Targeted recombination in active populations as a new mouse genetic model to study sleep‐active neuronal populations: Demonstration that Lhx6+ neurons in the ventral zona incerta are activated during paradoxical sleep hypersomnia

Abstract

The cFos immunostaining allowed the identification of multiple populations of neurons involved in the generation of paradoxical sleep. We adopted the transgenic (targeted recombination in active populations) mouse model, which following injection of tamoxifen, allows expression of Cre-dependent reporter constructs (i.e., mCherry) in neurons expressing cFos during waking or paradoxical sleep hypersomnia following automatic paradoxical sleep deprivation. Three groups of mice were subjected to two periods of waking, one period of waking and one of paradoxical sleep hypersomnia, or two periods of paradoxical sleep hypersomnia. A high percentage of doublelabelled neurons was observed in the lateral hypothalamic area and zona incerta of two periods of waking and two periods of paradoxical sleep hypersomnia in mice, but not in those of one period of waking and one of paradoxical sleep hypersomnia in animals. Melanin-concentrating hormone neurons in the lateral hypothalamic area and Lhx6+ cells in the zona incerta constituted 5.7 ± 1.5% and 8.8 ± 2.3% of all mCherry+ cells and 20.6 ± 4.8% and 24.6 ± 5.9% of all cFos+ neurons in two periods of paradoxical sleep hypersomnia in animals. In addition, melanin-concentrating hormone cells as well as Lhx6+ neurons rarely expressed mCherry (or cFos) in the waking condition, in contrast to orexin neurons, which constituted approximately 30% of mCherry+ and cFos+ neurons. Our results validate the TRAP methodology and open the way to use it for identifying the neurons activated during waking and paradoxical sleep hypersomnia. Furthermore, they indicate for the first time that Lhx6+ neurons in the zona incerta, like melanin-concentrating hormone cells in the lateral hypothalamic area, are activated during paradoxical sleep hypersomnia but not during waking. These results indicate that Lhx6+ neurons might play a role in the control of paradoxical sleep, like the melanin-concentrating hormone cells. K E Y W O R D S hypothalamic neuropeptides, sleep-wake cycle 2 of 11 | LEE Et aL. 1 | INTRODUC TI ON The cFos immunostaining has long been utilized to identify the neurons activated during waking (W), slow-wave sleep (SWS) and paradoxical (i.e., rapid eye movement [REM]) sleep (PS). By this means,

Granule cells in the infrapyramidal blade of the dentate gyrus are activated during paradoxical (REM) sleep hypersomnia but not during wakefulness: a study using TRAP mice

“…To resolve the specificity issue and determine whether reactivation of the same neurons occurs, we used the new 'targeted recombination in active populations (TRAP)' method that allows to permanently tag neurons activated by defined stimuli [6][7][8] . We already validated this method in our recent study on the activation of lateral hypothalamic neurons during PSR and W 9 . The transgenic TRAP2 mice express tamoxifen-dependent CreER T2 recombinase under cFos promoter.…”

“…CreER T2 can only undergo recombination when 4-hydroxytamoxifen (4-OHT, an active metabolite of tamoxifen) is present with a time window of approximately 4 h centered expression in the neurons. To maximize the amount of PS during the time-window of the CreER T2driven recombination, we took advantage of the homeostatic regulation of PS: the amount of PS is increased after the deprivation of PS 9 . To obtain a selective deprivation, we used an automatic PSD system that utilizes algorithm to detect PS and then send TTL signal to the recording chamber to interrupt the state [9][10][11] .…”

“…To maximize the amount of PS during the time-window of the CreER T2driven recombination, we took advantage of the homeostatic regulation of PS: the amount of PS is increased after the deprivation of PS 9 . To obtain a selective deprivation, we used an automatic PSD system that utilizes algorithm to detect PS and then send TTL signal to the recording chamber to interrupt the state [9][10][11] .…”

Neural and Homeostatic Regulation of REM Sleep

“…In the dorsomedial hypothalamic nucleus (DMH), a subpopulation of galanin-expressing GABAergic neurons that project to the raphe pallidus, a brainstem area containing serotonergic neurons with lowest activity during REM sleep (Trulson and Trulson, 1982), is preferentially active during REM sleep, and optogenetic activation of these projection neurons increases REM sleep (Chen et al, 2018). A separate GABAergic population in the zona incerta (ZI), which expresses the LIM homeodomain factor Lhx6, promotes both NREM and REM sleep as shown by chemogenetic activation and inhibition experiments (Liu et al, 2017) and express elevated levels of c-Fos during REM sleep hypersomnia (Lee et al, 2020). Lhx6 neurons inhibit nearby wake-promoting orexin/hypocretin and GABAergic neurons in the LH and their axons project to wake-promoting populations in the midbrain.…”

Evolutionary Origin of Distinct NREM and REM Sleep

“…In reptiles, MCH neurons reside in the periventricular and lateral hypothalamic area (Cardot et al, 1994). In mice, Lhx6-expressing neurons in the zona incerta are activated during REM sleep and are involved in regulating sleep (Liu et al, 2017;Lee et al, 2020). Lhx6 is a LIM homeodomain factor which is conserved in chicken and anuran (Medina et al, 2011), and thus likely also in reptiles.…”