Purkinje Cell Bodies Research Articles

Expression of serotonin 5-HT(2A) receptors in the human cerebellum and alterations in schizophrenia.

The occurrence of human cerebellar serotonin 5-HT(2A) receptors (5-HT(2A)R) is equivocal and their status in schizophrenia unknown. Using a range of techniques, we investigated cerebellar 5-HT(2A)R expression in 16 healthy subjects and 16 subjects with schizophrenia. Immunocytochemistry with a monoclonal antibody showed labelling of Purkinje cell bodies and dendrites, as well as putative astrocytes. Western blots showed a major band at approximately 45 kDa. Receptor autoradiography and homogenate binding with [(3)H]ketanserin revealed cerebellar 5-HT(2A)R binding sites present at levels approximately a third of that in prefrontal cortex. 5-HT(2A)R mRNA was detected by reverse transcriptase-polymerase chain reaction, with higher relative levels in men than women. Several aspects of 5-HT(2A)R expression were altered in schizophrenia. 5-HT(2A)R immunoreactivity in Purkinje cells was partially redistributed from soma to dendrites and was increased in white matter. 5-HT(2A)R mRNA was decreased in the male patients. 5-HT(2A)R measured by dot blots and [(3)H]ketanserin binding (B(max) and K(d)) were not significantly altered in schizophrenia. These data show that 5-HT(2A)R gene products (mRNA, protein, binding sites) are expressed in the human cerebellum at nonnegligible levels; this bears upon 5-HT(2A)R imaging studies which use the cerebellum as a reference region. 5-HT(2A)R expression is altered in schizophrenia; the shift of 5-HT(2A)R from soma to dendrites is noteworthy since atypical antipsychotics have the opposite effect. Finally, the results emphasise that expression of a receptor gene is a mutifaceted process. Measurement of multiple parameters is necessary to give a clear picture of the normal situation and to show the profile of alterations in a disease.

Elimination of bax expression in mice increases cerebellar purkinje cell numbers but not the number of granule cells

Cerebellar Purkinje cells and granule cells have been studied extensively as models for investigating neuron-target interactions and the regulation of cell numbers in the developing central nervous system. Recent studies of transgenic mice that overexpress a human Bcl-2 transgene in Purkinje cells suggest both that programmed cell death plays an unexpected role in regulating Purkinje cell number and that Purkinje cells influence the number of granule cells. The role of cell death-related proteins and Purkinje-granule cell interactions in cerebellar development was investigated further in this study by counting the number of Purkinje and granule cells in knockout mutants with a deletion in the proapoptotic gene, Bax. The total number of Purkinje cells was estimated using stereological counting principles in six adult wild type mice, four hemizygous Bax +/- controls, and six Bax -/- knockout mutants. The total number of granule cells per cerebellum was estimated in three adult wild type mice, three hemizygous Bax +/- controls, and three Bax -/- knockout mutants. The number of Purkinje cells increased significantly by over 30% in the Bax -/- knockout mutants compared with wild type and hemizygote controls, whereas the number of granule cells was unchanged in the Bax -/- mutants. There was no change in the volume of the cerebellar cortex or in the size of Purkinje cell bodies in the Bax -/- mutants, implying that Purkinje cell density was increased in the Bax -/- mutants. The increase in Purkinje cell numbers in the Bax -/- knockout mice supports previous evidence that Purkinje cells undergo a period of naturally occurring cell death that is mediated at least in part by the cell death proteins Bcl-2 and Bax. The lack of an effect of Bax gene expression on granule cell numbers indicates that Bax is not an obligate participant in naturally occurring cell death in granule cells.

A novel postsynaptic density protein: the monocarboxylate transporter MCT2 is co-localized with δ-glutamate receptors in postsynaptic densities of parallel fiber-Purkinje cell synapses

Confocal immunofluorescence microscopy showed strong monocarboxylate transporter 2 (MCT2) labeling of Purkinje cell bodies and punctate labeling in the molecular layer. By immunogold cytochemistry, it could be demonstrated that the MCT2 immunosignal was concentrated at postsynaptic densities of parallel fiber-Purkinje cell synapses. The distribution of MCT2 transporters within the individual postsynaptic densities mimicked that of the delta2 glutamate receptor, as shown by use of two different gold-particle sizes. The MCT2 distribution was also compared with the distributions of other monocarboxylate transporters (MCT1 and MCT4). The MCT1 immunolabeling was localized in the endothelial cells, while MCT4 immunogold particles were associated with glial profiles, including those abutting the synaptic cleft of the parallel fiber-spine synapses. The postsynaptic density (PSD) molecules identified so far can be divided into five classes: receptors, their anchoring molecules, molecules involved in signal transduction, ion channels, and attachment proteins. Here, we provide evidence that this list of molecules must now be extended to comprise an organic molecule transporter: the monocarboxylate transporter MCT2. The present data suggest that MCT2 has specific transport functions related to the synaptic cleft and that this transporter may allow an influx of lactate derived from perisynaptic glial processes. The expression of MCT2 in synaptic membranes may allow energy supply to be tuned to the excitatory drive.

A novel isoform of beta-spectrin II localizes to cerebellar Purkinje-cell bodies and interacts with neurofibromatosis type 2 gene product schwannomin.

We report the identification of a full-length novel beta-spectrin II gene (betaSpIIsigma2) in human brain. The betaSpIIsigma2 gene has 32 exons encoding an actin-binding domain, followed by 17-spectrin repeats, and a short COOH-terminal regulatory region that lacks the Pleckstrin homology (PH) domain. Pair-wise sequence analysis showed an additional 36 and 28 amino acids located at the NH2 and COOH-terminal regions of betaSpIIsigma2, respectively. Northern-blot analysis showed an abundant expression of betaSpIIsigma2 transcripts in brain, lung, and kidney. Western-blot analysis confirmed the predicted approximately 225 kD molecular size of betaSpIIsigma2 protein in these same tissues. In brain, immunofluorescent staining revealed that betaSpIIsigma2 was enriched in cerebellar neurons, with specific enrichment in Purkinje cell bodies, but not in dendrites. Of considerable interest, neurofibromatosis type 2 (NF2) gene product schwannomin was found to co-immunoprecipitate with betaSpIIsigma2 in cultured Purkinje cells. These results suggest that betaSpIIsigma2 may play an important role in the assembly of the specialized plasma membrane domain of Purkinje neurons and that schwannomin may be involved in actin-cytoskeleton organization by interacting with betaSpIIsigma2.

2. A novel postsynaptic density protein: the monocarboxylate transporter MCT2is colocalized with glutamate receptors in postsynaptic densities of parallel fiber-Purkinje cell synapses

Confocal immunofluorescence microscopy showed strong monocarboxylate transporter 2 (MCT2) labeling of Purkinje cell bodies and punctate labeling in the molecular layer. By immunogold cytochemistry, it could be demonstrated that the MCT2 immunosignal was concentrated at postsynaptic densities of parallel fiber–Purkinje cell synapses. The distribution of MCT2 transporters within the individual postsynaptic densities mimicked that of the δ2 glutamate receptor, as shown by use of two different gold-particle sizes. The MCT2 distribution was also compared with the distributions of other monocarboxylate transporters (MCT1 and MCT4). The MCT1 immunolabeling was localized in the endothelial cells, while MCT4 immunogold particles were associated with glial profiles, including those abutting the synaptic cleft of the parallel fiber-spine synapses. The postsynaptic density (PSD) molecules identified so far can be divided into five classes: receptors, their anchoring molecules, molecules involved in signal transduction, ion channels, and attachment proteins. Here, we provide evidence that this list of molecules must now be extended to comprise an organic molecule transporter: the monocarboxylate transporter MCT2. The present data suggest that MCT2 has specific transport functions related to the synaptic cleft and that this transporter may allow an influx of lactate derived from perisynaptic glial processes. The expression of MCT2 in synaptic membranes may allow energy supply to be tuned to the excitatory drive.

Cellular and sub-cellular localisation of GABA B1 and GABA B2 receptor proteins in the rat cerebellum

Following the recent discovery that GABA B receptors expressed in cell lines are only functional when both GABA B1 and GABA B2 are expressed, the present study reports on the development of polyclonal antisera specific for carboxyl-terminal portions of the two related GABA B receptor components respectively. Western blotting indicated the specificity of affinity-purified antibodies for native or recombinant expressed GABA BR1 and GABA BR2, with no cross-reactivity, both antisera detecting the heterodimer in rat cerebellar membranes. Immunohistochemistry revealed a distinct distribution of both receptor proteins in rat cerebellum. GABA B1 immunoreactivity was primarily located in the granule cell layer and Purkinje cells, with discrete immuno-positive cell bodies being present in the molecular layer. GABA B2 staining revealed intense immunoreactivity in the molecular layer, with weaker staining in the granule cell layer. Purkinje cell bodies were less intensely immuno-positive for GABA B2. Co-localisation of both receptor proteins was observed using double immunofluorescence techniques, consistent with the notion that both proteins are required for the formation of functional GABA B receptors in vivo. Immunofluorescence also indicated that GABA B receptors did not co-localise with glial fibrillary acid protein, confirming a neuronal localisation for GABA B receptors. Electron microscopic analysis of the molecular layer revealed that the distribution of immunolabelling for both GABA B1 and GABA B2 was mainly located on the membrane of Purkinje cell dendrites and spines and in parallel fibre terminals.

The Polyglutamine Expansion in Spinocerebellar Ataxia Type 6 Causes a β Subunit-Specific Enhanced Activation of P/Q-Type Calcium Channels in Xenopus Oocytes

Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited degenerative disorder of the cerebellum characterized by nearly selective and progressive death of Purkinje cells. The underlying mutation in SCA6 consists of an expansion of a trinucleotide CAG repeat in the 3' region of the gene, CACNA1A, encoding the alpha(1A) subunit of the neuronal P/Q-type voltage-gated calcium channel. Although it is known that this mutation results in an expanded tract of glutamine residues in some alpha(1A) splice forms, the distribution of these splice forms and the role of this mutation in the highly selective Purkinje cell degeneration seen in SCA6 have yet to be elucidated. Using specific antisera we demonstrate that the pathological expansion in SCA6 can potentially be expressed in multiple isoforms of the alpha(1A) subunit, and that these isoforms are abundantly expressed in the cerebellum, particularly in the Purkinje cell bodies and dendrites. Using alpha(1A) subunit chimeras expressing SCA6 mutations, we show that the SCA6 polyglutamine expansion shifts the voltage dependence of channel activation and rate of inactivation only when expressed with beta(4) subunits and impairs normal G-protein regulation of P/Q channels. These findings suggest the possibility that SCA6 is a channelopathy, and that the underlying mutation in SCA6 causes Purkinje cell degeneration through excessive entry of calcium ions.

Localization of P450scc and 5α-reductase type-2 in the cerebellum of fetal and newborn sheep

Prenatally, neuroactive steroids that modulate GABAergic activity may be synthesized de novo within the fetal brain. We have examined changes in immunoreactivity staining for the steroidogenic enzymes cholesterol P450 side-chain cleavage (P450scc), and 5α-reductase type-2 in the cerebellum of late gestation (130–145 days gestation) fetal sheep and newborn lambs (1–4 weeks of age). Both enzymes were predominantly localized in the Purkinje cell body and dendrites of the fetal and newborn cerebellum, with weaker immunoreactivity in a few cells of the inner granular layer. P450scc immunoreactivity was present in Purkinje neurons expressing either of the neuronal microtubule associated proteins MAP1b/5 or MAP2a/b, but was absent from GFAP and HNK-1 positive cells. Soma of Purkinje neurons were also immunopositive for 5α-reductase type-2 in the fetuses, but expression decreased to just detectable levels in the 1–2 and 2–4 week old lambs. Both MAP1b/5- and MAP2a/b-positive Purkinje neurons showed 5α-reductase type-2 expression in the fetus, whereas the residual 5α-reductase staining in the newborn lamb was present only in MAP2a/b-positive Purkinje neurons. Allopregnanolone in the cerebellum decreased from 21.8±1.9 ng/g wet weight in fetuses at 140–145 days gestation to 6.7±0.5 ng/g in 2–4 week old lambs ( P<0.05). Thus, synthesis of neuroactive steroids from cholesterol is possible in cerebellar neurons in late gestation and persists into neonatal life, 5α-reductase type-2 expression is greater in the fetus compared to the neonate, and allopregnanolone concentrations in the cerebellum decrease significantly after birth.

Immunostaining of ganglioside GD1b, GD3 and GM1 in rat cerebellum: cellular layer and cell type specific associations.

We have studied the cellular distribution of gangliosides GD1b, GD3 and GM1 in rat cerebellum by immunostaining, using monoclonal antibodies and confocal microscopy. Antibodies against astroglial, neuronal and synaptic vesicle associated molecules were used for colocalization analyses. In the gray matter, the anti-GD1b antibody stained thin strands in the molecular layer (ML), interpreted as Bergman glia fibers based on colocalized staining with anti-glial fibrillary acidic protein (GFAP). The neuropil in the granule (GL) and Purkinje (PL) cell layers was also anti-GD1b positive. The anti-GD3 antibody stained the ML, the neuropil in the GL and PL and also the granule and Purkinje cell bodies, appearing intracytoplasmically and vesicle associated. Anti-GD1b and anti-GD3 staining in the GL glomeruli were colocalized with anti-synaptophysin staining. The anti-GM1 antibody stained cell bodies in the ML but they could not be characterized in colocalization experiments. The GL and PL were not stained with the anti-GM1 antibody. In the white matter, different staining patterns were seen for the gangliosides, the anti-GM1 staining being the most intense. This study shows cellular layer and cell type specific associations of the investigated gangliosides and localization of GD1b and GD3 at synaptic sites, warranting further studies on their role in synaptic mechanisms.

Three-dimensional morphology of cerebellar climbing fibers. A study by means of confocal laser scanning microscopy and scanning electron microscopy.

The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze-fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4-64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing-over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high-resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.

Immunohistochemical localization of N-methyl-D-aspartate receptor NR1, NR2A, NR2B and NR2C/D subunits in the adult mammalian cerebellum.

The distributions of the N-methyl-D-aspartate (NMDA) receptor NR1, NR2A, NR2B and NR2C/D subunits were mapped in adult mouse cerebellum using subunit-specific antibodies. Immunostaining with anti-NR1 antibodies was prominent in cell bodies and dendritic arbors of Purkinje cells, was light to moderate in cerebellar granule cells, Golgi interneurons and interneurons in the molecular layer. Anti-NR2A subunit-specific antibody staining of mouse cerebellum was moderate in the granule cells, and moderate to dense in Purkinje neurons and Bergmann glia. However, Purkinje neurons were not immunolabelled in adult rat brain. Anti-NR2B subunit-specific immunostaining was prominent in Purkinje cell bodies and dendrites but absent from the granule cell layer. Anti-NR2C/D subunit-specific immunostaining was largely restricted to cerebellar granule cells. These studies reveal that NMDA receptor subunits display distinct but overlapping expression patterns in the adult mammalian cerebellum. Furthermore, we have observed some differences between rats and mice in terms of the NMDA receptor subunits expressed in specific cerebellar cell types.

Modulation of glutamate transporters (GLAST, GLT-1 and EAAC1) in the rat cerebellum following portocaval anastomosis

Glutamate transporters have the important function of removing glutamate released from synapses and keeping extracellular glutamate concentrations below excitotoxic levels. Extracellular glutamate increases in portocaval anastomosis (PCA), so we used a portacaval anastomosis model in rats to analyze the expression of glutamate transporters (GLAST, GLT-1 and EAAC1) in rat cerebellum, 1 and 6 months after PCA, using immunohistochemical methods. In controls, EAAC1 immunoreactivity in Purkinje cells and glial GLAST and GLT-1 immunoreactivities in the molecular layer (ML) increased from young to old rats. One month after PCA, Purkinje cell bodies were not immunostained for neuronal EAAC1 glutamate transporter, whereas glial glutamate transporter expressions (GLAST and GLT-1) were decreased when compared to young controls. In rats with long-term PCA (6 months post-PCA), neuronal and glial glutamate transporter expressions were increased. The expression of the neuronal glutamate transporter EAAC1 was less intense than old controls, whereas glial glutamate transporters (GLAST and GLT-1) increased more than their controls. Since the level of the neuronal glutamate transporter (EAAC1) in long-term PCA did not reach that of the controls, GLAST and GLT-1 glutamate transporters seemed to be required to ensure the glutamate uptake in this type of encephalopathy. EAAC1 immunoreactivity also was expressed by Bergmann glial processes in long-term PCA, but this increase did not suffice to reverse the alterations caused at the early stage. The present findings provide evidence that transitory alteration of glutamate transporter expressions could be a significant factor in the accumulation of excess glutamate in the extracellular space in PCA, which probably makes Purkinje cells more vulnerable to glutamate effect.

Detection of Disialoganglioside in Rat Cerebellar Cortex by Light and Electron Microscopy.

Gangliosides have been implicated in various neural activities, but their distribution or localization, especially at the subcellular level, has not fully been elucidated. Using immunohistochemistry on sections of rat cerebellum with a monoclonal antibody specific for disialoganglioside(GD2), we found that GD2 was expressed on the plasma membrane of Purkinje cell bodies and dendrites. Intermediate stacks of the Golgi apparatus of the Purkinje cell were also stained. In the neuropil surrounding Purkinje cell bodies and dendrites, there existed both immunohistochemically positive and negative nerve processes. In the positive processes, synaptic membranes and vesicles were stained. Granule cells and parallel fibers were negative. Golgi cells in the granular layer were also negative. In the molecular layer, basket cells and outer stellate cells were negative. In the cerebellar cortex, GD2 was restricted to Purkinje cells and some of the nerve processes and synapses adjacent to Purkinje cell bodies and dendrites. Our findings that there are GD2−positive as well as −negative afferent fibers and synapses relating to Purkinje cells suggest a possible role of GD2 in some specific neural transmission.

Electrophysiological Characterization of Voltage-Gated K+Currents in Cerebellar Basket and Purkinje Cells: Kv1 and Kv3 Channel Subfamilies Are Present in Basket Cell Nerve Terminals

To understand the processes underlying fast synaptic transmission in the mammalian CNS, we must have detailed knowledge of the identity, location, and physiology of the ion channels in the neuronal membrane. From labeling studies we can get clues regarding the distribution of ion channels, but electrophysiological methods are required to determine the importance of each ion channel in CNS transmission. Dendrotoxin-sensitive potassium channel subunits are highly concentrated in cerebellar basket cell nerve terminals, and we have previously shown that they are responsible for a significant fraction of the voltage-gated potassium current in this region. Here, we further investigate the characteristics and pharmacology of the voltage-dependent potassium currents in these inhibitory nerve terminals and compare these observations with those obtained from somatic recordings in basket and Purkinje cell soma regions. We find that alpha-DTX blocks basket cell nerve terminal currents and not somatic currents, and the IC(50) for alpha-DTX in basket cell terminals is 3.2 nM. There are at least two distinct types of potassium currents in the nerve terminal, a DTX-sensitive low-threshold component, and a second component that activates at much more positive voltages. Pharmacological experiments also reveal that nerve terminal potassium currents are also markedly reduced by 4-AP and TEA, with both high-sensitivity (micromolar) and low-sensitivity (millimolar) components present. We suggest that basket cell nerve terminals have potassium channels from both the Kv1 and Kv3 subfamilies, whereas somatic currents in basket cell and Purkinje cell bodies are more homogeneous.

Microwave-stimulated recovery of myosin-V immunoreactivity from formalin-fixed, paraffin-embedded human CNS

The lability of brain myosin-V (BM-V) to aldehyde-fixation has hindered immunohistochemical (IH) studies of this actin-based motor. We show here that BM-V immunoreactivity (IR) can be retrieved from formalin-fixed, paraffin-embedded human tissue. BM-V IR was optimally retrieved by boiling 5 μm cerebellar tissue sections in 10 mM sodium citrate buffer, pH 6, for 15 min, using a microwave oven set at 900 W and 2.45 GHz. A polyclonal, affinity purified anti-BM-V antibody, raised in rabbits against the tail domain of chicken BM-V, was shown here to recognize a single band in Western blots of human cortical homogenates. The combined use of this monospecific antibody and of the antigen retrieval (AR) method above allowed us to verify that BM-V IR is strongly expressed in human Purkinje cell bodies and dendrites, and in granule cells. The same pattern of BM-V IR expression was consistently and maximally detected in tissues stored in 10% formalin from 1 week to 2.5 months. The AR protocol for BM-V described here permits its IH study in formaldehyde-fixed tissues. It is a valuable tool to study BM-V in well fixed tissues, as occurs with the large collection of human archival tissue available.

Developmental expression of corticotropin-releasing factor in the postnatal murine cerebellum

Corticotropin-releasing factor (CRF) is present in climbing and mossy fibers and both have a distinct pattern of distribution in the adult cerebellar cortex. The intent of this developmental study is to determine when the lobular pattern of CRF distribution emerges, and to analyze the morphogenesis of CRF immunoreactive climbing and mossy fibers in individual cerebellar lobules. Between postnatal day (P)0 and P3, CRF-immunoreactive (IR) punctate elements are present throughout the cerebellum. By P3, there is a decrease in the density of staining in the white matter and punctate elements become concentrated within the developing cortex. Between P3 and P7 CRF-IR, varicosities circumscribe Purkinje cell bodies, and are present in the internal and external granule cell layers. Between P10 and P12, there is a major reduction in the density of CRF-IR puncta, especially in the internal and external granule cell layers. Varicosities remain around Purkinje cell bodies and some extend into the molecular layer. During this interval, CRF-IR profiles are first evident in axonal configurations characteristic of developing climbing fibers, although there are lobular differences in the degree of maturation of this afferent system. Axonal enlargements characteristic of immature mossy fibers can first be seen at P10 in lobules IX and X; they cannot be differentiated until P12–14 in more rostral or lateral lobules. CRF-IR fibers in lobules IX and X, the vestibulocerebellum, develop into mature climbing and mossy fibers before any other area of the cerebellum. In other lobules of the cerebellum the gradient of maturation for these axonal phenotypes is from medial to lateral and posterior to anterior. Between P10 and P12, CRF-IR climbing fibers are present in all lobules of the cerebellum. After P12, few climbing fibers are observed in the anterior lobe of the cerebellum at midvermal levels; those present are only faintly immunolabeled. Based on its early expression and uniform distribution between P0 and P10, CRF could have a role in cerebellar development. After this age, as climbing and mossy fiber terminal phenotypes mature, and the differential adult patterns of distribution emerge, CRF likely begins to function as a neuromodulator as has been shown in the adult cerebellum.

De novo expression of lipocortin-1 in reactive microglia and astrocytes in kainic acid lesioned rat cerebellum.

An understanding of the role of reactive glia in the neurodegenerative/regenerative process requires a knowledge of the molecules synthesised by these cells following trauma. We investigated the cellular localisation of lipocortin-1 (LC-1), a putative neuroprotective agent, in cryostat sections of normal and kainic acid lesioned rat cerebellum. In the normal cerebellum lipocortin-1 immunoreactivity was detected in Purkinje cell bodies and molecular layer interneurons. Following kainic acid (1 microg) induced lesions, it was rapidly upregulated in activated microglia, from which it appeared to be secreted. At later time points it was detected in activated astrocytes. LC-1 protein levels were quantified by a sensitive and specific ELISA. Compared to control cerebellum, LC-1 levels were dramatically elevated following lesion, peaking at 3 days: 760% of basal (unlesioned) levels. In situ hybridisation studies revealed a marked upregulation of LC-1 mRNA at 1 and 3 days following the lesion, indicating the transient de novo synthesis of this protein, consistent with a localisation to microglia. In vitro studies, on cultured astrocytes and microglia, demonstrated high levels of intracellular LC-1 in both cell types. LC-1 was detected in microglial but not astrocytic, conditioned media, confirming the in vivo observations that activated microglia may secrete LC-1. Our data show that at early time points following excitotoxic lesion to the cerebellum, it is activated microglia that synthesise and possibly secrete this protein, suggesting an important role of this cell type in immunosuppression and neuroprotection following damage to the central nervous system.

Developmental expression of γ-aminobutyric acid transporters (GAT-1 and GAT-3) in the rat cerebellum: evidence for a transient presence of GAT-1 in Purkinje cells

The cerebellar cortex contains several classes of GABAergic neurons. Previous studies have shown that most GABAergic neurons in this region possess the capacity for γ-aminobutyric acid (GABA) uptake. The present study determined the postnatal expression of two GABA transporters, GAT-1 and GAT-3, in the cerebellar cortex and deep nuclei of the rat by using immunocytochemistry. Immunoreactivity for GAT-1 and GAT-3 appears at postnatal day 7 (P7), emerges centroperipherally across the cerebellum during the following 2 weeks and reaches an adult-like pattern by P30. The mature patterns are fully established by P45, which for GAT-1 is characterized by immunolabeled profiles localized exclusively to neuropil, mostly in the molecular layer and the pinceaux deep to the Purkinje cell bodies, and for GAT-3 as immunoreactivity distributed in the neuropil of mainly the granular layer. Before the adult patterns are completed, GAT-1 immunoreactivity is present in the somata of Purkinje, Golgi, basket and stellate cells between P7 and P21, while GAT-3 immunoreactivity is distinct in astrocytic somata which are organized in regularly spaced clusters. During this period, there is also a banding pattern in the sagittal plane of GAT-1 immunoreactivity in developing Purkinje cells. The postnatal development of GAT-1 and GAT-3 in the rat cerebellar cortex shares a similar spatiotemporal pattern with other GABAergic parameters, including the GABA synthesizing enzyme, GABA content and uptake. Specifically, the transient expression of GAT-1 in the somata and dendrites of cerebellar GABAergic neurons appears to correlate with the supra-adult levels of whole-tissue GABA uptake capability during development. Further, GAT-1 expression in immature Purkinje cells may play a unique role in regulating GABA's function during development, since mature Purkinje cells do not express GAT-1 or take up GABA.

Histopathological and ultrastructural features of feline hereditary cerebellar cortical atrophy: a novel animal model of human spinocerebellar degeneration.

Human spinocerebellar degeneration is one of the intractable diseases. We studied the detailed neuropathology of cats with hereditary cerebellar degeneration obtained from the experimental breeding. The findings included almost total loss of Purkinje cells with an increase in Bergmann's glia in the cerebellar hemisphere, preservation of some Purkinje cells in the vermis and moderate neuronal depletion of the olive nucleus. Cerebellar and pontine nuclei were normal. The cerebrum and spinal cord as well as the peripheral nervous system appeared normal. Electron microscopic examination revealed swelling of the distal dendrites of Purkinje cells in the less-affected nodule of the vermis, and clusters of presynaptic boutons without any synaptic contact in the severely affected folia where Purkinje cell bodies and dendrites disappeared. Prolonged existence of presynapses in the molecular and Purkinje cell layers was confirmed by positive immunoreactivity to anti-synaptophysin. Quantitative analysis using electron microscopy demonstrated an apparent increase in the density and mean size of presynapses in the molecular layer of the severely affected folia. These findings indicate that degeneration of Purkinje cells started at the most distal part of the dendrite in this animal model of cerebellar degeneration, and that presynapses, axon terminals of the granular cells and basket cells can exist for a long time even after complete degeneration of the Purkinje cells. Further investigation of this novel animal model may promote a better understanding of pathogenesis of human hereditary cerebellar degeneration.

Immunohistochemical localisation of mGluR7 protein in the rodent and human cerebellar cortex using subtype specific antibodies

Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G protein coupled receptors that are linked to multiple second messenger systems to regulate neuronal excitability and synaptic transmission. To characterise the protein expression of the two mGluR7 receptor splice variants in human and rat cerebellar cortex, antibodies specific to mGluR7 were generated. Antibodies were raised against a glutathione- S-transferase fusion protein containing amino acid residues located in the extracellular domain common to both the human and rat mGluR7 splice variants. These antibodies specifically detected human mGluR7a in mammalian cells transfected with this receptor. In agreement with mGluR7 in situ hybridisation studies, immunohistochemistry performed at the light microscope level revealed that mGluR7 protein expression occurred most prominently in a particular population of nerve cells common to both the human and rat, located within the cerebellar cortex of gray matter contained within transverse folia. Moreover, strong mGluR7-like immunoreactivity was seen in Purkinje cell body cytoplasm of the Purkinje cell layer. In the most superficial cerebellar cortical layer, the molecular layer, immunostaining was observed in Purkinje cell associated proximal and distal dendritic trees. No detectable labelling was evident in intrinsic deep cerebellar nuclei known to contain GABAergic terminals of projecting Purkinje cell axons. These data are suggestive of a post-synaptic location of mGluR7 in this central nervous system structure. In the rodent, additional non-Purkinje cells thought to represent inhibitory interneurones were labelled at all levels in the molecular layer. mGluR7-like immunoreactivity was not associated with glial cells.