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.

Glutamate receptor-mediated calcium responses in acutely isolated hippocampal astrocytes

“…The response to 5-HT was blocked or partially inhibited by 1 µM ketanserin (Fig. 5B), which is consistent with previous studies in hippocampal primary cultures (Deecher et al, 1993). Note in Figure 5 that 50 µM Glu produced a decrease in [Ca 2ϩ ] i from the plateau of a Ca 2ϩ response to 5-HT.…”

“…It required about 30 min to obtain a positive response to 10 µM 5-HT after blockade by ketanserin. This was previously reported for primary hippocampal astrocyte cultures (Deecher et al, 1993).…”

Receptor‐activated Ca 2+ increases in vibrodissociated cortical astrocytes: A nonenzymatic method for acute isolation of astrocytes

“…5-HT receptors have previously been demonstrated on cultured astrocytes from different brain regions (Hansson et al, 1987;Deecher et al, 1993;Whitaker-Azmitia et al, 1993). In this study, astrocytic 5-HT receptors coupled to changes in Ca 2ϩ metabolism could not be found.…”

Astrocytic calcium signals induced by neuromodulators via functional metabotropic receptors in the ventral respiratory group of neonatal mice

“…The explanation might be that Kir4.1 channels in the neurons might be Kir4.1/Kir5.1 heteromers (Tanemoto et al, 2000;Yang et al, 2000) that, unlike Kir4.1 monomers, do not allow anomalous gating and calcium permeability (Hartel et al, 2007 release from intracellular stores (Hartel et al, 2007). Despite an oligodendroglial expression of Kir4.1 in several regions of the CNS (Kalsi et al, 2004;Neusch et al, 2001;Poopalasundaram et al, 2000), oligodendrocytespecific features of the intracellular calcium release machinery (Kirischuk et al, 1995;Simpson et al, 1998) Astrocytes in the CNS express a variety of metabotropic receptors including those for 5-HT that increase cytoplasmic Ca 21 concentration Deecher et al, 1993;Hagberg et al, 1998;Kimelberg et al, 1997;Nilsson et al, 1991;Verkhratsky and Steinhauser, 2000). TRH and SP receptors, identified functionally and by mRNA detection, are also present (McDermott et al, 1992;Too et al, 1994).…”