Glial fibrillary acid protein (GFAP)-positive astrocytes isolated from the cerebral cortices of 3-10-dayold rats frequently showed increased intracellular Ca 2c oncentration responses to L-glutamate and glutamate analogues. However, few of the acutely isolated cells responded to ATP, and no such cells responded to serotonm [5-hydroxytryptamine(5-HT)]. The same cell that failed to respond to ATP or 5-HT often responded to glutamate. Culturing acutely isolated cells in media containing horse serum decreased Ca2~responses to glutamate but increased the responses to ATP and induced responses to 5-HT. In primary cultures prepared from the cerebral cortices of 1-day-old rats and cultured in horse serum, fewer of the cells responded to glutamate, but almost all cells responded to ATP and 5-HT. The lack of, or limited response to, 5-HT or ATP in the acutely isolated cells seems unlikely to be due to selective damage to the respective receptors because acutely isolated GFAPnegative cells showed responses to ATP, several different proteases and mechanical dissociation yielded cells that also responded to glutamate but not to ATP, and exposure of primary cultures to papain did not abolish Ca2r esponses to several transmitters. The responses of the acutely isolated cells to glutamate but limited or lack of responses to ATP and 5-HT also correspond to what has been seen so far for astrocytes in situ. Thus, the present studies provide direct evidence that some of the receptors seen in primary astrocyte cultures may reflect a response to culture conditions and that, in the context of the relevant information so far available, acutely isolated astrocytes seem to reflect better the in vivo state.

Very little information is available concerning the pharmacology of type 2 astroglia. During the past decade it has become apparent that two distinct lineages of astroglial cells can be defined in vitro. These two lineages are commonly referred to as type 1 and type 2 and are distinguished from each other on the basis of their morphological features and antigenic phenotypes. In contrast to type 1 astroglia, very little is known about the pharmacology of type 2 astroglia. The lack of information concerning the responsiveness of these cells stems primarily from difficulties encountered in isolating large numbers of type 2 astroglia free of other cell types. In the present study video- and photometer-based imaging systems were used to monitor the influence of a series of neuroligands on the intracellular calcium levels of individual cerebral type 2 astroglia in order to assess their expression of calcium-mobilizing receptors. The responses of 85 immunocytochemically identified cerebral type 2 astroglia to bradykinin (BK), norepinephrine (NE), histamine (HIST), carbachol (CARB), 2-methyl-thio ATP (2MT-ATP), glutamate (GLUT), and serotonin (5-HT) were analyzed. Approximately 50% of cerebral type 2 astroglia responded to BK, NE, HIST, CARB, and 2MT-ATP whereas only 16% and 9% of the cells responded to GLUT and 5-HT, respectively. The number of neuroligands that increased calcium in individual cells ranged from 0 to 6. These responses are quite similar to those previously demonstrated in cultured cerebral type 1 astroglia. No pattern of receptor co-expression was observed for the different neuroligands tests.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioligand binding, Northern blot analysis, and changes in [Ca2+]i were used to study serotonin [5-hydroxytryptamine (5HT)] receptor subtypes in primary cultures of astrocytes from neonatal rat cerebral cortex. Radioligand binding studies revealed the presence of 5HT2, but not the 5HT1 or 5HT3 receptor subtypes. Radioligand binding was also used to show the presence of serotonin uptake sites, which had previously been shown to be present by [3H]-5HT uptake, and also alpha 1-adrenergic receptors as has previously been reported by binding studies. Northern blot analysis of cortical astrocyte mRNA demonstrated the presence of transcripts for 5HT2 receptors, but failed to identify mRNA for 5HT1a or 5HT1c receptors. Thus, results from Northern blot analysis correlated with the radioligand binding data which showed only 5HT2 receptors. Equilibrium saturation studies, using 125[I]-LSD to label 5HT2 receptors, yielded a KD of 9 nM and a Bmax of 177 fmol/mg protein. Radioligand binding studies or primary astrocyte cultures prepared from other brain regions also showed the presence of alpha 1-adrenergic, 5HT2 receptor, and 5HT-uptake sites, but no detectable 5HT1a receptors, which were the only 5HT1 receptors studied. Studies demonstrating 5HT-induced, spiperone- and ketanserin-sensitive increases in free [Ca2+]i as measured by FURA-2, showed that the 5HT2 receptors were functional in these cells. These data provide clear evidence for the existence of both 5HT2 receptors and 5HT-uptake sites in the same primary astrocyte cultures from neonatal rat cerebral cortex, with no detectable evidence of 5HT1a or 5HT1c subtypes.

Previous studies have established that rat primary astrocyte cultures prepared from several brain regions of 1-4-d-old rats exhibit high-affinity, Na(+)-dependent and fluoxetine-sensitive serotonin (5-HT) uptake with a Km for 5-HT of 0.4 microM and a Ki for fluoxetine of 23 nM, which correspond to the characteristics for this transport for other brain preparations. However, it is not known whether astrocytes in situ show such uptake. We addressed this question by performing 3H-5-HT uptake experiments on cortical astrocytes, within 4 hr of isolating them from 6- and 21-d-old rats by the tissue-print technique. Quantitative autoradiography was combined with GFAP and neurofilament (NF) immunocytochemistry to distinguish astrocytic from neuronal 3H-5-HT uptake. In composition, the tissue-printed (TP) cells and processes were 60-70% GFAP (+) and 10-15% NF(+). 3H-5-HT uptake (0.3 microM 5-HT, 3.4 microCi/ml) in both tissue-printed GFAP(+) astrocytes and NF(+) structures was sensitive to 1 microM fluoxetine and was also Na+ dependent. More than 90% of TP astrocytes from 6- and 21-d-old rats and 100% of NF(+) structures from 21-d-old rats showed positive 3H-5-HT uptake (defined as > or = 31 grains/10(3) microns2). The highest level of uptake (> or = 191 grains/10(3) microns2) was never observed in TP astrocytes but was exhibited by about half of the NF(+) structures. In other experiments were found that 3H-5-HT uptake by 6-d-old TP astrocytes was comparable to uptake by postnatal age-matched primary cultured astrocytes that were grown in fetal bovine serum (FBS). However, primary cultured astrocytes grown in horse serum showed lower uptake than that observed with FBS, a finding similar to previous results in cultures where 3H-5-HT uptake was measured per milligram of cell protein. These results imply that high-affinity, Na(+)-dependent and fluoxetine-sensitive 5-HT uptake occurs in rat cortical astrocytes in situ.

In this study we address the effects of methylmercuric chloride (MeHgCl), a metal that is preferentially sequestered in astrocytes, on 5-HT and glutamate/aspartate uptake by rat primary astrocyte cultures. Quantitative autoradiography (ARG) combined with glial acidic fibrillary protein (GFAP) immunocytochemistry, as well as intact-cell (bulk) measurements of radiolabel uptake of these neurotransmitters were performed in 7- and 21-day-old primary astrocyte cultures. MeHg (10 µM for 30 min) treatment of astrocytes (21 days in culture) significantly inhibited the Na⁺-dependent and fluoxetine-sensitive [³H]5-HT uptake. D-aspartate uptake in 7- and 21-day-old cultures was even more sensitive to MeHg, leading to >99% inhibition of D-aspartate uptake by astrocytes (30 min; 10 µM MeHg). These results imply that the Na⁺-dependent and fluoxetine-sensitive 5-HT uptake, as well as the Na⁺-dependent L-glutamate/D-aspartate uptake systems in primary astrocyte cultures are sensitive to low concentrations of MeHg. Since astrocytic removal of glutamate (and aspartate) and 5-HT from the extracellular space in situ is crucial to the maintenance of chemical homeostasis, MeHg-induced uptake inhibition of 5-HT and aspartate could have cytotoxic effects on neighboring neurons.