Organization of cerebellar cortex secondary to deficit of granule cells in weaver mutant mice

This study deals with some consequences of the early postnatal abnormalities of cerebellar Bergmann glial fibers and granule cell neurons. (1) Cerebellar size is mildly reduced in heterozygous weaver (+/wv) mice and markedly reduced in homozygotes (wv/wv), but the pattern of fissures is essentially normal. Comparison with other mutants displaying small cerebella suggests that cell proliferation rate in the external granular layer is a key deteminant of cerebellar cortical folding. (2) Mossy fiber terminals differentiate on schedule despite the reduced number and abnormal positions of granule cells. However, many of them enter the modified molecular layer, and as noted especially in noninbred wv/wv mice one to two years old, form synapses with dendrites of aberrant granule cells. Where granule cells are absent, mossy fibers form more than the normal number of synapses with dendrites of Golgi type II neurons. (3) Purkinje cells are only mildly affected by the disorder of neighbouring cells. Their dendrites grow abnormally into the territory occupied by external granule cells, reach the external surface, and may turn inward. They form few tertiary branches. Dendritic spines are present in profusion and show membrane thickenings akin to normal postsynaptic elements. Although they receive no axonal contacts, the spines persist, enveloped by glial processes, for at least two years. Apart from the absence of parallel fiber contacts, afferent and intrinsic axons form the normal classes of synaptic connections with Purkinje cells. (4) Interneurons of the molecular layer are generated on schedule. At the time of their earliest recognition, they reside in the external granular layer, where they receive synaptic contacts from climbing fibers and other interneurons. In the absence of parallel fibers, interneurons differentiate in situ but their dendrites are abortive and randomly oriented. Growth of their dendrites, in contrast to that of Purkinje cell dendrites, appears to be markedly influenced by the organization of the local cellular milieu.     

Reactive astroglia-neuron relationships in the human cerebellar cortex: A quantitative, morphological and immunocytochemical study in Creutzfeldt-Jakob disease

In order to investigate the role of neuron-glia interactions in the response of astroglial to a non-invasive cerebellar cortex injury, we have used two cases of the ataxic form of Creutzfeldt-Jakob disease (CJD) with distinct neuronal loss and diffuse astrogliosis. The quantitative study showed no changes in cell density of either Purkinje or Bergmann glial cells in CJ-1, whereas in the more affected CJ-2 a loss of Purkinje cells and an increase of Bergmann glial cells was found. The granular layer in both CJD cases showed a similar loss of granule cells (about 60% ) in parallel with the significant increase in GFAP+ reactive astrocytes. GFAP immunostaining revealed greater reactivity of Bergmann glia in CJ-2 than in CJ-1, as indicated by the thicker glial processes and the higher optical density. Granular layer reactive astrocytes were regularly spaced. In both CJD cases there was strict preservation of the spatial arrangement of all astroglial subtypes—Fañanas cells, Bergmann glia and granular layer astrocytes. Reactive Fañanas and Bergmann glial cells and microglia/macrophages expressed vimentin, while only a few vimentin+ reactive astrocytes were detected in the granular layer. Karyometric analysis showed that the increase in nuclear volume in reactive astrloglia was directly related with the level of glial hypertrophy. The number of nucleoli per nuclear section was constant in astroglial cells of human controls and CJD, suggesting an absence of polyploidy in reactive astroglia. Ultrastructural analysis revealed junctional complexes formed by the association of macula adherens and gap junctions. In the molecular layer numerous vacant dendritic spines were ensheathed by lamellar processes of reactive Bergmann glia. Our results suggest that quantitative (neuron/astroglia ratio) and qualitative changes in the interaction of neurons with their region-specific astroglial partners play a central role in the astroglial response pattern to the pathogenic agent of CJD.     

Dynamic transformation of Bergmann glial fibers proceeds in correlation with dendritic outgrowth and synapse formation of cerebellar Purkinje cells

Bergmann glia (BG) are unipolar cerebellar astrocytes, whose radial (or Bergmann) fibers associate with developing granule cells and mature Purkinje cells (PCs). In the present study, we investigated the morphodifferentiation of BG by immunohistochemistry for glutamate transporter GLAST and electron microscopy. GLAST was expressed widely in cerebellar radial glia/astrocytes during fetal and neonatal periods and became concentrated in BG postnatally. During the second postnatal week when PC dendrites grow actively, GLAST immunostaining revealed dynamic cytologic changes in Bergmann fibers in a deep-to-superficial gradient; Bergmann fibers traversing the external granular layer were stained as rod-like fibers, whereas in the molecular layer, the rod-like pattern was gradually replaced with a reticular meshwork. At postnatal day 10, the superficial rod-like domain was composed of glial fibrillary acidic protein (GFAP)-positive/GLAST-positive straight fibers, forming cytoplasmic swellings and short filopodia. Along this domain, the tip of growing PC dendrites ascended vertically and entered the base of the external granular layer. The deeper reticular domain of Bergmann fibers was characterized by active expansion of GFAP-negative/GLAST-positive lamellate processes, which surrounded PC synapses almost completely. Therefore, the transformation of Bergmann fibers proceeds in correlation with dendritic differentiation of PCs. The intimate PC-BG relationships during cerebellar development raise the possibility that a preexisting glial shaft could serve as a structural substrate that directs dendritic outgrowth toward the pial surface, whereas the successive formation of a reticular glial meshwork should lead to structural maturation of newly formed PC synapses.     

Developmental regulation of tyrosine kinase substrate p36 (calpactin heavy chain) in rat cerebellum

The tyrosine kinase substrate p36 (calpactin heavy chain) is a calcium-dependent membrane- and cytoskeletal-binding protein. Using an affinity-purified antiserum raised against the p36 heavy chain from bovine adrenal medulla, we have examined the cellular distribution of p36 in developing and adult cerebellum. From immunoblotting, the level of p36 in cerebellum was found to decline during development. In dissociated cell cultures of P4 cerebellum, all cell types were labeled by anti-p36. In vibratome sections from cerebella of P10 rats, anti-p36 stained Purkinje cell bodies strongly and all other cell types less strongly, with the exception of cells in the external germinal layer, which were unstained. By 18 days postnatally p36 was present at higher levels in Bergmann glia and astrocytes of the white matter. In sections of adult cerebella, anti-p36 staining was restricted to Bergmann glial processes and to the processes of a subclass of astrocytes in the granular layer and the white matter. At no developmental stage was anti-p36 staining detectable in axons or dendrites in vibratome sections. These results suggest that p36 plays a role in some aspect of cellular differentiation common to all cerebellar cell types and may have additional functions in astrocytes of the adult cerebellum.     

Localization and expression of GABA transporters in the suprachiasmatic nucleus

GABA is a principal neurotransmitter in the suprachiasmatic hypothalamic nucleus (SCN), the master circadian clock. Despite the importance of GABA and GABA uptake for functioning of the circadian pacemaker, the localization and expression of GABA transporters (GATs) in the SCN has not been investigated. The present studies used Western blot analysis, immunohistochemistry and electron microscopy to demonstrate the presence of GABA transporter 1 (GAT1) and GAT3 in the SCN. By using light microscopy, GAT1 and GAT3 were co‐localized throughout the SCN, but were not expressed in the perikarya of arginine vasopressin‐ or vasoactive intestinal peptide‐immunoreactive (‐ir) neurons of adult rats, nor in the neuronal processes labelled with the neurofilament heavy chain. Using electron microscopy, GAT1‐ and GAT3‐ir was found in glial processes surrounding unlabelled neuronal perikarya, axons, dendrites, and enveloped symmetric and asymmetric axo‐dendritic synapses. Glial fibrillary acidic protein‐ir astrocytes grown in cell culture were immunopositive for GAT1 and GAT3 and both GATs could be observed in the same glial cell. These data demonstrate that synapses in the SCN function as ‘tripartite’ synapses consisting of presynaptic axon terminals, postsynaptic membranes and astrocytes that contain GABA transporters. This model suggests that astrocytes expressing both GATs may regulate the extracellular GABA, and thereby modulate the activity of neuronal networks in the SCN.     

Developmental regulation of tyrosine kinase substrate p36 (calpactin heavy chain) in rat cerebellum

The tyrosine kinase substrate p36 (calpactin heavy chain) is a calcium-dependent membrane- and cytoskeletal-binding protein. Using an affinity-purified antiserum raised against the p36 heavy chain from bovine adrenal medulla, we have examined the cellular distribution of p36 in developing and adult cerebellum. From immunoblotting, the level of p36 in cerebellum was found to decline during development. In dissociated cell cultures of P4 cerebellum, all cell types were labeled by anti-p36. In vibratome sections from cerebella of P10 rats, anti-p36 stained Purkinje cell bodies strongly and all other cell types less strongly, with the exception of cells in the external germinal layer, which were unstained. By 18 days postnatally p36 was present at higher levels in Bergmann glia and astrocytes of the white matter. In sections of adult cerebella, anti-p36 staining was restricted to Bergmann glial processes and to the processes of a subclass of astrocytes in the granular layer and the white matter. At no developmental stage was anti-p36 staining detectable in axons or dendrites in vibratome sections. These results suggest that p36 plays a role in some aspect of cellular differentiation common to all cerebellar cell types and may have additional functions in astrocytes of the adult cerebellum. This work was supported by project grants to R.D.B. from the Medical Research Council of the United Kingdom.     

Creatine Kinase Isoenzymes in Chicken Cerebellum: Specific Localization of Brain-type Creatine Kinase in Bergmann Glial Cells and Muscle-type Creatine Kinase in Purkinje Neurons

Creatine kinase isoenzymes were localized in the chicken cerebellum by the use of isoenzyme-specific anti-chicken creatine kinase antibodies. Brain-type creatine kinase was found in high amounts in the molecular layer, particularly in Bergmann glial cells but also in other cells of the cerebellar cortex, e.g. in astrocytes and in the glomerular structures, as well as in cells of the deeper nuclei. A mitochondrial creatine kinase isoform was primarily localized to the glomerular structures in the granule cell layer and was also identified in Purkinje neurons. Surprisingly, a small amount of the muscle-type creatine kinase isoform was identified in cerebellar extracts by immunoprecipitation, immunoblotting and native enzyme electrophoresis, and was shown to be localized exclusively in Purkinje neurons. Cell type-specific expression of brain- and muscle-type creatine kinase in Bergmann glial cells and Purkinje neurons, respectively, may serve to adapt cellular ATP regeneration to the different energy requirements in these specialized cell types. The presence of brain-type creatine kinase in Bergmann glial cells and astrocytes is discussed within the context of the energy requirements for ion homeostasis (K⁺ resorption), as well as for metabolite and neurotransmitter trafficking. In addition, the presence of muscle-type creatine kinase in Purkinje neurons, which also express other muscle-specific proteins, is discussed with respect to the unique calcium metabolism of these neurons and their role in cerebellar motor learning.     

GABAA/benzodiazepine receptor-like immunoreactivity in rat and monkey cerebellum

The cellular and subcellular localization of GABAA/benzodiazepine receptor-like immunoreactivity in the rat and monkey cerebellum has been studied with a monoclonal antibody (E9) directed against the alpha-subunit of purified GABAA/benzodiazepine receptors. At both the light and electron microscopic level E9 immunoreactivity is located in all 3 layers of the cerebellar cortex and within the deep cerebellar nuclei. The reaction product accumulates within the cytoplasm of neurons and their dendrites but axons are not immunoreactive. Glial cells in the white matter and the cortical layers are also unlabeled, although in some instances Bergmann glia do contain reaction product. The overall distribution and cellular and subcellular localization of E9 immunoreactivity is identical for both monkey and rat cerebellum. On the basis of cell size, morphology, and location it is evident that E9 immunoreactivity occurs in examples of all 5 neuronal types in the cerebellar cortex: Purkinje cells, Golgi type II cells, granule cells, and stellate and basket cells. However, the distribution of the reaction product within the cells is more selective. For example, electron microscopy demonstrates that axonal processes and terminals are not E9 immunoreactive with the single exception of the mossy fiber terminals in the granular layer. Also, examples of unlabeled axon terminals resembling those derived from Golgi type II cells, basket cells, and stellate cells form synapses with immunoreactive dendrites and cell bodies in the cortical layers. Finally, in the deep cerebellar nuclei unreactive axon terminals make symmetric synapses with immunostained neurons and dendrites. These results show that E9 monoclonal antibodies label neurons and portions of their processes which are postsynaptic in GABA-mediated inhibitory circuits, and demonstrates that this antiserum can be used as a morphological marker for cells which make GABAA/benzodiazepine receptors.     

Autoradiographic analysis of sulfate metabolism in the cerebellum of the mouse

The metabolism of [³⁵S]sulfate in the cerebellum of young adult mice was studied by autoradiography. Labeled sulfate was concentrated in the cytoplasma near the nucleus of capillary endothelial cells, Bergmann glia, oligodendroglia, and Purkinje cells. A few neurons in the granular layer took up small amounts of radiosulfate. Utilization of inorganic sulfate by other cerebellar cells, with the possible exception of astrocytes, was negligible. This measns that production of sulfate materials in the cerebellum is not specifically a glial or neuronal function, but is a characteristic of certain glial cells and certain neurons, as well as the cells which line the capillary bed.

The sulfated compounds did not remain where they were produced. Instead, they were redistributed throughout all the layers of the cerebellum. Oligodendroglia deposited sulfated material in myelin, presumably in the form of sulfated lipids (sulfatides). In Purkinje cells the radioactive material (probably sulfated mucopolysaccharide) spread into the dendrites which ramify in the molecular layer. This indicates that the process of cytoplasmic flow occurs in dendrites as well as axons. Part of the labeling of the molecular layer could be accounted for by assuming a comparable process of cytoplasmic flow in the Bergmann glia.     

Cx36, Cx43 and Cx45 in mouse and rat cerebellar cortex: species‐specific expression,compensation in Cx36 null mice and co‐localization in neurons vs. glia

Electrical synapses formed by connexin36 (Cx36)‐containing gap junctions between interneurons in the cerebellar cortex have been well characterized, including those formed between basket cells and between Golgi cells, and there is gene reporter‐based evidence for the expression of connexin45 (Cx45) in the cerebellar molecular layer. Here, we used immunofluorescence approaches to further investigate expression patterns of Cx36 and Cx45 in this layer and to examine localization relationships of these connexins with each other and with glial connexin43 (Cx43). In mice, strain differences were found, such that punctate labelling for Cx36 was differentially distributed in the molecular layer of C57BL/6 vs. CD1 mice. In mice with EGFP reporter representing Cx36 expression, Cx36‐puncta were localized to processes of stellate cells and other cerebellar interneurons. Punctate labelling of Cx45 was faint in the molecular layer of wild‐type mice and was increased in intensity in mice with Cx36 gene ablation. The vast majority of Cx36‐puncta co‐localized with Cx45‐puncta, which in turn was associated with the scaffolding protein zonula occludens‐1. In rats, Cx45‐puncta were also co‐localized with Cx36‐puncta and additionally occurred along Bergmann glial processes adjacent to Cx43‐puncta. The results indicate strain and species differences in Cx36 as well as Cx45 expression, possible compensatory processes after loss of Cx36 expression and localization of Cx45 to both neuronal and Bergmann glial gap junctions. Further, expression of both Cx43 and Cx45 in Bergmann glia of rat may contribute to the complex properties of junctional coupling between these cells and perhaps to their reported coupling with Purkinje cells.     

Expression of Rho GTPases Rho-A and Rac1 in the adult and developing gerbil cerebellum

Rho GTPases proteins are essential for cytoskeletal reorganization and play important roles in the development of neuronal dendrites and axons. Several studies have implicated two members of the Rho GTPase family Rho-A and Rac1 activities in the neuronal polarization and the formation of axons and dendrites. In order to correlate cellular expressions of Rho-A and Rac1 with neuronal polarity (axons versus dendrite formation) in the central nervous system, the cerebellum and immunochemical techniques have been chosen. In the adult cerebellar cortex differential pattern of distribution between Rho-A and Rac1 was observed. While Rac1 expression was restricted to Purkinje cell (somata, dendrites and axons), Rho-A was ubiquitously distributed within the cerebellar cortex. Rac1 was localized in the Purkinje cell dendritic arborization (largest and tiny dendrites) and in their axons. This pattern of distribution was also observed during the postnatal development and followed the dendritic morphogenesis of Purkinje cell. Rho-A was highly expressed in the adult Purkinje cells somata, in cells of the granular layer, in glia within the white matter and in axons. Intense staining was observed in Bergmann glia cell bodies and processes. In the developing cerebellum, Rho-A was highly present in cells of the external and internal granule layers and in the Purkinje cell layer. Bergmann glia cell bodies and processes had the most intense staining during the development. The present study reveals a high expression of Rac1 and Rho-A during Purkinje cell neurites outgrowth period which occurred after birth in the cerebellum. In addition Rho-A is highly expressed in granule cell progenitor cells present in the external granular layer and therefore may play an important role in granule cell progenitor migration.     

Microglial response to the neurotoxicity of 6-hydroxydopamine in neonatal rat cerebellum

Depletion of noradrenaline in newborn rats by 6-hydroxydopamine (6-OHDA) affects the postnatal development and reduces the granular cell area in the neocerebellum (lobules V-VII). During the first postnatal month, Bergmann glial fibers guide the migration of immature granule cells to the internal granule cell layer. Microglia and Bergmann glia may play an important role in this process, but the exact mechanism behind this phenomenon is not known. We studied the effect of systemic administration of 6-OHDA on the expression and localization on microglia and Bergmann glia in the neonatal cerebellum by immunohistochemistry. In the neocerebellum, 6-OHDA treatment caused a significant increase in the number of activated microglia. The increase was observed mainly in the granule cell layer and the cerebellar medulla. Bergmann glial cells in treated brains were abnormally located, did not form intimate associations with Purkinje cells, and the glial fibers were structurally different. Our findings indicate that a noradrenergic influence may be necessary for the normal maturation and migration of granule cells, and abnormal migration may be the result of Bergmann glia destruction and the activation of microglia. Activated microglia in the granule cell layer may be used as a marker for an injured cerebellar area.     

Developmental expression of GABA transporter-1 and 3 during formation of the GABAergic synapses in the mouse cerebellar cortex

In the brain, gamma-amino butyric acid (GABA), released extrasynaptically and synaptically from GABAergic neurons, plays important roles in morphogenesis, expression of higher functions and so on. In the GABAergic transmission system, plasma membrane GABA transporters (GATs) mediate GABA-uptake from the synaptic cleft in the mature brain and are thought to mediate diacrine of cytosolic GABA in the immature brain. In the present study, we focused on two GATs (GAT-1 and GAT-3) in the mouse cerebellar cortex, which are widely localized in neural and glial cells. Firstly, we examined the localization of GATs in the dendrites and cell bodies of developing GABAergic neurons, where GABA is extrasynaptically distributed, to clarify the GABA-diacrine before synaptogenesis. Secondly, we examined the developmental changes in the localization of GATs to reveal the development of the GABA-uptake system. Neither transporter was detected within the dendrites and cell bodies of GABAergic neurons, including Purkinje, stellate, basket and Golgi cells, in the immature cerebellar cortex. GAT-1 was observed within the Golgi cell axon terminals after postnatal day 5 (P5) and presynaptic axons of stellate and basket cells after P7. GAT-3 was localized within the astrocyte processes, sealing the GABAergic synapses in the Purkinje cell and granular layers after P10. These results indicated that GABA-diacrine did not work in the mouse cerebellar cortex. The onset of GAT-1-expression was prior to that of GAT-3. GAT-1 started to be localized within the GABAergic axon terminals during synapse formation. GAT-3 started to be localized within astrocyte processes when they sealed the synapses.     

Modulation of AMPA receptor subunits GluR1 and GluR2/3 in the rat cerebellum in an experimental hepatic encephalopathy model

The immunohistochemical expression and distribution of the AMPA-selective receptor subunits GluR1 and GluR2/3 were investigated in the rat cerebellum following portocaval anastomosis (PCA) at 1 and 6 months. With respect to controls, GluR1 and GluR2/3 immunoreactivities increased over 1 to 6 months following PCA, although immunolabelling patterns for both antibodies were different at the two analysed times. GluR1 immunoreactivity was expressed by Bergmann glial cells, which showed immunoreactive glial processes crossing the molecular layer at 6 months following PCA. The GluR2/3 subunit was expressed by Purkinje neurons and moderately expressed by neurons of the granule cell layer. Immunoreactivity for GluR2/3 was detectable in cell bodies and dendrites of Purkinje cells in young control cerebella, whereas GluR2/3 immunoreactivity was scarce 1 month post PCA. However, despite a lack of immunoreactivity in the Purkinje somata and main processes of adult control rats, GluR2/3 immunoreactivity was strongly enhanced in Purkinje neurons following long-term PCA. These findings suggest that the localization of the GluR2/3 subunit in Purkinje cells undergoes an alteration and/or reorganization as a consequence of long-term PCA. The combination of enhanced GluR immunoreactivity in long-term PCA, both in Bergmann glial cells and in Purkinje neurons, suggests some degree of neuro-glial interaction, possibly through glutamate receptors, in this type of encephalopathy.     

HISTOCHEMICAL AND BIOCHEMICAL STUDIES OF BUTYRYLCHOLINESTERASE ACTIVITY IN ADULT AND DEVELOPING CEREBELLUM. EFFECTS OF ABNORMAL THYROID STATE AND UNDERNUTRITION

The cellular and subcellular localization of BuchE activity (EC.3.1.1.8) was studied in the developing and adult rat cerebellum at light and electron microscope levels. In the adult cerebellum, BuchE activity was exclusively localized to glial cells, myelin and endothelial cells. In the immature cerebellum, BuchE activity was additionally found transiently localized to the neuroblasts of the external germinative layer and in Purkinje cells of the nodulus. In both the immature and the adult animals, the main part of the activity seemed to be membrane-bound. The developmental pattern of cerebellar BuchE activity was assayed in developing normal, hypothyroid, thyroxine-treated and undernourished rats. In normal newborn rats, the specific activity was higher than in adults and it showed one characteristic peak at 6 days (1.8 times the adult value reached at 30 days). At the age of 5 days, the ratio of BuchE-containing astrocytes (numbered in the ganglionic layer) to Purkinje cells was the same as the ratio of Bergmann astrocytes to Purkinje cells determined at 35 days in Nissl preparations; their nucleus size already represented 80% of the adult value and their processes were well developed. The three experimental conditions modified the timing of BuchE development. During the early post-natal period, it was accelerated in the thyroxine-treated and undernourished animals, while in the hypothyroid rats it was delayed. During the same period, the number of labelled astrocytes per Purkinje cell was modified only by hypothyroidism and undernourishment. On the basis of these histochemical and biochemical results, BuchE can be considered as a good marker for the study of Bergmann glia development in the early post-natal period.     

On the plasticity of the cerebellar renin-angiotensin system: localization of components and effects of mechanical perturbation

This study focuses on the renin-angiotensin system (RAS) in the cerebellar cortex and changes within this system after mechanically induced cerebellar injury. Using radioactive and non-radioactive in situ hybridization and immunocytochemistry angiotensinogen mRNA, angiotensinogen, angiotensin II and, for the first time, N-terminal angiotensin fragment (1–7) immunoreactivities, respectively, were demonstrated in the rat cerebellum. Angiotensinogen mRNA and angiotensinogen immunoreactivity (IR) were both present in glial cell populations of all layers, especially in the Purkinje and granular cell layers and within the cerebellar nuclei. Angiotensin II IR was demonstrated in glial cell population in all layers using a monoclonal angiotensin II antibody, while with a polyclonal angiotensin II antiserum (Denise) some Purkinje cell bodies were labelled. After lesioning the cerebellar cortex mechanically by an injection cannula a strong increase in angiotensinogen gene expression as well as in angiotensin II and angiotensin (1–7) immunoreactivities were observed in the glial cell populations. Furthermore, putative Bergmann glial processes, as indicated from the morphological appearance became strongly angiotensin II and angiotensinogen immunoreactive in the region close to the mechanically induced lesion. It could inter alia be demonstrated for the first time using confocal laser microscopy of ANG II IR and GFAP IR that ANG II in vivo in the intact cerebellar cortex is present in astroglial processes in the molecular layer and presumably secreted into the extracellular space in form of small spheric bodies and/or taken up by other cell types. In contrast, the N-terminal fragment angiotensin (1–7) IR was restricted to the glial cell populations and appeared only after the lesion event. Thus, it is suggested that the cerebellar RAS shows marked changes in response to mechanically induced lesions. The expression of angiotensinogen as well as the production of angiotensinogen IR and angiotensin II like IR is even after mechanical lesion restricted to astrocytes, i.e., cerebellar astrocytes and putataive Bergmann glial cells, and in case of immunoreactivities it spreads to the radially oriented Bergmann glial processes in the molecular layer.     

Close homologue of adhesion molecule L1 promotes survival of Purkinje and granule cells and granule cell migration during murine cerebellar development

Several L1‐related adhesion molecules, expressed in a well‐coordinated temporospatial pattern during development, are important for fine tuning of specific cerebellar circuitries. We tested the hypothesis that CHL1, the close homologue of L1, abundantly expressed in the developing and adult cerebellum, is also required for normal cerebellar histogenesis. We found that constitutive ablation of CHL1 in mice caused significant loss (20–23%) of Purkinje and granule cells in the mature 2‐month‐old cerebellum. The ratio of stellate/basket interneurons to Purkinje cells was abnormally high (+38%) in CHL1‐deficient (CHL1?/?) mice compared with wild‐type (CHL1+/+) littermates, but the γ‐aminobutyric acid (GABA)ergic synaptic inputs to Purkinje cell bodies and dendrites were normal, as were numbers of Golgi interneurons, microglia, astrocytes, and Bergmann glia. Purkinje cell loss occurred before the first postnatal week and was associated with enhanced apoptosis, presumably as a consequence of CHL1 deficiency in afferent axons. In contrast, generation of granule cells, as indicated by in vivo analyses of cell proliferation and death, was unaffected in 1‐week‐old CHL1?/? mice, but numbers of migrating granule cells in the molecular layer were increased. This increase was likely related to retarded cell migration because CHL1?/? granule cells migrated more slowly than CHL1+/+ cells in vitro, and Bergmann glial processes guiding migration in vivo expressed CHL1 in wild‐type mice. Granule cell deficiency in adult CHL1?/? mice appeared to result from decreased precursor cell proliferation after the first postnatal week. Our results indicate that CHL1 promotes Purkinje and granule cell survival and granule cell migration during cerebellar development. J. Comp. Neurol. 513:496–510, 2009. © 2009 Wiley‐Liss, Inc.     

Actin-depolymerizing factor (ADF) in the cerebellum of the developing rat: a quantitative and immunocytochemical study.

A specific antiserum against actin-depolymerizing factor (ADF) was used in a quantitative and immunocytochemical study of ADF in the cerebellum of developing rats. The Triton-soluble ADF concentration remained stable throughout development. Light and electron microscopic immunocytochemistry showed that ADF was not detected in all cerebellar cells. ADF immunoreactivity was found in Purkinje cells, but not in granule cells. It was found in the Bergmann astrocytes and the astrocytes of the white matter, but not in the oligodendrocytes. The cell bodies and dendrites of Purkinje cells were immunoreactive for ADF but the axons were not. In contrast, the other axons of the white matter (mossy and climbing fibres) were labeled. Thus, ADF was not restricted to either the dendritic or axonal compartments. However, dendritic spines and postsynaptic densities were immunoreactive, whereas presynaptic varicosities were unlabeled. The immunoreactivities for ADF and actin were compared. ADF staining was uniformly distributed throughout the entire dendritic arborization of the Purkinje cell, while filamentous actin is highly concentrated in the dendritic spines, indicating that ADF activity might vary according to its cellular localization.     

Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electronmicroscopic study in Macacus Rhesus

The cytology of the postmitotic migratory granule cell and its relationship to Bergmann glial processes was examined with Golgi staining and electron microscopy in the three cardinal planes in the developing cerebellar cortex of Macacus rhesus at various late fetal and early postnatal ages. After final mitosis the granule cell body transforms from a nearly round shape in the superficial zone of the external granular layer to a horizontal bipolar form with elongated processes oriented longitudinally to the folium, at the outer border of the molecular layer. Another descending process develops, and the cell soma becomes a pyramid flattened in the plane longitudinal to the folium. The nucleus moves into the descending process and the cell soma assumes a vertically oriented spindle shape while migrating among previously formed parallel fibers deeper in the molecular layer, and finally attains a round shape again when it lies deep to the Purkinje cell layer. During these transformations, the cell cytoplasm becomes more voluminous and contains a prominent Golgi apparatus, numerous free ribosomes, mitochondria, multivesicular and dense bodies, and fascicles of microtubules Longitudinally oriented microtubules concentrated in the vertical leading process disappear by the time the cell soma enters the granular layer. The slender trailing process loss most of its cytoplasmic organelles, acquires microtubules and together with the horizontal processes forms the characteristic T-shaped axon. The axon forms synapses with Purkinje and stellate cell dendrites at a time when other granule cells are still migrating among them. During the entire course of their migration across the molecular layer, granule cells are directly apposed to vertically oriented Bergmann fibers belonging to the Golgi epithelial cells. The sequence of developmental stages indicates that Golgi epithelial cells are a type of protoplasmic astrocyte. The Bergmann fibers were present at all stages examined, and their constant apposition to granule cells suggests a role in the lartter's migration. Numerous electronluscent beady enlargements are seen along the fiber except at sites were the surface is in contact with migrating cells; probably these enlargements change position as the granule cells pass along the Bergmann fiber. Lamellate expansions also project from the main shaft of the glial fiber and envelop the synaptic sites on spines of Purkinje cell dendrites. These expansions seem more durable, and the young neuron appears to avoid collision with them by sprialling around the glial shaft during its descent to the granular layer. The neuron-glia relationship apparently provides the necessary conditions for the migration of the young granule cell. Especially at late developmental stages when the molecular layer is more than 250 μ wide and is densely packed with highly oriented cell processes that have already established synaptic connections.     

Tissue culture studies of neural plasticity

Exposure of cerebellar cultures derived from neonatal mice to cytosine arabinoside for the first 5 days in vitro results in destruction of cerebellar granule cells and inhibition of glial maturation. Such cultures undergo reorganizational changes, primary features of which are a sprouting of Purkinje cell recurrent axon collaterals and the formation of recurrent axon collateral-Purkinje cell dendritic spine synapses. Such heterotypical synapses are inhibitory, in contrast to the excitatory parallel fiber-Purkinje cell dendritic spine synapses normally present. If locus coeruleus neurons are included with the cerebellar cultures, the catecholaminergic axons also sprout, and tissue levels of catecholamines are increased. Purkinje cell survival is enhanced in Ara C-treated cultures, as the target field for Purkinje cell axonal projections is expanded. Oligodendrocyte inhibition results in failure of myelination, and astrocyte inhibition, when it occurs, is associated with a failure of Purkinje cell ensheathment and a hypennnervation of Purkinje cell somata by sprouted recurrent axon collateral terminals. Transplantation of such cultures with granule cells and glia reverses many of these changes. Parallel fiber-Purkinje cell dendritic spine synapses are formed, and most heterotypical synapses disappear. The Purkinje cell population is reduced to normal, and most of the sprouted recurrent axon collaterals are eliminated. However, sprouted catecholaminergic axons are not significantly reduced. Transplanted cultures become myelinated and Purkinje cells acquire astrocytic sheaths, with an associated reduction of axosomatic synapses. Transplantation with glia alone does not reduce the sprouted recurrent axon collaterals, but does result in astrocytic ensheathment of Purkinje cells and an associated decrease of axosomatic synapses. These tissue culture models illustrate some of the plastic changes that the nervous system may undergo following injury and transplantation.