Orexin A-induced extracellular calcium influx in prefrontal cortex neurons involves L-type calcium channels

ABSTRACT

Orexins, novel excitatory neuropeptides from the lateral hypothalamus, have beenstrongly implicated in the regulation of sleep and wakefulness. In this study, weexplored the effects and mechanisms of orexin A on intracellular free Ca2+ concentration([Ca2+]i) of freshly dissociated neurons from layers V and VI in prefrontalcortex (PFC). Changes in [Ca2+]i were measured with fluo-4/AM using confocallaser scanning microscopy. The results revealed that application of orexin A (0.1 ~1ìM) induced increase of [Ca2+]i in a dose-dependent manner. This elevation of[Ca2+]i was completely blocked by pretreatment with selective orexin receptor 1antagonist SB 334867. While depletion of intracellular Ca2+ stores by the endoplasmicreticulum inhibitor thapsigargin (2 ìM), [Ca2+]i in PFC neurons showed noincrease in response to orexin A. Under extracellular Ca2+-free condition, orexin Afailed to induce any changes of Ca2+ fluorescence intensity in these acutely dissociatedcells. Our data further demonstrated that the orexin A-induced increase of [Ca2+]iwas completely abolished by the inhibition of intracellular protein kinase C or phospholipaseC activities using specific inhibitors, BIS II (1 ìM) and D609 (10 ìM),respectively. Selective blockade of L-type Ca2+ channels by nifedipine (5 ìM) significantlysuppressed the elevation of [Ca2+]i induced by orexin A. Therefore, thesefindings suggest that exposure to orexin A could induce increase of [Ca2+]i in neuronsfrom deep layers of PFC, which depends on extracellular Ca2+ influx via L-typeCa2+ channels through activation of intracellular PLC-PKC signaling pathway bybinding orexin receptor 1(AU)