Probes for VGAT

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.

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.

Torpor is a naturally occurring, hypometabolic, hypothermic state engaged by a wide range of animals in response to imbalance between the supply and demand for nutrients. Recent work has identified some of the key neuronal populations involved in daily torpor induction in mice, in particular projections from the preoptic area of the hypothalamus (POA) to the dorsomedial hypothalamus (DMH). The DMH plays a role in thermoregulation, control of energy expenditure, and circadian rhythms, making it well positioned to contribute to the expression of torpor. We used activity dependent genetic TRAPing techniques to target DMH neurons that were active during natural torpor bouts in female mice. Chemogenetic reactivation of torpor-TRAPed DMH neurons in calorie-restricted mice promoted torpor, resulting in longer and deeper torpor bouts. Chemogenetic inhibition of torpor-TRAPed DMH neurons did not block torpor entry, suggesting a modulatory role for the DMH in the control of torpor. This work adds to the evidence that the POA and the DMH form part of a circuit within the mouse hypothalamus that controls entry into daily torpor.SIGNIFICANCEDaily heterotherms such as mice employ torpor to cope with environments in which the supply of metabolic fuel is not sufficient for the maintenance of normothermia. Daily torpor involves reductions in body temperature, as well as active suppression of heart rate and metabolism. How the central nervous system controls this profound deviation from normal homeostasis is not known, but a projection from the preoptic area to the dorsomedial hypothalamus has recently been implicated. We demonstrate that the dorsomedial hypothalamus contains neurons that are active during torpor. Activity in these neurons promotes torpor entry and maintenance, but their activation alone does not appear to be sufficient for torpor entry.

Background Cannabidiol (CBD) has received attention for the treatment of Substance Use Disorders. In preclinical models of relapse, CBD attenuates drug seeking across several drugs of abuse, including cocaine. However, in these models, CBD has not been consistently effective. This inconsistency in CBD effects may be related to presently insufficient information on the full spectrum of CBD dose effects on drug-related behaviors. Methods We address this issue by establishing a full dose-response profile of CBD’s actions using expression of cocaine-induced conditioned place preference (CPP) as a model for drug motivated behavior in male rats, and by concurrently identifying dose-dependent effects of CBD on underlying neuronal activation as well as distinct neuronal phenotypes showing dose-dependent activation changes. Additionally, CBD levels in plasma and brain were established. Results CBD produced linear increases in CBD brain/plasma concentrations but suppressed CPP in a distinct U-shaped manner. In parallel with its behavioral effects, CBD produced U-shaped suppressant effects on neuronal activation in the prelimbic but not infralimbic cortex or nucleus accumbens core and shell. RNAscope in situ hybridization identified suppression of glutamatergic and GABAergic signaling in the prelimbic cortex as a possible cellular mechanism for the attenuation of cocaine CPP by CBD. Conclusions The findings extend previous evidence on the potential of CBD in preventing drug motivated behavior. However, CBD’s dose-response profile may have important dosing implications for future clinical applications and may contribute to the understanding of discrepant CBD effects on drug seeking in the literature.

Relaxin-3 is a highly conserved neuropeptide abundantly expressed in neurons of the nucleus incertus (NI), which project to nodes of the septohippocampal system (SHS) including the medial septum/diagonal band of Broca (MS/DB) and dorsal hippocampus, as well as to limbic circuits. High densities of the Gi/o-protein-coupled receptor for relaxin-3, known as relaxin-family peptide-3 receptor (RXFP3) are expressed throughout the SHS, further suggesting a role for relaxin-3/RXFP3 signaling in modulating learning and memory processes that occur within these networks. Therefore, this study sought to gain further anatomical and functional insights into relaxin-3/RXFP3 signaling in the mouse MS/DB. Using Cre/LoxP recombination methods, we assessed locomotion, exploratory behavior, and spatial learning and long-term reference memory in adult C57BL/6J Rxfp3 (loxP/loxP) mice with targeted depletion of Rxfp3 in the MS/DB. Following prior injection of an AAV((1/2))-Cre-IRES-eGFP vector into the MS/DB to delete/deplete Rxfp3 mRNA/RXFP3 protein, mice tested in a Morris water maze (MWM) displayed an impairment in allocentric spatial learning during acquisition, as well as an impairment in long-term reference memory on probe day. However, RXFP3-depleted and control mice displayed similar motor activity in a locomotor cell and exploratory behavior in a large open-field (LOF) test. A quantitative characterization using multiplex, fluorescent in situ hybridization (ISH) identified a high level of co-localization of Rxfp3 mRNA and vesicular GABA transporter (vGAT) mRNA in MS and DB neurons (~87% and ~95% co-expression, respectively). Rxfp3 mRNA was also detected, to a correspondingly lesser extent, in vesicular glutamate transporter 2 (vGlut2) mRNA-containing neurons in MS and DB (~13% and ~5% co-expression, respectively). Similarly, a qualitative assessment of the MS/DB region, identified Rxfp3 mRNA in neurons that expressed parvalbumin (PV) mRNA (reflecting hippocampally-projecting GABA neurons), whereas choline acetyltransferase mRNA-positive (acetylcholine) neurons lacked Rxfp3 mRNA. These data are consistent with a qualitative immunohistochemical analysis that revealed relaxin-3-immunoreactive nerve fibers in close apposition with PV-immunoreactive neurons in the MS/DB. Together these studies suggest relaxin-3/RXFP3 signaling in the MS/DB plays a role in modulating specific learning and long-term memory associated behaviors in adult mice via effects on GABAergic neuron populations known for their involvement in modulating hippocampal theta rhythm and associated cognitive processes.