Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep

Abstract

Multiple populations of wake-promoting neurons have been characterized in mammals, but few sleep-promoting neurons have been identified1. 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) sleep2,3. 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.

The BRAIN Initiative Cell Census Consortium: Lessons Learned toward Generating a Comprehensive Brain Cell Atlas

“…For retrograde monosynaptic tracing, i.e., labeling neurons directly presynaptic to a defined population, the original rabies virus -based system (Wickersham et al, 2007b) has been refined in ways that range from improvements in the complementing helper viruses (Kim et al, 2016; Kohara et al, 2014; Liu et al, 2017; Miyamichi et al, 2013; Wall et al, 2010) to the use of a rabies virus strain with reduced toxicity (Reardon et al, 2016) and the addition of a destabilization domain to the viral nucleoprotein (Ciabatti et al, 2017). Although there are fewer tools available, anterograde monosynaptic tracing – i.e., labeling neurons directly postsynaptic to a starting population – based on the vesicular stomatitis virus or herpes simplex virus has been described (Beier et al, 2011; Zeng et al, 2017).…”

The cellular and molecular landscape of hypothalamic patterning and differentiation

“…These datasets have been used as the basis for genetic studies that selectively disrupt development of specific hypothalamic regions and/or cell types (20)(21)(22)(23)(24), leading in some cases to identification of novel functions for previously characterized hypothalamic regions or cell types (25,26). However, these datasets have important limitations: they do not provide cellular resolution of gene expression data, and they do not efficiently measure coexpression of multiple genes.…”

Monosynaptic tracing success depends critically on helper virus concentrations

“…Here we present results of our titration experiments to test the effects of using different dilutions of the helper viruses, to show the reasons for the specific concentrations that we recommend. We found that the two-helper combination described above and in Liu et al [18] gives much better results than the simpler single AAV which we described earlier [16], which did not allow independent optimization of transsynaptic tracing efficiency and minimization of background labeling in Cre-negative mice. We also found that excessively high titers of the helper viruses gave very poor results, suggesting that preventing toxicity due to overly high expression of the helper virus genes is as important as ensuring sufficient expression of them.…”

“…Having previously found Addgene's Viral Service (www.addgene.org/viral-service/aav-prep) to be an excellent source of high-quality AAVs, we authorized them to package and distribute three of our published Cre-dependent helper AAVs: the standalone helper virus AAV-syn-FLEX-splitTVA-EGFP-B19G from Kohara et al [16] (referred to below as the "tricistronic" helper virus) and the two viruses to be used in combination as described in Liu et al [18]: AAV-syn-FLEX-splitTVA-EGFP-tTA and AAV-TREtight-mTagBFP2-B19G. Although the resulting preparations, all with serotype 1 capsids, had much higher titers than earlier batches that we had previously used successfully for similar experiments, we nonetheless first tried using them "straight": undiluted except insofar as, for the two-helper-virus combination, the two viruses were combined in a 50/50 mixture by volume (see Methods).…”

“…Our current approach is to use a mixture of two AAVs, described first in Liu et al [18], which are coupled by the tetracycline transactivator system [26]. The first AAV is Cre-dependent and expresses, in Cre-expressing cells, TVA (transmembrane isoform [7; 27; 28]), EGFP [29], and the tetracycline transactivator ("tTA"); the second is not directly Cre-dependent but simply expresses both G (SAD B19 strain [30]) and the blue fluorophore mTagBFP2 [31] under the control of the tetracycline response element.…”