. Kaminski Schierle GS, Hansson O, Brundin P. Flunarizine improves the survival of grafted dopaminergic neurons. Neuroscience 1999; 94:17-20. 15. Karlson J, Love RM, Clarke DJ et al. Effects of anaesthetics and lazaroid U-83836E on survival of tranplanted rat dopaminergic neurones. Brain Res 1999; 821:546-550.

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Index Symbols (RS)-α-amino-3-hydroxy-5-methylisoxazole-4propionate 1, 3, 85, 91 [11C]nomifensine 1 [32P]oligonucleotide probes 46, 66, 67, 82 [3H]CNQX (6-cyano-7-nitro-quinoxaline-2,3dione) 85 [3H]CV 205-502 82, 83 [3H]DA uptake 22, 43, 75, 77, 78, 80, 92, 112 [3H]flunitrazepam 86 [3H]GBR 12909 79 [3H]GBR 12935 79, 80, 85 [3H]glutamate 85 [3H]mazindol 1, 24, 79, 81, 111, 112 [3H]muscimol 86 [3H]SCH 23390 23 [3H]spiperone 23, 81, 82, 92, 112 [3H]spiroperidol 23 [3H]thymidine 17, 19, 28 1-methyl-4-phenylpyridine (MPP+) 4 28 kDa calcium-binding protein (CaBP) 17, 45, 62 3,4-dihydroxyphenylalanine (L-DOPA) 3, 4 6-hydroxydopamine (6-OHDA) 4, 6, 37, 43, 44, 60, 75, 80, 82, 85, 89, 93, 94, 96-98, 106, 108-114 6-L-[18F]-fluorodopa 1, 75 ω-conotoxin GVIA 25

A Alternative splicing 46 Alzheimer’s disease 46, 47 American Society for Neural Transplantation and Repair (ASNTR) 7 AMPA receptor subunits 87, 91 Amphetamine 4, 30, 60, 82, 92-94, 97, 98, 100, 106, 114, 119 Apomorphine 30, 80, 93, 119 Apoptosis 119 Aprotinin 14 Area CA3 27 Astroglia 14, 26, 28, 41, 48, 109 Astrotactin 28 Asymmetrical synapses 56 Ataxia 30, 90

ATP-dependent glutamate uptake system 25 ATP-regulated K+ channels 25 Avian leukemia protooncogene (Ets-2) 13 Axonogenesis 24 Axotomy 47, 111

B β-amyloid protein precursor (βAPP) 13, 46-48 β2 chimerin 26 Balance rod tests 90, 100 Basic fibroblast-growth factor (bFGF) 108, 110, 111 Bergmann glia 26, 28 Basic fibroblast-growth factor receptor (bFGF-R) 110, 111 Bilateral graft 89, 90, 99, 100 Brain-derived neurotrophic factor (BDNF) 108, 111 Brainware engineering 6

C c-src protooncogene-encoded protein-tyrosine kinase 25 c-fms 108, 109 Calcicludine 26 Calcineurin 70 Catecholamine histofluorescence 22 Caudate nucleus 6, 20 Caudate-putamen complex 23, 37, 39, 41, 53, 68, 76, 79, 80, 82, 114 Cell suspension graft 38-43, 47, 54, 64-67, 69, 70, 75, 77, 91, 100 Cerebellar cortex 13, 26, 27, 29 Cerebellar graft 29, 67, 89 Cerebellomedullary cistern 29 Chemeiotactic affinity 64 Chemoaffinity 64 Cholecystokinin (CCK) 45, 62 Choline acetyltransferase (ChAT) 57 Cingulate cortex 63 Circling behavior 89 Climbing fibers 27 Cocaine 79 Concanavalin A 26

Index Corpus callosum 44, 99 Corpus striatum 1, 4 Corticostriatal pathway 86 Cytokines 108, 109

D D1.1 ganglioside 26 D3 receptor 112 DA β-hydroxylase (DBH) 39, 40 Decay constant 16 Delayed-cavity transplantation protocol 37 DeMyer silver 39, 40 Dendritic shaft 55, 56, 60 Dendritic spine 1, 27, 29, 55, 56, 60, 61 Dendritogenesis 20 Depth of reinnervation 53, 80 Desipramine 75, 79 Developmental cell death 44, 108 Differential ultracentrifugation 80 DNA fragmentation 108 Dopamine D1 receptors 23, 81 Dopamine D2 receptors 23, 78, 81, 82, 92, 112 Dopamine D3 receptors 82, 112 Dopamine uptake 1, 22, 23, 43, 76, 78-81, 92, 106, 111, 112 Dopaminergic dendrites 20, 58, 66, 92, 97, 98 Double graft 107, 113 Down syndrome 46

E Electron microscopy 14, 41, 58, 60 Entorhinal cortex 68, 82 Excitatory amino acid receptor 85 Exponential decay 15 External germinal layer (EGL) 23, 24, 26

121 GIRK2 14, 31 Girk2 14, 31 Globus pallidus 1, 44, 67 Golgi epithelial cells 28 Granule cells 14, 20, 23-30, 68 Growth-associated phosphoprotein GAP-43 26, 66-70 Guanine nucleotide-binding protein (G protein) 31

H Half-life of neurons 16 Hematoxylin-eosin 3, 40 Hippocampus 13, 27, 48, 64, 109 Hole-board matrix 29 Hole-board test 101 Human chromosome 21 46 Human-to-rat graft 6, 61

I Immortal cell lines 107 In situ hybridization histochemistry 46, 47, 66, 67, 69, 82, 85 In vivo microdialysis 6 In vivo voltammetry 6 Indomethacin 108 Inhibitory amino acid receptor 85, 86 Interleukin-1α (IL-1α) 108 Interleukin-2 (IL-2) 108, 109 Interleukin-6 (IL-6) 108, 109 Internal granular layer (IGL) 23, 26 Inward-rectifier K+ channel 14 Isles of Calleja 82, 112

J Juvenile Parkinson’s disease 2

F

K

Familial Parkinson’s disease 13 Frontal cortex 21, 22, 38, 63, 64

Kainate 1, 85 Kainic acid 47 Kunitz isoforms 47

G γ-aminobutyric acid (GABA) 1, 4, 66, 86, 114 GDNF (glial cell line-derived neurotrophic factor) 111, 112 Genetically modified cells 107

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Dopaminergic Neuron Transplantation in the Weaver Mouse Model of Parkinson’s Disease

L Laminin 14 Lateral hypothalamus 64 Lateral septal nucleus 21, 63, 82 Lateral ventricle 39, 40, 44, 53, 62, 114 Lewy body 1-3 Limbic system 14, 91 Linear regression 15 Lipopolysaccharide 108 Locus ceruleus 64, 68, 89 LY 171555 80

M Macrophage-colony stimulating factor-1 (MCSF) 108, 109 Macrophages 107 MAP2 67 Mesencephalic grafts 6, 37, 39, 41, 45, 46, 53, 56, 59, 60, 68, 75, 78, 79, 81, 82, 85, 86, 89-91, 95, 98, 99, 108, 110, 111 Met-enkephalin 45 Methamphetamine 58, 89, 90, 94, 95, 113 Microglia 109 Microtubule-associated protein 2 (MAP2) 20, 21, 26, 67, 68, 69, 70 Missense mutation 14 Mitotic division 6, 37 Mitral cells 68 Molecular layer 23, 28, 29, 63 Monoaminooxidase B 4 Monoclonal antibody OZ42 26 Monocyte chemoattractant protein-1 (MCP1) 109 Mosaïc chimeras 27 Mossy fibers 27 Mouse chromosome (Mmu) 16 13, 46, 67 Multiple systems atrophies 13

N N-methyl–D-aspartate (NMDA) 1, 3, 4, 85 N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 4, 37, 60, 111 Navigational behavior 29 Necrosis 107 Nerve growth factor 26 Nervous mutant (nr/nr) 30 Network of European CNS Transplantation and Restoration (NECTAR) 7

Neural cell adhesion molecule (N-CAM) 66, 67, 70 Neural stem cells 107 Neurite outgrowth 14, 28 Neuroblasts 24, 37 Neurogenesis 17 Neuromuscular junction 64 Neuron fallout 15 Neuronal migration 13, 27 Neuroprotective compounds 107 Neurotensin 45, 83, 84 Neurotensin receptors 83 Ni(NH4)2(SO4)2 intensification 57 Nigro-striato-nigral loop 107, 113 Nissl stain 40 Nitric oxide 107 Nucleus accumbens 22, 23, 38, 45, 62, 64, 76, 80-82, 85, 92, 112, 113 Nucleus basalis of Meynert 47, 63 Nucleus centralis superior 68 Nucleus interpeduncularis 15, 62 Nucleus mammillaris lateralis 62 Nucleus mammillaris posterior 62 Nucleus raphé dorsalis 68 Nucleus supramammillaris 62

O Olfactory tubercle 21, 38, 62, 76, 82 Olivopontocerebellar degeneration (Menzel type) 13 Open-field tests 29 Opiate receptor binding 45 Optokinetic nystagmus 29

P Paracrine action 58 Paraflocculus 24 Parallel fibers 26, 27, 29 Paralysis agitans 1 Parasympathetic nervous system 64 Pargyline 75 Parietal cortex 64 Parkinson’s disease 1-6, 13, 41, 45, 63, 79, 83, 86, 96, 106, 109, 114 PC12 cells 48 Pergolide 30 Phenocopy 13, 28 Phosphorylated neurofilaments 66, 67 Pituitary 82 Posteroventral pallidotomy 4

Index Prefrontal cortex 63 Primary degeneration of the granular layer of the cerebellum (Norman type) 13 Progenitor cells 107 Programmed (apoptotic) cell death 107, 108 Programmed cell death 108 Protein P-38 66 Purkinje cells 26, 27, 28, 29, 30 Putamen 6, 14, 21, 23, 37-41, 44, 45, 53, 55, 68, 70, 76, 78-80, 82, 83, 85, 93, 96, 99, 110, 113, 114 Pyramidal cells 27, 68

R Resting membrane potential 14 Retinotectal projection 64 Retrorubral nucleus 14, 15, 45 Rotational asymmetry 89, 91, 92, 97, 98, 102, 106, 113

S Saxitoxin-sensitive Na+ channels 25 Seizures 20, 30 Silver grains 22, 76 SKF 38393 80 SMI-31 66, 67 SMI-32 66, 67 Solid grafts 29, 37, 38, 43, 55, 66 Soluble superoxide dismutase (Sod-1) 13 Spinal motor neurons 112 Spiny branchlets 27 Spiny projection neurons 1, 93 Stereological correction 55, 56, 61 Stratum oriens 27 Stratum radiatum 27 Striatal eminence 114 Striatal matrix 62 Striatal patches 62 Subependymal plate 23, 45 Substantia grisea centralis 62 Substantia nigra pars compacta 15, 18, 46, 67, 68, 70, 93 Substantia nigra pars reticulata 18, 20, 21, 30, 43, 64, 67, 70, 93, 113 Sulfonylurea receptors 25 Symmetrical synapses 1, 55 Sympathetic nervous system 64 Synapsin I 29 Synaptogenesis 6, 55, 67, 108 Synaptophysin 66, 67, 70 Synaptosomal fractions 80

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T T lymphocytes 109 T-maze 102 Tectal grafts 64 Temporal mismatch 57, 83, 113 Time-lapse microcinematography 28 Tissue culture 27-29 Tissue plasminogen activator 14 Tonic/clonic convulsions 30 Transforming growth factor-β superfamily 111 Triton X-100 43 trk proto-oncogene 111 trkB 111 Trypan blue exclusion 44 Tumor necrosis factor-α (TNFα) 107 Turning behavior 81, 85, 89, 91-93, 97, 113 Tyrosine hydroxylase (TH, tyrosine monooxygenase) 5, 14, 15, 17-19, 22, 25, 39, 40, 42, 53, 54, 78, 91, 111 Tyrosine kinase receptor family 111

U Unilateral grafts 80, 89, 90, 106 Uvula vermis 29

V Varicosities 20, 27, 53, 60, 61, 76, 78 Ventral tegmental area 14, 15, 45-47, 67, 68, 81-83 Vermis 26, 29

W Wheat germ agglutinin 26 Wnt-3 protein 26 wv mutation 14, 20, 26, 31

X X-ray irradiation 28 Xenopus oocytes 14

Z Zona incerta 62