Changes in L1 Antigen Expression in the Rat Striatum After Substantia Nigra Lesions

L1 antigen promotes neurite outgrowth from dopaminergic neurons in tissue culture. In the present study, we examined the effects of dopaminergic deafferentation of the striatum on L1 expression. In the medial-periventricular part of the striatum, both complete and partial substantia nigra (SN) lesions decreased L1 expression. Complete lesions increased L1 expression in the dorso-medial and ventrolateral parts of the striatum on the lesioned side when compared with that on the non-lesioned side. The decrease in the ventro-lateral area was maintained in animals examined three months after the lesioning. Animals with partial SN lesions showed a different pattern of altered L1 expression. After frontal cortex lesions, changes in L1 expression also occur preferentially in the dorso-medial and periventricular striatum. Therefore, the results indicate a complex regulation of L1 expression after damage of striatal circuitry, manifested by a preferential occurrence of changes in periventricular regions.

Prenatal Exposure to Ethanol Causes Differential Effects in Nerve Growth Factor and its Receptor in the Basal Forebrain of Preweaning and Adult Rats

In this study we investigated nerve growth factor (NGF) levels in the cortex and hippocampus of the offspring of pregnant female Sprague-Dawley rats receiving a single intragastric administration of acute ethanol on the 15th day of gestation and compared them with a control group of rats that received an injection of sucrose. We also examined the distribution of the low-affinity NGF receptor, p75NGFR, on NGF-responsive neurons that are localized in the septum and the nucleus of Meynert, which receive the respective trophic support from the hippocampus and the cortex. In the ethanol-treated group, the results show that at post-natal age 15 days, the NGF septohippocampal pathways were markedly affected. At day 15, the NGF level was significantly higher in the offspring of ethanol-treated rats. By day 40, NGF values in both groups decreased to similar levels. At day 60, however, the NGF level in the ethanol-treated animals decreased to a significantly lower value than that of the control group, which remained essentially unchanged. In parallel, at day 60 the numbers of septal cholinergic neurons expressing p75NGFR were also significantly lower in ethanol-treated rats than in control animals. Because ethanol is known to induce neurological disorders, as well as deficits in cell proliferation and differentiation, the results suggest that one cause of the deleterious effects induced by ethanol is the low availability of NGF during certain stages of postnatal brain development.

A Method for Studying the Effects of Neurochemicals on Long-Term Compensation in Unilaterally Labyrinthectomized Rats

A new method has been developed to study the influence of drugs and toxicants on longterm recovery of dynamics in the horizontal vestibulo-ocular reflex of the rat after hemilabyrinthectomy (HL). HL was performed by injecting sodium arsanilate into the middle ear. The lesion was confirmed by histology. Eye movements elicited by sinusoidal vestibular stimulation, in both light and darkness, were recorded by a search-coil technique and then analyzed by a computer program created with virtual instrument soft-ware, which calculated the gain of the slow-phase velocity (SPV) and the saccades para-meters (frequency, amplitude, and peak velocity) to the lesioned side and to the intact side separately. During the 2-10 week period after HL, repeated analysis of the spontaneous long-term recovery of such parameters revealed a slight but significant reduction of the post-HL asymmetry between SPV gain to the lesioned side and to the intact side. During the follow-up period, a post-HL increase of the phase lead remained unchanged. The reduced number of saccades/min was not completely restored. To test the usefulness of the experimental model for neurochemical investigation of such adaptation, we administered baclofen and toluene to rats 8–12 wk after hemilabyrinthectomy. Baclofen, a specificGABABagonist, immediately restored the symmetry of SPV gain. By contrast, toluene, which has some effects on the central vestibular system that are related toGABABtransmission, aggravated the asymmetry in both the SPV gain and the number of saccades. We suggest that the experimental model would be useful for studying neurochemical mechanisms in vestibular adaptation processes.

Fetal Dopaminergic Neurons Transplanted to the Normal Striatum of Neonatal or Adult Rats and to the Denervated Striatum of Adult Rats

Fetal ventral mesencephalon from the 15th gestational day was grafted into the striatum of neonatal and adult rats. In one group of adult rats, fetal nigra was transplanted into normal striatum. In a second group, the tissue was transplanted at sites where dopaminergic fibers were denervated with 6-hydroxydopamine. The behavior of the dopaminergic neurons and glial reactions were studied by staining with cresyl violet to localize the transplants and by immunolabeling tyrosine hydroxylase (TH) and glial fibrillary acidic protein. In normal adults, the transplants were small. At the edge of the transplants, TH-positive neurons were packed into clusters, and an interface without any significant crossover of TH-positive fibers was present. Glial reaction was minimal in and around the transplant. In the denervated striatum, transplants were generally larger than those in normal striatum and surrounded by a glial scar. TH-positive neurons were both closely packed and loosely arranged at the periphery of the transplants. Processes could be clearly defined and could be traced to the adjacent host striatum through the TH-free denervated area. In neonates, the transplants were large and at times extended beyond the striatum. Most TH-positive neurons were arranged linearly along the periphery of the transplant. Cell bodies were widely separated and a well-developed neuropil was present. Fibers from the transplant mingled freely with the host striatum without any interface. In all three transplant groups, tracing the TH-positive neurites was easy because they were thicker and coarser than other elements. No apparent glial reaction occurred in the neonates. Thus, the growth and maturation of dopaminergic neurons seemed to vary in different environments. The most conducive environment appears to be neonatal brain in which growth factors are readily available.

Morphogenetic Plasticity of Neuronal Elements in Cerebellar Glomeruli during Deafferentation-Induced Synaptic Reorganization

Reorganization of the cerebellar glomerulus, the main synaptic complex within the granule cell layer, was investigated using quantitative morphological techniques. All afferents to the cerebellar cortex, including mossy-fibers, were surgically destroyed by undercutting the cerebellar vermis. Fifteen days after the operation, which resulted in the removal of the main excitatory afferent to the glomerulus, a significant reorganization of the whole synaptic complex was observed, whereas the structural integrity of the glomerulus was remarkably well preserved. This was indicated by the observation that the number of granule cell dendrites (≈50 per glomerulus), as well as the number of dendritic digits (≈210 per glomerulus) bearing most of the ≈230 synaptic junctions per glomerulus, did not change significantly after mossy-fiber degeneration. The total number of synapses in the reorganized glomerulus did not change either, despite the disappearance of two-thirds of (excitatory) synaptic junctions caused by mossy-fiber degeneration. In the reorganized glomeruli, however, the inhibitory, GABA-containing Golgi axonal varicosities became the dominant synaptic type—about four-fifths (≈200) of all synapses within the glomerulus—whereas the dendritic synapses between the granule cells represented only one-fifth of all synaptic junctions. The quantitative data of the reorganized cerebellar glomerulus demonstrate both a remarkable constancy and a plasticity of he excitatory granule cells and inhibitory Golgi neurons building up this synaptic complex. constancy (the preservation of certain specific structural features) is represented by an eventually unchanged number of dendrites and synaptic junctions within the deafferented lomerulus. Such constancy was made possible, however, by the morphogenetic plasticity of both nerve-cell types to produce new, dendrodendritic and axo-dendritic synapses to compensate for the loss of mossy-fiber synapses.

The Early Phase of Vascularization in Intraocular Telencephalic Transplants

The present study focused on the early events of vascularization of intraocular cerebral transplants. Telencephalic pieces of rat embryos (El5) were transplanted into the anterior eye chamber of adult rats in deep ketamine-xylazine narcosis. At 3-, 4-, 5-, 6-, or 7-day postoperative survival periods, the rats were perfused and the transplants, with their iridic beds, were processed into serial, semi-thin sections, In 3- and 4-day transplants, neither dilated (perfused) nor collapsed blood vessels were found, but tissue defects, without proper wall and filled by non-nucleated (mature, host) erythrocytes, were seen. On post-operative day 5, large sinusoids were seen lines by endothel and free of blood cells (as a consequence of perfusion). On days 6 and 7, the usual, although large, blood vessels were found. Our results suggest that the critical period of transplant vascularization is between postoperative days 4 and 5, and that the original vessels of donor tissue degenerate and disappear during the first postoperative days and thus, do not participate directly in transplant vascularization. Our hypothesis is that vascular invasion begins with the opening of host blood vessels into clefts formed by degeneration of graft tissue. For a period, a hemostasis occurs in these blood-filled lacunae, and then endothel invasion from host vessels forms the proper wall. The transplant vasculature develops from these large sinusoids. The results challenge the role of the pre-existing donor vessels in transplant vascularization. A possible explanation of such paradoxical results is that the donor tissue must reach a stage of maturation to receive the ingrowing vessels, either host vessels, and the presence of vessels in the donor brain is the sign of this stage of maturation but has no direct role in transplant vascularization

A Rapid Replacement of Vimentin-Containing Radial Glia by Glial Fibrillary Acidic Protein-Containing Astrocytes in Transplanted Telencephalon

The present study follows the early events in the development of astroglia in rat embryonic (El5) tissue grafted into the cortex of adult rats. Astroglial elements (radial glia and astrocytes) were studied by glial fibrillary acidic protein (GFAP) and vimentin immunohistochemistry on post-transplantation (PT) days 7, 11, 14, 17, and 21. At PT7, GFAP-immunopositive elements were only scarce fibers in the transplants. At PTll, a dense network of long, GFAP-immunopositive fibers enmeshed the entire transplant, and astrocytes were already recognized. The fibers also showed vimentin immunoreactivity. By PT14, astrocytes became the predominant GFAP-labeled elements, although a few long fibers persisted. When compared with in situ development, the grafts showed earlier GFAP-immunoreactivity and earlier appearance of astrocytes, as well as a more rapid transition from the immature to the mature form of the glial system.

Morphometric Analysis of Hippocampal Pyramidal Neuronsin situand in Grafts Developing in the Anterior Eye Chambers of Young and Aged Wistar Rats

We performed a morphometric analysis of the somatic and nuclear areas in the pyramidal neurons of the hippocampal fields CA1 and CA3in situand in grafts developing for six weeks in the anterior eye chambers of young (3-to-9 wk.) and of aged (18-to-19.5 mos.) Wistar rats. The mean areas of the CA1 pyramidal somata and nuclei were significantly decreased in the aged animalsin situ. The mean parameters of the CA3 pyramidal neurons were not changed, although their distribution was different (bimodalversusunimodal in the young animals). In both groups of recipients, the areas of CA1 neurons and of their nuclei were significantly larger in the grafted tissue than those foundin situ. The areas of CA3 neurons did not show any difference in aged recipients and demonstrated only slight hypertrophy in young recipients. We concluded that the area sizes of the pyramidal cell bodies and nuclei in CA1 neurons are more sensitive than those of CA3 neurons to both aging and transplantation. The age of recipients did not significantly influence the growth and development of grafted pyramidal cells.

Cerebellar Culture Models of Dendritic Spine Proliferation After Transplantation of Glia

Studies of Purkinje cell dendritic spine proliferation after transplantation of cytosine arabinoside (Ara C) treated organotypic cerebellar cultures with glia and granule cells, either separately and in combination, were reviewed. Exposure of cerebellar explants to Ara C for the first 5 days in vitro results in the destruction of granule cells, the only excitatory cortical neurons, and oligodendroglia, and functionally compromises surviving astrocytes so that they do not appose neuronal membranes. In the absence of granule cells, there is a sprouting of Purkinje cell recurrent axon collaterals, the terminals of which project to and form heterotypical synapses with Purkinje cell dendritic spines, which are usually occupied by terminals of granule cell axons (parallel fibers). After this reorganization has been achieved, the explants can be transplanted with the missing elements to induce a second round of reorganization, with approximate restoration of the usual interneuronal relationships. Addition of both granule cells and glia resulted in a proliferation of clusters of Purkinje cell dendritic spines, which formed synapses with axon terminals of transplanted granule cells, and as synapse formation progressed, the spine clusters became reduced. Transplantation of Ara C-treated cultures with glia alone resulted in a proliferation of clusters of Purkinje cell dendritic spines, but in the absence of granule cells the spines remained unattached, and the clusters persisted throughout the period of observation. Purkinje cell dendritic spine proliferation was induced by exposure of Ara C-treated cultures to astrocyte-conditioned medium. When Ara C-treated cerebella cultures were transplanted with granule cells in the absence of functional glia, parallel fiber- Purkinje cell dendritic spine synapses formed, but no clusters of Purkinje cell dendritic spines were observed. These findings suggest that Purkinje cell dendritic spine proliferation is induced by an astrocyte-secreted factor, resulting in an expansion of postsynaptic sites available for synaptogenesis.

Minor Immunoreactivity in GDNF-, BDNF-, or NT-3-Treated Substantia Nigra Allografts

Glial-cell-line-derived neurotrophic factor (GDNF) stimulates the survival of dopaminergic neurons. Little is known, however, about the possible immune sequelae of GDNF exposure or of exposure to other putative trophic factors. To address these questions, pieces of mesencephalic tissue, substantia nigra, from 15-day-old donor embryos were transplanted into the anterior chamber of the eye of adult male Sprague- Dawley recipient rats. At 5-day intervals, an aliquot (0.5 µg) of GDNF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or cytochrome-C (CC) was injected into the anterior chamber of the eye of the recipients, and the sizes of the transplants were measured. GDNF increased transplant survival and growth. On day 42, all rats were sacrificed, and the grafts were evaluated by cresyl-violet staining and by immunohistochemistry using antibodies raised against neurofilament (NF), tyrosine hydroxylase, or glial fibrillary acidic protein (GFAP), as well as the following monoclonai antibodies: OX-38 anti-CD4, OX-8 anti-CD8, OX-18 anti-MHC class I, OX-6 anti- MHC class II, OX-42 anti-CD11b, R-73 anti-a and anti-ß T-cell receptor, and EDI raised against monocytes/macrophages. BDNF-treated grafts showed only weak immunoreactivity, and even weaker reactions were seen in grafts treated with NT-3, GDNF, or CC. No single immune system marker was significantly elevated in grafts from any treatment group. We used OX-42 and EDI to study possible alterations of microglial components. Ramified microglial cells were found in GDNF-treated grafts and to a lesser extent in NT-3 and BDNF-treated grafts. EDl-labeled reactive microglial components were found in NT-3- and BDNF-treated grafts. Additionally, large and rounded OX-42-positive phagocytic cells were found in NT-3-treated grafts. Together with our previous finding that GDNF treatment of spinal cord transplants activates immune responses and leads to microglial activation, our data dempnstrate that although treatment with GDNF and to some degree with BDNF can enhance immune responses to immunogenic grafts, such as fetal spinal cord grafts, but the trophic factors per se do not elicit any marked response in non-immunogenic grafts like substantia nigra.