GABAergic disinhibition from the BNST to PNOCARC neurons promotes HFD-induced hyperphagia.

Activation of prepronociceptin (PNOC)-expressing neurons in the arcuate nucleus (ARC) promotes high-fat-diet (HFD)-induced hyperphagia. In turn, PNOCARC neurons can inhibit the anorexic response of proopiomelanocortin (POMC) neurons. Here, we validate the necessity of PNOCARC activity for HFD-induced inhibition of POMC neurons in mice and find that PNOCARC-neuron-dependent inhibition of POMC neurons is mediated by gamma-aminobutyric acid (GABA) release. When monitoring individual PNOCARC neuron activity via Ca2+ imaging, we find a subpopulation of PNOCARC neurons that is inhibited upon gastrointestinal calorie sensing and disinhibited upon HFD feeding. Combining retrograde rabies tracing and circuit mapping, we find that PNOC neurons from the bed nucleus of the stria terminalis (PNOCBNST) provide inhibitory input to PNOCARC neurons, and this inhibitory input is blunted upon HFD feeding. This work sheds light on how an increase in caloric content of the diet can rewire a neuronal circuit, paving the way to overconsumption and obesity development.

Hypothalamic Pomc Neurons Innervate the Spinal Cord and Modulate the Excitability of Premotor Circuits.

Locomotion requires energy, yet animals need to increase locomotion in order to find and consume food in energy-deprived states. While such energy homeostatic coordination suggests brain origin, whether the central melanocortin 4 receptor (Mc4r) system directly modulates locomotion through motor circuits is unknown. Here, we report that hypothalamic Pomc neurons in zebrafish and mice have long-range projections into spinal cord regions harboring Mc4r-expressing V2a interneurons, crucial components of the premotor networks. Furthermore, in zebrafish, Mc4r activation decreases the excitability of spinal V2a neurons as well as swimming and foraging, while systemic or V2a neuron-specific blockage of Mc4r promotes locomotion. In contrast, in mice, electrophysiological recordings revealed that two-thirds of V2a neurons in lamina X are excited by the Mc4r agonist α-MSH, and acute inhibition of Mc4r signaling reduces locomotor activity. In addition, we found other Mc4r neurons in spinal lamina X that are inhibited by α-MSH, which is in line with previous studies in rodents where Mc4r agonists reduced locomotor activity. Collectively, our studies identify spinal V2a interneurons as evolutionary conserved second-order neurons of the central Mc4r system, providing a direct anatomical and functional link between energy homeostasis and locomotor control systems. The net effects of this modulatory system on locomotor activity can vary between different vertebrate species and, possibly, even within one species. We discuss the biological sense of this phenomenon in light of the ambiguity of locomotion on energy balance and the different living conditions of the different species.

Differential Coassembly of α1-GABAARs Associated with Epileptic Encephalopathy.

GABAA receptors (GABAARs) are profoundly important for controlling neuronal excitability. Spontaneous and familial mutations to these receptors feature prominently in excitability disorders and neurodevelopmental deficits following disruption to GABA-mediated inhibition. Recent genotyping of an individual with severe epilepsy and Williams-Beuren syndrome identified a frameshifting de novo variant in a major GABAAR gene, GABRA1 This truncated the α1 subunit between the third and fourth transmembrane domains and introduced 24 new residues forming the mature protein, α1Lys374Serfs*25 Cell surface expression of mutant murine GABAARs is severely impaired compared with WT, due to retention in the endoplasmic reticulum. Mutant receptors were differentially coexpressed with ß3, but not with ß2, subunits in mammalian cells. Reduced surface expression was reflected by smaller IPSCs, which may underlie the induction of seizures. The mutant does not have a dominant-negative effect on native neuronal GABAAR expression since GABA current density was unaffected in hippocampal neurons, although mutant receptors exhibited limited GABA sensitivity. To date, the underlying mechanism is unique for epileptogenic variants and involves differential ß subunit expression of GABAAR populations, which profoundly affected receptor function and synaptic inhibition.SIGNIFICANCE STATEMENT GABAARs are critical for controlling neural network excitability. They are ubiquitously distributed throughout the brain, and their dysfunction underlies many neurologic disorders, especially epilepsy. Here we report the characterization of an α1-GABAAR variant that results in severe epilepsy. The underlying mechanism is structurally unusual, with the loss of part of the α1 subunit transmembrane domain and part-replacement with nonsense residues. This led to compromised and differential α1 subunit cell surface expression with ß subunits resulting in severely reduced synaptic inhibition. Our study reveals that disease-inducing variants can affect GABAAR structure, and consequently subunit assembly and cell surface expression, critically impacting on the efficacy of synaptic inhibition, a property that will orchestrate the extent and duration of neuronal excitability.

PNOCARC Neurons Promote Hyperphagia and Obesity upon High-Fat-Diet Feeding.

Calorie-rich diets induce hyperphagia and promote obesity, although the underlying mechanisms remain poorly defined. We find that short-term high-fat-diet (HFD) feeding of mice activates prepronociceptin (PNOC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC). PNOCARC neurons represent a previously unrecognized GABAergic population of ARC neurons distinct from well-defined feeding regulatory AgRP or POMC neurons. PNOCARC neurons arborize densely in the ARC and provide inhibitory synaptic input to nearby anorexigenic POMC neurons. Optogenetic activation of PNOCARC neurons in the ARC and their projections to the bed nucleus of the stria terminalis promotes feeding. Selective ablation of these cells promotes the activation of POMC neurons upon HFD exposure, reduces feeding, and protects from obesity, but it does not affect food intake or body weight under normal chow consumption. We characterize PNOCARC neurons as a novel ARC neuron population activated upon palatable food consumption to promote hyperphagia.

GABAAR isoform and subunit structural motifs determine synaptic and extrasynaptic receptor localisation.

GABAA receptors (GABAARs) are the principal inhibitory neurotransmitter receptors in the central nervous system. They control neuronal excitability by synaptic and tonic forms of inhibition mostly mediated by different receptor subtypes located in specific cell membrane subdomains. A consensus suggests that α1-3ßγ comprise synaptic GABAARs, whilst extrasynaptic α4ßδ, α5ßγ and αß isoforms largely underlie tonic inhibition. Although some structural features that enable the spatial segregation of receptors are known, the mobility of key synaptic and extrasynaptic GABAARs are less understood, and yet this is a key determinant of the efficacy of GABA inhibition. To address this aspect, we have incorporated functionally silent α-bungarotoxin binding sites (BBS) into prominent hippocampal GABAAR subunits which mediate synaptic and tonic inhibition. Using single particle tracking with quantum dots we demonstrate that GABAARs that are traditionally considered to mediate synaptic or tonic inhibition are all able to access inhibitory synapses. These isoforms have variable diffusion rates and are differentially retained upon entering the synaptic membrane subdomain. Interestingly, α2 and α4 subunits reside longer at synapses compared to α5 and δ subunits. Furthermore, a high proportion of extrasynaptic δ-containing receptors exhibited slower diffusion compared to δ subunits at synapses. A chimera formed from δ-subunits, with the intracellular domain of γ2L, reversed this behaviour. In addition, we observed that receptor activation affected the diffusion of extrasynaptic, but not of synaptic GABAARs. Overall, we conclude that the differential mobility profiles of key synaptic and extrasynaptic GABAARs are determined by receptor subunit composition and intracellular structural motifs. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.