Loss of adenomatous polyposis coli in Bergmann glia disrupts their unique architecture and leads to cell nonautonomous neurodegeneration of cerebellar Purkinje neurons

The tumor suppressor adenomatous polyposis coli (APC) is a multifunctional protein that inhibits the Wnt/beta-catenin signaling pathway and regulates the microtubule and actin cytoskeletons. Using conditional knockout (CKO) mice in which the APC gene is inactivated in glial fibrillary acidic protein (GFAP)-expressing cells, we show a selective and critical role for APC in maintaining the morphology and function of cerebellar Bergmann glia, which are specialized astroglia that extend polarized radial processes from the Purkinje cell layer to the pial surface. APC-CKO mice developed Bergmann glia normally until the accumulation of beta-catenin started around postnatal day 10 (P10). Their radial fibers then became shortened with a marked reduction of branching collaterals and their cell bodies translocated into the molecular layer followed by loss of their pial contact and transformation into stellate-shaped cells by P21. Purkinje neurons were normal in appearance and number at P21, but there was significant loss of Purkinje neurons and cerebellar atrophy by middle age. Outside the cerebellum, neither beta-catenin accumulation nor morphological changes were identified in GFAP-expressing astroglia, indicating region-specific effects of APC deletion and an essential role for APC in maintaining the unique morphology of Bergmann glia as compared with other astroglia. These results demonstrate that loss of APC selectively disrupts the Bergmann glial scaffold in late postnatal development and leads to cerebellar degeneration with loss of Purkinje neurons in adults, providing another potential mechanism for region-specific non-cell autonomous neurodegeneration.     

NG2 cells differentiate into astrocytes in cerebellar slices

NG2-glia are an abundant population of glial cells that have been considered by many to be oligodendrocyte progenitor cells (OPCs). However, growing evidence suggests that NG2-glia may also be capable of differentiating into astrocytes and neurons under certain conditions. Here, we have examined NG2-glia in cerebellar slices, using transgenic mice in which the astroglial marker glial specific protein (GFAP) drives expression of the reporter gene enhanced green fluorescent protein (EGFP). Immunolabelling for NG2 shows that NG2-glia and GFAP-EGFP astroglia are separate populations in most areas of the brain, although a substantial population of NG2-glia in the pons also express the GFAP-EGFP reporter. In the cerebellum, NG2-glia did not express EGFP, either at postnatal day (P)12 or P29–30. We developed an organotypic culture of P12 cerebellar slices that maintain cytoarchitectural integrity of Purkinje neurons and Bergmann glia. In these cultures, BrdU labelling indicates that the majority of NG2-glia enter the cell cycle within 2 days in vitro (DIV), suggesting that NG2-glia undergo a ‘reactive’ response in cerebellar cultures. After 2 DIV NG2-glia began to express the astroglial reporter EGFP and in some cases the respective GFAP protein. However, NG2-glia did not acquire phenotypic markers of neural stem cells or neurons. The results suggest that NG2-glia are not lineage restricted OPCs and are a potential source of astrocytes in the cerebellum.     

Developmental fates and migratory pathways of dividing progenitors in the postnatal rat cerebellum

To investigate the developmental fates and the migratory pathways of dividing progenitors in both the white matter (WM) and the external granule layer (EGL) in the early postnatal rat cerebellum, a replication-deficient retrovirus carrying the β-galactosidase gene (BAG) was injected into the deep cerebellar tissue or the EGL of postnatal rats to label dividing progenitors. After 1–3 days post-injection (1–3 dpi) of BAG into the deep cerebellar tissue of postnatal day 4/5 (P4/5) rats, labeled immature, unipolar cells were found mainly in the WM. From 4 to 6 dpi, similar cells appeared in the internal granule (IGL), Purkinje cell, and molecular layers, although about half of the labeled cells still resided in the WM and appeared immature. The first morphologically definable Bergmann glia, astrocytes, and oligodendrocytes were also observed. From 14 to 20 dpi, most labeled cells had developed into Bergmann glia, astrocytes, oligodendrocytes, and interneurons in their appropriate layers. When BAG injections were performed at P14, unipolar cells were initially observed, but the majority of these differentiated into myelinating oligodendrocytes in the WM and IGL by 17 dpi. Few immature cells were labeled by injections administered at P20, and these did not develop into mature glia, but into cells with lacy, fine processes, possible representing immature oligodendrocytes. In contrast, BAG-labeled progenitors of EGL produced only granule neurons. Thus, within the first 2 postnatal weeks, dividing progenitors in the WM migrate as immature cells into the cortex before differentiating into a variety of glia and interneurons. The genesis of oligodendrocytes continues through the 2nd postnatal week and largely ceases by P20. EGL cells do not produce glia, but only granule cells. © 1996 Wiley-Liss, Inc.     

Cellular localization of the full-length isoform of the type 2 corticotropin releasing factor receptor in the postnatal mouse cerebellar cortex

Corticotropin releasing factor (CRF) and its cognate receptors, defined as Type 1 and Type 2 have been localized within the cerebellum. The Type 2 CRF receptor (CRF-R2) is known to have both a full length (CRF-R2alpha) and a truncated (CRF-R2alpha-tr) isoform. A recent study documented CRF-R2alpha primarily in Bergann glia and astrocytes, as well as in populations of Purkinje cells in the adult cerebellum. The goal of the present study is to determine if CRF-R2alpha is present in the postnatal cerebellum, and if so to describe its cellular distribution. RT-PCR data showed that CRF-R2alpha is expressed in the mouse cerebellum from birth through postnatal day 21. Between birth and P14, CRF-R2alpha-immunoreactivity was localized within the somata of Purkinje cells, and migrating GABAergic interneurons. GFAP-immunoreactive astrocytes, including Bergmann glia, also expressed CRF-R2alpha-immunoreactivity from P3-P14. There is a change, however, in CRF-R2alpha immunolabeling within neurons as the cerebellum matures. Compared to its expression in the adult cerebellum, Purkinje cells, and GABAergic interneurons showed more extensive CRF-R2alpha immunolabeling during early postnatal development. We postulate that CRF-R2alpha could be involved in developmental events related to the survival and differentiation of Purkinje cells and GABAergic neurons, whereas in the adult, this isoform of the CRF receptor family is likely involved in modulating Bergmann glia that have been shown to play a role in regulating the synaptic environment around Purkinje neurons.     

Effects of prenatal ethanol exposure on the development of Bergmann glia and astrocytes in the rat cerebellum: an immunohistochemical study.

The consequences of prenatal ethanol exposure on the postnatal development of Bergmann glia and astrocytes in the rat cerebellum were investigated by using glial fibrillary acidic protein (GFAP) immunolabeling. Pregnant rats were either fed with an ethanol containing liquid diet (6.7% v/v) or pair-fed with an isocaloric diet throughout gestation. On postnatal day (PD) 15 and 22, parasagittal sections of the cerebellar vermis from female offspring were processed for GFAP immunohistochemistry to assess the development of Bergmann glia and astrocytes in lobules I, VII, and X and astrocytes in the central core of white matter. On PD 15, compared to control animals, ethanol exposed animals had fewer GFAP positive Bergmann glial fibers per unit length of molecular layer; a significantly greater percentage of morphologically immature Bergmann fibers; a significantly greater GFAP positive astrocytic area per unit area of internal granular layer and central white matter; and the astrocytic processes were wider and more closely packed. These glial changes were associated with significantly thicker external granular layer in all 3 lobules. However, no significant differences were seen between the ethanol exposed and control animals on PD 22, indicating "catch-up growth" in the ethanol exposed animals during the third postnatal week. These results suggest that prenatal ethanol exposure causes (1) delayed maturation of Bergmann glia, which in turn contributes to the delayed migration of granule cells; and (2) alterations in the normal postnatal development of astrocytes.     

Embryonic Purkinje Cells Grafted on the Surface of the Adult Uninjured Rat Cerebellum Migrate in the Host Parenchyma and Induce Sprouting of Intact Climbing Fibres

By grafting solid pieces of cerebellar anlage onto the surface of the adult rat cerebellum, we have investigated the problem of the interactions between embryonic and adult neurons in an intact brain. A few days after grafting, embryonic astrocytic processes crossed the graft-host interface and radiated into the recipient molecular layer. Several grafted Purkinje cells also migrated into the host brain along such processes as well as adult Bergmann glia. Adult climbing fibres, labelled by means of Phaseolus vulgaris leucoagglutinin (PHA-L), sprouted new collateral branches which terminated on embryonic Purkinje cells at both extra- and intraparenchymal levels. No sign of activation of host astroglia or microglia was evident in the host cerebellum in relation to these processes. Embryonic Purkinje cells which migrated into the host cerebellum developed an adult-like morphology. Intraparenchymal grafts of neocortical embryonic tissue induced conspicuous growth of host olivary axons, characterized by a pattern which was different from that observed following cerebellar grafts. By contrast, when neocortical tissue was placed onto the surface of the recipient cerebellum, graft-host interactions were limited and climbing fibre sprouting was rarely seen. These results show that (i) supernumerary Purkinje cells can penetrate and settle in the adult intact cerebellar cortex, (ii) adult climbing fibres are able to innervate these new targets in the absence of any injury or activation of non-neuronal cells of the adult brain, and (iii) in the absence of damage to the adult brain, the plasticity of adult olivary axons is specifically elicited and controlled by embryonic Purkinje cells.     

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.     

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.     

Chronological and immunohistochemical observations of cerebellar dysplasia and vermis defect in the hereditary cerebellar vermis defect (CVD) rat

Hereditary cerebellar vermis defect (CVD) rats, a new neurological mutant, developed both cerebellar vermis defect and cerebellar dysplasia. Developmental alterations in the cerebellum of the CVD rats were studied chronologically and immunohistochemically. The earliest architectural abnormality was a maldevelopment of the inferior cerebellar peduncle from embryonic day 17 (E17), leading to an indistinct separation between the cerebellum and the pons. From E19, the CVD rats lacked vermis development and, therefore, the cerebellar hemispheres were fused. After birth, Purkinje cells and external granule cells (EGCs) penetrated into the pontine tissue, but retained their normal position until postnatal day 10. Cerebellar lamination began to be disturbed due to abnormal perivascular aggregations of the EGCs, resulting in convoluted and occasionally perivascular lamination. There were no Bergmann glia in the heterotopic cerebellum of the pons, and abnormally arranged Bergmann glia were observed in the mildly disorganized cerebellar hemispheres. Immunohistochemistry for calbindin revealed that abnormal orientation of the Purkinje cells might be related to the perivascular EGCs. Parvalbumin-immunopositive microneurons were seen only in the disarranged molecular layers, and synaptophysin-immunopositive cerebellar glomeruli were present in the afflicted internal granular layers. These findings suggest that perivascular EGCs may play an important role in cerebellar dysplasia and the developmental plasticity in the altered cerebellogenesis. Received: 1 October 1996 / Revised: 24 April 1997 / Revised, accepted: 11 June 1997     

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.     

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.     

Role of astroglial cell clones in the survival and differentiation of cerebellar embryonic neurons

To investigate the role of astrocytes in the survival and differentiation of cerebellar neurons during development, we have used astroglial cell clones, derived from 8-day postnatal cerebellar explants and which might be the in vitro equivalents of the 3 main types of cerebellar astrocytes, the Golgi epithelial cells and their Bergmann processes, the velate protoplasmic and the fibrous astrocytes (F. Alliot and B. Pessac, Brain Res., 306 (1984) 283-291). Nearly all single cells, dissociated from 15-day embryonic mouse cerebella and seeded at low density, adhered to layers of each of the cerebellar astroglial cell clones as well as to other glial lines or artificial substrates. However, the cerebellar embryonic neurons survived well only on monolayers of either the 'Golgi-Bergmann'-like or the 'velate protoplasmic'-like clones. On these layers, 60-80% of the neurons were still present after 5 days of co-culture, while only less than 5% survived on the other types of substrates. The differentiation pattern of the neurons surviving on the 'Golgi-Bergmann' and the 'velate protoplasmic' astroglial clones was studied with markers of postmitotic granule cells, the major neuronal population in adult cerebellum. The velate protoplasmic-like clone was the only one able to support the coordinate acquisition by most surviving neurons of the phenotypic characteristics of granule cells, i.e. a distinct morphology, a specific epitope binding the monoclonal antibody 7-8 D2 and immunoreactivity to glutamate. These data show a broad heterogeneity in the capacity of astroglial cell clones to support embryonic cerebellar neurons. In addition, they indicate that neuronal survival per se is not sufficient for the acquisition of a differentiated neuronal phenotype.     

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.     

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.     

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     

Temporal control of gene recombination in astrocytes by transgenic expression of the tamoxifen-inducible DNA recombinase variant CreERT2

Inducible gene modification using the Cre/loxP system provides a valuable tool for the analysis of gene function in the active animal. GFAP-Cre transgenic mice have been developed to achieve gene recombination in astrocytes, the most abundant cells of the central nervous system, with pivotal roles during brain function and pathology. Unfortunately, these mice displayed neuronal recombination as well, since the GFAP promoter is also active in embryonic radial glia, which possess a substantial neurogenic potential. To enable the temporal control of gene deletions in astrocytes only, we generated a transgenic mouse with expression of CreERT2, a fusion protein of the DNA recombinase Cre and a mutated ligand-binding domain of the estrogen receptor, under the control of the human GFAP promoter. In offspring originating from crossbreedings of GFAP-CreERT2-transgenic mice with various Cre-sensitive reporter mice, consecutive intraperitoneal injections of tamoxifen induced genomic recombination selectively in astrocytes of almost all brain regions. In Bergmann glia, which represent the main astroglial cell population of the cerebellum, virtually all cells showed successful gene recombination. When adult mice received cortical stab wound lesions, simultaneously given tamoxifen induced substantial recombination in reactive glia adjacent to the site of injury. These transgenic GFAP-CreERT2 mice will allow the functional analysis of loxP-modified genes in astroglia of the postnatal and adult brain.     

Highly differential expression of SN1, a bidirectional glutamine transporter,in astroglia and endothelium in the developing rat brain

The transmitters glutamate and GABA also subserve trophic action and are required for normal development of the brain. They are formed from glutamine, which may be synthesized in glia or extracted from the blood. In the adult, the glutamine transporter SN1 is expressed in the astroglia. SN1 works in both directions, depending on the concentration gradients of its substrates and cotransported ions, and is thought to regulate extracellular glutamine and to supply the neurons with the transmitter precursor. In this article, we have quantified the expression and studied the localization of SN1 at different developmental stages. SN1 is expressed in astroglia throughout the CNS from embryonic stages through adulthood. No indication of SN1 staining in neuronal elements has been obtained at any stage. Quantitative immunoblotting of whole brain extracts demonstrates increasing expression of SN1 from P0, reaching a peak at P14, twice the adult level. A moderate and slower rise and fall of the expression levels of SN1 occurs in the cerebellum. Strong transient SN1-like staining is also found in Bergmann glia and vascular endothelium in the first postnatal weeks. Strong intracellular staining in the same time period suggests a high rate of SN1 synthesis in the early postnatal period. This coincides with the increasing levels of glutamate and GABA in the CNS and with the time course of synaptogenesis. This study suggests that the expression of SN1 is highly regulated, correlating with the demand for glutamine during the critical period of development.     

Binding of developing mouse cerebellar cells to fibronectin: a possible mechanism for the formation of the external granular layer

The role of the matrix glycoprotein fibronectin in the formation of the external granular layer of the developing mouse cerebellum was investigated by in vitro studies of the binding of cerebellar cells to a fibronectin-coated culture substratum and by in vivo immunocytochemical localization of antiplasma fibronectin antiserum in cerebellar tissue. The adhesion of cells dissociated from embryonic and early postnatal mouse cerebellum is developmental stage-specific when the cells are plated on tissue culture substrata derivatized with human plasma fibronectin. Cells dissociated from mouse cerebellum at embryonic day 13 form cellular aggregates on insoluble plasma fibronectin. In contrast, cells dissociated from embryonic day 16 through postnatal day 7 cerebellum form a monolayer. Time-lapse video recordings reveal extensive cell movement of late embryonic and early postnatal cerebellar cells on insoluble plasma fibronectin. Late embryonic and early postnatal cerebellar cells bind to fibronectin but do not degrade the fibronectin substratum. Immunocytochemical studies of the binding of antiplasma fibronectin antisera to cryostat sections of intact embryonic and early postnatal cerebellar tissue reveal a brightly stained region of endogenous fibronectin along the route of granule cell migration from the lateral caudal part of the neuroepithelium lining the fourth ventricle up onto the external surface of the cerebellar anlage. When the formation of the external granular layer is completed, the intense region of fibronectin is no longer visible.     

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.     

The extending astroglial process: development of glial cell shape, the growing tip, and interactions with neurons

To analyze how astroglial cells attain the complex shapes that support neuronal migration and positioning in vitro (Hatten et al., 1984; Hatten 1985), early postnatal mouse cerebellar cells were plated in microcultures, and glial process outgrowth was monitored by high-resolution time-lapse video microscopy combined with immunocytochemical localization of antisera to glial filament protein (GFP), and by electron microscopy. The 2 principal astroglial forms seen in these cultures, stellate and Bergmann-like (Hatten et al., 1984), begin to develop their distinctive shapes by the outgrowth of processes in the first 8 hr after the cells are plated. Glial process extension is most vigorous in this period, resulting predominantly in stellate forms. A second population of glial cells, having fewer, longer processes reminiscent of Bergmann glia in vivo, first appears about 5 hr after plating. During the next 16-24 hr, while the stellate cells only slightly increase their process length, the bipolar cells double their length. The most striking feature of the elongating glial process is its highly motile tip, which rapidly extends microspikes and lamellopodia. Unlike the neuronal growth cone, which is the expanded terminal of a thin neurite shaft, the glial growing tip forms the end of a wide, paddle-like process that is filled with motile mitochondria and masses of glial filaments, and is bordered by an undulating lamella fringed by microspikes. Soon after the emergence of glial processes, cell-cell interactions between the growing glial process tip and granule neurons occur. Within minutes of an initial encounter between the glial process and the neuron, contact relationships that are stable during the observation period form between the cells. Subsequently, many neurons extend a small neurite onto the glial process, and astroglial process extension continues by the movement of the glial growing tip out beyond the neuron. Thus, cerebellar astroglia in vitro develop complex shapes in the same fashion as do neurons: the outgrowth of processes tipped by a motile ending. The growing tips of astroglial processes interact with neurons, resulting in the stable association of neurons and glia.