Cytosolic calcium elevation induced by orexin/hypocretin in granule cell domain cells of the rat cochlear nucleus in vitro

Using rat brain slice preparations, we examined the effect of orexin on cytosolic Ca2+ concentrations ([Ca2+](i)) in the granule cell domain (GCD) cells of the cochlear nucleus that carry non-auditory information to the dorsal cochlear nucleus. Application of orexin concentration-dependently increased [Ca2+](i), and in two thirds of GCD cells these increases persisted in the presence of tetrodotoxin. There was no significant difference between the dose-response curve for orexin-A and that for orexin-B. Extracellular Ca2+ removal abolished the [Ca2+](i) elevation induced by orexin-B, whereas depletion of intracellular Ca2+ stores had no effect. The orexin-B-induced elevation of [Ca2+](i) was not blocked by inhibitors of reverse-mode Na+/Ca2+ exchanger (NCX) and nonselective cation channel, whereas it was blocked by lowering the extracellular Na+ or by applying inhibitors of forward-mode NCX and voltage-gated R- and T-type Ca2+ channels. The ORX-B-induced increase in [Ca2+](i) was also blocked by inhibitors of adenylcyclase (AC) and protein kinase A (PICA), but not by inhibitors of phosphatidylcholine-specific and phosphatidylinositol-specific phospholipase C. In electrophysiological experiments using whole-cell patch clamp recordings, half of GCD cells were depolarized by orexin-B, and the depolarization was abolished by a forward-mode NCX inhibitor. These results suggest that orexin increases [Ca2+](i) postsynaptically via orexin 2 receptors, and the increase in [Ca2+](i) is induced via the AC-PKA-forward-mode NCX-membrane depolarization-mediated activation of voltage-gated R- and T-type Ca2+ channels. The results further support the hypothesis that the orexin system participates in integrating neural systems that are involved in arousal, sensory processing, energy homeostasis and autonomic function. (C) 2010 Elsevier Inc. All rights reserved.