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

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

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.     

Thyroid hormone action: induction of morphological changes and protein secretion in astroglial cell cultures

The effects of triiodothyronine (T3) on cell morphology and protein secretion were examined in astrocytes cultured in a chemically defined medium devoid of other hormones and growth factors. The flat polygonal astrocytic cells treated with T3 (1-50 nM) and maintained in non-renewed medium cultures were progressively transformed into process-bearing cells. These changes were initially observed 3 days after the end of T3 treatment and accounted for more than 50% of the cells 7-8 days thereafter. The proteins secreted by the T3-stimulated cells were analyzed on SDS-PAGE after cell labeling for 4.5 h with [35S]methionine. The effect of T3 on protein secretion was dose-dependent. Half-maximal stimulation was reached with 0.2-0.5 nM hormone and the proteins of 46, 59, 67, 78, 85 and 140 kDa were over-secreted (greater than 300% of control). These results were only obtained when the cell medium was not renewed after T3 treatment.     

Morphologic heterogeneity of human adult astrocytes in culture: correlation with HLA-DR expression

Human adult astrocytes derived from brain surgical resections showed marked morphologic heterogeneity when cultured in vitro, ranging from a flat, fibroblast-like appearance to process-bearing cells with little soma cytoplasm. The majority of cells were intermediate in morphology, bearing a prominent cytoplasmic cell body with processes radiating from them. The morphologic heterogeneity was more extensive than that of adult rat astrocytes, and was not correlated with the extent of attendant gliosis in the surgical specimens, or the site of the surgical resection. None of the human astrocytes expressed A2B5, thus preventing their classification on basis of lineage into type 1 or type 2 astrocytes. However, functional differences appear to exist between subpopulations of human astrocytes, since the proportion of process-bearing human astrocytes that expressed HLA-DR in vitro was significantly greater than that found for flat astrocytes.     

Reversibility of ganglioside effects on astrocyte morphology

The B-subunit of cholera toxin (BCT) induces a morphological change in cultured rat cerebral astrocytes from flat (epithelioid) to stellate (process-bearing). This stellation is reversed by the gangliosides GM1 and GD1a at concentrations of 10 microM or higher. Upon changing to a ganglioside-free medium, the flat astrocytes reacquire the stellate morphology within 3 hr, indicating that the antistellation effect of gangliosides is reversible. The possibility that this reversibility was due to a loss of exogenously acquired gangliosides from the cell membrane can be ruled out since pretreatment with GM1, but not GD1a, which does not bind BCT, results in an increased responsiveness to BCT, which was identical whether measured immediately after withdrawal of the ganglioside or 3 hr later. Asialo-GM1, which neither binds BCT nor reverses BCT-induced stellation by itself, prevents the return to stellation after withdrawal of the gangliosides. These data suggest that while gangliosides remain associated with the cell, their effect on astrocytes can change from opposing to permitting the stellate morphology.     

Neuronal and non-neuronal catechol-O-methyltransferase in primary cultures of rat brain cells

Previous biochemical and histochemical studies have suggested that catechol-O-methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers. COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18-day-old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double-staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, flat cells displaying type-1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type-2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane-bound 28-kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.     

Astrocytic shape and glial fibrillary acidic protein immunoreactivity are modified by estradiol in primary rat hypothalamic cultures

Primary cultures from fetal rat hypothalamus (embryonic day 15-16) were grown for 9 days in a serum-free medium and then fixed and immunostained for glial fibrillary acidic protein (GFAP). The majority of the GFAP-immunoreactive astrocytes were flat, polygonal, without processes and showed a low intensity immunoreactivity which was restricted to the perinuclear region. Elongated, process-bearing astrocytes, with an intense immunoreactivity in the soma and processes, were also observed in a smaller proportion (30%). Addition of estradiol (10(-12) to 10(-8) M) to the culture medium 24 h before GFAP immunostaining resulted in an increased proportion (59-69%) of process-bearing, intense immunoreactive cells. This effect was blocked by tamoxifen (10(-8) M). The total number of GFAP-immunoreactive astrocytes was not modified by estrogen or tamoxifen. These results indicate that estradiol may modulate the cell shape and the distribution of GFAP in astrocytes in culture and suggest that astrocytes can be a target for sex steroids during development of the central nervous system.     

Differential modulation of the cholinergic activity of rat CNS neurons in culture.

Treatment of septal cultures prepared from 17-day-old embryos with two different antimitotic agents, cytosine arabinoside (ara C) and 5'-fluoro-2'-deoxyuridine (FUdR), caused a 2-fold increase in the level of choline acetyltransferase (CAT) activity and no change in the glutamic acid decarboxylase (GAD) activity. In these cultures, there was also a large decrease in the number of astrocytes as determined by immunofluorescence for glial fibrillary acidic protein (GFAP). Furthermore, when epidermal growth factor (EGF) was added to the septal cultures to increase the astrocyte population, the CAT activity decreased. Therefore, it would appear that the astrocytes are responsible for producing this down-regulation on cholinergic neurons. In order to determine whether all CNS cholinergic neurons can be inhibited in this manner, cultures were prepared from two other CNS regions that contain a high percentage of cholinergic neurons, i.e. the striatum and the ventral spinal cord. When these cultures were treated with the antimitotic agents, there was little modification of the CAT or GAD activities. These results suggest that the astrocytic microenvironment of the septal neurons exerts an inhibitory effect on the CAT activity either via a soluble factor or via cell-cell contact. Such studies are an important demonstration that non-neuronal cells may alter cholinergic properties during CNS development.     

Establishment of a permanent rat brain-derived glial cell line as a source of purified oligodendrocyte-type 2 astrocyte lineage cell populations

A permanent glial cell line (L3) has been established from mixed glial cultures obtained from neonatal rat forebrain by repetitive passaging and selection of the process-bearing cells growing on top of a flat cell monolayer. Continuous propagation of the process-bearing cells was supported by the flat cells, of presumed astroglial origin, which were present in negligible amounts following each passage but then grew and formed a basal, feeder layer. Throughout a culture period of over 2 years, the L3 cells have maintained a stable morphological and antigenic phenotype. In serum-containing culture medium, most of the process-bearing cells expressed at the same time features of immature oligodendrocytes (O4 positivity) and of astrocytes [glial fibrillary acidic protein (GFAP) positivity]. A smaller proportion of them was labeled by the monoclonal antibody LB1. LB1+ or O4+ cells were rarely GFAP-, and GFAP+ cells were rarely LB1- or O4-. GalC+ oligodendrocytes were seen only occasionally, but the proportion of these cells increased up to 30% upon culturing in chemically defined medium containing 0.5% fetal calf serum. The L3 process-bearing cells accumulated the neurotransmitter gamma-aminobutyric acid (GABA), expressed the proteoglycan chondroitin sulfate, and responded to the mitogenic action of platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). All these properties are characteristic of cells belonging to the O-2A (oligodendrocyte-type 2 astrocyte) cell lineage. The L3 flat cells were largely negative for the glial markers tested, but resembled type 1 astrocytes in their ability to support the growth of O-2A lineage cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of glutamate transporters in astrocytes: Evidence for a relationship between transporter expression and astrocytic phenotype

The astrocytic glutamate transporters, EAAT1 and EAAT2, remove released L-glutamate from the synaptic milieu thereby maintaining normal excitatory transmission. EAAT dysfunction during the excitotoxicity and oxidative stress of neurological insults may involve homoeostatic mechanisms associated with astrocytic function. We investigated aspects of EAAT function and expression in concert with astrocytic phenotype in primary cultures of cortical astrocytes and mixed cells of the spinal cord. In spinal cord mixed cultures, hydrogen peroxide (300 microM) reduced both EAAT activity and cellular viability to half of their basal values at 24 h post-treatment, but at 2 h EAAT activity was unaltered, while cellular viability was significantly decreased, suggestive of a mechanism for the maintenance of EAAT activity. Cytochemistry for MAP2, GFAP and propidium iodide revealed that neurons and astrocytes were damaged in a time-dependent manner. A change in astrocyte morphology was observed, with astrocyte cell bodies becoming larger and processes becoming more stellate and often shorter in length. EAAT1 immunoreactivity was reduced at 24 h post-treatment and a re-distribution of the protein was noted after 2 h treatment. In pure astrocytes, lipopolysaccharide (1 microg/ml, 3 d) increased [3H]D-aspartate uptake by 90%, as well EAAT1 immunoreactivity and astrocyte stellation, as shown by immunofluorescent labelling for GFAP. In both culture systems, prominent changes were noted in EAAT function and localization in conjunction with altered astrocytic phenotype. Our findings are indicative of a relationship between astrocytic phenotype and the level of EAAT activity that may be a vital component of astrocytic homeostatic responses in brain injury.     

Astroglia growth retardation and increased microglia proliferation by lithium and ornithine decarboxylase inhibitor in rat cerebellar cultures: Cytotoxicity by combined lithium and polyamine inhibition

Lithium, the most prevalent treatment for manic-depressive illness, might have a neuroprotective effect after brain injury. In culture, lithium can exert neurotoxic effects associated with reduction in polyamine synthesis but neuroprotective effects as cultured neurons mature. Cumulative evidence suggests that lithium may exert some of its effects on neurons indirectly, by initially acting on glial cells. We used rat cerebellar cultures to ascertain the effects of lithium on ornithine decarboxylase (ODC) activity, the enzyme catalyzing the first step in polyamine synthesis, and to compare effects of lithium with those of the ODC inhibitor alpha-difluoromethylornithine (DFMO) on neuron survival and glial growth. Switching cultures from high (25 mM) to low (5 mM) KCl concentrations served as the traumatic neuronal insult. The results indicate the following. 1) Whereas high depolarizing KCl concentration enhances neuron survival, it inhibits astroglial growth. 2) Lithium (LiCl; 1-5 mM) enhances neuronal survival but inhibits astroglial growth. 3) Lithium treatment leads to reduced ODC activity. 4) DFMO enhances neuron survival but inhibits astroglial growth. 5) Lithium and DFMO lead to transformation of astroglia from epithelioid (flat) to process-bearing morphology and to increased numbers of microglia. 6) Combined lithium plus DFMO treatment is cytolethal to both neurons and glia in culture. In conclusion, lithium treatment results in growth retardation and altered cell morphology of cultured astroglia and increased microglia proliferation, and these effects may be associated with inhibition of polyamine synthesis. This implies that direct effects on astrocytes and microglia may contribute to the effects of lithium on neurons.     

Autoradiographic quantitation of beta-adrenergic receptors on neural cells in primary cultures. II. Comparison of receptors on various types of immunocytochemically identified cells

We have developed a microcomputer-based video method to quantify neurotransmitter receptors on single, immunocytochemically labeled cultured cells. This method has been applied to determine whether beta-adrenergic receptors are more numerous on neurons, astroglia, oligodendroglia or fibroblasts in primary neural cell cultures, and to assess the heterogeneity of receptor expression within a single cell type. Dissociated cells from perinatal rat cerebral cortex were grown in very sparse cultures on polylysine-coated glass slides. The cultured cells were fixed and permeated, then stained with fluorescently labeled immunocytochemical markers for astroglia (glial fibrillary acidic protein), fibroblasts (fibronectin), oligodendroglia (galactocerebroside) or neurons (A2B5). beta-Adrenergic receptors were labeled with [125I]pindolol or [125I]cyanopindolol, and dry-mount autoradiography was carried out on the fixed cells. Cells were identified according to their morphology and cell-type specific staining, then autoradiographic grains associated with the defined cells were visualized by reflected polarized light microscopy and counted with a microcomputer-based video digitizing system. Using this technique, we have determined that fibroblasts have less than 15% of the number of beta-adrenergic receptors expressed by polygonal astroglia, whereas oligodendroglia and neurons had no detectable binding of 125I-labelled ligands. This suggests that in these mixed neural cell cultures, the great majority of beta-adrenergic receptors are associated with astroglia. Furthermore, we determined that process-bearing astroglia have less than 5% of the number of beta-adrenergic receptors expressed by polygonal astroglia. Since process-bearing astroglia are thought to be derived from polygonal astroglia, these results suggest that the beta-adrenergic receptor is lost from this population of astroglia during development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuron-glial interactions involved in the regulation of glutamine synthetase

Cocultures of rat cortical astrocytes with cerebellar granule cell neurons, but not a variety of other cell types tested, resulted in an induction of glutamine synthetase (GS) mRNA over the basal levels expressed in pure astrocyte cultures. This induction involved both contact- and noncontact-mediated events and may be a result of astroglial differentiation promoted by interactions with the primary neurons. Astrocytes grown in the presence of the granule neurons (but not the other cell types tested) exhibited a more complex, process-bearing morphology typical of more differentiated cells. In addition, glial cell proliferation was inhibited not only by the presence of live granule cells, but also by fixed neurons and neuronal membranes. Under the same experimental conditions, GS mRNA was increased (two- to threefold) compared with the expression observed in pure astrocyte cultures. Because of the role of GS in glutamate metabolism and the influence of the glutamatergic granule neurons on glial GS mRNA levels, the effect of exogenous glutamate was examined. The addition of 100 microM glutamate to the culture medium resulted in an increase in GS mRNA in the astrocyte cultures similar to that observed in the cocultures, where the addition of glutamate did not further increase GS mRNA levels. These results provide further evidence for the importance of neuron-glial interactions in the regulation of glial gene expression.     

Development of septal cholinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth, and expression of transmitter-specific enzymes

To characterize the role of NGF in the development of forebrain cholinergic neurons, we established primary cell culture systems to grow these cells under controlled in vitro conditions. Cultures of dissociated cells were prepared from the septal area of fetal (E17) rats, which contained part of the group of basal forebrain cholinergic neurons. Cultures were treated either with NGF (100 ng/ml) or with an antiserum against NGF (1:500 dilution). To assess the influence of non-neuronal cells, 2 types of high-density cultures were prepared: mixed neuronal-glial cultures and pure neuronal cultures. Cholinergic neurons were identified using choline acetyltransferase (ChAT) immunocytochemistry and AChE cytochemistry. Receptors for NGF (NGF-R) were located immunocytochemically using monoclonal antibodies against rat NGF-R. We report that, first, NGF-R are exclusively localized on cholinergic neurons in septal cultures. All neurons labeled with antibodies against NGF-R also contained AChE. Twenty-one percent of all AChE-positive neurons were not stained in NGF-R immunocytochemistry (AChE has earlier been shown to be colocalized with ChAT in septal cultures). Second, NGF treatment increases and anti-NGF treatment reduces the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF promotes survival of septal cholinergic neurons in these cultures. In cultures of high plating density, NGF increased the number of NGF-R and ChAT-positive neurons without affecting the number of AChE-positive neurons in these cultures. These results suggest that exogenous NGF is not required for survival of cholinergic neurons in high-density cultures but stimulates the expression of ChAT and NGF-R. Third, NGF stimulates fiber growth of septal cholinergic neurons, as assessed by computerized image analysis of AChE-positive neurons. Fourth, NGF specifically increases ChAT and AChE activities in septal cultures. These NGF-mediated increases in enzyme activities are more pronounced when neurons are grown together with glial cells. In pure neuronal cultures, NGF increased ChAT and AChE activities by 101 and 16%, and in mixed neuronal-glial cultures by 318 and 87%, respectively. Anti-NGF blocked the effects of NGF but failed to reduce ChAT and AChE activities below control levels in cultures of high plating density. Fifth, astrocytes attenuate the expression of ChAT and AChE by septal neurons in the absence of NGF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of astrocytic morphology, proliferation, marker profile and response to neurons in wild-type and weaver mutant mouse cerebella in culture

In serum-free monolayer cultures of early postnatal weaver (wv/wv) cerebellum granule neurons show decreased attachment, survival and neurite outgrowth when compared to wild-type (+/+) littermate cultures. wv/wv Astrocytes display a more epithelioid morphology and altered proliferation. However, both morphology and proliferation of wv/wv astrocytes were reversed to a normal phenotype by addition of purified small neurons from early postnatal cerebella from +/+ animals. Attachment of +/+ neurons to wv/wv astrocytes was not significantly different from that of +/+ astrocytes and antigenic marker profiles of wv/wv and +/+ astrocytes differed only slightly. Attempts failed to revert the abnormal wv/wv phenotype in neurons by addition of gangliosides, triiodothyronine T3, prostaglandin A2, medium containing 1% horse serum, conditioned medium from +/+ cerebellar cultures, or by cocultivation with +/+ astrocytes. We would like to suggest that the primary defect of the wv/wv mutation is predominantly an abnormality in granule cell neurons, but not of the vast majority of astrocytes.     

Neutral glycolipid and ganglioside composition of type-1 and type-2 astrocytes from rat cerebral hemisphere.

We reported previously that the major gangliosides in primary mixed-type astrocyte cultures are GM3 and GD3. To obtain more information regarding the exact distribution of glycosphingolipids in different types of astrocytes, we established a line of type-1 astrocytes that are characterized by a Ran-2 positive, broad flat morphology, and by the absence of binding to A2B5 antibodies. We also purified O-2A progenitor cells by immunopanning and cultured them in the presence of 10% newborn calf serum. They differentiated into type-2 astrocytes that were identified by immunostaining for each of GD3, A2B5, and GFAP. Using these cell cultures, we demonstrate that the major gangliosides were GM3 in type-1 astrocytes and GM3 and GD3 in type-2 astrocytes. In addition, a set of neutral glycolipids was identified based on the HP-TLC migration properties of CMH, CDH, CTH, and Glob, but the component distribution of these glycolipids is related to that of glycolipids of astrocytes. A marked increase in the expression of CTH and Glob was shown in type-2 astrocytes. The amount of neutral glycolipid-sugar was higher in the type-2 astrocytes than in the type-1 astrocytes. These results suggest that the increase in the total glycosphingolipid content and the change in the neutral glycolipid composition produced by type-2 astrocytes may be related to their biological functions and the cellular compositions.     

Neuroimmunology of gangliosides in human neurons and glial cells in culture

Gangliosides (sialic-acid-bearing glycolipids) have received attention in recent years because of their role in cell recognition phenomena, synaptic transmission, memory generation, and nerve regeneration in the fields of neurosciences. It is suggested that each brain region or each neural cell type may contain a specific and characteristic set of gangliosides. We have investigated the immunocytochemical localization of several classes of gangliosides that include GM1, GM4, GD3, and GQ gangliosides on the cell surface of various cell types found in human neural cell cultures with antibodies specific for these gangliosides. Cell cultures were obtained from adult human brains and fetal human dorsal root ganglia and spinal cord and cultured in vitro for the period up to 6 months and utilized for the ganglioside immunocytochemistry. It was demonstrated that GM1 ganglioside was present in all galactocerebroside-positive oligodendrocytes and most of glial fibrillary acid protein (GFAP)-positive astrocytes (80%), most of neurofilament-positive neurons (80%), 50-70% of Schwann cells, and 5-10% of fibronectin-positive fibroblasts; GM4 ganglioside could be detected in all oligodendrocytes, 80% of astrocytes, and 50% of Schwann cells, while no staining was found in neurons or fibroblasts; GD3 ganglioside was present in all oligodendrocytes and 5-10% of astrocytes but not in neurons, Schwann cells, or fibroblasts; and all of fetal CNS neurons and approximately 80-90% of fetal dorsal root ganglia (DRG) neurons and a small percentage of astrocytes (10-20% in fetal and less than 1% in adult astrocytes) was labeled by A2B5 antibody which is specific for GQ ganglioside, while this antibody did not stain cell surface of oligodendrocytes, Schwann cells, or fibroblasts. Three classes of gangliosides, GM1, GM4, and GD3 were found to be definite components of fetal and adult human oligodendroglial plasma membrane, while GM1 and GM4 gangliosides were detected on the surface of most astrocytes. Only a minor population of astrocytes from both fetal and adult human CNS contained GD3 and GQ gangliosides. Two classes of gangliosides, GM1 and GQ, were detected on the surface of fetal human neurons. More than half of fetal Schwann cells reacted to GM1 and GM4 antibodies but did not to GD3 or GQ antibodies. We recognized the presence of a specific and characteristic set of gangliosides on the cell surface of different human neural cell types and these findings should facilitate further investigation of the precise biological activity of these gangliosides.     

Beta-amyloid-induced reactive astrocytes display altered ability to support dendrite and axon growth from mouse cerebral cortical neurons in vitro

OBJECTIVES: The presence of beta-amyloid (betaA) deposition, induction of reactive gliosis and dystrophic neurites, is a characteristic feature of neuritic plaques in Alzheimer's disease. In vitro, betaA-exposed astrocytes become reactive, similar to astrocytes in contact with betaA plaques in vivo. How betaA-exposed reactive astrocytes support neuron process growth, however, is not well defined. Therefore, we used neuron/astrocyte co-cultures in which astrocytes had been grown on betaA, to assess whether process growth was altered. METHODS: Purified rat cortical astrocytes were plated on the betaA peptide's neurotoxic fragment (25-35), the scrambled (35-25) peptide, or poly-D-lysine alone and grown to confluency before mouse cortical neurons were seeded at low density onto the astrocyte monolayer. Cell survival was assessed using trypan blue, lactate dehydrogenase release and propidium iodide. Process growth was analyzed using specific antibodies against MAP2 and the 200 kDa neurofilament subunit (NF-H) to identify dendrites and axons, respectively. RESULTS: betaA-exposed astrocytes changed dramatically from their flat polygonal shape into stellate process-bearing morphology. Viability however, was not affected. Immunocytochemical analysis of neuronal processes using anti-MAP2 and anti-NF-H, demonstrated that betaA (25-35)induced reactive astrocytes had an altered ability to support dendrite and axon growth after 3 days in vitro. Indeed, primary dendrite number and axon length were decreased by 30 and 26%, respectively, compared with control astrocytes, whereas individual primary dendrite length increased by 20%. Astrocyte support of dendritic branching, however, was not affected by betaA. DISCUSSION: We conclude that an astrocyte reaction to betaA may contribute, in part, to neuronal dystrophy associated with betaA plaques.     

新生小鼠端脑组织神经干细胞诱导分化为胆碱能神经元的研究

目的探讨新生小鼠端脑组织神经干细胞是否能够分化成胆碱能神经元。方法取新生小鼠端脑组织．用无血清方法分离培养神经干细胞；用克隆培养的方法检验培养细胞的干细胞特性；用免疫荧光细胞化学的方法检测神经干细胞标志巢蛋白（nestin）及干细胞诱导分化后神经元标志微管相关蛋白2（MAP2）、星形胶质细胞标志胶质纤维酸性蛋白（GFAP）、胆碱能标志胆碱乙酰转移酶（CHAT）；比较不同的诱导分化条件（5％胎牛血清、5％胎牛血清＋碱性成纤维细胞生长因子）对胆碱能神经元分化的影响。结果从新生小鼠端脑组织分离培养出具有自我更新、扩增能力的神经球；各培养基中神经球均为nestin阳性。诱导分化后均能够产生MAP2阳性神经元、GFAP阳性星形胶质细胞以及ChAT阳性的胆碱能神经元。分化培养中加入碱性成纤维细胞生长因子能够提高胆碱能神经元分化的比例。结论新生小鼠端脑组织神经干细胞能够分化成胆碱能神经元。     

Nuclear and cytoplasmic localization of basic fibroblast growth factor in astrocytes and CA2 hippocampal neurons.

Fibroblast growth factors (FGFs) are known to stimulate mitogenesis in a variety of non-neuronal cell types and to support the survival in vitro of many neuronal cell types. The physiological role of FGFs in the CNS is currently not known. The present study determined the distribution in the rat CNS of a prominent member of the FGF family, basic FGF (bFGF). Immunohistochemical analysis showed that bFGF immunoreactivity was found predominantly in astrocytes throughout all regions of the CNS. In contrast, only a few neuronal populations were found to contain bFGF immunoreactivity, most prominent among them, neurons in the CA2 area of the hippocampus. This predominant localization of bFGF to astrocytes was confirmed by two other observations: (1) highly enriched cultures of astrocytes contained bFGF immunoreactivity and bioactivity, whereas highly enriched cultures of cerebral cortical neurons contained no detectable bFGF, and (2) neonatal rat cerebral cortex, which contains only a few differentiated astrocytes, also contained no detectable bFGF immunoreactivity and only low amounts of bFGF bioactivity. Immunocytochemical analysis also suggested that bFGF immunoreactivity was present in the nucleus as well as the cytoplasm of astrocytes and CA2 neurons. This nuclear localization was confirmed by EM analysis of the intracellular distribution of the immunoperoxidase reaction product. In addition, preparations of both nuclear and soluble fractions of brain extracts contained bFGF immunoreactivity and bioactivity. These data suggest that bFGF might be involved in mediating astrocytic influences on the late postnatal maturation and plasticity in the CNS, and that the nuclear localization of bFGF within astrocytes may play an important role in the differentiation of these cells. In addition, bFGF may play a similar role in a few specific neuronal populations, such as CA2 hippocampal neurons.