Expression of serotonin transporter (SERT) and receptors 5-HTR1A and 5-HTR2A in an animal model of hypothermia

Abstract

The role of anterior hypothalamus and preoptic area in thermoregulation has been studied since the late 19^th century (Charles Richet 1884, Aronsohn&Sachs 1885), and thermosensitive neurons have been recorded in these areas. Numerous animal studies with injection of serotonergic agonists or antagonists proved activation of thermoregulatory responses, and complex serotonergic circuits with origin in dorsal raphe nucleus (DRN) and projection in hypothalamus and preoptic area (POA), nucleus accumbens (Acc), and central amygdala were identified. We assessed the expression of serotonin transporter SERT, 5-HT1A and 5-HT2A receptors at mRNA and protein level in several brain regions: brainstem raphe, hypothalamus, Acc and POA, amygdala, piriform cortex (Pir) in adult male CD1 mice kept 4 h/day at 4 °C or normal temperature (control) for 1–2 months. For qRT-PCR total RNA was extracted from fresh samples with a GenElute™ kit (Sigma), followed by RT and qPCR with hydrolysis probes (Applied Biosystems). For immunofluorescence 200 µm slices were cut from fixed brains and stained with primary antibodies ASR-021, ASR-033, AMT-004 (Alomone Labs) and secondary antibody N2404-Ab635P-L (NanoTag), co-stained with phalloidin-AlexaFluor488 and DAPI. We obtained statistically significant (two-tailed t test) increases in mRNA expression in hypothermia vs. control for SERT and 5-HT2A in the brainstem raphe and hypothalamus. Fluorescence intensities (averaged over small relevant regions and normalized to DAPI fluorescence) were higher in hypothermia for the two receptors in DRN and Acc, and similar to control levels in Pir and amygdala, proving activation of central serotonergic thermoregulatory circuits.