Region-specific regulation of preproenkephalin mRNA in cultured astrocytes.

Regulation of preproenkephalin (PPE) mRNA was examined in astrocytes cultured from several regions of the neonatal rat brain. Astrocytes from these regions expressed differing levels of PPE mRNA, with higher levels in astrocytes from the hypothalamus followed by frontal cortex and striatum. Further, PPE mRNA was regulated differently in hypothalamic than in striatal glia. Treatment of striatal astrocytes with the beta-adrenergic agonist, isoproterenol, or with agents which directly increased intracellular cAMP (forskolin or 8-bromo-cAMP) elevated levels of PPE mRNA. By contrast, none of these treatments altered levels of PPE mRNA in hypothalamic astrocytes despite increasing cAMP levels 60-fold. These observations indicate that there is striking regional heterogeneity in the expression and regulation of PPE mRNA by astrocytes, suggesting that proenkephalin or its derived peptides help to mediate region-specific brain functions.     

Morphological differentiation of cultured astrocytes is blocked by cadmium or cobalt

The morphological transformation of astrocytes in primary culture from flat to process bearing is induced by cyclic adenosine monophosphate (cAMP). Calcium currents in cultured astrocytes are also enhanced by cAMP. This correlation suggests the hypothesis that cAMP-induced calcium entry causes the morphological differentiation. The experiments reported here show that cadmium or cobalt, which block astrocyte calcium channels, also block the cAMP-induced morphological differentiation. These results support the hypothesized link between calcium entry and astrocyte differentiation.     

Effect of ATP on astrocyte stellation is switched from suppressive to stimulatory during development

Adenosine 5′-triphosphate (ATP) functions as a neurotransmitter or neuromodulator in the brain. To understand the role of ATP during brain development, we investigated the effects of ATP on morphology of cultured astrocytes obtained from the cerebral cortices of embryonic day 18 (E18) and postnatal day 2 (PN2) rats. In E18 astrocytes, ATP (10–1000 μM) alone did not affect astrocyte morphology, but significantly suppressed astrocyte stellation induced by the β-adrenoceptor agonist isoproterenol or the membrane-permeable cyclic AMP analog dibutyryl cyclic AMP. The suppressive effect of ATP in embryonic astrocytes was selectively mimicked by P_2U purinoceptor agonists. ATP had no effect on stellation induced by the protein kinase C (PKC) activator phorbol ester. It is probable that ATP, via P_2U purinoceptors, suppresses cyclic AMP-dependent regulatory mechanism for stellation in embryonic astrocytes. On the other hand, PN2 astrocytes differentiated into stellate cells in response to ATP. The ATP-stimulated stellation in PN2 astrocytes was mimicked by adenosine, and blocked by P₁ purinoceptor antagonists. It is probable that ATP is broken down into adenosine, which stimulates P₁ purinoceptors, inducing stellation in postnatal astrocytes. These findings suggest that the effect of ATP on astrocyte stellation is switched from suppressive (P_2U purinoceptor-mediated) to stimulatory (P₁ purinoceptor-mediated) during late embryonic to neonatal stages. ATP may be a critical factor that determines timing of astrocyte differentiation during development.     

Thyroid hormone and conditioned medium effects on astroglial cells from hypothyroid and normal rat brain: Factor secretion,cell differentiation,and proliferation

The effects of triiodothyronine (T₃)on cell morphology were examined in cerebral hemisphere and cerebellar astrocyte cultures obtained from normal and hypothyroid neonatal rats. T₃-treatment induced morphological changes in astrocytes from cerebral hemispheres. This morphological effect was produced earlier if astrocytes were treated with conditioned medium obtained from cerebral hemisphere astrocyte cultures previously exposed to 50 nM T₃. T₃ or conditioned medium-treatment produced faster morphological changes in hypothyroid rat cerebral hemisphere astrocyte monolayers. Cerebellar astrocytes from normal brain did not respond to thyroid hormone with morphological changes, but proliferated after T₃-treatment. However, hypothyroid cerebellar astrocyte cultures exhibited morphological changes, differently than normal cells. We verified that T₃ may induce astrocyte secretion of factor(s) that promotes morphological differentiation in cerebral hemisphere astroglial cultures and stimulates the proliferation of cerebellar astrocytes. Astrocytes obtained from hypothyroid animals were more sensitive to secreted factors than normal cells. These results emphasize the heterogeneity and the importance of glial cells to normal brain development and open new questions about thyroid hormone therapy in hypothyroidism. © 1995 Wiley-Liss, Inc.     

Uptake of [3H]serotonin and [3H]glutamate by primary astrocyte cultures. II. Differences in cultures prepared from different brain regions.

Regional astrocyte cultures were derived by dissecting six regions; brain stem, cerebellum, mesencephalon, basal ganglia plus diencephalon, cerebral cortex, and hippocampus, from 3 to 4-day-old neonatal rat brains. Glial fibrillary acidic protein (GFAP) immunocytochemistry was used to confirm the astrocyte composition of the cultures. The percentage of GFAP (+) cells between regions varied from 75% to 100%. Once confluent these cultures were incubated with radiolabeled serotonin or glutamate for uptake and autoradiographic studies. For the different brain regions Na(+)-dependent, [3H] L-glutamate, and fluoxetine-sensitive [3H] 5-HT uptake varied markedly. The relative order of uptake for [3H] 5-HT was MS (mesencephalon) greater than CC (cerebral cortex) greater than BG + DI (basal ganglia + diencephalon) greater than HP (hippocampus) greater than BS (brain stem) greater than CB (cerebellum). For [3H] L-glutamate the order was HP greater than CC greater than BG + DI greater than MS = BS greater than CB. For [3H] 5-HT this essentially corresponds to the reported order of binding in situ of the [3H] 5-HT-specific uptake ligand [3H] citalopram. For [3H] L-glutamate regional variation of the uptake for the different cultures corresponds to the regional uptake reported for different regions of rat brain. Double-label studies with GFAP and radiolabeled neurotransmitters were also used to study uptake into GFAP(+) astrocytes by autoradiography. Flat GFAP cells with or without processes comprised 65-98% of the cultures and represented most of the uptake. The percentage of all GFAP(+) cells that were positive for uptake of ARG varied from 50% to 90% and also showed differences in grain density both intra- and inter-regionally. These differences in transmitter uptake by GFAP(+) astrocytes in primary culture, which are dependent on the region of origin and correspond to regional differences in situ, suggest that such uptake in vitro may reflect uptake by astrocytes in vivo. Implied in this is that uptake by astrocytes represents a significant component of serotonin uptake in vivo.     

L-glutamate activates RhoA GTPase leading to suppression of astrocyte stellation

The actin cytoskeleton is known to support cellular morphological changes. Rho family small GTPases function as switching molecules to promote the convergence of both extracellular and intracellular signals in regulating cytoskeletal organization. Evidence indicates that L-glutamate suppresses morphological changes of astrocytes over a broad spectrum. To test the possibility that L-glutamate affects cytoskeletal reorganization, we investigated its effect on morphological changes induced by manganese exposure. L-glutamate concentration-dependently prevented and reversed manganese-induced astrocyte stellation and cytoskeletal disruption. The suppressive effect of L-glutamate on manganese-induced stellation was mediated by the activation of the glutamate transporter rather than ionotropic or metabotropic glutamate receptors. Pharmacological and biochemical approaches revealed the involvement of Ras homolog gene family, member A (RhoA) activation in L-glutamate-mediated suppression of manganese-induced stellation. The activation of RhoA by L-glutamate was partly through the up-regulation of guanine nucleotide exchange factor phosphorylation and was abrogated by competitive nonsubstrate inhibitors. Furthermore, the hyperphosphorylation of myosin light chain and cofilin through the activation of RhoA following L-glutamate treatment synergistically stabilized actin stress fibres. These results suggest that manganese-induced stellation is suppressed by a mechanism involving glutamate transporters. Our in vitro findings also strongly indicate that astrocyte morphological plasticity is under the control of RhoA and that manganese and L-glutamate regulate astrocyte morphology by modulating this switching molecule under culture conditions.     

Actin cytoskeleton remodeling governs aquaporin-4 localization in astrocytes

Aquaporin-4 (AQP4) is constitutively concentrated in the plasma membrane of the perivascular glial processes, and its expression is altered in certain pathological conditions associated with brain edema or altered glial migration. When astrocytes are grown in culture, they lose their characteristic star-like shape and AQP4 continuous plasma membrane localization observed in vivo. In this study, we differentiated primary astrocyte cultures with cAMP and lovastatin, both able to induce glial stellation through a reorganization of F-actin cytoskeleton, and obtained AQP4 selectively localized on the cell plasma membrane associated with an increase in the plasma membrane water transport level, but only cAMP induced an increase in AQP4 total protein expression. Phosphorylation experiments indicated that AQP4 in astrocytes is neither phosphorylated nor a substrate of PKA. Depolymerization of F-actin cytoskeleton performed by cytochalasin-D suggested that F-actin cytoskeleton plays a primary role for AQP4 plasma membrane localization and during cell adhesion. Finally, AQP4 knockdown does not compromise the ability of astrocytes to stellate in the presence of cAMP, indicating that astrocyte stellation is independent of AQP4.     

Dynamic reorganization of the astrocyte actin cytoskeleton elicited by cAMP and PACAP: a role for phosphatidylInositol 3-kinase inhibition

Cyclic AMP (cAMP)-raising agents induce astrocytes grown in vitro to adopt a stellate morphology resembling their in vivo appearance, through the depolymerization of actomyosin stress fibres. The signalling pathways responsible for cAMP-induced astrocyte stellation have thus far remained largely elusive. We showed in this study that the neurotrophic peptide PACAP (pituitary adenylate cyclase-activating polypeptide) mimicked the effect of forskolin, a direct activator of adenylate cyclase, on the actin cytoskeleton of primary rat astrocytes. The depolymerization of stress fibres induced by PACAP or forskolin was prevented by the expression of a constitutively active mutant of RhoA, but not by a protein kinase A (PKA) blocker, indicating that cAMP-raising agents act upstream of RhoA, in a PKA-independent manner. In addition, PACAP and forskolin inhibited basal Akt phosphorylation, and basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3-kinase (PI 3-K) activities. Incubation with a PI 3-K blocker resulted in the depolymerization of stress fibres. This effect was blocked by the expression of a constitutively active mutant of RhoA, indicating that PI 3-K inhibition acted upstream of RhoA. Together, these data demonstrate for the first time that depolymerization of stress fibres, and the resulting astrocyte stellation, induced by stimulation of cAMP production involves the inhibition of the PI 3-K-RhoA pathway.     

Adenosine stimulates stellation of cultured rat cortical astrocytes

We investigated the effect of adenosine on astrocyte morphology by using cell cultures prepared from the cerebral cortices of neonatal rats. Cultured rat cortical astrocytes exhibited flattened, polygonal morphology in the absence of stimulation, but differentiated into process-bearing stellate cells in response to adenosine (1–1000 μM). Adenosine-induced astrocyte stellation was abolished by treatment with microtubule inhibitors, colchicine and paclitaxel, indicating the involvement of cytoskeletal elements. The effect of adenosine was mimicked by other adenosine receptor agonists, and blocked by adenosine receptor antagonists and guanosine 5′-O-(2-thiodiphosphate), indicating that the effect of adenosine is mediated by G protein-coupled adenosine receptors. Although adenosine receptors are known to be linked to adenylate cyclase or phospholipase C, adenosine did not change intracellular cyclic AMP level nor intracellular Ca²⁺ concentration in astrocytes. Alternatively, adenosine-induced stellation was abolished by tyrosine phosphatase inhibitors, orthovanadate and phenylarsine oxide, suggesting that adenosine causes astrocyte stellation through tyrosine dephosphorylation. Adenosine may function as a factor regulating astrocyte differentiation.     

Brain astrocytes express region-specific surface glycoproteins in culture

Astrocytes derived from the mouse brain mesencephalon and striatum regulate neuronal morphogenesis in a region-specific manner in vitro. To begin defining molecular mechanisms that may underlie this functional heterogeneity, lectin probes were used to compare surface glycoproteins expressed by astrocytes from different brain regions. These experiments demonstrated marked differences in surface glycoproteins depending on the anatomic origin of the astrocytes. In particular, mesencephalic and cerebellar astrocytes express a fucosylated glycoprotein with an apparent molecular weight of 190 kD that is absent or rarely expressed by striatal or cortical astrocytes. These findings raise the possibility that carbohydrate diversity of astrocyte surface molecules may play a role in the heterogeneity of region-specific neuron-glial interactions.     

Astrocytes cultured from specific brain regions differ in their expression of adrenergic binding sites

We sought to characterize regional heterogeneity of astrocytes using adrenergic receptor sites as cellular markers. Primary cultures made from 6 regions of neonatal rat brain consisted almost exclusively of astrocytes. Membranes from astrocytes cultured 1-3 weeks were prepared for radioligand binding assays of beta- and alpha 2-adrenergic sites using the ligands [3H]dihydroalprenolol and [3H]p-aminoclonidine, respectively. Receptor expression was not affected by time in culture. Astrocytes from different brain regions varied up to 3-fold with respect to number but not affinity for both classes of adrenergic binding site with a rank order of cerebral cortex = superior colliculus greater than hippocampus = ventral midbrain greater than or equal to caudate nucleus greater than or equal to hypothalamus. Binding to beta- and alpha 2-adrenergic receptors was positively correlated across brain regions. Astrocytic receptor binding in each region did not correspond to total receptor levels assessed by quantitative autoradiography. We conclude that: (a) astrocytes are markedly heterogeneous between major brain regions with respect to expression of adrenergic binding sites; (b) regional variations in the density of adrenergic binding sites in brain reflect, in part, local specialization of astrocytes; and (c) a substantial proportion of the adrenergic binding sites in some brain regions may be on astrocytes.     

Differential migration of astrocytes grafted into the developing rat brain

Fetal and neonatal astrocytes migrate in specific patterns when transplanted into the adult rat host brain. However, it is unclear whether these astrocytes demonstrate the same degree of mobility during early brain development. In the present study, neonatal cortical, hippocampal, and hypothalamic astrocytes were collected from the brains of 1- to 3-day-old rats and placed in tissue culture. After 14 to 21 days, cultures enriched in astrocytes were harvested and labelled with either the fluorescent dye Fast Blue or fluorescein-labelled latex beads. They were then transplanted into the right frontal cerebrum of neonatal rats at 2,5,8, and 11 days postpartum. Seven days after transplantation, animals were sacrificed and their brains were fixed by immersion in aldehydes, sectioned on a cryostat, and examined with fluorescence microscopy. Transplanted astrocytes migrated along the corpus callosum, internal capsule, glial limitans, ventricular linings, and hippocampal structure. Labelled cells were also found in the contralateral hemisphere in day 2 brains. Migration in a radial fashion from the injection site toward the periphery was a particularly obvious pattern, and was most pronounced in these younger hosts. In days 5 and 8 rat brains, astrocyte migration became more restricted to the hemisphere of implantation. In 11-day-old host brains hemispheric restriction and other region-specific influences became manifest and specifically modulated migration. Radial migration was absent in the 11-day-old host group except for cells of cortical origin. The observed results demonstrate that neonatal cortical, hippocampal, and hypothalamic astrocytes transplanted into the neonatal cerebrum migrate in patterns that are more extensive than in the adult brain. This suggests that cellular migration in the neonatal brain is governed by factors that are less restrictive than those regulating migration in the adult brain. In particulur, our observations imply that radial glia may provide migratory substrates for transplanted astrocytes, and region-specific regulation of migration may begin around 11 days after birth. © 1993 Wiley-Liss, Inc.     

Astrocyte stellation in saline media lacking bicarbonate: possible relation to intracellular pH and tyrosine phosphorylation

Primary cultures of astrocytes exhibit a polygonal morphology, but on treatment with agents that increase cAMP they change to stellate cells. We found that astrocyte stellation also occurred on replacing the culture medium with saline buffered with HEPES. However, stellation did not occur when the medium was replaced with saline buffered with bicarbonate/CO(2) provided Ca(2+) was present. Since exposure of astrocytes to media lacking bicarbonate results in a decrease in intracellular pH (pH(i)) we sought evidence for an association between pH(i) and morphology. Astrocytic pH(i) was monitored for 60 min after transferring the cells to HEPES or bicarbonate-buffered saline. HEPES-induced stellation was associated with transient acidification which coincided with the morphological changes. Acidification was not observed in cells transferred to bicarbonate-saline. However when cytoplasmic acidification of cells in bicarbonate-saline was induced pharmacologically, rapid stellation occurred. Stellation induced by cAMP is reversed by activation of the RhoA pathway with lysophosphatidic acid (LPA). Here we found that LPA inhibited HEPES-induced stellation, but only with Ca(2+) present. Inhibition of stellation by LPA+Ca(2+) was associated with transient acidification followed by modest alkanization. A close association of tyrosine phosphorylation with stellation and pH(i) was observed. Thus incubation of astrocytes in HEPES-saline with orthovanadate to inhibit dephosphorylation abolished stellation and acidification; conversely incubation of cells in bicarbonate-saline with genistein to inhibit tyrosine kinases caused stellation and major acidification. Acidification may be one of several factors resulting in stellation, but it is not a necessary factor since stellation without acidification was observed in bicarbonate-saline lacking Ca(2+).     

Gabaergic signaling mediates the morphological organization of astrocytes in the adult rat forebrain

Previous studies have provided evidence that the morphological organization of immature astrocytes is influenced by the inhibitory neuronal transmitter gamma amino-butyric acid (GABA). The present study was designed to determine whether the occurrence of differential organization of mature astrocytes throughout various regions of the adult brain is related to differential GABAergic signaling. For this we first used Western blotting and high-performance liquid chromatography to quantify the levels of the astrocytic protein glial fibrillary acidic protein (GFAP) and GABA, respectively, within the same tissue punches taken from different forebrain regions of the adult rat, as well as immunocytochemistry for GFAP, GABA, or glutamate decarboxylase to visualize the morphological organization of astrocytes and of GABAergic axons in these regions. These data indicate that GFAP and GABA contents are correlated throughout the different forebrain regions analyzed, and that the regions containing the highest densities in GABAergic terminals are those that contain astrocytes exhibiting the highest degree of stellation. Secondly, we chronically increased GABAergic signaling in vivo by the systemic administration of an inhibitor of GABA transaminase or by the intracerebroventricular infusion of muscimol, a potent agonist of GABA(A) receptors. Our data show that in both cases, the GFAP content of the different forebrain regions is significantly augmented, in close association with a marked increase in the number of astrocytic processes and with their degree of branching. Taken together, these data strongly suggest that GABAergic signaling mediates the morphological organization of astrocytes and their expression of GFAP in the adult brain.     

ＥＧb７６１对星形胶质细胞抗凋亡作用的研究

研究无糖对星形胶质细胞的影响和ＥＧb７６１（Ｇinkgobiloba Extract 761)对细胞损伤后的有效保护浓度及其抗凋亡作用。实验选用２４h后分组：无糖组、ＥＧb７６１药物组和正常组,继续培养72h后,①测定LDH,药物组的LDH释放量低于无糖组和正常组,经样本均数间的两两比较q检验有显著性差异（P＜0.01)。②二倍体细胞比例测定,药物组的凋亡峰明显低于无糖组和正常组,三组间的t检验P＜0.01。③光镜下免疫学观察：体外无糖培养20d的星形胶质细胞的胸体比正常组细胞胞体明显增大,突起断裂,细胞胞浆内出现空泡状物,细胞增殖速度减慢。药物组细胞形态与正常组相比无明显变化。结果提示：一定浓度的ＥＧb７６１对无糖损伤的星形胶质细胞具有保护作用,对正常条件下体外培养的星形胶质细胞有抗凋亡作用。     

Bystin as a novel marker for reactive astrocytes in the adult rat brain following injury

Bystin has been identified as a protein which mediates cellular interactions between trophoblastic and endometrial epithelial cells by forming complexes with two partners, trophinin and tastin, during embryo implantation. However, the presence of bystin in the central nervous system has not been demonstrated. Here, we report the cloning of the full-length cDNA of the rat bystin gene from adult brain. Immunohistochemical and RT-PCR analysis showed that the levels of bystin expression were markedly up-regulated in the both 6-hydrodopamine-lesioned rat nigrostriatum and stab-lesioned cerebral cortex in adult rats. Double immunofluorescence staining revealed that most bystin-expressing glial cells were astrocytes (immature or mature). To determine the mechanisms for the up-regulation of bystin expression in glial cells, primary cultures of postnatal cortical astrocytes were employed. Western blot analysis showed that the expression of bystin was elevated by treatment with pro-inflammatory mediators lipopolysaccharide and interleukin-1 beta. Nerve growth factor known to be released after brain injury also induced bystin expression in the cultures. Exposure of astrocyte cultures to the differentiating agent forskolin resulted in up-regulation of bystin followed by a pronounced astrocytic stellation. The results suggest that the injury in the adult brain induces spatiotemporal up-regulation of bystin and it could be influenced, at least in part, by elevation of intracellular cAMP level. Bystin expressed by reactive astrocytes may be involved in their differentiation during the inflammatory processes following brain injury. The reappearance of bystin may also indicate that some reactive astrocytes have the capacity to recapitulate early developmental stages.     

Effects of diabetes mellitus on astrocyte GFAP and glutamate transporters in the CNS

Diabetes mellitus increases the risk of central nervous system (CNS) disorders such as stroke, seizures, dementia, and cognitive impairment. The cellular mechanisms responsible for the increased risk of these disorders are incompletely understood. Astrocytes are proving critical for normal CNS function, and alterations in their activity could contribute to diabetes-related disturbances in the brain. We examined the effects of streptozotocin (STZ)-induced diabetes in rats on the level of the astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP), number of astrocytes, and levels of the astrocyte glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate/aspartate transporter (GLAST), in the cerebral cortex, hippocampus, and cerebellum by Western blotting (WB) and immunohistochemistry (IH). Studies were carried out at 4 and 8 weeks of diabetes duration. Diabetes resulted in a significant decrease in GFAP protein levels (WB) in the hippocampus and cerebellum at 4 weeks and in the cerebral cortex, hippocampus and cerebellum by 8 weeks. Attenuated GFAP immunoreactivity (IH) was evident in the hippocampus, cerebellum and white matter regions such as the corpus callosum and external capsule at both 4 and 8 weeks of diabetes. Astrocyte cell counts of adjacent sections immunoreactive for S-100B were not different between control and diabetic animals. No significant differences were noted in astrocyte glutamate transporter levels in the cerebral cortex, hippocampus, or cerebellum at either time period (WB, IH). With the expanding list of astrocyte functions in the CNS, the role of astrocytes in diabetes-induced CNS disorders clearly warrants further investigation.     

Pituitary adenylate cyclase-activating polypeptide induces astrocyte differentiation of precursor cells from developing cerebral cortex

Ciliary neurotrophic factor and bone morphogenetic proteins induce astrocytogenesis in the developing rat brain by stimulating STAT- and Smad-dependent signaling, respectively. We previously found that stimulation of the cAMP-dependent signaling pathway also triggers differentiation of cerebral cortical precursor cells into astrocytes, providing an additional mechanism to promote astrocyte differentiation. In this study, we show that pituitary adenylate cyclase-activating polypeptide (PACAP), but not the related vasoactive intestinal peptide, induces astrocyte differentiation of cortical precursor cells, even after a transient exposure. Cortical precursors were found to express predominantly the short isoform of the PACAP-specific PAC1 receptor, which couples to adenylate cyclase. Consistent with this notion, we determined that exposure of cortical precursors to PACAP resulted in a dose-dependent increase in cAMP production. Pretreatment of cells with the cAMP antagonist Rp-cAMPS prevented astrocyte differentiation. Thus, PACAP acts as an extracellular signal to trigger cortical precursor cell differentiation into astrocytes via stimulation of intracellular cAMP production.     

Spinal cord astrocytes in vitro: Phenotypic diversity and sodium channel immunoreactivity

The expression of sodium channels in morphologically and antigenically distinct astrocytes derived from neonatal rat spinal cords was examined at various times in culture. During the course of this study [2-40 days in vitro (DIV)], nine morphologies of glial fibrillary acidic protein (GFAP)⁺ cells were distinguished: (1a) flat, fibroblast-like; (1b) elongated, with generally few, short processes; (1c) triangular soma with three short, stubby processes; (1d) bipolar with long, slender processes; (1e) bipolar with broad, flared processes; (1f) stellate with radially oriented slender processes extending from a small to moderate-sized soma; (1g) multiple short, stubby processes extending from a moderate-sized soma; (1h) flat, roundish shape with either a smooth edge (“pancake”-like) or numerous very short processes; and (1i) broad, elongated cell body with orthogonally oriented short, spike-like processes. Not all cell types were present at all times in culture. Each type of astrocyte displayed sodium channel immunoreactivity at some time in culture; however, different types of astrocytes exhibited different patterns, over time, of sodium channel staining. Sodium channel immunoreactivity in all astrocyte types was reduced to low levels by 14 DIV, and was not detectable at 40 DIV. Except for types 1b and 1e, A2B5 staining was present on all astrocyte morphologies at some time in culture, and was generally attenuated with longer times in vitro; in contrast to cultures derived from neonatal rat optic nerve, A2B5 staining does not distinguish unequivocally between the various classes of morphologically different astrocytes derived from spinal cord. O4 immunoreactivity was consistently observed only on bipolar, elongated, and process-bearing astrocytes, though not all process-bearing astrocytes were O4⁺. These results demonstrate that astrocytes derived from neonatal spinal cord are morphologically and antigenically heterogeneous. Moreover, while spinal cord astrocytes express sodium channels, these astrocytes exhibit a time-course of channel expression that is different from astrocytes derived from several other CNS regions where sodium channel staining is maintained even for extended times in culture, suggesting a regional modulation of astrocyte function.