Serotonergic Pre-treatments Block In Vitro Serotonergic Phase Shifts of the Mouse Suprachiasmatic Nucleus Circadian Clock

The suprachiasmatic nucleus (SCN) contains a circadian clock that maintains its time-generating and phase-modulating capacities in vitro. Previous studies report clear differences in the ability of serotonergic stimuli to phase-shift the SCN clock when applied directly to the SCN either in vivo or in vitro: while mice and rat circadian clocks are readily phase-shifted by serotonin (5-HT) or 5-HT agonists applied in vitro, hamster and mice circadian clocks respond inconsistently to 5-HT agonists injected directly into the SCN in vivo. Here we have investigated one possible explanation for these differences: that the SCN isolated in vitro experiences reduced endogenous 5-HT signaling, which increases clock sensitivity to subsequent 5-HT stimulation. For these experiments we treated mouse SCN brain slices with low concentrations of compounds that increase serotonin signaling: 5-HT, a 5-HT agonist (8-OH-DPAT), the 5-HT precursor, l-tryptophan, or the 5-HT re-uptake inhibitor, fluoxetine. Pretreatment with each of these substances completely blocked subsequent phase-shifts induced by mid-subjective day treatment with either 5-HT or 8-OH-DPAT, while they did not block phase-shifts induced by the adenylate cyclase activator, forskolin. Time-course data on l-tryptophan-induced inhibition are consistent with this treatment inducing receptor internalization, while timing of the recovery from inhibition is consistent with receptor reinsertion. Together these data support the hypothesis that SCN clock sensitivity to serotonergic phase modulation is affected by the amount of prior serotonin signaling present in the SCN, and that this signaling alters the density of surface 5-HT receptors on SCN clock neurons.