Dissociating orexin-dependent and -independent functions of orexin neurons using novel Orexin-Flp knock-in mice

Abstract

Uninterrupted arousal is important for survival during threatening situations. Activation of orexin/hypocretin neurons is implicated in sustained arousal. However, orexin neurons produce and release orexin as well as several co-transmitters including dynorphin and glutamate. To disambiguate orexin-dependent and -independent physiological functions of orexin neurons, we generated a novel Orexin-flippase (Flp) knock-in mouse line. Crossing with Flp-reporter or Cre-expressing mice showed gene expression exclusively in orexin neurons. Histological studies confirmed that orexin was knock-out in homozygous mice. Orexin neurons without orexin showed altered electrophysiological properties, as well as received decreased glutamatergic inputs. Selective chemogenetic activation revealed that both orexin and co-transmitters functioned to increase wakefulness, however, orexin was indispensable to promote sustained arousal. Surprisingly, such activation increased the total time spent in cataplexy. Taken together, orexin is essential to maintain basic membrane properties and input-output computation of orexin neurons, as well as to exert awake-sustaining aptitude of orexin neurons.

Effects of sex and estrous cycle on sleep and cataplexy in narcoleptic mice

“…OREX neurons express other neurotransmitters in addition to OREX such as dynorphin, glutamate or galanin [48], suggesting that these latter neuropeptides might only mildly intervene in the sleep phenotype. In agreement with the similarity of these two mouse models, a recent study [49] has demonstrated that OREX neuropeptide is essential for the maintenance of neuronal electrophysiological properties. OREX neurons without OREX neuropeptides have lower spontaneous firing frequencies, lower input resistances, and a hyperpolarized membrane potential, suggesting that these neurons are less excitable and function poorly in the absence of OREX neuropeptides.…”

GABA neurons in the ventral tegmental area regulate non-rapid eye movement sleep in mice

“…Thus, we wondered whether VTA Gad67+ neurons mediate their sleep-promoting effect through the inhibition of LH orexin neurons. To test this, we generated a bigenic orexin-Flippase (Flp); Gad67-Cre mouse, in which orexin neurons exclusively express Flp recombinase and Gad67+ neurons express Cre recombinase (Figure 6a–c) (Chowdhury et al, 2019). We injected a Cre-inducible AAV expressing the blue light-gated cation channel channelrhodopsin2 (E123T/T159C; ChR2) (Berndt et al, 2011) in the VTA as well as a Flp-inducible AAV expressing tdTomato in the LH of orexin-Flp; Gad67-Cre mice (Figure 6a–c).…”

The Impacts of Age and Sex in a Mouse Model of Childhood Narcolepsy

“…An increase in dark period REM sleep was expected as the orexin neurons are thought to normally suppress REM sleep during the dark period (Kantor et al, 2009). Kantor et al (2009) demonstrated this disinhibition of REM sleep during the subjective night in mice housed in constant darkness, and this effect has also been shown in orexinataxin mice and orexin knockout mice maintained on a 12:12 light:dark cycle (Hara et al, 2001;Kantor et al, 2009;Roman et al, 2018;Chowdhury et al, 2019).…”

“…Orexins are wake-promoting neuropeptides necessary for the maintenance of long periods of wakefulness and the regulation of REM sleep ( Saper et al, 2001 ; Lu et al, 2006 ; Branch et al, 2016 ; Chowdhury et al, 2019 ). Narcolepsy is caused by severe loss of the orexin-producing neurons in the hypothalamus, and the resulting symptoms of narcolepsy include excessive daytime sleepiness, the occurrence of REM sleep at any time of day, and cataplexy – episodes of muscle atonia during wakefulness that are likely produced by some of the same neural mechanisms that produce atonia during REM sleep ( Mahoney et al, 2019 ).…”