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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

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 double‐labelled 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.