Abstract
Multiple populations of wake-promoting neurons have been characterized in mammals, but few sleep-promoting neurons have been identified. Wake-promoting cell types include hypocretin and GABA (γ-aminobutyric-acid)-releasing neurons of the lateral hypothalamus, which promote the transition to wakefulness from non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Here we show that a subset of GABAergic neurons in the mouse ventral zona incerta, which express the LIM homeodomain factor Lhx6 and are activated by sleep pressure, both directly inhibit wake-active hypocretin and GABAergic cells in the lateral hypothalamus and receive inputs from multiple sleep-wake-regulating neurons. Conditional deletion of Lhx6 from the developing diencephalon leads to decreases in both NREM and REM sleep. Furthermore, selective activation and inhibition of Lhx6-positive neurons in the ventral zona incerta bidirectionally regulate sleep time in adult mice, in part through hypocretin-dependent mechanisms. These studies identify a GABAergic subpopulation of neurons in the ventral zona incerta that promote sleep.
Highlights
All experimental animal procedures were approved by the Johns Hopkins UniversityLhx6-creERT2 knock-in mice were generated by Z
Lhx6+ cells represent 45% and 33% of all Slc32a1+ and Gad1+ cells in the ventral zona incerta (VZI) (Fig. 1c, d)
We showed that 6-h sleep deprivation from zeitgeber time (ZT)[0] to ZT6—either alone or in combination with 1 h of recovery sleep from ZT6 to ZT7—increased Fos expression in Lhx6+ VZI neurons (Fig. 2f–h), demonstrating that Lhx6+ VZI neurons are activated by high sleep pressure
Summary
Lhx6-creERT2 knock-in mice were generated by Z. The conditional Lhx[6] allele was generated through homologous recombination using a targeting construct in which loxP sites were placed in non-coding regions that are 5′ to coding exon 1b and 3′ to coding exon 3. For the Lhx6-targeting vector, the 2-kb genomic fragment between the homology regions is replaced by a 4-kb cassette, containing the following: (1) the 1b, 2 and 3 coding exons flanked by loxP sites; (2) the neomycinresistance gene under the control of the phosphoglycerate kinase (PGK) promoter Open field test—Mice were placed in the open field chamber and time spent in the centre and periphery was assessed for 30 min, using a Photobeam activity system to measure beam breaks. Three trials were performed for each mouse with a 1-min resting period between trials
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