Weaver mouse cerebellar granule neurons fail to migrate on wild-type astroglial processes in vitro

To study the regulation of glial-guided neuronal migration, we have analyzed the behavior of cerebellar granule neurons purified from the homozygous weaver (wv/wv) B6CBA-w mouse, an autosomal recessive genetic mutation that suffers a failure of granule cell migration along Bergmann glial processes (Rakic and Sidman, 1973a, b; Rezai and Yoon, 1972), on the processes of astroglia purified from homozygous normal B6CBA-Aw-J-wv (+/+) mouse cerebella. When co-cultured with normal astroglia, weaver granule neurons failed to form neuron-glia contacts characteristic of migrating neurons and impaired normal astroglial morphological differentiation. Normal astroglial cells co-cultured with weaver granule cells had enlarged cell somata with stunted processes and enlarged endfeet compared to normal astroglia co-cultured with normal granule cells. In contrast, normal neurons associated with weaver astroglia, forming tight appositions seen for migrating neurons in vivo, and enhanced weaver astroglial morphological differentiation. Weaver astroglia co-cultured with normal granule cells contained a more normal complement of glial filaments and had a smaller perikaryon with longer, more tapered processes than their counterparts co-cultured with weaver neurons. These results suggest, in agreement with the study of Goldowitz and Mullen (1982) on heterozygous mutant chimeras, that the granule neuron is a primary site of action of the weaver gene, and further support our previous findings that neuron-glia interactions regulate astroglial morphological differentiation (Hatten, 1985).     

Riding the glial monorail: a common mechanism for glial-guided neuronal migration in different regions of the developing mammalian brain.

In vitro studies from our laboratory indicate that granule neurons, purified from early postnatal mouse cerebellum, migrate on astroglial fibers by forming a 'migration junction' with the glial fiber along the length of the neuronal soma and extending a motile 'leading process' in the direction of migration. Similar dynamics are seen for hippocampal neurons migrating along hippocampal astroglial fibers in vitro. In heterotypic recombinations of neurons and glia from mouse cerebellum and rat hippocampus, neurons migrate on astroglial processes with a cytology and neuron-glia relationship identical to that of homotypic neuronal migration in vitro. In all four cases, the migrating neuron presents a stereotyped posture, speed and mode of movement, suggesting that glial fibers provide a generic pathway for neuronal migration in developing brain. Studies on the molecular basis of glial-guided migration suggest that astrotactin, a neuronal antigen that functions as a neuron-glia ligand, is likely to play a crucial role in the locomotion of the neuron along glial fibers. The navigation of neurons from glial fibers into cortical layers, in turn, is likely to involve neuron-neuron adhesion ligands.     

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.     

Co-cultivation of astroglial and neuronal primary cultures from rat brain

A technique is described for a two-cell co-cultivation system which permits in vitro evaluation of neuron-glia interactions. Primary astroglial enriched cultures from newborn rat cerebral hemispheres, striatum or cerebral cortex, grown for 3 days, were co-cultivated with primary neuron-containing cultures from 15- to 17-day rat embryo cerebral hemispheres, substantia nigra or brainstem, respectively, grown for 10 days on polylysine-coated surfaces. The neuronal cells were identified morphologically and immunohistochemically by antibodies to neuron-specific enolase. The two cultures were grown together for 7 days, separated by a U-formed 1 mm glass-rod. The results show that neurons exert a morphogenetic effect on astroglial cells in the form of extension of cell processes. The co-culture system allows investigation of potent local humoral interactions between astroglial cells and neurons.     

Differences in neuronal and glial cell phenotypic expression in neuron-glia cocultures: influence of glia-conditioned media and living glial cell substrata.

Neuron-glia cocultures were prepared using, as a source for glial cells, either C6 glia (2B clone) of early (2B23) or late (2B111) passages or advanced passages of glial cells derived from primary cultures prepared from aged mouse cerebral hemispheres (MACH). Six-day-old chick embryo cerebral hemispheres (E6CH) were the source of neuron-enriched cultures. Glutamine synthetase (GS) activity was used as a marker for astrocytes and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity was used as a marker for oligodendrocytes. GS activity was markedly enhanced in cocultures of E6CH neurons and 2B23 glioblastic cells, whereas GS activity was reduced in cocultures of E6CH neurons and 2B111 astrocytic glia. In contrast, CNP activity was enhanced in cocultures of C6 glial cells with E6CH neurons. Glial cells from aged mouse brain did not respond to coculturing with E6CH neurons. It appears from these findings that neuronal input enhances the differentiation of glioblastic cells to either astrocytic or oligodendrocytic expression, whereas it decreases the activity of committed astrocytes. In contrast, glial cells from aged mouse brain do not respond to neuronal input. Choline acetyltransferase (ChAT) activity, a marker for cholinergic neurons, was enhanced only when E6CH cultures were grown in conditioned medium (CM) from 2B23 glioblastic cells. In contrast, ChAT activity was markedly diminished when E6CH neurons were cocultured with MACH glial cells but not when grown in CM from MACH glial cells. Thus, humoral factors from immature glial cells appear to enhance cholinergic neuronal phenotypic expression whereas cell-cell membrane contacts with aged glial cells diminish cholinergic phenotypic expression. The findings present supportive evidence that neuron-glia interrelationships are age dependent.     

Neuron–Glia Interaction in the Suprachiasmatic Nucleus: A Double Labeling Light and Electron Microscopic Immunocytochemical Study in the Rat

The morphological interactions between astroglial and neuronal elements were elucidated in the rat suprachiasmatic nucleus (SCN) by light and electron microscopic immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), vasoactive intestinal peptide (VIP) and arginine-vasopressin (AVP). Throughout the SCN, particularly in its ventral portion, GFAP-like-immunoreactive (GFAP-LI) astroglial elements were found. These astrocytes displaying GFAP-like immunoreactivity occasionally contained fairly well-developed organelles. Some of these astrocytes were found as satellite cells in close contact with non-immunoreactive neuronal perikarya and processes. Around the neurons, GFAP-LI astroglial processes were also observed to cover some portions of presynaptic and postsynaptic elements. In addition, these astroglial elements were seen between two neuronal somata and pericytes of blood capillaries as glial endfeet. By double labeling immunoelectron microscopy using antibodies against GFAP/VIP and GFAP/AVP, some portions of VIP-like-immunoreactive or AVP-like-immunoreactive neuronal somata and processes were found to be engulfed by GFAP-LI astroglial processes. The possible functional roles of the morphological interactions between astroglial and neuronal elements are discussed.     

Activation of protein kinase A induces neuronal differentiation of HiB5 hippocampal progenitor cells

Cyclic AMP-dependent protein kinase (PKA) signaling has been shown to be a critical regulator for neuronal or glial differentiation in the developing brain and several neuronal cell lines. However, the involvement of the PKA signaling cascade in hippocampal neuronal development and differentiation is poorly understood. The present study was performed to investigate whether activation of the PKA pathway directly regulates differentiation of hippocampal progenitor cell line, HiB5. Treatment of hippocampal HiB5 cells with 0.5 mM dibutyryl-cyclic AMP (dbcAMP) at 39 degrees C in N2 medium caused dramatic morphological changes including neurite outgrowth within 24 h and an inhibition of proliferation. During these processes, PKA activity as well as phosphorylation of the cAMP responsive element binding protein (CREB) were augmented. To characterize dbcAMP-induced differentiation of HiB5 cells, the expressions of several neuronal marker genes were investigated. After 24 h of dbcAMP treatment, the expression of NF-H and NF-M neuronal makers increased with a concomitant decrease in nestin (a marker for neural precursor cells) and GFAP an astrocyte marker expression, suggesting that HiB5 cells can develop a neuronal phenotype. Using the doxycycline-inducible, enhanced GFP-fused PKA catalytic subunit alpha (PKAcalpha-EGFP) overexpression system, we found that overexpressed PKAcalpha-EGFP induces neurite outgrowth in HiB5 cells. Taken together, these pharmacological and genetic transfection studies provide compelling evidence for the role of PKA activation on neuronal differentiation in HiB5 hippocampal progenitor cells.     

Polysialylated neural cell adhesion molecule expression by neurons and astrolial molecule in the rat dentate gyrus declines dramatically with increasing age

The expression of polysialylated neurons in the dentate gyrus of the hippocampal formation of young (postnatal day 40), mature (postnatal day 80) and aged (postnatal day 540) male Wistar rats has been investigated by immunohistochemical techniques employing a monoclonal antibody specific for neural cell adhesion molecule-linked α2,8 polysialic acid. A strong immunoreactivity was found on the cell bodies, dendrites and axons of granule-like neuronal cells at the border between the hilar region and the granule cell layer of the young rat. In the mature animal the number of immunoreactive neurons declined dramatically and were virtually absent in the aged group. Using an alternative fixation procedure, glial fibrillary acidic protein-positive and polysialylated astroglia processes were found in close proximity to the dendrites of the polysialylated granule-like cells. The number of astroglial processes traversing the granule cell layer showed a similar age-dependent decline to that observed with the polysialylated neurons. Glial fibrillary acidic protein-positive and polysialylated stellate astroglia were present throughout the hippocampal formation, but did not show the marked age-dependent decline observed with the astroglial processes in the granule cell layer.The neuronal dendrites and astroglial processes exhibited a strict numerical ratio in the young and mature animal and, in double immunofluorescence studies with anti-polysialic acid and anti-glial fibrillary acidic protein, the astroglial processes exhibited apparent points of cell and/or dendritic contact. These findings suggest that loss of polysialylated astroglial processes precedes the decline in polysialylated dentate neurons.     

Heterochronous maturation of regional brain astroglia: neuronal modulation of striatal glial cells differentiation ex vivo

Subcultured astroglial cells from striatum, cerebral cortex and ventral mesencephalon obtained from primary cultures of fetal (E14, E17 and E21) or postnatal (days 5-6) rats showed different regional, age-dependent morphological response (stellation) to cyclic AMP. While most of the cerebral cortex and ventral mesencephalic astroglial cell population was responsive at all ages tested, striatal cells at E14 and E17 were not. At age E21 striatal astroglia showed a significant shift toward a mature-like type of response to cyclic AMP. Postnatal striatal astroglia responded to cyclic AMP as the cortical and ventral mesencephalic astroglia did, with generalized stellation. Prenatal striatal astroglia was characterized immunocytochemically as A2B5+, fibronectin+, vimentin+, S-100+ and GFAP-. Failure of early prenatal (E14, E17) striatal astroglia to differentiate in response to cyclic AMP, was overcome by previous (5-7 days) co-culture with primary cell dissociates from postnatal-, but not from prenatal donors, from all brain regions tested including a non-target region for striatal cells, such as septum. This effect was duplicated when striatal astroglia was co-cultured with cell populations enriched in neurons through Percoll gradients. Only cell-to-cell contact co-cultures were able to induce a change in the studied response. Dead neuron-enriched populations obtained following various types of physical treatments were also able to change significantly striatal cell response toward cyclic AMP. Enriched astroglial populations from postnatal donors did not change striatal astroglial response toward cyclic AMP, except for ventral mesencephalic astroglia which induced a comparatively reduced but significant increase in striatal cell responsiveness. It is concluded that astroglial maturation and potential for phenotype expression during brain development proceeds with regional heterochrony. Also, that maturation of prenatal striatal astroglia responsiveness toward cyclic AMP is inducible by non-diffusible factors, probably of neuronal origin, expressed in live or dead primary cultures from various, homotopic and heterotopic, postnatal brain regions. It is further suggested that striatal afferents and/or mature local striatal neurons express membrane associated molecules that regulate responsiveness for phenotype expression of striatal glial cells, thus reinforcing the concept of a highly interactive, continuous neuron-glial developmental process that takes place during brain organization.     

Radial glial cells as neuronal precursors: a new perspective on the correlation of morphology and lineage restriction in the developing cerebral cortex of mice

Radial glia is a ubiquitous cell type in the developing central nervous system (CNS) of vertebrates, characterized by radial processes extending through the wall of the neural tube which serve as guiding cables for migrating neurons. Radial glial cells were considered as glial precursor cells due to their astroglial traits and later transformation into astrocytes in the mammalian CNS. Accordingly, a hypothetical morphologically distinct type of precursor was attributed the role of neurogenesis. Recent evidence obtained in vitro and in vivo, however, revealed that a large subset of radial glia generates neurons. We further demonstrate here that the progeny of radial glial cells does not differ from the progeny of precursors labeled from the ventricular surface, implying that there is no obvious relation between precursor morphology and neuron-glia lineage decisions in the developing cerebral cortex of mice. Moreover, we show that many radial glial cells seem to maintain their process during cell division and discuss the implications of this observation for the orientation of cell division. These new data are then related to radial glial cells in other non-mammalian vertebrates persisting into adulthood and suggest that radial glia are not only neurogenic during development, but also in adulthood.     

Organization of the embryonic and early postnatal murine hippocampus. I. Immunocytochemical characterization of neuronal populations in the subplate and marginal zone

Immunocytochemical techniques were used to characterize the neuronal populations in the hippocampal subplate and marginal zone from embryonic day 13 (E13) to postnatal day 5 (P5). Sections were processed for the visualization of microtubule-associated protein 2 (MAP2) and other antigens such as neurotransmitters, neuropeptides, calcium-binding proteins and a synaptic antigen (Mab SMI81). At E13–E14, only the ventricular zone and the primitive plexiform layer were recognized. Some cells in the later stratum displayed MAP2-, γ-aminobutyric acid (GABA)-and calretinin immunoreactivities. From E15 onwards, the hippocampal and dentate plates became visible. Neurons in the plexiform layers were immunoreactive at E15–E16, whereas the hippocampal and dentate plates showed immunostaining two or three days later. Between E15 and E19 the following populations were distinguished in the plexiform layers: the subventricular zone displayed small neurons that reacted with MAP2 and GABA antibodies; the subplate (prospective stratum oriens) was poorly populated by MAP2- and GABA-positive cells; the inner marginal zone (future stratum radiatum) was heavily populated by multipolar GABAergic cells; the outer marginal zone (stratum lacunosum-moleculare) displayed horizontal neurons that showed glutamate- and calretinin immunoreactivities, their morphology being reminiscent of neocortical Cajal-Retzius cells. Thus, each plexiform layer was populated by a characteristic neuronal population whose distribution did not overlap. Similar segregated neuronal populations were also found in the developing dentate gyrus. At perintal stages, small numbers of neurons in the plexiform layers began to express calbindin D-28K and neuropeptides. During early postnatal stages, neurons in the subplate and inner marginal zones were transformed into resident cells of the stratum oriens and radiatum, respectively. In contrast, calretinin-positive neurons in the stratum lacunosum-moleculare disappeared at postnatal stages. At E15–E19, SMI81-immunoreactive fibers were observed in the developing white matter, subplate and outer marginal zone, which suggests that these layers are sites of early synaptogenesis. At PO-P5, SMI81 immunoreactivity became homogeneously distributed within the hippocampal layers. The present results show that neurons in the hippocampal subplate and marginal zones have a more precocious morphological and neurochemical differentiation than the neurons residing in the principal cell layers. It is suggested that these early maturing neurons may have a role in the targeting of hippocampal afferents, as subplate cells do in the developing neocortex. © 1994 Wiley-Liss, Inc.     

Effect of growth factors and steroids on transglutaminase activity and expression in primary astroglial cell cultures

Type-2 transglutaminase (TG-2) is a multifunctional enzyme involved in the regulation of cell differentiation and survival that recently has been shown to play an emerging role in astrocytes, where it is involved in both proliferation and differentiation processes. Growth factors (GFs) such as EGF, basic fibroblast growth factor, insulin-like growth factor-I (IGF-I), and insulin (INS) are trophic and mitogenic peptides that participate in neuron-glia interactions and stimulate neuronal and astroglial proliferation and differentiation. Steroid hormones such as glucocorticoids and estrogens also play a pivotal role in neuronal and astroglial proliferation and differentiation and are key hormones in neurodegenerative and neuroprotective processes. We investigated the effects of the interaction of GFs with dexamethasone (DEX) or 17beta-estradiol (E(2)) on TG-2 activity and their expression in cultured astrocytes. We observed a significant increase in TG-2 activity and expression in astroglial cells treated for 24 hr with IGF-I, EGF, or INS. Priming of the cells with DEX or E(2), for 48 hr also led to an increase in TG-2 levels. When growth factors were present in the last 24 hr of the steroid treatment, a reduction in TG-2 expression and activity and a different subcellular TG-2 distribution were found. Our data indicate that steroid hormone-GF interaction may play an important role in astroglial function. The effect on TG-2 could be part of the regulation of intracellular pathways associated with the astrocyte response observed in physiological conditions and, possibly, also in neuropathological diseases.     

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.     

Development of beta-adrenergic receptors and their function in glia-neuron communication in cultured chick brain

The beta-receptors of intact neuronal and glial cells of chick embryonic brain were studied via the specific binding of the beta-antagonist [3H]dihydro-L-alprenolol ( [3H]DHA). Cells were cultivated in either highly homogeneous or mixed populations; the neuronal cells were also grown under the influence of glial conditioned medium (GCM) or 10(-11)-10(-10) M L-norepinephrine or L-isoproterenol. The beta-receptors of both neuronal and glial cells proved to be positively cooperative (n = 2.5) and of high affinity, with a Kdapp of 98 and 44 pM, respectively. The Kdapp value was influenced only slightly by the different culture conditions. The receptor concentration was relatively low in the homogeneous neuronal and glial cultures (Bmax = 6.4 and 3.3 fmol/10(6) cells, respectively). It increased by a factor of 2-3 if development of the neuron-glia contacts in the culture was possible (mixed cultures). GCM and beta-agonists elevated the number of beta-receptors of the neuronal cells approximately 4-fold, even in the absence of glial cells. This receptor-number change was preceded by a well observable morphological differentiation. Both the morphological and the beta-receptor effects of L-norepinephrine were antagonized by L-propranolol. The beta-receptor number increased about 2-fold during a 10-day in vitro development, even in neuron-glia mixed cultures.     

Development of microglia in the prenatal rat hippocampus

The distribution and appearance of microglial cell precursors in the prenatal hippocampus were examined in embryonic day 14 (E14) to E21 rats by nucleoside diphosphatase histochemistry. For comparison, the differentiation of astroglial cells was analyzed from E17 by vimentin and glial fibrillary acidic protein immunohistochemistry. Based on morphologic features, nucleoside diphosphatase-positive microglial cell precursors were classified as ameboid microglial cells and primitive ramified microglial cells. Ameboid microglia were present in the hippocampal primordium on E14. As the hippocampus developed, however, ameboid microglia gradually transformed into primitive ramified microglia, first recognized at E19. Microglial cell precursors, often related to nucleoside diphosphatase-labeled blood vessels, were particularly observed next to the pial surface on days E14 and E17 and in the highly vascularized area around the hippocampal fissure from E19. Within the brain parenchyma, the microglial cell precursors tended to be located within the differentiating cell and neuropil layers rather than in the germinative zones. The late developing dentate gyrus remained almost devoid of microglial cell precursors before birth. Vimentin-positive astroglial processes with radial orientation were observed throughout the hippocampal subregions from E17. In contrast, glial fibrillary acidic protein-positive, radial processes were barely discernible in the fimbria and the dentate gyrus before E19. The results are discussed in relation to the possible interactive role of microglial cells in central nervous tissue development and histogenesis. Regarding the origin of hippocampal microglial cell precursors, the present observations support the view that these cells may well originate from different mesodermal sources depending on time and localization. J. Comp. Neurol. 377:70-84, 1997. © 1997 Wiley-Liss, Inc.     

Influence of endogenous ciliary neurotrophic factor on neural differentiation of adult rat hippocampal progenitors

Ciliary neurotrophic factor is the only known neurotrophic factor that can promote differentiation of hippocampal neural progenitor cells to glial cells and neurons in adult rats. This process is similar to spontaneous differentiation. Therefore, ciliary neurotrophic factor may be involved in spontaneous differentiation of neural stem cells. To verify this hypothesis, the present study isolated neural progenitor cells from adult male rats and cultured them in vitro. Results showed that when neural progenitor cells were cultured in the absence of mitogen fibroblast growth factor-2 or epidermal growth factor, they underwent spontaneous differentiation into neurons and glial cells. Western blot and immunocytochemical staining showed that exogenous ciliary neurotrophic factor strongly induced adult hippocampal progenitor cells to differentiate into neurons and glial cells. Moreover, passage 4 adult hippocampal progenitor cells expressed high levels of endogenous ciliary neurotrophic factor, and a neutralizing antibody against ciliary neurotrophic factor prevented the spontaneous neuronal and glial differentiation of adult hippocampal progenitor cells. These results suggest that the spontaneous differentiation of adult hippocampal progenitor cells is mediated partially by endogenous ciliary neurotrophic factor.     

Neuron-associated astroglial cells express beta- and alpha 1-adrenergic receptors in vitro

In vivo, astroglial cells are closely interrelated with neurons. The present studies were undertaken to determine if certain astroglial properties are influenced when maintained in a heterogeneous cellular environment. Astrocytes and neurons were co-cultured from hippocampal tissue and the parameters examined included astroglial morphology and expression of beta- and alpha 1-adrenergic receptors (AR). Astroglial cells exhibit an extremely elongated morphology when growing in direct contact with neurons. Astroglia growing under the same culture conditions, but not in direct contact with neurons, exhibited a polygonal morphology. One hundred percent of the elongated astroglia expressed the beta-AR with an average density of 1320 binding sites/1000 microns 2. In contrast, only 40-50% of these elongated astroglia expressed alpha 1-ARs. Our results indicate that astroglia, maintained in the presence of neurons, continue to express beta- and alpha 1-ARs. These results suggest that under in vivo conditions, where astroglia normally exist in close contact with neurons, astrocytes may express surface receptors which enable them to sense neuronal activity and to selectively respond to such activity. The elongated astroglia described here may exhibit morphological features more reminiscent to that which exists in vivo.     

Changes in Glial Fibrillary Acidic Protein Immunoreactivity in Response to Experimental Hepatic Encephalopathy in the Rat Hippocampus

The response of astroglial cells in the hippocampus to long-term portacaval anastomosis (PCA), an experimental model of hepatic encephalopathy, was studied in adult male rats and compared with controls. Six months after PCA, the rat hippocampi were processed for glial fibrillary acidic protein (GFAP). GFAP-immunopositive astroglial profiles were observed in all hippocampal layers in PCA rats, but GFAP distribution differed in PCA rats and controls. In PCA rats, cell bodies and cell processes immunostained with GFAP were observed mainly in the CA1-CA3 layers in relation to pyramidal neurons, whereas the number and length of the astroglial processes decreased in the rest of the hippocampal regions. In addition, decreased GFAP immunoreactivity in the perivascular processes was observed in PCA rats compared with controls. These results show that long-term PCA elicited different responses in GFAP expression in different hippocampal regions, which might suggest a regional variation in glial sensitivity.     

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.