Two input pathways to the suprachiasmatic nucleus (SCN) of the hypothalamus are the glutamatergic retinohypothalamic tract and the serotonergic afferent from the midbrain raphe nucleus. To determine whether these two temporal signaling pathways can converge at the cellular level, we have investigated the effects of serotonin on glutamate-induced calcium responses of individual SCN neurons isolated in cell culture. Dispersed cultures were formed from the SCN of neonatal rats. The calcium indicator Fura-2 acetoxymethyl ester was used to assess the changes in [Ca2+]i by recording the 340-nm/380-nm excitation ratio. Application of glutamate (5 μM) to the culture caused a rapid (within 10 s) increase in the fluorescence ratio of neurons indicating a marked increase in the concentration of intracellular free calcium. However, when 5-hydroxytryptamine (5-HT; 5 μM) was coapplied with glutamate, 31% of neurons showed an overall 61% reduction in the peak of the glutamate-induced calcium increase. Application of the 5-HT7/1A receptor agonist, (±)-8-hydroxy-2-(di- n-propylamino)tetralin [(±)-8-OH-DPAT] (1 μM), also reduced the calcium elevation this time by 80% in 18% of the neurons tested. When the 5-HT7/2/1C receptor antagonist, ritanserin (800 nM), was coapplied with serotonin, it blocked modulation of the glutamate responses. Further support for the involvement of the 5-HT7receptor was provided by the ability of the adenylate cyclase activator, forskolin (10 μM), and the cAMP analogue, 8-Br cAMP (0.5 mM), to mimic the suppressive effect of serotonin. Blocking spike-mediated cell communication with tetrodotoxin (1 μM) did not prevent the serotonergic suppression of glutamate-induced responses. These results support the hypothesis that the serotonergic modulation of photic entraining signals can occur in SCN neurons.
Light exposure induces short- and long-term changes in the excitability of retinorecipient neurons in suprachiasmatic nucleus
