0133 OREXIN MEDIATES FEED-FORWARD INHIBITION OF VLPO SLEEP-ACTIVE NEURONS - A MECHANISM FOR CONTROLLING AROUSAL

Abstract

The ventrolateral preoptic area (VLPO) plays an essential role in the initiation and maintenance of sleep. It contains a cluster of sleep-active neurons that are GABAergic and co-expresses galanin (VLPOGABA/Gal). VLPO is innervated by wake-promoting neurons and the VLPOGABA/Gal neurons are strongly inhibited by noradrenaline, carbachol and serotonin. VLPO also receives input from orexin neurons and administration of orexin in VLPO arouses mice from sleep suggesting that orexin might be inhibiting the sleep-active VLPOGABA/Gal neurons. However, orexin is an excitatory peptide, thus orexin’s effect in the VLPO remains unclear. In this study we investigate the effect of orexin on VLPO neurons in brain slices. We recorded VLPO neurons in brain slices from WT and Vgat-IRES-cre mice. We filled the recorded cells with biocytin for anatomical localization. We recorded from VLPO GFP-labelled GABAergic neurons in Vgat-IRES-cre mice that were injected in VLPO with an AAV-flex-GFP. We identify VLPO-sleep active neurons based on the inhibitory responses to noradrenaline or carbachol and/or by the presence of VGAT and galanine mRNAs using single cell RT-PCR. We found a dual response to orexin in VLPO. About 60% of VLPO neurons were excited by orexin but 40% were inhibited through release of GABA. The VLPO neurons that were inhibited by orexin were also inhibited by carbachol or noradrenaline suggesting that they could be the VLPOGABA/Gal sleep-active neurons. We tested this hypothesis by recording VLPO GABAergic neurons (GFP-labelled). Orexin increased the frequency of spontaneous IPSCs in 50% of the VLPO GABAergic neurons and these neurons expressed galanin mRNA. Orexin inhibits VLPOGABA/Gal sleep-active neuron by increasing GABAergic afferent input. This GABAergic input could originate from the local neurons that are directly activated by orexin. We propose that during wakefulness VLPOGABA/Gal sleep-active neurons are strongly and directly inhibited by wake-promoting signals, such as noradrenaline, carbachol and serotonin. Whereas, orexin could act by activating local GABAergic neurons that in turn produce feed-forward inhibition of VLPOGABA/Gal sleep-promoting neurons. R01NS091126 and P01HL095491.