A Novel Population of Wake-Promoting GABAergic Neurons in the Ventral Lateral Hypothalamus

Abstract

The largest synaptic input to the sleep-promoting ventrolateral preoptic area (VLPO) [1] arises from the lateral hypothalamus [2], a brain area associated with arousal [3-5]. However, the neurochemical identity of the majority of these VLPO-projecting neurons within the lateral hypothalamus (LH), as well as their function in the arousal network, remains unknown. Herein we describe a population of VLPO-projecting neurons in the LH that express the vesicular GABA transporter (VGAT; a marker for GABA-releasing neurons). In addition to the VLPO, these neurons also project to several other established sleep and arousal nodes, including the tuberomammillary nucleus, ventral periaqueductal gray, and locus coeruleus. Selective and acute chemogenetic activation of LH VGAT(+) neurons was profoundly wake promoting, whereas acute inhibition increased sleep. Because of its direct and massive inputs to the VLPO, this population may play a particularly important role in sleep-wake switching.

Figure 1. Conditional anterograde tracing from LH VGAT+ neurons

A) Schema of ChR2-eYFP construct and viral targeting to the LH of VGAT-ires-Cre mice. B1) Immunolabelling for eYFP (brown) in cell bodies in the LH, counterstained with Nissl (blue), arrows indicate ChR2-eYFP cell bodies. Scale: 200 μm. B2) Low magnification photomicrograph indicating injection site of ChR2-eYFP (green) in a LH brain section immunolabelled against MCH (magenta) and orexin-A (blue). eYFP labeled cells did not stain for either orexin-A or MCH. B3) High magnification of boxed area in B2, arrows indicate ChR2-eYFP cell bodies. Scale: 100 μm. C1–I1) eYFP immunoreactivity of ChR2-eYFP terminals in the VLPO, TMN, vPAG, LC, RTN (anterior; H1, posterior; I1) and PZ. Scale bar: 100 μm. C2–F2: High magnification of boxed areas shown in panels to the right. Scale bar: 20 μm. H2–I2: High magnification of dorsal boxed areas shown in H1 and I1. Scale bar: 20 μm. H3–I3: High magnification of ventral boxed areas shown in H1 and I1. Scale bar: 20 μm. Abbreviations: 3V; 3^rd ventricle, 4V; 4^th ventricle, 7N; facial nerve, aq; cerebral aqueduct, BSTS; bed nucleus of stria terminalis supracapsular part, cp; cerebellar peduncle, ec; external capsule, ic; internal capsule, fx; fornix, LC; locus coeruleus, LV; lateral ventricle, Me5; mesencephalic trigeminal nucleus, mt; mammillothalamic tract, opt; optic tract, ox; optic chiasm, PZ; parafacial zone, RTN; thalamic reticular nucleus, scp; superior cerebellar peduncle, VLPO; ventrolateral preoptic area, vPAG; ventral periaqueductal gray, vTMN; ventral tuberomammillary nucleus xscp; decussation of the superior cerebellar peduncle. See also Figure [S1].

Figure 2. Conditional activation of LH VGAT+ neurons drives wakefulness

A) Schema of hM3Dq-mCherry construct and viral targeting to the LH of VGAT-ires-Cre mice. B) Dual immunolabelling against mCherry (brown) and c-Fos in LH VGAT+ cell bodies following injection of saline (B1) or CNO (B2) at 10AM, 90 minutes prior to sacrifice demonstrates that CNO activates neurons transduced with hM3Dq-mCherry, scale: 200μm. c) Percentage 3-hourly wake (C1), NREM sleep (C2) and REM sleep (C3) quantities following injection (n=8). Repeated measures 2 –way ANOVA followed by Sidak post-hoc test. D) FFT analysis of wake during CNO activation (n=8), showing a ratio of power at each frequency post-injection compared with baseline. Shaded area indicates SEM. Inserts show averaged frequency bands. Repeated measures 2 –way ANOVA followed by Sidak post-hoc test. E) Quantification of behaviors observed in mice during 1 hour following 10am CNO injection or 7pm saline injection. Bars represent the mean percent of time (± SEM) that mice (n=8) spent carrying out each behavior. 2-way ANOVA followed by Sidak post-hoc test. F) Quantification of c-Fos immunostaining in the VLPO, TMN and LC following saline injection at 10AM (n = 11) or CNO injection at 10AM (n = 12). Mean ± SEM shown. 2-way ANOVA followed by Sidak post-hoc test. G) Heat map showing overlapping regions of transfected neurons from mice in which unilateral LH VGAT+ activation increased wakefulness., * p < 0.05,** p < 0.01, **** p < 0.0001, See also Figure [S2] and [S3]. Abbreviations: 3V; 3^rd ventricle, ec; external capsule, fx; fornix, LC; locus coeruleus, LH; lateral hypothalamus, mt; mammillothalamic tract, ic; internal capsule, opt; optic tract, TMN; tuberomammillary nucleus, VLPO; ventrolateral preoptic area.

Figure 3. Conditional inhibition of LH VGAT+ neurons increases sleep

A) Schema of hM4Di-mCherry construct and viral targeting to the LH of VGAT-ires-Cre mice. B) Dual immunolabelling against mCherry (brown) and c-Fos in LH VGAT+ cell bodies following injection of saline (B1) or CNO (B2) at 7PM, 90 minutes prior to sacrifice. Red arrows show c-Fos and mCherry immunolabelled neurons, white arrows show neurons immunolabelled for mCherry only. CNO nearly eliminated c-Fos expression in LH VGAT+ neurons, indicating that they were inhibited, scale: 200μm. Percentage 3-hourly wake (C1), NREM sleep (C2) and REM sleep (C3) quantities following injection (n=9). Repeated measures 2 –way ANOVA, followed by Sidak post-hoc test. d) FFT analysis of EEG During wake, showing the ratio of power at each frequency post-injection compared to baseline (n = 9). Shaded area indicates SEM. Inserts show averaged frequency bands. Repeated measures 2 –way ANOVA, followed by Sidak post-hoc test, ** p < 0.01, **** p < 0.0001, R. Abbreviations: 3V; 3^rd ventricle, ec; external capsule, fx; fornix, LH; lateral hypothalamus, mt; mammillothalamic tract, ic; internal capsule, opt; optic tract.