Detection of 5‐hydroxytryptamine₂ receptors by radioligand binding, northern blot analysis, and Ca²⁺ responses in rat primary astrocyte cultures

Abstract

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.