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Cerebellar diseases causing substantial cell loss often lead to severe functional deficits and restoration of cerebellar function is difficult. Neurotransplantation therapy could become a hopeful method, but there are still many limitations and unknown aspects. Studies in a variety of cerebellar mutant mice reflecting heterogeneity of human cerebellar degenerations show promising results as well as new problems and questions to be answered. The aim of this work was to compare the development of embryonic cerebellar grafts in adult B6CBA Lurcher and B6.BR pcd mutant mice and strain-matched healthy wild type mice. Performance in the rotarod test, graft survival, structure, and volume was examined 2 months after the transplantation or sham-operation. The grafts survived in most of the mice of all types. In both B6CBA and B6.BR wild type mice and in pcd mice, colonization of the host's cerebellum was a common finding, while in Lurcher mice, the grafts showed a low tendency to infiltrate the host's cerebellar tissue. There were no significant differences in graft volume between mutant and wild type mice. Nevertheless, B6CBA mice had smaller grafts than their B6.BR counterparts. The transplantation did not improve the performance in the rotarod test. The study showed marked differences in graft integration into the host's cerebellum in two types of cerebellar mutants, suggesting disease-specific factors influencing graft fate.

For many degenerative cerebellar diseases, currently, no effective treatment that would substantially restore cerebellar functions is available. Neurotransplantation could be a promising therapy for such cases. Nevertheless, there are still severe limitations for routine clinical use. The aim of the work was to assess volume and morphology and functional impact on motor skills of an embryonic cerebellar graft injected in the form of cell suspension in Lurcher mutant and wild-type mice of the B6CBA and C3H strains after a 6-month survival period. The grafts survived in the majority of the mice. In both B6CBA and C3H Lurcher mice, most of the grafts were strictly delimited with no tendency to invade the host cerebellum, while in wild-type mice, graft-derived Purkinje cells colonized the host's cerebellum. In C3H Lurcher mice, but not in B6CBA Lurchers, the grafts had smaller volume than in their wild-type counterparts. C3H wild-type mice had significantly larger grafts than B6CBA wild-type mice. No positive effect of the transplantation on performance in the rotarod test was observed. The findings suggest that the niche of the Lurcher mutant cerebellum has a negative impact on integration of grafted cells. This factor seems to be limiting for specific functional effects of the transplantation therapy in this mouse model of cerebellar degeneration.

Background & Objective: Neurotransplantation has been recently the focus of interest as a promising therapy to substitute lost cerebellar neurons and improve cerebellar ataxias. However, since cell differentiation and synaptic formation are required to obtain a functional circuitry, highly integrated reproduction of cerebellar anatomy is not a simple process. Rather than a genuine replacement, recent studies have shown that grafted cells rescue surviving cells from neurodegeneration by exerting trophic effects, supporting mitochondrial function, modulating neuroinflammation, stimulating endogenous regenerative processes, and facilitating cerebellar compensatory properties thanks to neural plasticity. On the other hand, accumulating clinical evidence suggests that the self-recovery capacity is still preserved even if the cerebellum is affected by a diffuse and progressive pathology. We put forward the period with intact recovery capacity as "restorable stage" and the notion of reversal capacity as "cerebellar reserve"., Conclusion: The concept of cerebellar reserve is particularly relevant, both theoretically and practically, to target recovery of cerebellar deficits by neurotransplantation. Reinforcing the cerebellar reserve and prolonging the restorable stage can be envisioned as future endpoints of neurotransplantation., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Clinical trials testing the hypothesis that fetal dopamine grafts would provide antiparkinsonian benefit in patients who had already developed side effects from their long-term use of L-dopa revealed, in some cases, the presence of dyskinesias even in the absence of L-dopa. The form, intensity, and frequency of these dyskinesias were quite variable, but their manifestation slowed the clinical development of cell replacement therapies. Rodent models of graft-induced dyskinesias (GIDs) have been proposed, but their accuracy in modeling GIDs has been questioned because they usually require amphetamine for their presentation. The present study attempted to model GIDs in parkinsonian monkeys and, for the first time, to test the effect of grafts on previously dyskinetic monkeys. Toward this end, monkeys were rendered parkinsonian with n-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and dyskinetic with levodopa. They then received intraputamenal grafts of fetal dopaminergic cells, control cerebellar cells, or vehicle bilaterally and were studied for 18 months. Dopaminergic cells were grafted in a manner designed to produce either "hot spot" or "widespread" striatal innervation. Although levodopa-induced dyskinesias could be elicited postoperatively, GIDs were never observed in any animal at any time after grafting. Grafted monkeys were also challenged with levodopa but did not show any greater responses to these challenges than before grafting. These studies support the development of future dopamine neuron cell transplantation therapy-based approaches, indicating that in relevant primate models with appropriate cell preparation methodology, with successful graft survival and putamenal dopamine innervation, there is no evidence of graft-induced dyskinesias. J. Comp. Neurol. 525:498-512, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Hereditary cerebellar ataxias are severe diseases for which therapy is currently not sufficiently effective. One of the possible therapeutic approaches could be neurotransplantation. Lurcher mutant mice are a natural model of olivocerebellar degeneration representing a tool to investigate its pathogenesis as well as experimental therapies for hereditary cerebellar ataxias. The effect of intracerebellar transplantation of embryonic cerebellar solid tissue or cell suspension on motor performance in adult Lurcher mutant and healthy wild-type mice was studied. Brain-derived neurotrophic factor level was measured in the graft and adult cerebellar tissue. Gait analysis and rotarod, horizontal wire, and wooden beam tests were carried out 2 or 6 months after the transplantation. Higher level of the brain-derived neurotrophic factor was found in the Lurcher cerebellum than in the embryonic and adult wild-type tissue. A mild improvement of gait parameters was found in graft-treated Lurcher mice. The effect was more marked in cell suspension grafts than in solid transplants and after the longer period than after the short one. Lurcher mice treated with cell suspension and examined 6 months later had a longer hind paw stride (4.11 vs. 3.73 mm, P < 0.05) and higher swing speed for both forepaws (52.46 vs. 32.79 cm/s, P < 0.01) and hind paws (63.46 vs. 43.67 cm/s, P < 0.001) than controls. On the other hand, classical motor tests were not capable of detecting clearly the change in the motor performance. No strong long-lasting negative effect of the transplantation was seen in wild-type mice, suggesting that the treatment has no harmful impact on the healthy cerebellum.

SCA2 transgenic mice are thought to be a useful model of human spinocerebellar ataxia type 2. There is no effective therapy for cerebellar degenerative disorders, therefore neurotransplantation could offer hope. The aim of this work was to assess the survival and morphology of embryonic cerebellar grafts transplanted into the cerebellum of adult SCA2 mice. Four month-old homozygous SCA2 and negative control mice were treated with bilateral intracerebellar injections of an enhanced green fluorescent protein-positive embryonic cerebellar cell suspension. Graft survival and morphology were examined three months later. Graft-derived Purkinje cells and the presence of astrocytes in the graft were detected immunohistochemically. Nissl and hematoxylin-eosin techniques were used to visualize the histological structure of the graft and surrounding host tissue. Grafts survived in all experimental mice; no differences in graft structure, between SCA2 homozygous and negative mice, were found. The grafts contained numerous Purkinje cells but long distance graft-to-host axonal connections to the deep cerebellar nuclei were rarely seen. Relatively few astrocytes were found in the center of the graft. No signs of inflammation or tissue destruction were seen in the area around the grafts. Despite good graft survival and the presence of graft-derived Purkinje cells, the structure of the graft did not seem to promise any significant specific functional effects. We have shown that the graft is available for long-term experiments. Nevertheless, it would be beneficial to search for ways of enhancement of connections between the graft and host., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Lurcher mutant mice represent a natural model of olivocerebellar degeneration. They serve as a tool to study pathogenesis, the functional impact of the degeneration as well as therapeutic approaches. Wild type littermates are used as healthy controls. Neurotransplantation may be a promising method of therapy for neurodegenerative diseases. The aim of this work was to compare the long-term survival rate of the solid embryonic cerebellar graft in adult Lurcher mutant and wild type mice of the B6CBA strain and to assess the fundamental structural features of the graft. The graft was obtained from 12-day-old GFP mouse embryos. The brains of host mice were examined histologically 6 months after the transplantation. The graft was identified according to its GFP fluorescence. The graft presence and structure was assessed. The graft survived in all 14 Lurcher mice and in 12 of the 14 wild type mice. Cell migration and fibre sprouting from the graft were poor. No marked differences in the graft morphology between Lurcher mutant and wild type mice were found. The graft survival and appearance were similar to those after a shorter period described in a previous study. This suggests that during the 6 months, no intensive or commonly occurring processes changing the graft had proceeded and that the Lurcher mutant cerebellum niche had no strong influence over the fate of the solid cerebellar graft., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

This study has tested the feasibility of using physical delivery methods, employing static and oscillating field "magnetofection" techniques, to enhance magnetic nanoparticle-mediated gene transfer to rat oligodendrocyte precursor cells derived for transplantation therapies. These cells are a major transplant population to mediate repair of damage as occurs in spinal cord injury and neurological diseases such as multiple sclerosis. We show for the first time that magnetic nanoparticles mediate effective transfer of reporter and therapeutic genes to oligodendrocyte precursors; transfection efficacy was significantly enhanced by applied static or oscillating magnetic fields, the latter using an oscillating array employing high-gradient NdFeB magnets. The effects of oscillating fields were frequency-dependent, with 4 Hz yielding optimal results. Transfection efficacies obtained using magnetofection methods were highly competitive with or better than current widely used nonviral transfection methods (e.g., electroporation and lipofection) with the additional critical advantage of high cell viability. No adverse effects were found on the cells' ability to divide or give rise to their daughter cells, the oligodendrocytes-key properties that underpin their regeneration-promoting effects. The transplantation potential of transfected cells was tested in three-dimensional tissue engineering models utilizing brain slices as the host tissue; modified transplanted cells were found to migrate, divide, give rise to daughter cells, and integrate within host tissue, further evidencing the safety of the protocols used. Our findings strongly support the concept that magnetic nanoparticle vectors in conjunction with state-of-the-art magnetofection strategies provide a technically simple and effective alternative to current methods for gene transfer to oligodendrocyte precursor cells., (© 2011 American Chemical Society)

Mesenchymal stem cells have been proven to be potentially effective in the treatment of a large variety of diseases, including neurodegenerative disorders. Of these, cerebellar ataxia is a group of disorders characterized by the degeneration of the cerebellum, particularly the Purkinje cells, responsible for motor coordination and control of the motor functions. To analyze the possibility of using bone marrow-derived mesenchymal stem cells in treating ataxia, we transplanted these cells in the cerebellum of newborn Lurcher mutant mice, a very aggressive mouse model characterized by the selective early post-natal death of Purkinje cells in the cerebellum. Two months after the surgical procedure, the treated mice presented significant improvements in the motor behavior tests performed. Histological analysis of the cerebellum indicated that the donor cells had migrated throughout the cerebellum, as well as a significant increase in the number of Purkinje cells. Many grafted stem cells were located adjacent to the Purkinje cell layer, and expressed BDNF, NT-3 or GDNF, neurotrophic factors implicated in Purkinje cell survival. Also, a small percentage of the grafted stem cells had fused with Purkinje cells. Thus, we have shown that mesenchymal stem cells are capable of integrating into the central nervous system, migrate towards the areas where neurodegenerative processes are occurring, and rescue the degenerating cells through cell trophic effects. This is an adequate and feasible model that could be translated into a therapeutic approach for clinical assays in neurodegenerative diseases., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Lurcher mutant mice represent a model of olivocerebellar degeneration. They suffer from complete loss of Purkinje cells and a reduction of granule cells and inferior olive neurons. Their wild type littermates serve as healthy controls. The aim of the work was to compare solid embryonic cerebellar graft survival within a period of 9 weeks after their transplantation in adult Lurcher mutant and wild type mice of the B6CBA strain. The solid grafts were injected through a hole in the occipital bone. Host mice were sacrificed 3, 6, or 9 weeks after the transplantation and their cerebella and brain-stems were examined histologically to assess graft presence and structure. We did not find significant differences in graft survival rates between Lurcher mutant and wild type mice. The frequency of graft presence did not differ between mice examined 3, 6, and 9 weeks after the transplantation, neither in Lurchers nor in wild type mice. The grafts were of various sizes. In some cases, only small residua of the grafts were found. Nerve fiber sprouting and cell migration from the graft to the host tissue were observed more often in wild type mice than in Lurchers when examined 6 weeks after surgery. In the period 3-9 weeks after transplantation, massive dying out of the grafts was not observed despite regressive processes in some of the grafts. The degenerative changes in the Lurcher mutant cerebellum do not have strong impact on the fate of the solid cerebellar graft., ((c) 2009 Wiley-Liss, Inc.)

Lurcher mutant mice represent a model of olivocerebellar degeneration. They are used to investigate cerebellar functions, consequences of cerebellar degeneration and methods of therapy influencing them. The aim of the work was to assess the effect of foetal cerebellar graft transplantation, repeated enforced physical activity and the combination of both these types of treatment on motor skills, spontaneous motor activity and spatial learning ability in adult B6CBA Lurcher mice. Foetal cerebellar grafts were applied into the cerebellum of Lurchers in the form of solid tissue pieces. Enforced motor activity was realised through rotarod training. Motor functions were examined using bar, ladder and rotarod tests. Spatial learning was tested in the Morris water maze. Spontaneous motor activity in the open field was observed. The presence of the graft was examined histologically. Enforced physical activity led to moderate improvement of some motor skills and to a significant amelioration of spatial learning ability in Lurchers. The transplantation of cerebellar tissue did not influence motor functions significantly but led to an improvement of spatial learning ability. Mutual advancement of the effects of both types of treatment was not observed. Spontaneous motor activity was influenced neither by physical activity nor by the transplantation. Physical activity did not influence the graft survival and development. Because nerve sprouting and cell migration from the graft to the host cerebellum was poor, the functional effects of the graft should be explained with regard to its trophic influence rather than with any involvement of the grafted cells into neural circuitries.

Possible effect of trophic factors from embryonic cerebellar graft transplanted in adult Lurcher mutant mice on LTP as electrophysiological marker of learning and memory process was studied. Also the combination of the transplantation and long-term forced motor training was investigated. An evaluation of LTP ability in four animal groups (transplanted, sham-operated, with and without forced motor activity) and comparison among them showed the highest LTP improvement in the group with combination of both influences (ie. transplantation and motor training).

Possible influence of embryonic cerebellar graft transplanted into the adult neurodegenerative brain in Lurcher mutant mice on long-term potentiation (LTP) in hippocampus was investigated. Evaluation of LTP ability and comparison with the tests of motor learning suggests similarities between magnitude of LTP and criteria of motor learning. Also interstrain differences were described. Our results support ideas about tight cooperation among brain structures which are involved in mechanisms of learning and memory.

Lurcher mutant mice suffer from complete loss of cerebellar Purkinje cells. The aim of the work was to compare the solid embryonic cerebellar graft survival in adult Lurcher mutant mice derived from strains C3H and C57Bl/7 and to assess the morphology of the grafts. Embryonic cerebellar tissue was obtained from 12-13 days mice embryos expressing green fluorescent protein (GFP). Embryonic cerebellum was injected with a glass microcapillary into the cerebellum of adult Lurcher mutant mouse. Host mice were sacrificed 2-12 weeks after the transplantation. Brainstems and cerebella were examined histologically. The graft and graft derived GFP-positive cells were detected according to their green fluorescence. To visualise the structure of the graft Nissl staining was used. Graft survival percentage was evaluated in groups of mice sacrificed during the first, second or third month after the transplantation. The graft was found in all C57Bl/7 mice and in 90.9% of C3H mice examined within one month after the transplantation. In the second month the graft was present in 83.3% of C57Bl/7 and 50.0% of C3H mice. Till the third month the graft survived in 68.2% of C57Bl/7 mice and 22.2% of C3H mice. In C57Bl/7 mice a cerebellar structure was developed in the graft and migration of graft derived-cells to the host tissue was observed more often than in C3H mice. C567Bl/7 mice seem to be more suitable for experiments testing functional consequences of transplantation into the cerebellum requiring good long-term graft survival.

In the last decades, there have been many efforts directed to gain a better understanding on adult neuron-target cell relationships. Embryonic grafts have been used for the study of neural circuit rewiring. Thus, using several donor neuronal tissues, such as cerebellum or striatum, developing grafted cells have been shown to have the capability of substituting neural cell populations and establishing reciprocal connections with the host. In addition, different lesion paradigms have also led to a better understanding of target dependence in neuronal cells. Thus, for example, axotomy induces profound morphofunctional changes in adult neurons, including the loss of synaptic inputs and discharge alterations. These alterations are probably due to trophic factor loss in response to target disconnection. In this review, we summarize the different strategies performed to disconnect neurons from their targets, and the effects of target substitution, performed by tissue grafting, upon neural properties. Using the oculomotor system-and more precisely the abducens internuclear neurons-as a model, we describe herein the effects of disconnecting a population of central neurons from its natural target (i.e., the medial rectus motoneurons at the mesencephalic oculomotor nucleus). We also analyze target-derived influences in the structure and physiology of these neurons by using cerebellar embryonic grafts as a new target for the axotomized abducens internuclear neurons.

Study Objectives: The sleep disorder narcolepsy is now considered a neurodegenerative disease because there is a massive loss of neurons containing the neuropeptide, hypocretin, and because narcoleptic patients have very low cerebrospinal fluid levels of hypocretin. Transplants of various cell types have been used to induce recovery in a variety of neurodegenerative animal models. In models such as Parkinson disease, cell survival has been shown to be small but satisfactory. Currently, there are no data indicating whether hypocretin neurons can survive when grafted into host tissue. Here we examined the survival of hypocretin-containing neurons grafted into the pontine reticular formation, a region traditionally regarded to be key for rapid eye movement sleep generation., Design: In 2 experiments, a suspension of cells from the posterior hypothalamus of 8- to 10-day old rat pups was injected into the pons (midline, at the level of the locus coeruleus) of adult rats. Control rats received cells from the cerebellum, tissue that is devoid of hypocretin neurons. In the first experiment (n = 33), the adult rats were sacrificed 1, 3, 6, 12, 24, or 36 days after transplant, and cryostat-cut coronal sections of the brainstem were examined for presence of hypocretin-immunoreactive neurons. In the second experiment (n = 9), the transplant medium was modified to include agents that stimulate cell growth, and recipient rats were sacrificed 9, 12, and 36 days after receiving the graft., Settings: Basic neuroscience research laboratory., Measurements and Results: In the first experiment, clearly defined hypocretin-immunoreactive containing somata and varicosities were visible in pons of rats sacrificed 1 day after grafting of posterior hypothalamic cells but not in rats receiving cerebellum tissue. The hypocretin-immunoreactive somata were not visible in rats sacrificed at 12, 24, or 36 days, indicating that the neurons had died. However, in the second experiment, where enriched transplant medium was used, clearly defined hypocretin-immunoreactive somata with processes and varicosities were present in the graft zone 36 days after implant. These somata were similar in size and appearance to adult rat hypocretin-immunoreactive neurons., Conclusions: These results indicate that hypocretin neurons obtained from rat pups can be grafted into a host brain, and efforts should be made to increase survival of these neurons.

The loss of afferent synaptic boutons is a prominent alteration induced by axotomy on adult central neurons. In this work we attempted to prove whether synapse loss could be reverted by reconnection with a new target. We severed the medial longitudinal fascicle of adult cats and then transplanted embryonic cerebellar primordia at the lesion site immediately after lesion. As previously shown, the transected axons from abducens internuclear neurons penetrate and reinnervate the graft [J Comp Neurol 444 (2002) 324]. By immunocytochemistry and electron microscopy we studied the synaptology of abducens internuclear neurons under three conditions: control, axotomy and transplant (2 months of survival time). Semithin sections of the abducens nucleus were immunostained against calretinin, to identify abducens internuclear neurons, and either synaptophysin (SF), to label synaptic terminals, or glial fibrillary acidic protein (GFAP) to detect the astrocytic reaction. Optical and linear density of SF and GFAP immunostaining were measured. Data revealed a significant decrease in the density of SF-labeled terminals with a parallel increase in GFAP-immunoreactive elements after axotomy. On the contrary, in the transplant group, the density of SF-labeled terminals was found similar to control, and the astrocytic reaction induced by lesion was significantly reduced. At the ultrastructural level, synaptic coverage and linear density of boutons were measured around the somata of abducens internuclear neurons. Whereas a significant reduction in both parameters was found after axotomy, cells of the transplant group received a normal density of synaptic endings. The ratio between F- and S-type boutons was found similar in the three groups. Therefore, these findings indicate that the grafting of a new target can prevent the loss of afferent synaptic boutons produced by the axotomy.

The different cerebellar phenotypes are generated according to a precise time schedule during embryonic and postnatal development. To assess whether the differentiative potential of cerebellar progenitors is progressively restricted in space and time we examined the fate of embryonic day 12 (E12) or postnatal day 4 (P4) cerebellar cells after heterotopic-heterochronic transplantation into the embryonic rat brain in utero or into organotypic CNS explants in vitro. Donor cells, isolated from transgenic mice overexpressing the enhanced-green fluorescent protein under the control of the beta-actin-promoter, engrafted throughout the host brainstem and diencephalon, whereas they rarely incorporated into specific telencephalic structures. In any recipient site, the vast majority of transplanted cells could be recognized as cerebellar phenotypes, and we did not obtain clear evidence that ectopically located cells adopted host-specific identities. Nevertheless, the two donor populations displayed different developmental potentialities. P4 progenitors exclusively generated granule cells and molecular layer interneurons, indicating that they are committed to late-generated cerebellar identities and not responsive to heterotopic-heterochronic environmental cues. In contrast, E12 precursors had the potential to produce all major cerebellar neurons, but the repertoire of adult phenotypes generated by these cells was different in distinct host regions, suggesting that they require instructive environmental information to acquire mature identities. Thus, cerebellar precursors are able to integrate into different foreign brain regions, where they develop mature phenotypes that survive long after transplantation, but they are committed to cerebellar fates at E12. Embryonic progenitors are initially capable, although likely not competent, to generate all cerebellar identities, but their potential is gradually restricted toward late-generated phenotypes.

The reestablishment of topographically organized connections is a necessary prerequisite to obtain a full anatomical repair following brain injury. One system where such an issue can be addressed is the olivocerebellar system, where, normally, clusters of inferior olive neurons project to neurochemically heterogeneous Purkinje cell compartments defined by the expression of cell-specific markers, such as zebrin II. To assess whether adult injured olivocerebellar axons that regenerate into cerebellar transplants are able to establish target-specific innervation of grafted Purkinje cells, we made surgical transections in the white matter of adult rat cerebella and placed solid grafts from the embryonic cerebellar anlage into the lesion site. The transplanted tissue developed highly organized minicerebella, in which Purkinje cells were distributed into distinct clusters of zebrin II-immunopositive or -immunonegative neurons, mimicking the cortical compartments present in the normal adult cerebellum. Olivocerebellar axons, labeled by biotinylated dextran amine tracing, regenerated into the transplants where they formed discrete patches made of several terminal arbors impinging upon Purkinje cell dendrites. Among 401 such climbing fiber patches, 96% exclusively innervated Purkinje cells of either phenotype and stopped at the border of the zebrin II(+/-) Purkinje cell clusters, whereas only 4% were extended across this boundary and innervated both zebrin II-positive and -negative Purkinje cells. The results obtained support the view that the embryonic cerebellar tissue provides target-specific information that can be decoded by ingrowing adult olivocerebellar axons in order to establish appropriate innervation patterns with zebrin II-positive or -negative Purkinje cell compartments., (©2002 Elsevier Science (USA).)

Macrophage colony stimulating factor (M-CSF) is known to be the most effective growth factor for macrophage and microglial proliferation. In the brain tissue system, M-CSF is mainly produced in astrocytes and microglia, but is not known to occur in neurons. In the present paper, we examined the distribution of neurons expressing M-CSF in the mouse brain by immunohistochemistry and in situ hybridization. We observed M-CSF immunoreactivity in both the cerebellum and the olfactory bulb. These positive cells were found to be Purkinje cells in the cerebellum, and mitral cells in the olfactory bulb. M-CSF mRNA expression was also confirmed to occur in these cells. Purkinje cells of reeler and weaver mutants showed M-CSF expression as seen in wild-type mice; however, those in the staggerer mutant did not. This expression in wild-type mice first appeared at postnatal day 7 and continued stably thereafter. When Purkinje cells were deprived of their climbing fibre innervation by inferior cerebellar pedunculotomy or by transplantation of cerebellar anlagen into the anterior eye chamber, the expression of M-CSF remained unchanged. These data indicate that expression of M-CSF in Purkinje cells is controlled by an intrinsic mechanism and could, therefore, be a new marker of postnatal development in rodent cerebella. The absence of M-CSF expression in the staggerer mutant is possibly due to developmental arrest in the early postnatal period.

The ability of in vitro-expanded neural precursor cells or cell lines to differentiate following transplantation has significant implications for current research on central nervous system repair. Recently, interest has been focussed on grafts of such neural precursors implanted also into the eye or retina. Here, we demonstrate with a non-traumatizing subretinal transplantation method, that grafts of the two immortalized brain-derived cell lines C 17-2 (from postnatal mouse cerebellum) and RN33B (from the embryonic rat medullary raphe) survive for at least up to four weeks, after implantation into the adult normal rat retina. For both cell lines, implanted cells gradually integrate into all major retinal cell layers, including the retinal pigment epithelium, and judged by the morphology differentiate into both glial- and neuron-like cells, as shown by thymidine autoradiography, mouse-specific in situ hybridization, and using immunohistochemistry to detect the reporter gene LacZ. Our results suggest that these and other similar neural cell lines could be very useful in the continuous experiments in models of retinal disorders to further assess both the cell replacement and ex vivo gene therapy approaches., (Copyright 2001 Academic Press.)

Spinocerebellar ataxia type 1 (SCA-1) is one of several neurodegenerative diseases, including Huntington's disease, spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, and SCA-2, SCA-3, SCA-6, and SCA-7, each caused by an expanded number of CAG repeats in the coding region of their respective genes. The mechanism by which the resulting proteins are pathogenic is unknown. Clinical trials of neural transplants in Huntington's disease patients are under way. While initial reports are encouraging, definitive evidence of graft survival in patients despite the ongoing disease process is not possible with current imaging techniques. Transplants in primates have shown long-term survival of striatal grafts and recovery of function, but have used lesioning to model Huntington's phenotypically. Studies of striatal grafts in a transgenic mouse model of Huntington's have not yet shown a behavioral benefit. We describe a behavioral benefit of cerebellar grafts in a transgenic model of SCA-1 in which the ataxic phenotype results from expression of an expanded ataxin-1 protein. Mice were transplanted at an age when their ataxic phenotype is just becoming evident. Compared with sham-operated littermates, grafted mice showed better performance on multiple behavioral tests of cerebellar function. Differences persisted for 10 to 12 weeks posttransplant, after which there was a progressive decline in motor performance. At 20 weeks postsurgery, donor Purkinje cell survival was evident in 9 of 12 graft recipients. These results indicate that transplants can have behavioral benefits and grafts can survive long-term despite the ongoing pathological process in a brain actively expressing an expanded polyglutamine protein., (Copyright 1999 Academic Press.)

Injection of chick cerebellar membranes, rich in kainate binding sites, into Xenopus oocytes resulted in the structural integration of chick membrane patches into the oocyte plasma membrane that could be easily identified by specific immunofluorescent staining. Application of kainate to the oocyte perfusion medium, under voltage-clamp conditions, induced dose-dependent (EC50 = 87+/-14 microM) inward currents, confirming the functional incorporation to the oocyte of kainate-driven channels. Responses to kainate were consistently nondesensitizing and strongly potentiated by cyclothiazide, suggesting the selective involvement of alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionate (AMPA)-preferring receptors. Binding experiments with (S)-[3H]AMPA confirmed the presence in the chick membrane preparation of low-affinity AMPA receptors (K(D) = 278 nM) amounting to <2% of the total population of kainate binding sites. A tenfold concentration of guanine nucleotides, with different degrees of phosphorylation, blocked the responses to 100 microM kainate by approximately 90%. In the case of GMP, additional concentration-inhibition studies yielded an IC50 of 180+/-11 microM. Our results illustrate the apparent failure of kainate-binding proteins to form functional channels, even when maintaining their own native membrane environment, and confirm the antagonistic behavior of guanine nucleotides, including GMP, toward glutamate receptors, in agreement with previous results of ligand-binding experiments and, more interestingly, with the marked neuroprotective effects of some guanine nucleotides in different excitotoxicity experimental paradigms.

This study was designed to explore the efficacy of a human clone cell line as an alternative neural graft source and to validate the practice of cryopreservation and xenografting as logistical approaches toward conducting neural transplantation. We investigated the biological effects of transplanting cultured human neurons (NT2N cells) derived from a well-characterized embryonal carcinoma cell line into the brains of rats subjected to transient, focal cerebral ischemia induced by embolic occlusion of the middle cerebral artery. At 1 month and extending throughout the 6-month posttransplantation test period, ischemic animals that were transplanted with NT2N cells and treated with an immunosuppressive drug displayed a significant improvement in a passive avoidance task as well as a normalization of asymmetrical motor behavior compared to ischemic animals that received rat fetal cerebellar cell grafts or vehicle alone. Remarkably, cryopreserved NT2N cell grafts compared with fresh NT2N cell grafts, remained viable in the immunosuppressed rat brain and effective in producing behavioral recovery in immunosuppressed ischemic animals. The long-term viability of cryopreserved NT2N cell xenografts in vivo and their sustained effectiveness in promoting behavioral recovery suggest potential utilization of xenografting and cryopreservation as useful protocols for establishing clone cell lines as graft source in neural transplantation therapies for central nervous system disorders.

In the cerebellum, up- and downregulation of specific GABAA and NMDA receptor subunits coincide with granule cell migration and differentiation. In this study, in situ hybridization techniques with GABAA and NMDA receptor subunit specific probes were employed to assess whether the molecular phenotype of heterotopically grafted cerebellar granule cells corresponds to that of normal cerebellum. The cerebellar anlage of rat fetuses was stereotactically grafted into the rostral striatum of adult rats. Eight weeks after transplantation, analysis demonstrated acquisition of an adult differentiation status reflected by abundant GABAA alpha 6 and NR2C mRNA expression in granule cells. Complete lack of NR2B transcripts, molecular markers of immature granule cells, argues against persistence of undifferentiated cells. The data suggest that intrinsic cell-autonomous factors largely determine the molecular commitment of granule cells and that a restricted specific environment is not necessary to promote granule cell differentiation.

Embryonic striatal precursors grafted into the lesioned adult host striatum show limited integration with little migration and restricted efferent projections. In the present study, the influence of an immature striatal environment on the integrative capacity of grafted neuroblasts was examined after transplantation of striatal progenitors into the striatum at different stages of postnatal development. Mouse progenitors, derived from embryonic day 13.5-14 lateral or medial ganglionic eminence or the cerebellar primordium, were transplanted as a single cell suspension into the developing postnatal day 1, 7 and 21 rat striatum. The grafted cells and their axonal projections were visualized using antibodies raised against the mouse-specific neural markers, M6 and M2. Cells from the lateral (but not the medial) ganglionic eminence showed a remarkable capacity to innervate selectively the striatal target structures, globus pallidus, entopeduncular nucleus and substantia nigra, reminiscent of endogenous striatal neurons, which is not observed after grafting into adult hosts. M6 and M2-immunopositive cellular profiles from both the lateral and medial ganglionic eminences were observed to have migrated extensively away from the injection site, in contrast to the cerebellar precursors which remained clustered at the implantation site. Cells from the lateral ganglionic eminence were largely confined within the striatal complex where they developed striatal characteristics, displaying expression of DARPP-32, the 32,000 mol. wt dopamine- and cyclic AMP-regulated phosphoprotein, whereas cells from the medial ganglionic eminence had migrated caudally along the internal capsule and were observed predominantly in the globus pallidus and thalamus, in addition to the striatum. The cells located outside the striatum were all DARPP-32 negative. The improved integration and increased projection capacity of the lateral ganglionic eminence precursors grafted into postnatal day 1 hosts gradually declined as the host advanced into later stages of development (postnatal day 7), and in postnatal day 21 hosts the grafted striatal precursors behaved similarly to grafts implanted into adult recipients. These results demonstrate the specific capacity of embryonic striatal progenitors to integrate into the developing basal ganglia circuitry during early postnatal development, and that the extent of neuronal and glial integration and graft host connectivity declines when the host has developed beyond the first postnatal week.

Cerebellar anlagen from rat embryos were grafted into the cerebellum of intact adult rats. Most of the grafts survived and formed 'minicerebella'. The location of the grafts were varied, which provided various types of host/graft interface in laminar configuration: Hw/Gg, Hw/Gp, Hw/Gm, Hg/Gw, Hg/Gg, Hm/Gw, Hm/Gg, Hm/Gp, and Hm/Gm that comprised of the granule cell layer (g), the Purkinje cell layer (p), the molecular layer (m), or the white matter (w) of the host (H) or graft (G). The manner of entrance of mossy and climbing fibers through the host/graft interface and their outgrowth in the graft which has various subset of laminar organization of the cerebellum were examined 28-158 days after grafting by means of anterograde Phaseolus vulgaris leucoagglutinin (PHA-L) labeling. Each cerebellar afferent entered the graft through specific types of interface and grew in specific layers. Mossy fibers passed through Hg/Gg and Hw/Gg, grew in Gg, and mostly terminated there like normal fibers. Fibers in Gg, though rarely, grew further outside Gg like in development. Climbing fibers passed through Hm/Gg, Hw/Gg, and Hw/Gm, proceeded in cortical layers, and terminated in Gm. The outgrowth of climbing fibers in Gm showed selectivity for the direction of Gm with respect to the polarity of Purkinje cells; they permeated Gm to form terminal arbors similar to normal in the direction from the side of Purkinje cell somata to dendrites but not in the reverse direction. Occasionally a single fiber innervated neighboring multiple Purkinje cells. These results indicate that mature cerebellar afferents have potential to regrow and innervate the extraneous cerebellar anlage by finding paths and targets in a manner similar to normal ontogenesis.

Investigations into the relationship between the cerebellum and nonmotor processing have produced a substantial body of evidence which seems to require a revision of accepted notions about the functional role of the cerebellum. This chapter presents a perspective on the contemporary and possible future therapeutic and research implications of these findings. These include the need for patients and their families to know of the behavioral consequences of cerebellar disease processes; potential approaches for improvement through rehabilitation therapies; and future treatment strategies, such as electrical stimulation of the cerebellum and psychosurgical approaches applied to the cerebellum. In addition, some areas of basic science investigation that could prove informative in understanding this relationship are addressed. It will be important to obtain a more complete characterization of the anatomy, physiology, and functional topography of the cerebellum in humans and in animal models, and a greater understanding of the clinical consequences of cerebellar lesions.

Activated Schwann cells such as those in the distal stump of a cut peripheral nerve, or those cultured in vitro, develop a molecular phenotype very different from that of quiescent Schwann cells, and express high levels of the transcription factor c-jun. We studied the expression of c-jun messenger RNA, by in situ hybridization, and Jun-like immunoreactivity of Schwann cells in segments of peripheral nerve, or in cell suspensions grafted into the adult rat brain. Schwann cells rapidly lost their Jun immuno-positivity, and down-regulated expression of c-jun messenger RNA once implanted into the brain, and only the Schwann cells contained in the portion of peripheral nerve which remained outside the brain maintained Jun-like immunopositivity. c-jun messenger RNA was also down-regulated in the grafts, but more slowly than the protein; however, a proximodistal gradient in the level of expression of c-jun messenger RNA along the graft, comparable to that found for Jun immunoreactivity, was not detected. Schwann cells transplanted into the lesioned central nervous system promote regeneration of some injured central nervous system axons, but this regenerative response is always much more limited than peripheral nervous system regeneration. We suggest a correlation between the limited regeneration of central nervous system axons into peripheral nerve grafts and the loss of c-jun expression in Schwann cells following exposure to the central nervous system environment.

To gain insight into cellular and molecular mechanisms subserving neuronal cell migration in the adult mouse forebrain, we have first investigated the cellular composition of the subventricular zone-olfactory bulb pathway (SVZ-OB). The pathway was essentially composed of cells with neuronal and astrocytic identities, neuronal cells being four times more numerous than astrocytes. Neuronal cells (precursors and some young postmitotic neurons) formed continuous cellular strands of migratory cells from the anterior horn of the lateral ventricle to the olfactory bulb. These chains of migrating cells moved within channels formed by the processes of a special subpopulation of astrocytes. The neuronal cells expressed the embryonic form of polysialic acid neural cell adhesion molecule, and the astrocytes were tenascin-C positive, thus preserving an embryonic cellular environment. Through transplantation experiments, the second part of this study attempted to analyze the functional properties of the adult SVZ-OB pathway. Early postnatal (P2-13) cerebellar progenitor cells, taken from a transgenic mouse line in which cerebellar granule cells and molecular layer interneurons (basket/stellate cells) expressed the reporter gene lacZ, were implanted in the SVZ-OB pathway of adult wild-type mice. Unlike grafted SVZ cells that migrate all along the pathway, none of the cerebellar precursors reached the olfactory bulb, although some of them were able to migrate along the caudal one-third of the pathway. The majority (over 67%) of the migrating cells were progenitors that acquired the phenotype of basket/stellate cells. Granule cell progenitors and most granule cells did not survive transplantation. These results show that the adult SVZ-OB pathway is not a "passive generic guidance" for all classes of premigratory neurons. From the two types of grafted cerebellar progenitors, only those with migratory capability and that do not follow radial glial axes are able to translocate along the SVZ-OB pathway. Furthermore, the basket/stellate cell progenitors are specified at the time of grafting: Neither their identity nor the pace of expression of their major distinctive features are influenced by local signals emanating from the adult forebrain.

The effects of neurotransplantation on development of audiogenic seizures (AS) were studied in Krushinskiĭ-Molodkina (KM) rats genetically predisposed to AS and in Wistar rats especially selected for AS. Within 24 weeks after bilateral combined transplantation of the newborn striatum and cerebellum into the parietal cortex, the AS didn't develop in Wistar rats in 74% of tests. In control Wistar rats with cortical grafts or saline injections into the parietal cortex there were no significant changes in the AS development. In the experimental KM rats the grafting led to the 4-5-times increase in the AS latency. In 50% of rats it decreased the AS severity by 1-2. In the control KM rats these parameters didn't change. It was concluded that it was possible to suppress or decrease the AS development by the transplantation of the newborn striatal and cerebellar tissue into the cortex of rats predisposed to this kind of seizures.

The mechanism of the behavioral improvement observed in parkinsonian primates that receive intrastriatal transplants of fetal dopamine neurons has not been firmly established. Dopamine production by grafted neurons may be the basis of the behavioral recovery. Alternatively, stimulation of the host dopamine system by the transplant procedure itself may be central to the outcome. The present study examined whether dopamine concentration was raised in the caudate nucleus of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated primate following grafting, and if so, whether the elevation was dependent on either (i) the introduction of the implantation cannula (sham), (ii) the brain region that was grafted, or (iii) the gestational age of fetal tissue that was grafted. Transplantation of early gestational age fetal ventral mesencephalon (embryonic days 40-50) was associated with significant elevation of caudate nucleus dopamine concentration to a mean of approximately 20% of control values in the vicinity (within 2 mm) of the graft, compared with more distant sites in the caudate nucleus. With early gestational age fetal ventral mesencephalon, the ratio of homovanillic acid/dopamine concentration near the graft site was normalized compared to the elevated value found in the caudate nucleus distant from the graft site. Grafts of later stage fetal ventral mesencephalon, or fetal cerebellum, or sham implantation did not increase dopamine concentration or lower homovanillic acid/dopamine ratio near the graft site. Biochemical and histochemical evidence suggests that host dopamine neurons terminating in the nucleus accumbens are not the source of the changes. Numerous tyrosine hydroxylase-positive neurons at the graft site were only observed in the MPTP-treated monkeys that received grafts of early gestational age fetal ventral mesencephalon. These data lend strong support to the hypothesis that dopamine derived from grafted dopamine neurons is the major basis for behavioral recovery observed following intrastriatal transplantation in our MPTP-treated monkeys.

Fetal grafts of normal cerebellar tissue were implanted into the cerebellum of Purkinje cell degeneration mutant mice (pcd/pcd), a model of adult-onset recessively inherited cerebello-olivary atrophy, in an attempt at correcting their cellular and motor impairment. Donor cerebellar cells engrafted in the appropriate sites, as evidenced by the pattern of expression of insulin-like growth factor-I (IGF-I) system genes. Bilateral cerebellar grafts led to an improvement of motor behaviors in balance rod tests and in the open field, providing evidence for functional integration into the atrophic mouse cerebellum and underscoring the potential of neural transplantation for counteracting the human cerebellar ataxias.

A synopsis is presented of the recent history of cerebellar tissue transplantation over the past 25 years. The properties of growth and differentiation of cerebellar grafts placed intraocularly or intracranially are reviewed, as well as the interaction of heterotopic and orthotopic grafts with the host brain. Particular emphasis is placed on the use of ataxic mouse mutants as recipients of donor cerebellar tissue for the correction of their structural deficits and the functional recovery of behavioural responses.

Regeneration of severed central axons is supposed to depend on two factors: a permissive local environment and the particular intrinsic properties of axotomized neurones. To assess the role of each of these factors in axonal regeneration, the capability of two particular axon populations of the adult mouse cerebellum to grow into target-specific (cerebellum) and target-unspecific (neocortex) embryonic grafts was determined. Purkinje cell and inferior olivary axons were transected by passing a microscalpel through the axial white matter of the cerebellar folia, particularly those of the anterior lobe. Immediately after the injury, solid transplants were placed in the lesion cavity. Purkinje cell axons were labelled by using anticalbindin immunocytochemistry, and olivocerebellar fibres were visualized by biotinylated dextran amine anterograde axonal tracing. Following axotomy, Purkinje cell axons appeared as thickened processes ending with large terminal clubs. Their morphology and number did not change up to the longest survival time considered (2 months), thereby confirming previous demonstrations that Purkinje cells survive axon injury (I. Dusart and C. Sotelo, 1994, J. Comp. Neurol. 347:211-232). Inferior olivary axons were thinner and bore smaller terminal bulbs. When embryonic cerebellar grafts, containing cortical and deep nuclear precursors, were placed close to the injured axons, olivocerebellar fibres vigorously regenerated into the transplants and ended in new climbing fibres along the dendrites of grafted Purkinje cells. By contrast, host Purkinje cell axons never showed any outgrowth towards the graft. Similarly, these axons failed to regenerate into grafts containing solely the rostromedial portion of the cerebellar anlage, mostly consisting of deep nuclear neurones, their main targets. Comparable results were obtained by transplanting embryonic neocortical tissue: inferior olivary axons also regenerated into the grafts, although with distinct terminal arbours without the climbing fibre phenotype, whereas Purkinje cell axons always failed to grow. These results provide the first direct demonstration that severed inferior olivary axons are able to regenerate. In addition, they show that the growth-permissive/-promoting conditions created by embryonic nervous tissue are not sufficient to induce the regeneration of every axonal type and allow us to hypothesise that successful regeneration depends on the interplay between environmental cues and intrinsic properties of the axotomized neurones.

The purpose of this study was to determine whether loss of the reproductive cycle after lesions of the medial basal hypothalamus can be reversed by transplantation of the embryonic olfactory placode (OP) into female rhesus monkeys. Seven adult female rhesus monkeys with regular menstrual cycles received bilateral radiofrequency lesions in the arcuate nucleus and the median eminence. After confirmation of anovulation in these monkeys, four monkeys were stereotaxically implanted with the OP obtained from monkey fetuses on embryonic days 35-36. The remaining three monkeys were similarly implanted with embryonic cerebellum (CB) as a control. Fetuses were delivered by cesarean section, and the OP and CB were immediately dissected out using a stereomicroscope. Fetal tissue was then cut into small pieces (< 1 mm3), mixed with artificial cerebrospinal fluid containing small pieces of Gelfoam, and stereotaxically injected into the infundibular recess of the third ventricle. The recovery of ovulatory cycles in recipient monkeys was observed for at least 6 months; sex skin color changes and menstrual records were obtained daily, and serum samples for LH, estrogen, and progesterone were obtained twice a week. Three of four OP-transplanted monkeys resumed their ovulatory cycles within 2 months, whereas the fourth monkey, an elderly female, failed to recover her cycle. In contrast, none of the three CB-transplanted monkeys resumed ovulatory cycles. Histological examination indicated that 1) lesion scars were present in the median eminence-stalk region as well as the medial basal portion of the arcuate nucleus of all seven brains; and 2) cartilage was present in the third ventricles of the OP-implanted brains. Moreover, immunocytochemical staining revealed that in all OP monkeys, small, round, and immature LHRH-positive cells with fine short processes were found in the third ventricle and/or median eminence-stalk region, whereas no similar LHRH cells were found in CB-transplanted monkeys. It is concluded, therefore, that implantation of LHRH neurons derived from the fetal OP can result in resumption of the ovulatory cycle in female monkeys whose own LHRH pulse-generating mechanisms were impaired. Moreover, the results suggest that LHRH neurons derived from embryonic OP possess the physiological functions necessary for the stimulation of gonadotropin secretion.

Acrylamide (30 mg/kg body wt.) administered intraperitoneally daily to young adult male rats, five times a week for 3 consecutive weeks, affected the cerebellar functions, as exhibited by a significant reduction in rotarod performance, spontaneous locomotor activity, glutathione-S-transferase activity, and 3H-flunitrazepam binding in cerebellum. Transplantation of dissociated fetal cerebellar cells (E14) to cerebellum resulted in a significant recovery in behavioral and neurochemical parameters evaluated 9 weeks after transplantation. Light- and electron-microscopic studies confirmed the viability and specificity of cerebellar grafts.

Neonatal mouse cerebellar cultures depleted of granule cells and functional glia by exposure to cytosine arabinoside were transplanted with either granule cells and glia or with granule cells in the absence of functional glia. Myelination was evident in cultures transplanted with granule cells and glia, excess sprouted cortical neurites were reduced, Purkinje cells acquired astrocytic sheaths and had a near normal complement of axosomatic synapses, and homotypical parallel fiber-Purkinje cell dendritic spine synapses were present in a 2.4:1 ratio to heterotypical recurrent axon collateral-Purkinje cell dendritic spine synapses. Cultures transplanted with granule cells were not myelinated, sprouted cortical neurites were not reduced. Purkinje cells lacked astrocytic sheaths and their somata remained hyperinnervated, and the ratio of homotypical to heterotypical dendritic spine synapses was 1.4:1. In the absence of functional glia there was a greater persistence of heterotypical recurrent axon collateral-Purkinje cell dendritic spine synapses. These results are consistent with a previously described astrocytic role in the regulation of axosomatic synapse density on glially ensheathed neurons, and suggest astrocytic participation in the reduction of heterotypical axospinous synapses. Astrocyte-mediated synapse reduction may be an important mechanism for circuit reorganization after transplantation or during development.

Chick/quail chimeras with partial cerebellar grafts have been performed to obtain further information about the origin and migratory movements of cerebellar cortical neurons. The grafts were performed by exchanging between these two species a precise, small portion of the E2 cerebellar primordium, as defined in Martinez and Alvarado-Mallart (Eur. J. Neurosci. 1:549-560, 1989). All grafts were done unilaterally. The chimeric cerebella, fixed at various developmental stages, were analyzed in serial Feulgen-stained preparations to map the distribution of donor and host cells in the ependymal layer (considered to be reminiscent of the primary germinative neuroepithelium) and in the various cortical layers. In some of the oldest cases, we also used antiquail immunostaining to recognize quail cells. In the ependymal layer, it has been possible to conclude that each hemicerebellar primordium undergoes a morphogenetic rotation that changes its rostrocaudal axis to a rostromedio-caudolateral direction. However, important individual variations were observed among the chimeric embryos with respect to the ependymal area expected to be formed by donor cells. These variations cannot be explained solely on the basis of microsurgical procedure; however, they suggest the existence of important reciprocal interaction between host and grafted neuroepithelia. Therefore, it was not possible to draw a precise fate map of the E2 cerebellar primordium. Nevertheless, the dispersion of grafted cells in the cerebellar cortex, when compared to the real extent of the ependymal grafted area in each particular case, provided important data: (1) The external granular layer (EGL), the secondary germinative epithelium, seems not to originate exclusively from the "germinative trigone," as is usually considered the case. It emerges from a larger but restricted portion of the primary cerebellar matrix extending about the caudal fourth or third of the ventricular epithelium, as defined after its morphogenetic rotation. (2) The Purkinje cells (PCs) develop from all areas of the cerebellar epithelium. Although the distribution of donor PCs parallels the grafted ventricular layer mediolaterally, donor PCs extend more in the rostrocaudal dimension. The PC layer is formed mainly by donor cells in the lobules underlain by the grafted ependymal layer. However, donor PCs are also observed in cortical lobules surmounting the host ventricular layer. In these lobules, the donor PCs form clusters of various widths interrupting the host PCs. Reciprocally, clusters of host PCs are also found in the lobules formed mainly by donor PCs. The alternate small clusters of donor or host PCs are surrounded by Bergmann fibers of the other species' origin.(ABSTRACT TRUNCATED AT 400 WORDS)

Pieces of cerebellar primordia were obtained from G16 (day 16 of gestation) rat fetuses and stereotaxically injected into the striatum of adult Wistar rats. The transplants were allowed to integrate with the host brain for 2 h up to 6 months after implantation. Ninety four out of 105 transplants perfectly integrated with the host brain (90%) and established the typical trilaminar histoarchitecture of cerebellar cortex. The transplants were sufficiently vascularized. Vessels seen within the grafts provided all ultrastructural elements of a blood-brain barrier. Light microscopic evaluation of graft development showed no considerable retardation of cerebellar histogenesis. Electron microscopic examination disclosed normal ultrastructure of cerebellar neurons, as well as elements of regular synaptic organization. The topic of efferent graft-to-host projections was investigated 2.5 months after transplantation using the monoclonal Purkinje cell marker anti-Leu-4 (CD3). This method allowed us to detect immunoreactive, morphologically intact axons of grafted Purkinje cells running over long distances (at least 500 microns) within the host striatum. Whilst afferent but in no case efferent connections of heterotopic cerebellar transplants had been demonstrated elsewhere, we could now prove the reciprocal modus of graft-host interaction with heterotopic cerebellar grafts.

Solid cerebellar grafts, taken from normal mouse embryos (gestational day 12-14), were transplanted into the cerebellum of adult Lurcher mice. The degree of Purkinje cell replacement was analysed one to three months after transplantation by means of immunocytochemistry (antibodies against calbindin, cGMP-dependent protein kinase and neurofilament proteins) and electron microscopy. Grafted Purkinje cells succeed in moving out of the graft and migrate into the host cerebellar cortex. They are present next to the graft in the granule cell and molecular layers, and far from the graft remnant, only in the molecular layer, indicating that, although both layers subserve Purkinje cell migration, the molecular layer is the ultimate target. In the host molecular layer, axons of transplanted Purkinje cells form thick bundles running in the frontal plane over long distances. Most of them terminate in the upper granule cell layer by enlarged bulbs resembling collapsed growth cones. Axons reaching their normal targets (the neurons of the deep cerebellar nuclei) are observed only in cases where the granule cell layer is disrupted and/or grafted Purkinje cells remain in the white matter. The projection is massive only from grafts lying in the close vicinity of the target neurons. Electron-microscopic analysis of grafted Purkinje cells populating the host cerebellar cortex reveals that their synaptic investment is abnormal. In the molecular layer, where the normal inputs are reduced, the compartmentation in proximal and distal dendritic segments is severely affected, climbing fibre synapses only form on a minority of grafted cells and "pinceau" formations are absent. In the granule cell layer, the synaptic investment is similar to that of Purkinje cells in agranular cerebellum, and even heterelogous synapses with mossy fibres have been observed. These results, compared to those previously obtained with grafting experiments in Purkinje cell degeneration mutant mouse, allow us to conclude that: (i) the Purkinje cell-deficient molecular layer of the host, despite its severe atrophy and reactive gliosis, still exerts a positive neurotropism specific for grafted Purkinje cells; (ii) the unlesioned host granule cell layer underlying the molecular layer containing grafted Purkinje cells, even if almost depleted of granule cells, remains an obstacle for the re-establishment of a corticonuclear projection; and (iii) the degree of synaptic integration of grafted Purkinje cells is directly related to the nearby presence of available host axon terminals. Hence, owing to the atrophy of the Lurcher cerebellum, the postgrafting restoration of the cerebellar cortical circuit is much less complete in this mutant.

Cerebellum of rat embryos at day 17 of gestation was transplanted into sensomotor cortex of adult syngenic rats. DNA content in granule and Purkinje cells of the transplant was determined cytophotometrically on day 30 after surgery. It is shown that granule cells are diploid; about 3% Purkinje cells contain hyperdiploid and tetraploid nuclei which corresponds to the content of such cells in the adult cerebellum. The conclusion is drawn that cell hyperploidy does not depend on the functional load but is essential property of the tissue.

Anatomical and biochemical changes in cerebellum after the onset of pcd gene defects have revealed in addition to Purkinje cell degeneration, the occurrence of secondary degenerative events in granule neurons, neurons of the inferior olivary complex and deep cerebellar nuclei. Transplantation in young mutant animals thereby presents a prospect that not only the missing Purkinje cells may be replaced but the subsequent transneuronal losses may be minimized. The present study has evaluated the outcome of implanting embryonic cerebellar cell suspensions into pcd mutants at the age of 45 day-old, a stage marked by the end of Purkinje cell degeneration. Immunocytochemistry (ICC) was used to monitor the extent of gliosis in host cerebellum and to trace the development of embryonic Purkinje cells grafted. The results suggest that the donor cells have no apparent difficulty in survival during reactive gliosis. Migration of these neuroblasts from the grafted zone into adjacent host molecular layer and significant expression of calcium binding protein (CaBP) by grafted Purkinje cells were noted within 9 days after transplantation. Dendritic patterns of grafted Purkinje cells are well-developed by day 17 post transplantation up to 3 months. In addition, synaptic contacts between host fiber endings and the CaBP-positive dendritic spines are present in the molecular layer of folia containing the graft, suggesting synaptic integration between graft and host.

One aspect of integration of implanted neurons into the neuronal circuitry of a defective host brain is the re-establishment of a host-to-graft afferent innervation. We addressed this issue by using the adult cerebellum of 'Purkinje cell degeneration' (pcd) mutant mice, which lack virtually all Purkinje cells after postnatal day (P) 45. Purkinje cells constitute one of the cerebellar cell types being innervated by axons of raphé serotonin (5-HT) neurons. In normal mice, 5-HT-immunoreactive fibers are distributed to all cerebellar folia. Following Purkinje cell loss in pcd mice, cerebellar 5-HT-immunoreactive fibers persist. Cerebellar cell suspensions were prepared from embryonic day (E) 11-13 normal mouse embryos and were intraparenchymally grafted into the cerebellum of pcd mutants either directly or after pre-treatment with 5,7-dihydroxytryptamine (5,7-DHT) to selectively remove 5-HT cells of donor origin. The state of Purkinje cells and 5-HT axons was monitored in alternate sections by 28-kDa Ca(2+)-binding protein (CaBP) and 5-HT immunocytochemistry, respectively. Serotonin-immunoreactive axons were seen in the grafts from 5 to 32 days after transplantation. In some of the grafts which had not been pre-treated with 5,7-DHT, a small number of 5-HT-immunoreactive cell bodies was found, indicating that part of the 5-HT fiber innervation of the graft could actually derive from donor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the hypothesis that neural transplantations can alter seizure susceptibility in a genetic animal model of epilepsy, 93 pubescent genetically epilepsy-prone rats with stage 9 seizures received either bilateral inferior colliculi (N = 21) or lateral ventricle (N = 42) transplants or sham transplants (N = 30). The grafts consisted of embryonic locus ceruleus, neocortical, or cerebellar tissue. Starting 2 days after the transplantation the rats were subjected to audiogenic stimulations every other day for 61 days. Latency to the running and tonic phase, seizure severity score, and duration of the tonic and clonic phase were compared in the neural transplant and sham-operated controls. Rats that received transplants had a longer latency to the tonic phase and a shorter duration of the clonic phase than the controls. At age 110 days the rats had electrodes implanted bilaterally into the angular bundle and were kindled. No difference in kindling rate was found between the rats that received neural grafts and the sham-operated controls. Cerebrospinal fluid concentration of norepinephrine was not altered by the transplants. This study demonstrates that the anticonvulsant effects of neural transplants, using the genetically epilepsy-prone model of epilepsy, are mild.

Monoclonal M6 antibody binds to the surface of murine central nervous system neurons as well as to apical surfaces of epithelial cells in the choroid plexus and proximal tubules of the kidney. M6 antigen is expressed in the central nervous system as early as embryonic day 10, most strongly in the marginal zone of the neural tube, and remains detectable in adulthood. IgG or Fab fragments of M6 antibody interfere with the extension of neurites by cultured cerebellar neurons. Effects of the antibody on neurite extension are readily detectable after 24 h. No reduction of cell viability is detected during the first 3 days of antibody treatment. Cultures maintained in the presence of antibody for longer than 5 days exhibit reduced viability of neurons. This reduction in long-term viability in the presence of M6 antibody is largely avoided when 25 mM KCl is included in the culture medium. The antibody-mediated perturbation of neurite outgrowth is not blocked by the presence of elevated KCl. The unusually short and flattened appearance of neurites in these cultures suggests that the M6 antibody selectively affects neurite extension. Time-lapse cinematography of anti-M6-treated neurons reveals no apparent effect on movement of lamellipodia and filopodia of growth cones. Only the overall extension of the neurite appears to be inhibited. M6 antigen is a 35 kD glycoprotein that can be isolated from a deoxycholate- (DOC) solubilized membrane fraction from adult mouse brain.