Hypothalamic
orexin (
hypocretin) neurons play crucial roles in arousal control. Their involvement in
anesthesia and analgesia remains to be better understood. In order to enhance our view on the neuroanatomy, we systematically mapped the projections of
orexin neurons with confocal microscope and light sheet microscope. We specifically expressed optogenetic
opsins tagged with fluorescence markers in
orexin neurons through adeno-associated
viral infection in the mouse brain. The imaging results revealed fine details and novel features of the
orexin projections throughout the brain, particularly related to the nuclei regulating arousal and
pain. We then optogenetically activated
orexin neurons in the lateral hypothalamus to study the effects on
anesthesia-related behaviors. cFos staining showed that optogenetic stimulation can activate
orexin neurons in the ChR2-mCherry group, but not the control mCherry group (62.86 ± 3.923% vs. 7.9 ± 2.072%; P < 0.0001). In behavior assays, optogenetic stimulation in the ChR2-mCherry group consistently elicited robust arousal from light
isoflurane anesthesia (9.429 ± 3.804 s vs. 238.2 ± 17.42 s; P < 0.0001), shortened the emergence time after deep
isoflurane anesthesia (109.5 ± 13.59 s vs. 213.8 ± 21.77 s; P = 0.0023), and increased the paw withdrawal latency in a hotplate test (11.45 ± 1.185 s vs. 8.767 ± 0.7775; P = 0.0317). The structural details of
orexin fibers established the neuroanatomic basis for studying the role of
orexin in
anesthesia and analgesia.