Title: Orexin Neurons Are Directly and Indirectly Regulated by Catecholamines in a Complex Manner

We previously reported that orexin neurons are directly hyperpolarized by noradrenaline (NA) and dopamine. In the present study, we show that NA, dopamine and adrenaline all directly hyperpolarized orexin neurons. This response was inhibited by the α2 adrenergic receptor (α2-AR) antagonist, idazoxan or BRL44408, and was mimicked by the α2-AR selective agonist, UK14304. A low concentration of Ba inhibited NA-induced hyperpolarization, suggesting that activation of G-protein coupled inward rectifier potassium channels is involved in the response. In the presence of a high concentration of idazoxan, NA induced depolarization or inward current. This response was inhibited by α1-AR antagonist, prazosin, suggesting the existence of α1-ARs on the orexin neurons along with α2-AR. We also examined the effects of NA on glutamatergic and GABAergic synaptic transmission. NA application dramatically increased the frequency and amplitude of spontaneous inhibitory synaptic currents (sIPSCs) and inhibited excitatory synaptic currents (sEPSCs) in orexin neurons. However, NA decreased the frequency of miniature EPSCs (mEPSCs) and IPSCs and the amplitude of evoked EPSCs and IPSCs through the α2-AR since the NA response on mPSCs was inhibited by idazoxan. These results suggest that the NA-induced increase in sIPSC frequency and amplitude is mediated via α1-ARs on the somata of GABAergic neurons that innervate the orexin neurons. Calcium imaging using orexin/YC2.1 transgenic mouse brain revealed that NA-induced inhibition of orexin neurons is not altered by sleep deprivation or circadian time in mice. The evidence presented here revealed that orexin neurons are regulated by catecholamines in a complex manner. Page 2 of 61