Circadian Phase Shifts to Neuropeptide Y<i>In Vitro</i>: Cellular Communication and Signal Transduction

Abstract

Mammalian circadian rhythms originate in the hypothalamic suprachiasmatic nuclei (SCN), from which rhythmic neural activity can be recorded in vitro . Application of neurochemicals can reset this rhythm. Here we determine cellular correlates of the phase-shifting properties of neuropeptide Y (NPY) on the hamster circadian clock in vitro . Drug or control treatments were applied to hypothalamic slices containing the SCN on the first day in vitro . The firing rates of individual cells were sampled on the second day in vitro . Control slices exhibited a peak in firing rate in the middle of the day. Microdrop application of NPY to the SCN phase advanced the time of peak firing rate. This phase-shifting effect of NPY was not altered by block of sodium channels with tetrodotoxin or block of calcium channels with cadmium and nickel, consistent with a direct postsynaptic site of action. Pretreatment with the glutamate receptor antagonists ( dl -2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione disodium) also did not alter phase shifts to NPY. Blocking GABA A receptors with bicuculline (Bic) had effects only at very high (millimolar) doses of Bic, whereas blocking GABA B receptors did not alter effects of NPY. Phase shifts to NPY were blocked by pretreatment with inhibitors of protein kinase C (PKC), suggesting that PKC activation may be necessary for these effects. Bathing the slice in low Ca 2+ /high Mg 2+ can block phase shifts to NPY, possibly via a depolarizing action. A depolarizing high K + bath can also block NPY phase shifts. The results are consistent with direct action of NPY on pacemaker neurons, mediated through a signal transduction pathway that depends on activation of PKC.