Hypocretin-1 causes G protein activation and increases ACh release in rat pons

Abstract

The effects of the arousal-promoting peptide hypocretin on brain stem G protein activation and ACh release were examined using 16 adult Sprague-Dawley rats. In vitro[35S]GTPgammaS autoradiography was used to test the hypothesis that hypocretin-1-stimulated G protein activation is concentration-dependent and blocked by the hypocretin receptor antagonist SB-334867. Activated G proteins were quantified in dorsal raphe nucleus (DR), locus coeruleus (LC) and pontine reticular nucleus oral part (PnO) and caudal part (PnC). Concentration-response data revealed a significant (P < 0.001) effect of hypocretin-1 (2-2000 nm) in all brain regions examined. Maximal increases over control levels of [35S]GTPgammaS binding were 37% (DR), 58% (LC), 52% (PnO) and 44% (PnC). SB-334867 (2 micro m) significantly (P < 0.002) blocked hypocretin-1 (200 nm)-stimulated [35S]GTPgammaS binding in all four nuclei. This is the first autoradiographic demonstration that hypocretin-1 activates G proteins in arousal-related brain stem nuclei as a result of specific receptor interactions. This finding suggests that some hypocretin receptors in brain stem couple to inhibitory G proteins. In vivo microdialysis was used to test the hypothesis that PnO administration of hypocretin-1 increases ACh release in PnO. Dialysis delivery of hypocretin-1 (100 micro m) significantly (P < 0.002) increased (87%) ACh release. This finding is consistent with the interpretation that one mechanism by which hypocretin promotes arousal is by enhancing cholinergic neurotransmission in the pontine reticular formation.