Abstract
The hypothalamic suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, undergoes serotonergic regulation, but the underlying mechanisms remain obscure. Here, we generated a subclone of an SCN progenitor cell line expressing Ca2+ sensors (SCN2.2YC) and compared its 5-HT receptor signalling with that of rat SCN neurons in brain slices. SCN2.2YC cells expressed 5-HT1A/2A/2B/2C, but not 5A/7, while all six subtypes were expressed in SCN tissues. High K+ or 5-HT increased cytosolic Ca2+ in SCN2.2YC cells. The 5-HT responses were inhibited by ritanserin and SB-221284, but resistant to WAY-100635 and RS-127445, suggesting predominant involvement of 5-HT2C for Ca2+ mobilisations. Consistently, Ca2+ imaging and voltage-clamp electrophysiology using rat SCN slices demonstrated post-synaptic 5-HT2C expression. Because 5-HT2C expression was postnatally increased in the SCN and 5-HT-induced Ca2+ mobilisations were amplified in differentiated SCN2.2YC cells and developed SCN neurons, we suggest that this signalling development occurs in accordance with central clock maturations.
Highlights
The hypothalamic suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, undergoes serotonergic regulation, but the underlying mechanisms remain obscure
Because 5-HT2C expression was postnatally increased in the SCN and 5-HT-induced Ca21 mobilisations were amplified in differentiated SCN2.2YC cells and developed SCN neurons, we suggest that this signalling development occurs in accordance with central clock maturations
Since retinohypothalamic tract (RHT) projections are known to be increased during a similar postnatal period, we analysed the possible interaction with retinal inputs to the SCN
Summary
The hypothalamic suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, undergoes serotonergic regulation, but the underlying mechanisms remain obscure. We generated a subclone of an SCN progenitor cell line expressing Ca21 sensors (SCN2.2YC) and compared its 5-HT receptor signalling with that of rat SCN neurons in brain slices. Because 5-HT2C expression was postnatally increased in the SCN and 5-HT-induced Ca21 mobilisations were amplified in differentiated SCN2.2YC cells and developed SCN neurons, we suggest that this signalling development occurs in accordance with central clock maturations. C-amino-butyric acid (GABA)-A receptors mediate excitatory synaptic signal transduction in neonatal brains[11], but are switched to reversible (i.e., excitatory and inhibitory) functions in SCN neurons during postnatal development[12]. C-Fos expression in the SCN induced by subcutaneous injection of a 5HT2A/2C agonist (2,5-dimethoxy-4-iodoamphetamine; DOI) was increased in a slightly different time frame to RHT development in rats[16], suggesting that differential developmental mechanisms may underlie these systems. SCN2.2 cells display (i) extended growth potential without evidence of transformed or tumorigenic properties, (ii) expression of E1A protein within all cell nuclei and (iii) www.nature.com/scientificreports circles). n 5 4–7 rats per group. *P , 0.05, **P , 0.01 (black asterisks for intact rats and grey asterisks for eyeless rats) versus the day 0 levels by oneway ANOVA followed by Duncan’s multiple range test
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