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PubMed2001

Induction of astrocyte differentiation by endothelial cells.

Here we have investigated the mechanisms that control astrocyte differentiation within the developing rat optic nerve. Astrocytes are normally generated by astrocyte precursor cells within the embryonic optic nerve. We show that there is a close temporal and spatial correlation between endothelial and astrocyte differentiation. We tested the potential role of endothelial cells in inducing astrocyte differentiation by developing an immunopanning method to highly purify endothelial cells from developing optic nerves. We show that the purified endothelial cells, but not other embryonic optic nerve cell types, strongly induce the differentiation of purified astrocyte precursor cells into astrocytes in vitro. Leukemia inhibitory factor (LIF) and LIF receptors have been implicated previously in astrocyte differentiation in vivo. We show that purified endothelial cells express LIF mRNA and that their ability to induce astrocyte differentiation is prevented by a neutralizing anti-LIF, but not anti-ciliary neurotrophic factor, antiserum. These findings demonstrate a role for endothelial cells in inducing astrocyte differentiation. The induction of astrocyte differentiation by endothelial cells makes sense phylogenetically, anatomically, and functionally, because astrocytes evolved concurrently with brain vasculature and ensheathe capillaries throughout the brain. The ability to purify and culture astrocytes and endothelial cells should provide an excellent model system for future studies of blood-brain barrier development.

Animals↗
PubMed2005

[Neuroprotective effect of exogenous vascular endothelial growth factor on anoxic rat spinal cord astrocyte and the underlying mechanism].

OBJECTIVE: To observe the effect of hypoxia on primary cultured rat spinal cord astrocyte and investigate the neuroprotective effect of exogenous vascular endothelial growth factor (VEGF) on anoxic astrocyte in vitro. METHODS: The rat embryonic spinal cord astrocytes were cultured and identified by cellular morphology and glial fibrillary acidic protein (GFAP). The astrocytes were treated with VEGF at different concentrations (10 ng/ml-100 ng/ml) as well as with VEGF receptor (Flk-1) inhibitor SU1498. Then the astrocytes were exposed to hypoxia for different time. The light microscope, MTT assay, TUNEL labeling and SABC immuno-histochemistry were used to measure and observe the changes of the astrocytes in cell morphology, growth, proliferation, GFAP identified that 98% of the purified 1-week cultured cells apoptosis, VEGF and Flk-1 expression. RESULTS: were astrocytes. 8-12 hours hypoxia obviously induced the activity of astrocyte. The cytobody became bigger, the cytoskeleton grew thicker. The activity of proliferation decreased but apoptosis index increased. The GFAP, VEGF and Flk-1 expression increased. The treatment with 50 ng/ml VEGF 20-24 hours before hypoxia apparently enhanced the activity, decreased the apoptosis index of the astrocytes, and hence improved the hypoxia-injuried astrocytes. Howerver, 700 ng/ml SU1498 obviously inhibited the neuroprotective effect of VEGF on anoxic astrocytes. CONCLUSION: Hypoxia induces reactive activity of astrocyte. The exogenous VEGF exerts neuroprotective effect on astrocytes via VEGF receptor-Flk-1.

Animals↗
PubMed1989

Astrocytes in Alzheimer's disease gray matter express alpha 1-antichymotrypsin mRNA.

The serine protease inhibitor alpha 1-antichymotrypsin (ACT) has been shown to be tightly associated with the amyloid found in plaque cores and blood vessels in the brains of patients with Alzheimer's disease (AD). Although the ACT found in plaques could be derived from the high levels of ACT in serum, previous Northern analysis revealed that ACT mRNA is produced locally in AD gray matter at much higher levels than in control gray matter. To determine which brain cells express ACT mRNA, we conducted in situ hybridization with 35S-labeled cRNA probes on hippocampal sections from four AD and three control cases. To identify astrocytes unequivocally, some of the hybridized sections were immunostained for glial fibrillary acidic protein, which is astrocyte-specific. Our results showed numerous astrocytes that were intensely labeled by the probe for ACT mRNA throughout the subicular gray matter of the AD cases. In contrast, astrocytes in control gray matter were rarely labeled by the probe for ACT mRNA. Examination of plaque cores in the AD subiculum showed that some astrocytes intensely labeled by the probe for ACT mRNA were closely associated with virtually every plaque core. Our results also showed many astrocytes in both AD and control white matter that were intensely labeled by the probe for ACT mRNA, and a small fraction of the astrocytes in a juvenile cerebellar astrocytoma that we examined were found to produce high levels of ACT mRNA. In every area in which astrocytes expressing ACT mRNA were found, astrocytes producing no detectable ACT message were also present. Our findings indicate that astrocytes produce the increased ACT mRNA in AD gray matter observed by Northern analysis, but they also show that ACT mRNA expression by astrocytes is not unique to AD. The presence of astrocytes expressing ACT mRNA near, and extending processes towards, plaque cores strongly suggests that some if not all of the ACT associated with amyloid plaque cores is produced by astrocytes surrounding the cores.

Alzheimer Disease↗
PubMed1999

CD95 (Fas/Apo-1) as a receptor governing astrocyte apoptotic or inflammatory responses: a key role in brain inflammation?

Astrocytes are a major cellular component of the brain that are capable of intense proliferation and metabolic activity during diverse inflammatory brain diseases (such as multiple sclerosis, Alzheimer's dementia, tumor, HIV encephalitis, or prion disease). In this biological process, called reactive gliosis, astrocyte apoptosis is frequently observed and could be an important mechanism of regulation. However, the factors responsible for apoptosis in human astrocytes are poorly defined. Here, we report that short term cultured astrocytes derived from different brain regions express significant levels of CD95 at their surface. Only late passage astrocytes are sensitive to CD95 ligation using either CD95 mAb or recombinant CD95 ligand. Blocking experiments using caspase inhibitors with different specificities (DEVD-CHO, z-VAD-fmk, and YVAD-cmk), an enzymatic activity assay, and immunoblotting show that CPP32/caspase-3 play a prominent role in CD95-induced astrocyte death. In contrast, early passage astrocytes are totally resistant to death, but a significant increase in astrocytic IL-8 secretion (p < 0.001, by Wilcoxon's test for paired samples) is observed after CD95 triggering. Production of IL-8 contributes to the resistance of astrocytes to CD95 ligation. Furthermore, in the presence of IFN-gamma, resistant astrocytes became sensitive to CD95-mediated death. These data suggest that microenvironmental factors can influence the consequences of CD95 ligation on astrocytes. Therefore, we propose that CD95 expressed by human astrocytes plays a pivotal role in the regulation of astrocyte life and death and may be a key factor in inflammatory processes in the brain, such as reactive gliosis.

Adjuvants, Immunologic↗
PubMed1996

Astrocyte survival and HSP70 heat shock protein induction following heat shock and acidosis.

Although severe acidosis is an important mediator of brain infarction, recent evidence suggests that mild acidosis may protect ischemic cells. The HSP70 heat shock protein is induced by acidosis in cultured cells and in ischemic brain and protects cells against many types of injury. Therefore, this study determined whether induction of heat shock proteins protects cultured astrocytes against acidosis. Brief exposure of cultured cortical astrocytes to acid (pH 5.2 for 40 min) or heat shock (45 degrees C for 40 min) markedly induced hsp70 mRNA and HSP70 protein. HSP70 protein was detected with the C92 monoclonal antibody (Welch and Suhan: J Cell Biol 103:2035, 1986), which has been shown to recognize the protein product of the full-length rat hsp70 cDNA (Longo et al: J Neurosci Res 36:325, 1993). Heat shock of the cultured cortical astrocytes completely protected the astrocytes from an otherwise lethal heat exposure 24 h later (45 degrees C for 4 h). In contrast, heat pretreatment sensitized the astrocytes to injury from acidosis 24 h later. Acid pretreatment, which markedly induced the HSP70 protein without producing astrocytic cell death, similarly sensitized the cells to injury from acidosis 24 h later (60% survival following pH 5.2 for 3 h versus 90% survival in controls; P < 0.0001). Surprisingly, heat shock pretreatment protected astrocytes against exposure to acid 48 h later (P < 0.05, 1.5-3 h), whereas acid pretreatment had no effect on astrocyte survival 48 h later. Since heat shock did not protect against acidosis at 24 h when HSP70 induction was maximal but did protect at 48 h when HSP70 was markedly diminished, the protective effect of heat shock at 48 h may be related to stress proteins present at 48 h. It is concluded that induction of HSP70 and other heat shock proteins by heat shock protects astrocytes against subsequent lethal heat shock. However, heat shock and acid treatment increase the vulnerability of astrocytes to acidosis 24 h later in spite of the induction of HSP70 heat shock proteins. The finding that heat shock protected astrocytes against acidosis 2 days later may suggest that delayed induction of stress proteins partially protects the astrocytes against damage produced by high concentrations of hydrogen ions.

Acidosis↗
PubMed1997

Leptomeningeal cells modulate the neurite growth promoting properties of astrocytes in vitro.

Leptomeningeal cells migrate into the lesion cavity after stab wounds to the adult mammalian central nervous system (CNS) and interact with astrocytes that form a new glia limitans. However, it is not known if leptomeningeal cells alter the ability of astrocytes near the lesion to support axon growth. In this study, we have used an in vitro approach to assess leptomeningeal cell-astrocyte interactions in a model that resembles the interactions of these cells in vivo. We cultured rat cortical astrocytes on top of monolayers of leptomeningeal cells or astrocytes. Differences in the morphology, neurite growth promoting properties, and expression of various extracellular matrix molecules and beta 1-integrin were assessed. Astrocytes acquired a long slender morphology when plated on leptomeningeal cells. Functionally, astrocytes cultured on top of leptomeningeal monolayers supported less neurite growth. Similar results were also obtained when astrocyte monolayers were treated with leptomeningeal cell-conditioned medium. Quantitative immunofluorescence labeling showed a reduction in cell surface bound laminin on astrocytes plated on leptomeningeal monolayers. Qualitative assessment of the immunofluorescence labeling showed an increase in matrix-like deposits of tenascin-C and chondroitin sulfate proteoglycan under similar culture conditions. This study provides the first direct evidence that leptomeningeal cells reduce the neurite growth promoting properties of astrocytes. These results suggest that interactions with leptomeningeal cells may 1) induce the formation of the slender astrocyte processes that form parallel to the lesion wall after penetrating injuries to the CNS; and 2) contribute along with other factors to alter astrocytes near the site of injury to a state that is less permissive for axon growth and regeneration.

Animals↗
PubMed1999

Mature hippocampal astrocytes exhibit functional metabotropic and ionotropic glutamate receptors in situ.

Astrocytes closely contact neurons where they respond to neuronally released glutamate in immature brain slices. In previous studies, neither metabotropic nor ionotropic glutamate receptor-mediated responses were detected by imaging Ca2+ in astrocytes from mature (P21-P42) animals, suggesting astrocyte glutamate receptors only contribute to hippocampus physiology during development. In contrast to Ca2+ imaging, published electrophysiological experiments suggest P30-P35 astrocytes have alpha-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. For this study, we imaged astrocytes in P31-P38 hippocampal slices to determine if metabotropic and ionotropic glutamate receptor activation elevates intracellular calcium in mature astrocytes. Drugs were perfused while [Ca2+]i was monitored (confocal imaging) in cells loaded with Calcium Green 1-AM. Imaged cells were subsequently identified as astrocytes by GFAP/S-100 immunostaining. Astrocytic Ca2+ increased after glutamate application in the presence of a glutamate uptake inhibitor. An agonist at group I/II metabotropic glutamate receptors, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), elicited Ca2+ increases as did group I agonist 3,5-dihydroxyphenylglycine (DHPG), suggesting that mature astrocytes respond to glutamate via metabotropic glutamate receptors. AMPA also elicited Ca2+ elevations that were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and occurred after treatment with omega-conotoxin MVIIC to block neurotransmitter release. These results demonstrate that astrocytes in mature hippocampus have functional ionotropic and metabotropic glutamate receptors that regulate astrocytic calcium levels. Glutamatergic regulation of astrocytic [Ca2+]i may be involved in synapse modeling, long-term potentiation, excitotoxicity and other events dependent on glutamatergic transmission in adult hippocampus.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2000

Inward rectifier K(+) channel Kir2.3 (IRK3) in reactive astrocytes from adult rat brain.

Astrocytic inward rectifying K(+) channels that participate in K(+) spatial buffering in the central nervous system have been extensively investigated, but specific gene products have not been fully identified. We studied primary cultured reactive astrocytes of stellate and polygonal morphology from adult rat brains, as well as stellate astrocytes from neonatal rat brains. Single-channel recordings of cell-attached patches revealed that polygonal reactive astrocytes expressed only one hyperpolarization-activated single-channel conductance of 11-15 pS whose open probability was independent of voltage, whereas stellate reactive and stellate neonatal astrocytes exhibited two conductances, 11-15 pS and 24-27 pS. All three subtypes of astrocytes exhibited a hyperpolarization-activated macroscopic inward K(+) current that was strongly rectifying and was abrogated by 1 mM intracellular Mg(2+) introduced during conventional but not perforated patch whole-cell recording. This Mg(2+)-sensitive current comprised the total inward rectifier current in polygonal reactive astrocytes, but only a fraction of the inward rectifier current in stellate reactive and stellate neonatal astrocytes. Because a strongly rectifying, inward rectifier K(+) channel with a single-channel conductance of 11-15 pS that is voltage independent is consistent with features of Kir2.3 (IRK3), we performed immunofluorescence experiments with anti-Kir2.3 and anti-glial fibrillary acidic protein antibodies. Both antibodies co-localized to all three subtypes of astrocytes in primary culture and to reactive astrocytes in situ within brain and gelatin sponge implants. Our data indicate that astrocytes of both polygonal and stellate morphology, from both adult and neonatal rat brain, express Kir2.3 both in vivo and in vitro. Constitutive expression of Kir2.3 regardless of cell morphology or age of origin of the source tissue suggests an important functional role for this channel in astrocytes.

Age Factors↗
PubMed2001

Correlation between neurological progression and astrocyte apoptosis in HIV-associated dementia.

The pathogenesis of HIV-associated dementia (HIVD) has been postulated to be due to the indirect effects of HIV infection, including the aberrant central nervous system production of cytokines and other neurotoxins. A correlation between the severity of dementia and production of neurotoxins in HIVD has been demonstrated. We have previously identified nonproductive HIV infection of astrocytes. Because astrocytes participate in the inactivation of neurotoxins, we hypothesize that HIV nonproductive infection of astrocytes may lead to an environment in which there is a significant level of astrocyte apoptosis and a consequent increase in the levels of neurotoxins and that this results in more rapidly progressing dementia. Postmortem brain tissue was examined from clinically well-characterized HIV-positive demented patients, HIV-positive nondemented patients, and HIV-seronegative nondemented control subjects. The HIVD group was further categorized into subjects with rapid and those with slow progression of dementia. Tissue was paraformaldehyde fixed and paraffin embedded, and 6-microm sections from the basal ganglia and mid-frontal gyrus were processed to detect apoptosis by in situ transferase dUTP nick end labeling. Astrocytes were co-localized using immunohistochemical techniques. In situ polymerase chain reaction (PCR) techniques were utilized to detect HIV DNA in astrocytes. The density of apoptotic astrocytes was significantly greater in the HIV-positive groups than in the HIV-negative group (p < 0.01). The HIVD rapid progressors had a significantly greater number of apoptotic astrocytes in the basal ganglia than did the HIVD slow progressors (p < 0.05). In addition, there were a greater number of HIV DNA-positive astrocytes, as demonstrated by in situ PCR, in the HIVD rapid progressors than in the slow progressor and HIV-nondemented groups. These data suggest that there is an increased rate of astrocyte loss in the subjects with rapidly progressive dementia, in association with an increased number of HIV DNA-positive astrocytes. The results emphasize the importance of understanding more completely the role of HIV infection of astrocytes in the neuropathogenesis of HIVD.

AIDS Dementia Complex↗
PubMed1989

Origin of retinal astrocytes in the rat: evidence of migration from the optic nerve.

To test recent ideas on the origin of retinal astrocytes, we have studied the spread of astrocytes in the developing retina of the albino rat. Astrocytes were identified with antibodies to their intermediate filaments, glial fibrillary acidic protein (GFAP). Astrocytes were first detected at E(embryonic day) 18, forming a corona of processes around the optic disc. Over subsequent days, astrocytes extended over the retina, covering approximately 35% of the retina at birth (typically E21-22) and reaching the edge of the retina by P(postnatal day)8. As they spread, astrocytes were closely associated with the developing vasculature, spreading ahead of patent vessels by a small but distinct margin. The most peripheral astrocytes assumed a bipolar morphology and extended processes towards the margin of the retina. Astrocytes nearer the optic disc showed the stellate shape characteristic of mature cells. The appearance of astrocytes at the optic disc at E18, 2 days after the appearance of type-1 astrocytes in the optic nerve (Miller et al.: Dev. Biol. 111:35-41, '85), suggests that retinal astrocytes may be type-1 astrocytes generated in the optic nerve. Watanabe and Raff (Nature 332:834-837, '88) have recently reported an independent study supporting the same conclusions.

Aging↗
PubMed1995

Heterogeneity of astrocytes in human optic nerve head.

Previous studies demonstrated regional differences in the synthesis of extracellular matrix by astrocytes during optic nerve head (ONH) maturation and in glaucomatous optic neuropathy, suggesting heterogeneity of astrocytes. To characterize different types of glial cells in human fetal and adult ONH, we used a variety of neural cell markers such as HNK-1/N-CAM, A2B5, galactocerebroside (GalC), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP). Cryostat or paraffin sections were prepared from fetal (16-25 weeks) and mature (8 months to 75 years old) ONH and processed for standard single/double immunocytochemistry. Two subpopulations of type 7 astrocytes were present in the mature prelaminar and laminar regions. Glial cells expressing only GFAP were identified as type 1A astrocytes at the edges of the cribriform plates. Cells forming the glial columns and lining the cribriform plates expressed both GFAP and HNK-1/N-CAM and were identified as type 1B astrocytes. In the myelinated nerve, type 1A astrocytes form the glial limiting membrane. Cells labeled with GFAP and A2B5 were identified as type 2 astrocytes, and GFAP-negative cells labeled with GalC, MBP, and HNK-1/N-CAM were identified as oligodendrocytes. In fetal ONH, all glial cells expressed HNK-1/N-CAM. In older fetal ONH, some glial cells also expressed GFAP. No type 2 astrocytes or oligodendrocytes were present in the fetal ONH. In conclusion, at least two subpopulations of type 1 astrocytes exist in human ONH: Type 1A astrocytes may serve as structural support for axons; type 1B astrocytes, which retain the developmental neural marker HNK-1/N-CAM, may have a more complex function by interfacing between blood vessels and other connective tissue surfaces. These findings demonstrate the heterogeneity of astrocytes in the human ONH and suggest differential regional responses to changes in their microenvironment.

Adult↗
PubMed2003

Effect of endothelin-1 on astrocytic protein content.

The astrocytic endothelin (ET) receptors, ET(A) and ET(B), modulate calcium signaling and the astrocytic gap junctional network. The nonselective ET receptor ligand ET-1 inhibits gap junction permeability, an effect that can be blocked by tolbutamide. This mechanism may play a role in pathophysiological conditions such as ischemic stroke, characterized by elevated tissue ET-1 levels and hypertrophic-appearing reactive astrocytes. Therefore, the effect of ET-1 on cellular protein content was investigated in confluent once-passaged rat astrocyte cultures under serum-free conditions, by the Lowry method. Gap junction permeability was determined by the dye transfer technique. ET-1 prevented the decrease in astrocytic protein content observed in controls. The effect of ET-1 on cellular protein content was most pronounced in cultures seeded at high density, but it was attenuated in ET(B)-deficient (sl/sl) astrocytes. This effect could be blocked by the nonselective ET antagonist LU 302872 (10 micro M), as well as by the protein synthesis inhibitor cycloheximide (10 micro M). This increase in astrocytic protein content was inhibited by the ATP-sensitive K(+) channel blocker tolbutamide, which also antagonized the ET-1-induced reduction of gap junction permeability and reversed the morphological changes observed in astrocytes upon ET-1 treatment. Cytosine arabinoside (10 micro M), a DNA synthesis blocker, inhibited the ET-1-induced BrdU uptake without affecting the ET-1-induced increase in astrocytic protein content. To conclude, ET-1 induces an increase in astrocytic protein content as well as changes in astrocyte morphology in vitro. This hypertrophic response involves uncoupling of the astrocytic gap junctional network and is not dependent on DNA synthesis.

Animals↗
PubMed1990

Electrical coupling, without dye coupling, between mammalian astrocytes and oligodendrocytes in cell culture.

Evidence of electrical and dye coupling between oligodendrocytes and astrocytes was sought in cultures of mouse spinal cord. Cell identity was verified using cell specific antigenic markers. In most experiments current was injected into oligodendrocytes while recording voltage in nearby astrocytes. Nine of 17 oligodendrocyte-astrocyte cell pairs showed weak electrical coupling; the average estimated coupling ratio was 0.03 +/- 0.06 (cf. 0.11 for oligodendrocyte-oligodendrocyte and 0.44 for astrocyte-astrocyte pairs; Kettenmann and Ransom: Glia, 1: 64-73, 1988). Application of 0.5 mM BaCl2 or 44.6 mM CsCl depolarized astrocytes and oligodendrocytes and was estimated to increase the coupling ratio between these cells 3-5-fold; these effects were rapid in onset and completely reversible. In 5 of 7 cases, oligodendrocyte-astrocyte pairs that appeared uncoupled in normal solution exhibited coupling during Ba++ or Cs+ exposure. The actions of these cations are believed to be mediated by blockade of glial K+ channels. Depolarization, per se, as induced by increasing [K+]o, did not increase coupling ratio. The fluorescent dye lucifer yellow (LY) was injected into 10 oligodendrocytes, 8 of which were electrically coupled to nearby astrocytes, and never passed into astrocytes in detectable quantities. Likewise, astrocytes injected with LY stained other astrocytes, but never oligodendrocytes. These findings document the presence of weak electrical coupling between astrocytes and oligodendrocytes, in the absence of dye coupling. Weak coupling of this sort could subserve metabolic interactions between these cells mediated by the passage of small but important molecules such as cyclic AMP, but would not allow strong electrical interactions. If such coupling among glial cells is widespread, it would constitute a "metabolic syncytium" that could serve to coordinate glial behavior.

Animals↗
PubMed1991

Retinal neurite growth on astrocytes is not modified by extracellular matrix, anti-L1 antibody, or oligodendrocytes.

Two factors that may influence the course of axonal regeneration in the central nervous system (CNS) are extracellular matrix (ECM) and cell surface molecules that may enhance or inhibit neurite outgrowth. Whereas cultured astrocytes have been reported to be a good substratum for neurite outgrowth, there is recent evidence that cultured oligodendrocytes are inhibitory. To test the influences of 1) ECM components, 2) the L1 adhesion molecule, and 3) the inhibitory potential of mature oligodendrocytes in the astrocytic environment, we have utilized a culture system in which neurites from embryonic rat retina grow vigorously on astrocyte monolayers. The major ECM components were assembled in neonatal rat cortical astrocyte-retina co-cultures only when the medium contained serum. In electron microscopic studies of serum containing cultures, retinal neurites were seen to be related to astrocyte surfaces but rarely were found in contact with ECM; in serum-free medium the association between neurites and astrocytes was similar. In addition, the growth of neurites was vigorous whether ECM was present or absent. Presence of antibodies against the cell surface adhesion molecule L1 did not inhibit retinal neurite elongation on glial fibrillary acidic protein-positive astrocytes. When oligodendrocytes from adult rat spinal cord were combined with the astrocytes, retinal neurites grew as well on the mixed glial population as on astrocytes alone. Immunostaining for galactocerebroside showed many oligodendrocyte processes to be aligned in the direction of neurite growth, suggesting association between the two cell types. This association was verified by electron microscopy. Furthermore, retinal explants extended neurites among myelin basic protein-positive oligodendrocytes cultured without astrocytes. Thus, the astrocyte surface is a strong promoter of neurite growth from embryonic rat retina. This growth did not depend upon either ECM or the L1 adhesion molecule. Because neurites grew on astrocytes in the presence of mature oligodendrocytes or among oligodendrocytes alone, we conclude that oligodendrocytes do not inhibit neurite growth under certain conditions.

Animals↗
PubMed1992

Expression of adrenergic receptors in individual astrocytes and motor neurons isolated from the adult rat brain.

Attempts to show the distribution of adrenergic receptors (ARs) in autoradiographs of a brainstem motor nucleus following elimination of motor neurons yielded the unexpected result of an increase in beta-AR density. This increase was related to the gliosis accompanying the motor neuron degeneration. To determine the cells on which the AR subtypes were located, we dissociated cells from various regions of the adult rat brain and subsequently identified astrocytes by glial fibrillary acidic protein (GFAP) immunofluorescence. Slides containing the astrocytes were prepared for autoradiography using the nonselective beta ligand 125I-iodocyanopindolol (125ICYP) or the alpha 1 ligand 125IBE 2254 (125I-HEAT). The addition of the selective beta 1 blocker betaxolol or the beta 2 blocker ICI 118.551 to the incubation medium to displace 125ICYP binding was used to determine the binding of beta-AR subtypes. The great majority (greater than 88%) of isolated astrocytes sampled from the trigeminal motor nucleus, cerebral cortex, striatum, and cerebellum showed beta-AR binding. Astrocytes from the first three regions had similar average densities of beta-ARs, whereas the density in cerebellar astrocytes was 2- to 3-fold greater. The beta 2-AR subtype was proportionally greater than the beta 1 subtype in each region. Reactive astrocytes isolated from the trigeminal motor nucleus after degeneration of motor neurons showed a beta-AR density nearly 2-fold greater than resting astrocytes from the same region, with the beta 1 subtype showing the greater proportional increase. There was no beta-AR binding on trigeminal motor neurons. Astrocytes also showed a significant level of alpha 1-AR binding. No differences in alpha 1-AR binding were found in normal astrocytes isolated from the different regions, nor was there an increase in reactive astrocytes. In contrast, trigeminal motor neurons had an alpha 1-AR density nearly 10 times greater than astrocytes. In terms of the NE modulation of synaptic responses in motor neurons, the distribution of ARs would permit NE to act indirectly through alpha 1 and beta receptors on astrocytes and directly through alpha 1 receptors on motor neurons.

Animals↗
PubMed1994

Cortical type 2 astrocytes are not dye coupled nor do they express the major gap junction genes found in the central nervous system.

The O-2A progenitor cell first described from the rat optic nerve is a bipotential precursor of oligodendrocytes and type 2 astrocytes. Each cell expresses specific markers that distinguish them as unique cell types. O-2A progenitors cultured in high serum preferentially differentiate into type 2 astrocytes and when exposed to defined medium or low serum develop along the oligodendrocyte lineage. We analyzed the gap junction gene expression of type 2 astrocytes to determine if they are coupled to form a syncytium, like their type 1 astrocyte counterparts. Dye coupling experiments demonstrated that cortical type 2 astrocytes are not coupled, while type 1 astrocytes in the same culture dish are highly coupled. Immunocytochemistry revealed the presence of Cx43 in type 1 astrocytes but we could not detect Cx26, 32, or 43 protein in type 2 astrocytes. In situ hybridization did not detect mRNA for any of the three connexin genes in type 2 astrocytes. These data demonstrate that type 2 astrocytes do not express the major gap junction genes found in the central nervous system. The precise function of type 2 astrocytes is not known but the lack of gap junction genes expression suggests that their functions are different from the spatial buffering capacity of type 1 astrocytes.

Animals↗
PubMed1995

Prolactin induced expression of interleukin-1 alpha, tumor necrosis factor-alpha, and transforming growth factor-alpha in cultured astrocytes.

Prolactin (PRL) is a potent mitogen in cultured astrocytes. Because one of the major effects of astrocyte proliferation is the expression of inflammatory cytokines, we examined the effect of PRL-induced mitogenesis on the expression of interleukin-1 (IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-alpha (TGF-alpha) in cultured astrocytes. Astrocytes were stimulated with PRL or growth hormone (GH), and the expression of cytokines was determined by immunohistochemistry and Western blot analysis. Following incubation of astrocytes with 1 nM PRL for 6 h, strong positive staining of IL-1 alpha and TNF-alpha, but not TGF-alpha, was found. No detectable staining for the above cytokines was found in vehicle, or GH treated astrocytes. When astrocytes were incubated in the presence of 1 nM PRL for 18 h, strong positive staining for IL-1 alpha and TGF-alpha was found. Immunocytochemical analysis of the expression of TNF-alpha and IL-1 alpha in PRL stimulated astrocytes suggested that the expression of IL-1 alpha preceded the expression of TNF-alpha. To confirm this observation, Western blot analyses were performed on extracts from astrocytes incubated with 1 nM PRL in unstimulated astrocytes, IL-1 alpha levels were not detectable. In astrocytes stimulated with 1 nM PRL, expression of IL-1 alpha was clearly detected after 1 h of incubation, and IL-1 alpha levels continued to increase during the course of the experiment (6 h). In contrast, in astrocytes stimulated with 1 nM PRL, an increase in the expression of TNF-alpha was first apparent after 2 h of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Expression of neuronal antigens by astrocytes derived from EGF-generated neuroprogenitor cells.

Previous studies have demonstrated that astrocytes reacting to CNS injury can express antigens normally associated with neurons. The origin of the reactive astrocytes, i.e., whether they are newly differentiated glial cells or preexisting astrocytes somehow triggered to express neuronal markers, remains difficult to determine using an in vivo model system. An in vitro model may prove more manageable. In the present study, primary brain cultures and EGF-generated neuroprogenitor cells were used to study the expression of neuronal antigens by established (primary) and nascent astrocytes, respectively. Astrocytes derived directly from dissociated mouse brains exhibited a flat morphology typical of type 1 astrocytes. These cells were nestin and GFAP positive and, in most cases, the antigens were colocalized. Primary astrocytes did not appear to express the putative neuronal markers GABA, Tau, or MAP2. Nascent astrocytes derived from EGF-generated progenitor cells showed a similar pattern of GFAP and nestin immunoreactivity. Contrary to primary astrocytes, many GFAP-intensive, stellate astrocytes exhibited Tau and MAP2. These cells also exhibited an intense nestin immunoreactivity. These data suggest that the reactive astrocytes expressing neuronal antigens in response to CNS trauma may be derived from neural progenitor cells rather than from previously differentiated astrocytes.

Animals↗