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.     

Evolution of Bergman glia in developing human fetal cerebellum: A Golgi, electron microscopic and immunofluorescent study

Astrogliogenesis in the human fetal cerebellum was examined in 46 cerebella obtained from hysterotomy specimens ranging between 9 and 20 weeks of ovulation age. By correlating the results obtained by rapid Golgi and Golgi-Cox methods, the indirect immunofluorescence technique for glial fibrillary acidic protein, and electron microscopy, it was possible to ensure identification of cells and obtain a comprehensive view of the ontogenesis of cerebellar astroglia, in particular Bergmann fibers. Radial fibers were present at 9 weeks of ovulation age, with features of astroglial differentiation. In the cerebellar hemisphere radial fibers arising near the ventricular zone did not reach all the way to the pial surface but terminated in vascular walls of the intermediate zone. A second set of glial cells located in the intermediate zone gave rise to long, tapering processes oriented radially to the pia, some reaching to the pial surface and terminating there in conical swellings. Radial glia with these features were observed in cerebella at all fetal ages examined, indicating their availability for guidance of external granular cells as they migrate inward.

With advancing fetal age, the segment of those radial glia traversing the molecular layer demonstrated an increasing resemblance to Bergmann fibers, though the cell bodies giving rise to these processes were still located below the Purkinje cells. Transitional forms between radial glial processes and fibers beginning to resemble Bergmann fibers were observed in numerous specimens impregnated with the Golgi methods. Astrogliogenesis in human fetal cerebellum occurs earlier than formerly believed, and Bergmann fibers are a final stage in the development of a defined group of radial glia in the cerebellum.     

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.     

Embryonic cerebellar astroglia in vitro

Three types of astroglia appear during cerebellar development--radial glia and Bergmann glia, which are thought to facilitate neuronal migration, and astrocytes, which are thought to compartmentalize mature granule neurons. Cells resembling Bergmann glia and astrocytes have been described in cultures of cerebellar cells harvested from early postnatal cerebellum. In this study, we have used cell-type specific antisera to visualize embryonic forms of cerebellar astroglia and their interaction with embryonic neurons in vitro. When cells were dissociated from mouse cerebellum on the thirteenth embryonic day (E13), 3 forms of cells were stained with antisera raised against purified glial filament protein ( AbGF ), all of which had more elongated processes and less complex shapes than astroglia from postnatal day 7. The vast majority of embryonic cerebellar neurons did not contact these immature forms of astroglia.     

Astroglia and microglia in cerebellar neuronal migration disturbances

Between the neuronal and glial cells there is a close relationship conditioning a tight morphological correlation and proper functional interplay. Disturbed interaction between glial and neuronal components leads to inappropriate neural circuits. The reflection of the failure of neural circuit organisation is the picture of morphological changes of neurons and glia. The appearance of microglia and astroglia was analysed in a defectively formed cellular network due to cerebellar neuronal migration disturbances. Focal disruption of neuron migration leads to their differentiation in an abnormal position manifested as heterotopias and cortical anomalies. Neurons that had lost their proper migratory way and heterotopically settled in the white matter were encircled by GFAP-positive astrocytes, with morphology appropriate for surrounding white matter. The microglial cells infiltrated the parenchyma within the heterotopic neurons playing a role in their elimination. In the cerebellar cortical malformations astrocytes were grouped near the Purkinje cells. In the minimal cortical dysplasia the increased number of astrocytes supported the neurons. Impaired morphological components of the glial-pial barrier were observed. In the massive cortical malformations a few degenerated astrocytes followed the disarranged Purkinje cells, while microglia and Bergmann glia fibres were not present. Absence of cells supporting and organizing the cerebellar cortex had an effect on loss of Purkinje cell shape, their disorientation and abnormal position. The appearance and localisation of the astroglia and microglia in the abnormal cerebellar circuitry due to migration disturbances is dependent on the pathomechanism of the anomalies.     

Immunoperoxidase localization of glial fibrillary acidic protein in radial glial cells and astrocytes of the developing rhesus monkey brain

Peroxidase-antiperoxidase (PAP) immunohistochemical staining, utilizing a specific antibody to the glial fibrillary acidic protein (GFA), was employed to analyze gliogenesis in the central nervous system of rhesus monkeys ranging in age from embryonic day 38(E38) to birth (E165) and through the second postnatal month. All major subdivisions of the brain contain glial cells, recognized by the presence of dark brown horseradish peroxidase (HRP) reaction product. Neuronal elements are not stained with this immunocytochemical technique. The first class of glial cell to appear during development are the radial glial cells; the radial glial fibers fan out from the ventricular and subventricular zones, where their cell bodies reside, to the pial surface where they terminate with conical endfeet. These glial cells appear within the first third of gestation, being present in the spinal cord and brainstem by E41; in the diencephalon by E45; and in the telencephalon and cerebellum by E47. The next class of glia to appear is the Bergmann glial cell of the cerebellar cortex, which can be stained by E54. Bergmann glial cells located below the Purkinje cell layer issue parallel processes which extend up to the pial surface. Within each major subdivision of the brain, massive numbers of elongated glial fibers continually alter their distinctive patterns to maintain constant ventricular-pial surface relationships during the major tectogenetic changes which occur throughout embryonic development. In Nissl-counterstained sections columns of migrating neurons are observed juxtaposed to GFA-positive radial and Bergmann glial fibers. Radial glial cells assume a variety of transitional forms during the process of their transformation into mature astrocytes. This transformation occurs in each structure at specific embryonic ages and is initiated after neuronal migration has begun to subside. The number of astroglial cells increases at an accelerated pace after neurogenesis is complete. The immunohistochemical localization of radial glial fibers at relatively early stages of embryonic development indicates that glial cells are present concomitantly with neurons, raising the possibility that at least two distinct populations of cell precursors compose the proliferative zones. Furthermore, the demonstration of large numbers of radial glial cells in all brain regions during the peak of neuronal migration and a close structural relationship between elongated glial fibers and migrating neurons support the concept that glia play a significant role in the guidance and compartmentalization of neuronal elements during development.     

Rapid appearance of pathological changes of neurons and glia cells in the cerebellum of microsphere-embolized rats

Neuropathological changes in the cerebellar cortex of microsphere-embolized rats were studied at 30 min and 3 h after the embolism. Necrotic processes including a sponge-like vacuolation in the molecular layer, a vague outline of some Purkinje cells, and a few pyknotic granule cells having small and dark profiles were identified at sometime between 30 min and 3 h after microsphere-induced embolism in Nissl staining. Glial fibrillary acidic protein staining shows an apparent reduction in the number of Bergmann glial processes in some of the areas where there was necrosis of the molecular layer and poor astroglia processes in the areas subjacent to the pyknotic granule cells. These data demonstrate that within a short time, microsphere-induced cerebral ischemia produces necrosis of cerebellar neurons (i.e. Purkinje and granule cells) and changes in cerebellar glia cells (i.e. Bergmann and astroglia cells), and that these neuropathological changes are secondary phenomenon caused by microsphere blockage of cerebellar blood flow.     

The toppler mouse: a novel mutant exhibiting loss of Purkinje cells

We describe the genetic and neurological features of toppler, a spontaneous autosomal mutation that appeared in a colony of FVB/N mice and that manifests as severe ataxia appearing at around 12 days of age, worsening with age. The lifespan of affected mice is 8-12 months, with occasional mice living longer. Both homozygous males and females are fertile, and females are able to nurture litters. Histological examination of brain revealed no striking abnormalities other than the loss of cerebellar Purkinje cells. The toppler mutation was mapped to mouse chromosome 8, and to assess whether it was novel or a recurrence of a previously described chromosome 8 mouse mutant, toppler mice were crossed with the nervous and tottering mouse mutants. These studies demonstrate that toppler is a unique mouse mutation. Purkinje cell abnormalities in toppler mice were obvious around postnatal day (P) 14, i.e., toppler Purkinje cells already exhibited abnormal morphology. Staining for calbindin, a calcium binding protein enriched in Purkinje cells, showed altered dendritic morphology. Between P14 and P30, dramatic Purkinje cell loss occurred, although there were differences in the degree of Purkinje cell loss in each lobule. At P30, the surviving Purkinje cells expressed zebrin II. From P30 through 6 months, many of the remaining Purkinje cells gradually degenerated. Purkinje cell loss was analyzed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL), and Purkinje cells were TUNEL-positive most abundantly at P21. In addition, Bergmann glia were TUNEL positive at P21, and they expressed activated caspase-3 at earlier time points. Interestingly, despite the apparent death of some Bergmann glia, there was up-regulation of glial fibrillary acidic protein, expressed in astrocytes as well as Bergmann glia. Given the changes in both Purkinje cells and glia in toppler cerebellum, this may be a very useful model in which to investigate the developmental interaction of Purkinje cells and Bergmann glia.     

Glial pathology in development of cerebellar dysplasia in the hereditary cerebellar vermis defect (CVD) rat

The cerebellar vermis defect (CVD) rat is a new neurological mutant characterized by a cerebellar vermis defect and dysplasia in the cerebellum, especially at the cerebellopontine junctions. In this study, the cytokinetics of glia in terms of the development of cerebellar dysplasia in the CVD rat was investigated using glial fibrillary acidic protein (GFAP) and vimentin immunohistochemistry. In the cerebellar hemispheres, dislocation of the Bergmann glia was observed from postnatal day 5 (P5) in lesions with abnormally aggregated external granule cells (EGCs). Rearranging Bergmann glia were often seen around the EGCs penetrating into the white matter. In the cerebellopontine junctional areas, Bergmann glia were induced after penetration of the Purkinje cells, identified with calbindin immunohistochemistry, and EGCs into the pons from P10. Bergmann fibers were frequently arranged perivascularly. In the clusters of Purkinje cells without EGC settlement in the pons, a small number of Bergmann fibers were observed and their alignment was completely disturbed. These findings suggest that morphological changes in the Bergmann glia depend on their contact with Purkinje cells, but that the orientation of their processes may be influenced by EGC settlement. These glial fibers in the CVD rat may play an important role in the aberrant migration of EGCs, resulting in the development of cerebellar dysplasia. Received: 13 April 1999 / Revised, accepted: 20 July 1999     

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.     

Differential neuronal and glial expression of GluR1 AMPA receptor subunit and the scaffolding proteins SAP97 and 4.1N during rat cerebellar development

In neurons, AMPA glutamate receptors are developmentally regulated and selectively targeted to synaptic sites. Astroglial cells also express AMPA receptors, but their developmental pattern of expression and targeting mechanisms are unknown. In this study we investigated by immunocytochemistry at the light and electron microscopy level the expression of GluR1 and its scaffolding proteins SAP97 (synapse-associated protein) and 4.1N during cerebellar development. In cerebellar cortex the GluR1 AMPA receptor subunit is expressed exclusively in Bergmann glia in the adult rodent. Interestingly, we observed that GluR1 was expressed postsynaptically at the climbing fibers (CF) synapse at early ages during Purkinje cell dendritic growth and before the complete ensheathment of CF/Purkinje cell synapses by Bergmann glia. However, its expression changed from neurons to Bergmann glia once these glial cells had completed their enwrapping process. In contrast, GluR2/3 and GluR4 AMPAR subunits were stably expressed in both Purkinje cells (GluR2/3) and Bergmann glia (GluR4) throughout postnatal development. Our data indicate that GluR1 expression undergoes a developmental switch from neurons to glia and that this appears to correlate with the degree of Purkinje cell dendritic growth and their enwrapping by Bergmann glia. SAP97 and 4.1N were developmentally regulated in the same pattern as GluR1. Therefore, SAP97 and 4.1N may play a role in the transport and insertion of GluR1 at CF/Purkinje cell synapses during early ages and at Bergmann glia plasma membrane in the adult. The parallel fiber (PF)/Purkinje cell synapse contained GluR2/3 but lacked GluR1, SAP97, and 4.1N at the time of PF synaptogenesis.     

Preferential transport and metabolism of glucose in Bergmann glia over Purkinje cells: A multiphoton study of cerebellar slices

Knowing how different cell types handle glucose should help to decipher how energy supply is adjusted to energy demand in the brain. Previously, the uptake of glucose by cultured brain cells was studied in real‐time using fluorescent tracers and confocal microscopy. Here, we have adapted this technique to acute slices prepared from the rat cerebellum by means of multiphoton microscopy. The transport of the fluorescent glucose analogs 2NBDG and 6NBDG was several‐fold faster in the molecular layer of the cerebellar cortex than in Purkinje cell somata and granule cells. After washout of free tracer, it became apparent that most phosphorylated tracer was located in Bergmann glia, which was confirmed by counterstaining with the glial marker sulforhodamine 101. The effective recovery of fluorescence after photobleaching showed that 2NBDG‐P can diffuse horizontally across the molecular layer, presumably through gap junctions between Bergmann glial cells. Our main conclusion is that in acute cerebellar slices, the glucose transport capacity and glycolytic rate of Bergmann glia are several‐fold higher than those of Purkinje cells. Given that the cerebellum is largely fueled by glucose and Purkinje neurons are estimated to spend more energy than Bergmann glial cells, these results suggest substantial shuttling of an energy‐rich metabolite like lactate between glial cells and neurons. © 2008 Wiley‐Liss, Inc.     

Identification of radial glial cells within the developing murine central nervous system: studies based upon a new immunohistochemical marker

The monoclonal antibody RC2 was generated in mouse by conventional hybridoma methodology. The antigen recognized by RC2 is robust, allowing aldehyde fixation appropriate to high resolution light and electron microscopic analyses. From the neural tube stage of fetal development the antibody delineates throughout the central nervous system a subpopulation of neuroepithelial cells which have a radial bipolar morphology. A descending process extends to the ventricular margin, and an ascending process contacts the glial limiting membrane by one or more endfeet varicosities. The persistence of these cells through the neurogenetic period allows their identification as radial glial. From as early as E9-10 the fibers appear to be organized in simple straight fascicles. Later in fetal development these fascicles show marked region-specific transformations in density and trajectory, particularly in association with cerebral corticogenesis and with cerebellar and basal ganglia development. The bipolar forms continue to stain with RC2 until they disappear in the postnatal period. Concurrently with a progressive perinatal loss of stained bipolar radial glia, RC2 identifies multipolar cell forms at various levels of the brain wall, as consistent with the transformation of radial glia into astrocytes. RC2 also recognizes monopolar cell forms in the spinal cord and the cerebellum as early as E15, and in the dentate gyrus of the hippocampal formation from the day of birth. Monopolar forms in the cerebellum are inferred to be progenitors of Bergmann glia. Although Bergmann glia are known to persist in adult life, these cells do not stain with RC2 beyond the 2nd postnatal week. The robustness of the antigen recognized by RC2 makes this probe a valuable tool to study the morphological transformations of the bipolar radial glia during their mitotic turnover. It also provides a sensitive stain for the study of the organization and the histogenetic role of the overall radial fiber system.     

Metabotropic glutamate receptor 2/3 immunoreactivity in the developing rat cerebellar cortex.

In adult rat cerebellar cortex, the metabotropic glutamate receptors (mGluRs) 2 and 3 (mGluR2/3) are present in somata, dendrites, and terminals of Golgi cells as well as in presumed glial processes (Ohishi et al. [1994], Neuron 13:55-66). In the present study, spatiotemporal changes in immunostaining for mGluR2/3 were examined in postnatal rat cerebellar cortex. mGluR2/3-immunoreactive Golgi cell somata appeared first in the internal granular layer at postnatal day 3 (P3) and were restricted to lobules IX and X; however, by P5, they were present in all lobules. Immunoreactive Golgi cell axons were adult-like, appearing as tortuous fibers with clusters of varicosities. They were observed first in the internal granular layer at P7 and increased in number and complexity with time. It was confirmed that mGluR2/3-immunoreactive Golgi cell axon terminals belong to the synaptic glomerulus by P10. Immunoreactive Golgi cell dendrites extending into the molecular layer became prominent after P15. By that time, the immunostaining pattern was characteristic of Golgi cells, as seen typically in adults. Many intensely immunoreactive radial processes existed at birth (P0). These traversed the molecular and external granular layers, reaching the pial surface in every cerebellar lobule. Because they showed coimmunoreactivity for glial fibrillary acidic protein, they were confirmed to be Bergmann glial fibers. After P9, they began to lose immunoreactivity at the portion corresponding to the molecular layer, while an immunostained granular pattern appeared in that layer. Immunoreactive radial processes, however, remained in the external granular layer, and finally, at P21, they disappeared together along with the external granular layer. Granular staining in the molecular layer reached background levels at this time. These spatiotemporal changes in mGluR2/3 distribution suggested that there may be distinct roles for mGluR2/3 in Golgi cells and Bergmann glial cells during the early postnatal period. mGluR2/3 in Golgi cells might be associated closely with systemic maturation, whereas mGluR2/3 in Bergmann glia might be needed for neuron-glia interactions related to granule cell development.     

Spatial, temporal, and cellular distribution of the activated extracellular signal regulated kinases 1 and 2 in the developing and mature rat cerebellum

The extracellular signal regulated kinases 1 and 2 (ERK1/2) are important members of an intracellular signaling cascade that is involved in many aspects of the cellular physiology and development of neurons and glia. ERK1/2 are expressed in many brain regions including the cerebellum; however, their role during cerebellar development is poorly understood. Immunohistochemical approaches using phosphorylation-state specific antiserum that recognizes only the activated-ERK1/2 (pERK) were used to characterize the spatial and temporal patterns of activated-ERK in the developing and adult rat cerebellum. The distribution and cell type-specificity of pERK-immunoreactivity (IR) followed an age-related pattern, with the density of pERK-IR Purkinje cells decreasing between P6 and P15 and increasing at later times. Immunopositive granule cell neurons increased from P6 to P12, became decreased during much of late postnatal cerebellar development, and absent in adults. Co-localization of pERK with glial fibrillary acidic protein or the neuronal marker beta-tubulin revealed that activated ERK is present in maturing Purkinje and granule cells, and the soma of Bergmann glia on P4, P10 and P15; pERK was detected in astrocytes on P10 and P15. Associated with weaning, there was a general increase in activated-ERK in all cell types on P22. In adults, pERK-IR was confined to the Purkinje cell layer and scattered cells in the corpus medullare. In summary, a high degree of developmental plasticity was observed in the spatiotemporal distribution of cerebellar pERK-IR suggesting that the ERK-pathway plays a dynamic role in regulating neuronal and glial migration, proliferation and differentiation in the developing cerebellum. In the mature cerebellum, ERK signaling may also mediate postsynaptic information processing.     

Neuronal influence on antigenic marker profile, cell shape and proliferation of cultured astrocytes obtained by microdissection of distinct layers from the early postnatal mouse cerebellum

To study the cellular heterogeneity of astrocytes from early postnatal mouse cerebellum in culture, Bergmann glia were enriched by hand-dissection of Purkinje, molecular and external granular layers ('outer' layer) and fibrous astrocytes of white matter and deep cerebellar nuclei ('inner' layer). Both populations of GFA protein and vimentin-positive astrocytes express N-CAM and the L2/HNK-1 epitope, but not tetanus toxin receptors or A2B5 antigen, at levels detectable by indirect immunofluorescence procedures. The two astrocyte populations are thus indistinguishable from each other. Expression of tetanus toxin receptors and A2B5 antigen in these astrocytes can, however, be induced by removal of neurons. The expression of tetanus toxin receptors is again reduced by readdition of purified populations of small cerebellar neurons. Morphology and proliferation of astrocytes from both layers is also dependent on the presence of neurons: removal of neurons leads to an epithelioid, rather than star-shaped morphology and a severalfold increase in proliferation. Readdition of neurons induces astrocytes to return to their star-shaped morphology. Epidermal growth factor increases proliferation in both populations of astrocytes. We conclude that neither antigenic marker profile, morphology nor proliferative responses serve to distinguish between enriched Bergmann glia and enriched fibrous astrocytes.     

The distribution and cellular localization of CRF-R1 in the vermis of the postnatal mouse cerebellum

The distribution of corticotropin-releasing factor (CRF), the development of CRF-binding sites, and the age at which application of CRF elicits a physiological response have been described previously in the postnatal mouse cerebellum. The intent of the present study was to determine the cellular and subcellular distribution of the CRF type 1 receptor (CRF-R1) in the vermis of the postnatal mouse cerebellum and to correlate these data with those presented in previous studies. On P0, CRF-R1 is present in the apical processes of migrating Purkinje cells. Between P0 and P8, CRF-R1 immunostaining is confined to a supranuclear position in Purkinje cell bodies. Between P9 and P14, the receptor immunolabeling circumscribes Purkinje cell nuclei and extends into their primary dendrites. An adult-like distribution is achieved between P16 and P21. Between P0 and P14, the CRF-R1 antibody also labels processes of migrating GABAergic interneurons that are directed toward the pial surface. By P12, labeling begins to circumscribe the nucleus of GABAergic cells in the internal granule cell layer. Finally, astrocytic processes in the white matter, as well as radial glial processes, show focal labeling with the CRF-R1 antibody beginning at P3 and throughout postnatal development. A previous study demonstrated that CRF does not elicit a physiological response in Purkinje cells until P9. This observation, together with the data presented in this study, suggests that the binding of CRF to the type 1 receptor may be involved in regulating the development of cerebellar neurons and glia immediately after birth, before CRF assumes its function as a neuromodulator later in postnatal development and in the adult.     

Neuronal migration defects in cerebellum of the<Emphasis Type="Italic">Large</Emphasis><Superscript>myd</Superscript> mouse are associated with disruptions in Bergmann glia organization and delayed migration of granule neurons

The Large gene encodes a putative glycosyltransferase that is required for normal glycosylation of dystroglycan, and defects in either Large or dystroglycan cause abnormal neuronal migration. The mechanism for this effect is not fully understood. This study analyzes the Largemyd mouse cerebellum during postnatal cerebellar development. Large is shown to be expressed most strongly in the Bergmann glial cells and Purkinje cells throughout cerebellar development, which is similar to what is known for dystroglycan expression. Discontinuities of the pial surface of the developing Largemyd mouse cerebellum correlate with disruption of the normal organization of the external granule cell layer and Bergmann glial fibers. At early time points, granule neurons express differentiation markers normally, both temporally and spatially, and show no defects in neurite outgrowth in in vitro assays. However, granule neuron migration is delayed within the external granule and molecular layers, resulting in granule neurons undergoing their intrinsically programmed differentiation in inappropriate locations. Consequently, cells expressing mature granule neuron markers become stranded within these layers. The cause of the less efficient migration is likely due to both physical disruption of the glial-guide scaffolding, as well as to suboptimal neuronal-glial guide interactions during migration.     

Developmental change and function of chondroitin sulfate deposited around cerebellar Purkinje cells

Chondroitin sulfate is a long sulfated polysaccharide with enormous structural heterogeneity that binds with various proteins, such as growth factors, in a structure-dependent manner. In this study, we analyzed the expression of chondroitin sulfate in the postnatally developing cerebellar cortex by using three monoclonal antibodies against chondroitin sulfate, MO-225, 2H6, and CS-56, which recognize different structural domains in this polysaccharide. During the first postnatal week, the patterns of immunohistochemical staining made by these antibodies were quite similar, and the molecular layer, the granule cell layer, and Bergmann glial fibers in the external granular layer were densely stained. After postnatal day 12 (P12), the expression of 2H6 epitopes was down-regulated in the molecular layer, and the expression of CS-56 epitopes in this layer was also reduced after P16. On the other hand, the strong expression of MO-225 epitopes, GlcA(2S)beta1-3GalNAc(6S) (D unit)-containing structures, remained until adulthood. These chondroitin sulfate epitopes were observed around Purkinje cells, including cell soma and dendrites. Detailed immunohistochemical analysis suggested that chondroitin sulfate was deposited between Purkinje cell surfaces and the processes of Bergmann glia. Furthermore, the amount of pleiotrophin, a heparin-binding growth factor, in the cultured cerebellar slices was remarkably diminished after treatment with chondroitinase ABC or D unit-rich chondroitin sulfate. With the previous findings that pleiotrophin binds to D unit-rich chondroitin sulfate, we suggest that the D-type structure is important for the signaling of pleiotrophin, which plays roles in Purkinje cell-Bergmann glia interaction, and that the structural changes of chondroitin sulfate regulate this signaling pathway.     

Combined pre- and postnatal ethanol exposure alters the development of bergmann glia in rat cerebellum

The development and maturation of Bergmann glial cells in the rat cerebellum was evaluated on postnatal day 15 by glial fibrillary acidic protein (GFAP) immunocytochemistry, following combined gestational and 10-day postnatal ethanol exposure (a full three trimester human equivalency). GFAP-positive Bergmann glial fibers of lobules I, III, VIb, VII and X of the cerebellar vermis were examined and counted in the molecular layer (ML), the external granular layer (EGL) and the external limiting membrane (ELM). Ethanol exposure reduced:

1. (1) the number of GFAP-positive fibers (per unit length of folia surface) at all three levels;

2. (2) the percentage of mature fibers; and

3. (3) the cross-sectional area in all lobules examined. When data from the five lobules were pooled, there were 7% fewer GFAP-positive fibers in the ML, 15% fewer in the EGL and 20% fewer in the ELM; the percentage of mature fibers was reduced by 16%; and the cross-sectional areas of lobules were reduced by 16%. The altered development of Bergmann glia could be one of the factors causing delayed migration of granular neurons and reductions in the number of granule cells reported in other studies following developmental ethanol exposures and could help to explain some of the motor dysfunctions reported in FAS victims.