Figure 4
5-HT2A signaling responses to the agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) are attenuated in vivo and in vitro by genetic deletion of caveolin-1. (a, b) Littermate wild-type (WT) and caveolin-1 (Cav-1) knockout (KO) mice were injected with 1.0 mg kg−1 DOI, 1 h later frontal cortex was isolated, mRNA extracted and transcript levels of c-Fos and egr-1 determined by real-time PCR. DOI-induced c-Fos and egr-1 gene expression in frontal cortex was decreased in mice heterozygous for Cav-1; DOI-induced gene expression was prevented in KO mice (panels a and b, mean±s.e.m., n=5 littermate mice for each group; *P<0.05 versus saline-treated mice in each group). (c) Neuronal 5-HT2A receptor calcium signaling is decreased by genetic deletion of Cav-1. Primary cortical neurons were isolated from WT and Cav-1 KO mice and cultured in 96-well plates. Neurons were infected with lentiviruses encoding 5-HT2A receptors and intracellular calcium release was determined by fluorescence calcium dye imaging using high-content imaging microscopy. Images of cells are shown before (left panels) and after (middle panels) an automated addition of the 5-HT2A agonist DOI. WT neurons expressing 5-HT2A receptors (middle panels) exhibited a robust increase in intracellular calcium dye fluorescence after DOI. In comparison, KO neurons expressing 5-HT2A receptors (lower panels) displayed a reduced intracellular calcium dye fluorescence after agonist. The calcium dye fluorescence in individual responding cells in the wells (segmenting) was determined. (d) Quantification of calcium dye fluorescence in individual cells is shown and expressed as fold over initial fluorescence; KO neurons exhibited a reduced peak and total calcium response (Figure 4d, mean fluorescence intensity±s.e.m. from >90 cells per group from n=3 cell preparations).
