Receptor-activated Ca2+ increases in vibrodissociated cortical astrocytes: a nonenzymatic method for acute isolation of astrocytes.

A new nonenzymatic method for the acute isolation of astrocytes from rat cerebral cortex is described. A vibratory device was used to dissociate the cells from thin brain slices, and the method yielded fresh and relatively well-preserved astrocytes without previous enzyme incubation. These cells were examined in a microspectrofluorometric system for measurement of changes in intracellular free calcium concentrations ([Ca2+]i), and their expression of various neurotransmitter receptors was determined. Acutely isolated glial fibrillary acidic protein (GFAP)-positive astrocytes (p7-p18) were seen to respond to the metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 10(-4) M) with increases in [Ca2+]i, and this response was blocked by (RS)-1-aminoindan-1,5 dicarboxylic acid (AIDA, 10(-3) M), an antagonist to group 1 metabotropic glutamate receptors. The delta-opioid receptor agonist D-Pen2, D-Pen5-enkephalin (DPDPE, 10(-6) M) evoked [Ca2+]i increases that were blocked by the delta-opioid antagonist ICI 174.388 (10(-5) M). The astrocytes failed to respond to 5-hydroxytryptamine (5-HT, 10(-5) M), although the same cells subsequently were found to respond to other agonists. Furthermore, [Ca2+]i responses evoked by phenylephrine (10(-5) M) were blocked by prazosin (0.2x10(-6) M), suggesting the expression of alpha1-adrenergic receptors on the acutely isolated astrocytes. The cells were also shown to react with [Ca2+]i increases in response to depolarization with high extracellular potassium concentrations (50x10(-3) M). The signals induced by depolarization were not seen in Ca2+-free buffer, indicating the presence of voltage-activated calcium channels in these cells. Thus, the present study confirms some of the results earlier obtained in cell cultures, suggesting that cortical astrocytes in vivo express glutamate, opiate, and adrenergic receptors, coupled to increases in [Ca2+]i, whereas no receptors for 5-HT could be detected.