Glial gene expression during aging in rat striatum and in long-term responses to 6-OHDA lesions

The male rat striatum was examined for age-related changes in mRNAs expressed in astrocytes and microglia in two rat genotypes that differ by 35% in mean and maximum life spans: F344 and the longer-lived F1 (BN x F344) hybrid. The findings extend the established age-related increases in GFAP (glial fibrillary acidic protein) to other glial mRNAs: two lipoprotein mRNAs that are predominantly expressed in striatal astrocytes, apoE (apolipoprotein E) and apoJ (apolipoprotein J, clusterin, CLI, or SGP-2), and two mRNAs expressed in striatal microglia, TGF-beta1 and complement C1qB. By Northern blot hybridization, both genotypes showed progressive increases of GFAP mRNA to > 2.5-fold by the lifespan. Although the rat strains differed 35% in life span, the slope of the GFAP mRNA regression on age did not differ. Relative to GFAP, the increases of apoE, apoJ, C1q, and TGF-beta1 mRNAs were smaller, < or = 1.5-fold. Because prior studies showed that acute damage to striatal afferents induced astrocyte gene expression increases resembling those that also occur during aging, we examined long-term effects of damage to substantia nigra neurons on striatal astrocyte changes during aging. Young F344 rats were given 6-OHDA lesions that cause striatal dopamine deficits and induce GFAP. When examined 15 months later at age 18 months, there was no effect during prior lesions on the age-related elevation of GFAP mRNA. We conclude that aging changes in striatal GFAP mRNAs do not interact with loss of dopaminergic output to the striatum from 6-OHDA lesions and may be independent of the relatively modest dopaminergic losses during normal aging.     

S100b counteracts effects of the neurotoxicant trimethyltin on astrocytes and microglia

Central nervous system degenerative diseases are often characterized by an early, strong reaction of astrocytes and microglia. Both these cell types can play a double role, protecting neurons against degeneration through the synthesis and secretion of trophic factors or inducing degeneration through the secretion of toxic molecules. Therefore, we studied the effects of S100B and trimethyltin (TMT) on human astrocytes and microglia with two glial models, primary cultures of human fetal astrocytes and a microglia cell line. After treatment with 10(-5) M TMT, astrocytes showed morphological alterations associated with an increase in glial fibrillary acidic protein (GFAP) expression and changes in GFAP filament organization. Administration of S100B before TMT treatment prevented TMT-induced changes in morphology and GFAP expression. A decrease in inducible nitric oxide synthase expression was observed in astrocytes treated with TMT, whereas the same treatment induced iNOS expression in microglia. In both cases, S100B prevented TMT-induced changes. Tumor necrosis factor-alpha mRNA expression in astrocytes was not modified by TMT treatment, whereas it was increased in microglia cells. S100B pretreatment blocked the TMT-induced increase in TNF-alpha expression in microglia. To trace the mechanisms involved in S100B activity, the effect of BAY 11-7082, an inhibitor of nuclear factor-kappaB (NF-kappaB) activation, and of PD98059, an inhibitor of MEK-ERK1/2, were investigated. Results showed that the protective effects of S100B against TMT toxicity in astrocytes depend on NF-kappaB, but not on ERK1/2 activation. These results might help in understanding the role played by glial cells in brain injury after exposure to chemical neurotoxicants and support the view that S100B may protect brain cells in case of injury. (c) 2005 Wiley-Liss, Inc.     

Immunolocalization of GLUT1 and GLUT3 glucose transporters in primary cultured neurons and glia

Immunofluorescence analysis was used to study the cellular localization of glucose transporters 1 and 3 (GLUT1 and GLUT3) in primary rat neuronal and glial cultures. In primary cultured cerebellar granule neurons and cortical neurons, GLUT3 was detected in a pattern consistent with a generalized cell surface distribution. GLUT3 distribution corresponded most closely with the neural cell adhesion molecule (NCAM), and showed overlapping but distinct distributions compared to synaptophysin, microtubule-associated protein 2 (MAP2), neurofilament protein, and growth-associated protein (GAP43). Culture of neurons in the presence of glia did not alter the cellular localization of GLUT3. GLUT1 was detectable in primary cerebellar granule neurons both at the cell surface and in the cytoplasm, and appeared decreased in neurons cocultured with glia. GLUT1, but not GLUT3, was detected in glial fibrillary acidic protein (GFAP)-positive astrocytes present in mixed neuronal-glial cultures derived from cerebellum and cerebral cortex, as well as in cortical astrocyte cultures. GLUT1, but not GLUT3, was also detected in microglia and oligodendrocytes present in these cultures. This study indicates a generalized cell surface expression of the glucose transporters expressed in neurons and glia, rather than selective targeting to different cellular domains or subcellular locations. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Aging and glial responses to lipopolysaccharide in vitro: greater induction of IL-1 and IL-6, but smaller induction of neurotoxicity

Glial activation during aging was analyzed in primary glia cultured from brain regions sampled across the life span. An initial study showed that phenotypes of activated astrocytes and microglia from aging rat cerebral cortex persisted in primary cultures (Neurobiol. Aging 19 (1998), 97). We extend these findings by examining effects of age on the activation of glial cultures from adult rat brain in response to lipopolysaccharide (LPS), an inflammatory stimulus. Mixed glia from F344 male rats, aged 3 and 24 months, cultured from cerebral cortex (Cx), hippocampus (Hc), and striatum (St), were assayed for cytokines implicated in Alzheimer's disease: IL-1alpha, IL-1beta, IL-6, and TNF-alpha. Regional differences across all age groups included consistently lower expression of these cytokines in glia derived from Cx than Hc and St. Aging increased basal IL-6 mRNA and secretion by >or=3-fold in glia from Cx and Hc. Aging also increased LPS-induced IL-1 and IL-6 in Hc more than in Cx, whereas no significant effects of age were seen in St-derived glial cytokines. TNF-alpha secretion did not differ by donor age (basal or LPS-induced). Nitric oxide production by microglia from Cx of aging brains showed a smaller induction in response to LPS, with proportionately less neurotoxicity. Thus, glial activation during aging shows regional selectivity in cytokine expression, with opposite effects of aging on the increased inducibility of IL-1 and IL-6 vs the decreased production of nitric oxide.     

Protein tyrosine kinase inhibitors suppress the production of nitric oxide in mixed glia, microglia-enriched or astrocyte-enriched cultures

Nitric oxide (NO) produced by glial cells has been implicated in the neuropathogenesis of various diseases. However, the signaling transduction pathway(s) for the production of NO in these cells is not well understood. To test whether protein tyrosine kinases (PTKs) are required for signaling events of NO production in glial cells, this study examined the effects of genistein and tyrphostin A25, two potent inhibitors of PTKs, on the production of NO in mouse primary mixed glia, microglia-enriched or astrocyte-enriched cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and interferon-γ (IFNγ). LPS induced a dose-dependent increase in NO production from the mixed glia cultures. The LPS-induced NO production was significantly enhanced by stimulating the cells with IFNγ. Genistein or tyrphostin A25 inhibited the production of NO in both LPS- and IFNγ/LPS-stimulated mixed glia cultures. The production of NO in the stimulated microglia-enriched or astrocyte-enriched cultures was also inhibited by tyrphostin A25. To verify the cellular sources of NO, immunocytochemical staining of inducible NO synthase (iNOS) was followed by staining with the microglia marker Mac-1 or the astrocyte marker glial fibrillary acid protein (GFAP) in microglia-enriched or astrocyte-enriched cultures. The expression of iNOS and the production of NO in microglia-enriched cultures were significantly higher than those in the identically stimulated astrocyte-enriched cultures. These results demonstrate that PTKs are involved in the signaling events of LPS-induced NO production in microglia and astrocytes, and that microglia are more responsive than astrocytes to stimuli which induce NO. These results may provide insights into therapeutic interventions in the pathway for NO production in the brain.     

Trehalose rescues glial cell dysfunction in striatal cultures from HD R6/1 mice at early postnatal development

The pathological hallmark of Huntington disease (HD) is the intracellular aggregation of mutant huntingtin (mHTT) in striatal neurons and glia associated with the selective loss of striatal medium-sized spiny neurons. Up to the present, the role of glia in HD is poorly understood and has been classically considered secondary to neuronal disorder. Trehalose is a disaccharide known to possess many pharmacological properties, acting as an antioxidant, a chemical chaperone, and an inducer of autophagy. In this study, we analyzed at an early postnatal development stage the abnormalities observed in striatal glial cell cultures of postnatal R6/1 mice (HD glia), under baseline and stressing conditions and the protective effects of trehalose.Our data demonstrate that glial HD alterations already occur at early stages of postnatal development. After 20 postnatal days in vitro, striatal HD glia cultures showed more reactive astrocytes with increased expression of glial fibrillary acidic protein (GFAP) but with less replication capacity, less A₂B₅⁺ glial progenitors and more microglia than wild-type (WT) cultures. HD glia had lower levels of intracellular glutathione (GSH) and was more susceptible to H₂O₂ and epoxomicin insults. The amount of expressed GDNF and secreted mature-BDNF by HD astrocytes were much lower than by WT astrocytes. In addition, HD glial cultures showed a deregulation of the major proteolytic systems, the ubiquitin–proteasomal system (UPS), and the autophagic pathway. This produces a defective protein quality control, indicated by the elevated levels of ubiquitination and p62 protein.Interestingly, we show that trehalose, through its capacity to induce autophagy, inhibited p62/SQSTM1 accumulation and facilitated the degradation of cytoplasmic aggregates from mHTT and α-synuclein proteins. Trehalose also reduced microglia activation and reversed the disrupted cytoskeleton of astrocytes accompanied with an increase in the replication capacity. In addition, trehalose up-regulated mature-BDNF neurotrophic factor expression and secretion, probably mediating cytoskeletal organization and helping in vesicular BDNF transport.Together, these findings indicate that glia suffers functional early changes in the disease process, changes that may contribute to HD neurodegeneration. Trehalose could be a very promising compound for treatment of HD and other diseases with abnormal protein aggregates. Furthermore our study identifies glial cells as a novel target for trehalose to induce neurotrophic and neuroprotective actions in HD.     

Distribution of glial fibrillary acidic protein (GFAP) in the cochlear nucleus of adult and aged rats

The age-related change in glial fibrillary acidic protein (GFAP) immunoreactivity was analyzed in young (3 months) and old (24 months) adult rat cochlear nuclei (CN). Quantitative analyses show a significant increase with age, in the number of GFAP positive astrocytes and processes in the old adult when compared with the young adult rat. There was also a differential distribution of GFAP immunoreactivity in the young adult CN where it predominates in the granular cell region, whereas in old rats, the GFAP immunoreactivity distribution was homogeneous in all parts of the nucleus. There was no change in the total number of neurons between these two stages in any part of the nucleus except for the antero-ventral CN, where a decrease in neuronal number was observed in the aged rats. The increase in GFAP immunoreactivity was related to an increase of both GFAP positive astrocyte number and processes. The increase of GFAP positive astrocytes may be due either to an alteration of auditory nerve fibers, changing the trophic interactions with post-synaptic cells, or to intrinsic alterations of CN neurons and local circuits reflecting aging of the CN.     

Quantitative proteomic profiling of the rat substantia nigra places glial fibrillary acidic protein at the hub of proteins dysregulated during aging: Implications for idiopathic Parkinson's disease

There is a strong correlation between aging and onset of idiopathic Parkinson's disease, but little is known about whether cellular changes occur during normal aging that may explain this association. Here, proteomic and bioinformatic analysis was conducted on the substantia nigra (SN) of rats at four stages of life to identify and quantify protein changes throughout aging. This analysis revealed that proteins associated with cell adhesion, protein aggregation and oxidation-reduction are dysregulated as early as middle age in rats. Glial fibrillary acidic protein (GFAP) was identified as a network hub connecting the greatest number of proteins altered during aging. Furthermore, the isoform of GFAP expressed in the SN varied throughout life. However, the expression levels of the rate-limiting enzyme for dopamine production, tyrosine hydroxylase (TH), were maintained even in the oldest animals, despite a reduction in the number of dopamine neurons in the SN pars compact(SNc) as aging progressed. This age-related increase in TH expression per neuron would likely to increase the vulnerability of neurons, since increased dopamine production would be an additional source of oxidative stress. This, in turn, would place a high demand on support systems from local astrocytes, which themselves show protein changes that could affect their functionality. Taken together, this study highlights key processes that are altered with age in the rat SN, each of which converges upon GFAP. These findings offer insight into the relationship between aging and increased challenges to neuronal viability, and indicate an important role for glial cells in the aging process.     

Inducible nitric oxide synthase expression is selectively induced in astrocytes isolated from adult human brain

Inducible nitric oxide synthase (iNOS) expression has been shown to be differentially regulated among different cell types and species. In cultures of primary human fetal glial cells, we have shown that astrocytes rather than microglia express iNOS. In the present study, we extended these findings to primary cultures of astrocytes and microglia derived from adult human brains. Mixed cultures of adult brain tissue were stimulated with IL-1β and IFNγ, a combination known to induce iNOS maximally in human fetal cells, and the expression of iNOS was determined by immunocytochemistry. Cell types were determined by morphology as well as immunocytochemistry for GFAP (astrocytes) and CD68 (microglia). The results showed that in cultures of adult human glia, iNOS was expressed following stimulation with cytokines, and the expression was restricted to astrocytes. Astrocyte iNOS immunoreactivity was detected both in the cytosol and in a discrete paranuclear region, a pattern noted in human fetal astrocytes. These results demonstrate that the ability to express iNOS is common to both fetal and adult human astrocytes.     

The effect of microglia on embryonic dopaminergic neuronal survival in vitro: diffusible signals from neurons and glia change microglia from neurotoxic to neuroprotective

When embryonic dopaminergic neurons are transplanted into the adult brain, approximately 95% die within a few days. To assess whether microglia activated during transplantation might be responsible for this rapid death, we examined the effect of microglia on rat embryonic dopaminergic neurons in vitro. Conditioned medium from 7-day-old microglia was found to decrease the number of dopamine neurons surviving in primary culture, but activation of the microglia with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or Zymosan A did not increase the toxicity of the conditioned medium. We next tested the effect of coculturing microglia and dopaminergic neurons by placing microglia in semipermeable well inserts over the neuronal cultures. The presence of microglia now increased dopaminergic neuronal survival, microglial activation again having no effect. To increase yet further the possible interactions between microglia and neurons, the mesencephalic cells and microglia were mixed together and placed as a tissue in three-dimensional culture, and here again the presence of microglia increased dopaminergic neuronal survival with no effect of activation. Contact of microglia with the mesencephalic cells therefore converted them from being toxic to dopaminergic neurons to promoting their survival. The change in microglial effect from toxic to protective was caused by soluble molecules secreted by cells in the neuronal cultures, as conditioned medium derived from microglia-neuronal cocultures also had a dopaminergic neuron survival effect, indicating that microglia in cocultures behave differently from microglia removed from neuronal and glial influence. Microglia cocultured with either neurons or astrocytes downregulated inducible nitric oxide synthase (iNOS), indicating a decrease in the production of nitric oxide and possibly other toxic molecules. These findings indicate that in their natural environment, microglia are likely to be beneficial for the survival of embryonic dopaminergic grafts.     

Astrocytes and Microglia Respond to Estrogen with Increased apoE mRNAin Vivoandin Vitro

This study examined the regulation of apolipoprotein E (apoE) by 17β-estradiol (E2) in brain glia, using rats with regular ovulatory cycles as anin vivomodel and cultured astrocytes and mixed glia asin vitromodels. Two brain regions were examined which had demonstrated transient synaptic remodeling during the estrous cycle. In the hippocampal CA1 region and the hypothalamic arcuate nucleus, apoE mRNA was elevated at proestrus when plasma E2 was high and synaptic density was increasing. Both astrocytes and microglia contributed to this increase in apoE mRNA.In vitro,E2 treatment had no effect on apoE mRNA levels in monotypic cultures of either astrocytes or microglia. In contrast, mixed glial cultures responded to E2 with increased apoE mRNA and protein, suggesting that heterotypic cellular interactions are important in the brain response to estrogens.In situhybridization in combination with cell-specific markers showed that E2 increased apoE mRNA levels in both astrocytes and microglia. These results, which are the first evidence of apoE mRNA localization to microgliain vivoand the control of apoE expression in brain cells by estrogens, are discussed in terms of the possible protective role of E2 in Alzheimer's disease and prior findings that emphasize the expression of apoE mRNA in astrocytes within the brain.     

STIMULATED MICROGLIA SUPERNATANTS INDUCED OVEREXPRESSION OF NEURONAL NITRIC OXIDE GENE IN ASTROCYTES

The glia- glia, glia-neuron interactions are very complex and have not yet been clearly understood. Microglia gain reactivity in almost every type of brain insult and by affecting astrocytes and neurons, they determine the fate of damaged brain. In the present study we aimed to see the effect of stimulated microglia on nNOS and iNOS expression of astrocytes. The microglia cultures were stimulated with zymosanA and astrocytes were treated with the medium of microglia for 48 h. The results revealed that the astrocytes treated with microglia medium expressed more nNOS and iNOS than the ones treated with normal medium.­­     

Nestin expression in hippocampal astrocytes after injury depends on the age of the hippocampus

Analysis of the expression of nestin in reactive astrocytes facilitates quantification of the extent of activation of astrocytes after injury in the mature CNS. We hypothesize that the capability of astrocytes for re-expressing nestin in response to CNS injury diminishes as a function of age. We quantified astrocytes positive for S-100beta protein, glial fibrillary acidic protein (GFAP) and nestin in the hippocampus of young adult, middle-aged, and aged Fischer 344 rats after an intracerebroventricular kainic acid (KA) administration. In all age groups, KA administration induced degeneration of CA3 pyramidal neurons, which led to a significant deafferentation in the CA1 region. The KA-induced neurodegeneration and deafferentation resulted in an increased population of astrocytes positive for S-100beta and glial fibrillary acidic protein (GFAP) in all age groups. Interestingly, these increases were highly comparable across the three age groups. However, in areas of both neurodegeneration and deafferentation, the overall numerical density of nestin-positive reactive astrocytes varied depending on the age at the time of injury with noticeably decreased numerical density in the injured middle-aged and aged hippocampus. In contrast, nestin-immunoreactive radial glia framework after lesion is not impaired with aging in the ependymal lining of the CA3 region.     

Dynamic cell type‐specific expression of Nrf2 after traumatic brain injury in mice

Nrf2 plays a pivotal role in antioxidant response and anti‐inflammation after traumatic brain injury (TBI), and its deletion aggravates TBI‐induced brain damage. Previous studies have demonstrated that Nrf2 is activated post TBI, but dynamic changes in expression and cell type‐specific characteristics remain unclear. In this study, the Feeney weight‐drop contusion model was conducted to mimic TBI, and the ipsilateral cerebral cortex was collected at 1, 3, 7 and 14 days post TBI (dpi). Nrf2 protein levels were observed by western blot. Cell type‐specific localization of Nrf2 after TBI was detected at different time intervals by double immunofluorescence staining. NeuN, GFAP, IBA1 and NG2 were used as cell type‐specific markers to neurons, astrocytes, microglia and NG2 glia, respectively. After TBI, Nrf2 protein levels peaked at 1 dpi. Robust transient Nrf2 accumulation was co‐localized with neurons, which was predominant at 1 dpi. Continuous weak Nrf2 expression was detected in activated astrocytes, and the number of double positive cells peaked at 7 dpi. Inducible widespread immunostaining of Nrf2 was observed in the nucleus of the microglia, and the number of Nrf2+ microglia peaked at 7 dpi. In addition, we also explored colocalization of Nrf2 in NG2 glia, in which the percentage of Nrf2+ in NG2 glia reached a climax at 3 dpi. This study reveals that the accumulation of endogenous Nrf2 might mediate different pathophysical roles in neurons and glias after TBI, the cell‐type specific and time‐dependent expression provide insights to explain the roles of Nrf2 in different neural cells.     

Estradiol promotes cell shape changes and glial fibrillary acidic protein redistribution in hypothalamic astrocytes in vitro: a neuronal-mediated effect.

We have previously shown that in hypothalamic mixed neuronal-glial cultures both astrocytic shape and distribution of glial fibrillary acidic protein (GFAP) are modified by estradiol. In the present study, we have investigated whether or not the presence of neurons is necessary for these hormonal effects. In mixed neuronal-glial hypothalamic cultures the proportion of process-bearing GFAP-immunoreactive cells was significantly increased after treatment for 30 min with 10(-12) M 17 beta estradiol. This effect was present for at least 1 day and was reverted by incubating the cells in estradiol-free medium. Estradiol incubation resulted in a progressive differentiation of GFAP-immunoreactive cells from a flattened epithelioid morphology to bipolar, radial, and stellate shapes. This effect was not observed in pure hypothalamic glial cultures. Furthermore, incubation of hypothalamic glial cells with medium conditioned by estradiol-treated mixed hypothalamic cultures did not affect the shape of GFAP-immunoreactive astrocytes. In contrast, addition of hypothalamic neurons, but not cerebellar neurons or fibroblasts, to established hypothalamic glial cultures affected the development of estradiol sensitivity in astrocytes. These results indicate that estradiol induction of shape changes in hypothalamic astrocytes is not only dependent on the presence of hypothalamic neurons, but that physical contact between astrocytes and neurons is necessary for the manifestation of the effect of this hormone.     

Neuronal loss in the rostral ventromedial medulla in a rat model of neuropathic pain

Cell death has been reported in the CNS in models of neuropathic pain (Sugimoto et al., 1990; Whiteside and Munglani, 2001; Scholz et al., 2005; Fuccio et al., 2009). In our present study, we examined the effects of spinal nerve ligation (SNL) on the number of neurons in the rostral ventromedial medulla (RVM), a brainstem region involved in modulation of nociception. In rats receiving SNL, we found that the number of RVM neurons decreased by 23% in the side ipsilateral to the surgery. The loss of RVM neurons was also associated with a bilateral increase in the number of glia as well as bilateral activation of both astrocytes and microglia. Administration of tauroursodeoxycholic acid (TUDCA), which reportedly inhibits apoptosis, significantly reduced the loss of neurons, the increase in glia, and the mechanical hypersensitivity induced by SNL. Among RVM neurons, we found that serotonergic (5-hydroxytryptamine, 5-HT) neurons decreased by 35% ipsilateral to SNL. Consistent with these findings, the density of 5-HT-immunoreactive varicosities in the superficial dorsal horn of the spinal cord was 15-30% lower, ipsilateral to SNL. To test the function of the remaining 5-HT neurons, we administered the 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT). Interestingly, after 5,7-DHT, mechanical withdrawal thresholds increased significantly. We conclude that nerve injury induces death of antinociceptive RVM neurons that can be reduced or abolished by TUDCA. We propose that the loss of RVM neurons shifts the balance of descending control from pain inhibition to pain facilitation.     

Expression of indoleamine 2,3-dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons

There is good evidence that the kynurenine pathway (KP) and one of its end products, quinolinic acid (QUIN) play a role in the pathogenesis of several major neurological diseases. While QUIN has been shown to be produced in neurotoxic concentrations by macrophages and microglia, the capacity of astrocytes and neurons to produce QUIN is controversial. Using interferon gamma (IFN-gamma)-stimulated primary cultures of human mixed brain cells, we assayed expression of the KP regulatory enzyme indoleamine 2,3-dioxygenase (IDO) and QUIN production by immunocytochemistry. Using IFN-gamma-stimulated purified cultures of neurons, astrocytes, microglia and macrophages, we studied IDO expression by RT-PCR and production of QUIN using mass spectrometry. We found that astrocytes, neurons, and microglia expressed IDO but only microglia were able to produce detectable amounts of QUIN. However, astrocytes and neurons had the ability to catabolize QUIN. This study also provides the first evidence of IDO expression and lack of production of QUIN in culture of primary human neurons.     

Two types of Na(+)-currents in cultured rat optic nerve astrocytes: changes with time in culture and with age of culture derivation.

Na+ channel expression was studied in cultures of rat optic nerve astrocytes using whole-cell voltage-clamp recordings. Astrocytes from postnatal day 7 rat optic nerve (RON) expressed two distinct types of Na+ currents, which had significantly different h infinity curves. Stellate, GFAP+/A2B5+ astrocytes showed currents with h infinity curve midpoints close to -65 mV, similar to Na+ currents in most neurons. In contrast, flat fibroblast-like GFAP+/A2B5- astrocytes showed Na+ currents with h infinity midpoints around -85 mV, almost 20 mV more hyperpolarized than in neurons or A2B5+ astrocytes. Interestingly, Na+ current expression was maintained in A2B5+ astrocytes but began to decrease in A2B5- astrocytes after 6 days in vitro (DIV) and fell to or below the level of detection (i.e., 1 pA/pF) at 12 DIV. Astrocytes cultured from neonatal rats (P0) are almost exclusively GFAP+/A2B5-. These cells did not display measurable Na+ currents when studied at 2 DIV; however, Na+ current was observed after 5 DIV in A2B5- astrocytes from these neonatal (P0) cultures. These findings were substantiated by immunocytochemical experiments using 7493, an antibody raised against purified rat brain Na+ channels; in P0-derived astrocyte cultures 7493 antibody staining was initially lacking (up to 3 DIV), but it was prominent in cultures after 5 DIV, suggesting that Na+ current expression in RON astrocytes occurs postnatally.