Probes for GAL

Humans and animals lacking orexin neurons exhibit daytime sleepiness, sleep attacks, and state instability. While the circuit basis by which orexin neurons contribute to consolidated wakefulness remains unclear, existing models posit that orexin neurons provide their wake-stabilizing influence by exerting excitatory tone on other brain arousal nodes. Here we show using in vivo optogenetics, in vitro optogenetic-based circuit mapping, and single-cell transcriptomics that orexin neurons also contribute to arousal maintenance through indirect inhibition of sleep-promoting neurons of the ventrolateral preoptic nucleus. Activation of this subcortical circuit rapidly drives wakefulness from sleep by differentially modulating the activity of ventrolateral preoptic neurons. We further identify and characterize a feedforward circuit through which orexin (and co-released glutamate) acts to indirectly target and inhibit sleep-promoting ventrolateral preoptic neurons to produce arousal. This revealed circuitry provides an alternate framework for understanding how orexin neurons contribute to the maintenance of consolidated wakefulness and stabilize behavioral state.

Single-cell RNA sequencing data can unveil the molecular diversity of cell types. Cell type atlases of the mouse spinal cord have been published in recent years but have not been integrated together. Here, we generate an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework. We report a hierarchical structure of postnatal cell type relationships, with location providing the highest level of organization, then neurotransmitter status, family, and finally, dozens of refined populations. We validate a combinatorial marker code for each neuronal cell type and map their spatial distributions in the adult spinal cord. We also show complex lineage relationships among postnatal cell types. Additionally, we develop an open-source cell type classifier, SeqSeek, to facilitate the standardization of cell type identification. This work provides an integrated view of spinal cell types, their gene expression signatures, and their molecular organization.

Light regulates daily sleep rhythms by a neural circuit that connects intrinsically photosensitive retinal ganglion cells (ipRGCs) to the circadian pacemaker, the suprachiasmatic nucleus. Light, however, also acutely affects sleep in a circadian-independent manner. The neural circuits involving the acute effect of light on sleep remain unknown. Here we uncovered a neural circuit that drives this acute light response, independent of the suprachiasmatic nucleus, but still through ipRGCs. We show that ipRGCs substantially innervate the preoptic area (POA) to mediate the acute light effect on sleep in mice. Consistently, activation of either the POA projecting ipRGCs or the light-responsive POA neurons increased non-rapid eye movement (NREM) sleep without influencing REM sleep. In addition, inhibition of the light-responsive POA neurons blocked the acute light effects on NREM sleep. The predominant light-responsive POA neurons that receive ipRGC input belong to the corticotropin-releasing hormone subpopulation. Remarkably, the light-responsive POA neurons are inhibitory and project to well-known wakefulness-promoting brain regions, such as the tuberomammillary nucleus and the lateral hypothalamus. Therefore, activation of the ipRGC-POA circuit inhibits arousal brain regions to drive light-induced NREM sleep. Our findings reveal a functional retina-brain circuit that is both necessary and sufficient for the acute effect of light on sleep.

Sodium deficiency elevates aldosterone, which in addition to epithelial tissues acts on the brain to promote dysphoric symptoms and salt intake. Aldosterone boosts the activity of neurons that express 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a hallmark of aldosterone-sensitive cells. To better characterize these neurons, we combine immunolabeling and in situ hybridization with fate mapping and Cre-conditional axon tracing in mice. Many cells throughout the brain have a developmental history of Hsd11b2 expression, but in the adult brain one small brainstem region with a leaky blood-brain barrier contains HSD2 neurons. These neurons express Hsd11b2, Nr3c2 (mineralocorticoid receptor), Agtr1a (angiotensin receptor), Slc17a6 (vesicular glutamate transporter 2), Phox2b, and Nxph4; many also express Cartpt or Lmx1b. No HSD2 neurons express cholinergic, monoaminergic, or several other neuropeptidergic markers. Their axons project to the parabrachial complex (PB), where they intermingle with AgRP-immunoreactive axons to form dense terminal fields overlapping FoxP2 neurons in the central lateral subnucleus (PBcL) and pre-locus coeruleus (pLC). Their axons also extend to the forebrain, intermingling with AgRP- and CGRP-immunoreactive axons to form dense terminals surrounding GABAergic neurons in the ventrolateral bed nucleus of the stria terminalis (BSTvL). Sparse axons target the periaqueductal gray, ventral tegmental area, lateral hypothalamic area, paraventricular hypothalamic nucleus, and central nucleus of the amygdala. Dual retrograde tracing revealed that largely separate HSD2 neurons project to pLC/PB or BSTvL. This projection pattern raises the possibility that a subset of HSD2 neurons promotes the dysphoric, anorexic, and anhedonic symptoms of hyperaldosteronism via AgRP-inhibited relay neurons in PB.

To understand how sleep-wakefulness cycles are regulated, it is essential to disentangle structural and functional relationships between the preoptic area (POA) and lateral hypothalamic area (LHA), since these regions play important yet opposing roles in the sleep-wakefulness regulation. GABA- and galanin (GAL)-producing neurons in the ventrolateral preoptic nucleus (VLPO) of the POA (VLPOGABA and VLPOGAL neurons) are responsible for the maintenance of sleep, while the LHA contains orexin-producing neurons (orexin neurons) that are crucial for maintenance of wakefulness. Through the use of rabies virus-mediated neural tracing combined with in situ hybridization (ISH) in male and female orexin-iCre mice, we revealed that the vesicular GABA transporter (Vgat, Slc32a1)- and galanin (Gal)-expressing neurons in the VLPO directly synapse with orexin neurons in the LHA. A majority (56.3 ± 8.1%) of all VLPO input neurons connecting to orexin neurons were double-positive for Vgat and Gal Using projection-specific rabies virus-mediated tracing in male and female Vgat-ires-Cre and Gal-Cre mice, we discovered that VLPOGABA and VLPOGAL neurons that send projections to the LHA received innervations from similarly distributed input neurons in many brain regions, with the POA and LHA being among the main upstream areas. Additionally, we found that acute optogenetic excitation of axons of VLPOGABA neurons, but not VLPOGAL neurons, in the LHA of male Vgat-ires-Cre mice induced wakefulness. This study deciphers the connectivity between the VLPO and LHA, provides a large-scale map of upstream neuronal populations of VLPO→LHA neurons, and reveals a previously uncovered function of the VLPOGABA→LHA pathway in the regulation of sleep and wakefulness.SIGNIFICANCE STATEMENT We identified neurons in the ventrolateral preoptic nucleus (VLPO) that are positive for vesicular GABA transporter (Vgat) and/or galanin (Gal) and serve as presynaptic partners of orexin-producing neurons in the lateral hypothalamic area (LHA). We depicted monosynaptic input neurons of GABA- and galanin-producing neurons in the VLPO that send projections to the LHA throughout the entire brain. Their input neurons largely overlap, suggesting that they comprise a common neuronal population. However, acute excitatory optogenetic manipulation of the VLPOGABA→LHA pathway, but not the VLPOGAL→LHA pathway, evoked wakefulness. This study shows the connectivity of major components of the sleep/wake circuitry in the hypothalamus and unveils a previously unrecognized function of the VLPOGABA→LHA pathway in sleep-wakefulness regulation. Furthermore, we suggest the existence of subpopulations of VLPOGABA neurons that innervate LHA.