key: cord-0320544-yy8x8l6u
authors: Han, Yong; He, Yang; Harris, Lauren; Xu, Yong; Wu, Qi
title: Reversal of obesogenic feeding and hypometabolism by a bifurcating GABAergic neural circuit
date: 2022-01-24
journal: bioRxiv
DOI: 10.1101/2022.01.23.477433
sha: 1df58c956b178cf003c4609b6669d0d275769c7b
doc_id: 320544
cord_uid: yy8x8l6u

Diet-induced obesity is characterized by unsatiated consumption of energy-dense diets and impaired metabolism, whereby anti-obesity effect of the high-level of circulating leptin is unknowingly blunted. Emerging evidence suggests that the leptin receptor (LepR) signaling system, residing within the agouti-related protein (AgRP) neurons of the hypothalamus, critically contributes to obesogenic feeding, nutrient partitioning, and energy metabolism. However, the neural circuit mechanism underlying the leptin-dependent control of obesogenic feeding and energy balance remains largely elusive. Here, we show that two distinct subgroups of LepR-expressing AgRP neurons send non-collateral, GABAergic projections to the dorsomedial hypothalamic nucleus (DMH) and to the medial part of the medial preoptic nucleus (MPO) for the differential control of metabolic homeostasis and obesogenic feeding, respectively. We found that the AgRPLepR-DMH neural circuit plays a significant role in leptin-dependent control of metabolic homeostasis through the α3-containing GABAA receptor signaling on the melanocortin 4 receptor neurons within the DMH (MC4RDMH). In contrast, the AgRPLepR-MPO neural circuit elicits dominant effects on the appetitive response to high-fat diet through the α2-containing GABAA receptors on the MC4RMPO neurons. Consistent with these behavioral results, the post-synaptic GABAA neurons located within the DMH and MPO displayed differentiated firing responses under various feeding and nutrient conditions. Our results demonstrate that these novel GABAergic neural circuits exert differentiated control of metabolic hemostasis and obesogenic feeding via distinct post-synaptic targets of leptin-responsive AgRP neurons. The findings of two genetically and anatomically distinct GABAA receptor signaling pathways within the DMH and MPO would undoubtedly accelerate the development of targeted, individualized, anti-obesity therapy.

examined the effect of leptin on the firing of postsynaptic ZsGreen neurons in the DMH.

We found that leptin treatment significantly enhanced neural activities of DMH neurons 157 (Fig.3k,l) . To reveal the hierarchical structure of this neural circuit, results derived from 158 HSV-based dual retrograde tracing showed that two distinct subgroups of AgRP neurons 159 project to the DMH and MPO, respectively ( Fig.3m-p) . Taken together, these results 160 implicated potential functional segregation of the bifurcating AgRP→DMH/MPO neural 161 circuit. 162 To examine the role of the AgRP → DMH circuit in regulation of feeding and 163 metabolism, we performed optogenetic manipulation in Agrp Cre ::Ai32 mice where ChR2-164 eYFP was selectively expressed in AgRP neurons and axonal terminals ( Fig.4a) 29, 30 . 165 Photostimulation of AgRP fibers in the DMH promoted feeding of chow diet but not high- To better understand the contribution of the AgRP→DMH circuit to the control of 177 leptin-mediated functions, we bilaterally injected the AAV9-fDIO-WGA-nsCre virus into 178 the ARC of Npy Flp ::Gad1 lox/lox ::Gad2 lox/lox ::Rosa26 tdTomato mice followed with treatment of 179 NB124 into the DMH, a strategy which can specifically inactivate the GABA signaling from 180 those DMH neurons directly innervated by AgRP neurons. We showed that acute ablation 181 of GABA signaling from the AgRP-projected DMH neurons significantly increased feeding 182 of chow diet without affecting intake of HFD (Fig.4m) . Under the treatment of chow diet, 183 these GABA signaling-deficient mice displayed a moderate but significant increase in 184 body weight coupled with impaired glucose intolerance (Fig.4n,o) . More importantly, inactivation of post-synaptic GABA signaling in the AgRP →DMH circuit blunted the actions of systemically administered leptin on chow diet feeding, body weight, and 187 glucose tolerance (Fig.4m-o) . Overall, these data demonstrate that the GABA signaling 188 within the downstream targets of the AgRP→DMH neural circuit plays a significant role in 189 control of leptin-mediated metabolic homeostasis.

To better understand the physiological responses of these GABAergic neurons in 191 the context of feeding and metabolic regulation, we employed in vivo opto-tetrode system 192 to reveal the dynamic activities of MC4R DMH neurons 73 . With the injection of AAV2- ChR2-GFP into the DMH of Mc4r Cre mice, the ChR2 neurons can be identified by the 194 latencies of evoked spikes accurately following high-frequency photostimulation, as well 195 as the identical waveforms of evoked and spontaneous spikes 73 . We investigated the signaling in the MC4R DMH neurons reduces feeding of chow diet and body weight while 217 the glucose tolerance was significantly improved (Fig.5j -l and Extended Data Fig.2 ).

Meanwhile, genetic deficiency in α3-GABAA signaling significantly decreased feeding 219 efficiency and WAT ( Fig.5m-o) . On the other hand, the gain-of-function study showed that 220 overexpression of α3-GABAA within the same MC4R DMH neurons manifested the exact 221 opposite phenotypes, including moderately increased chow diet feeding and body weight, 222 significantly increased feeding efficiency and WAT adiposity, and profoundly decreased 223 RQ ( Fig.5m-o) . These results suggest that the MC4R DMH neurons play a significant role 224 in control of glucose tolerance, nutrient partitioning, and feeding efficiency through the 225 3-GABAA signaling. To identify the critical GABAA receptor components contributed to these 242 phenotypes, we examined the expression profile of the major regulatory α1-α6 subunits 

It has been demonstrated that GABAA receptor antagonists can prevent 307 abnormalities in leptin actions on paraventricular hypothalamic neurons, which indicates 308 GABAA signaling seems to contribute to a persistently reduced negative feedback of 309 adiposity signals in obese models. Moreover, acute bicuculline administration seems to 310 be able to suppress food intake and prevent the obesity phenotype 77 . Further studies 311 identified the differential roles of subunits of GABAA receptors in the regulation of feeding 312 and body weight. Studies using genetic knock-in mice revealed a strong correlation 313 between individual GABAA subunits and specific phenotypes in BDZ treatment: the α1 314 subunit with sedative and amnesic effects; the α2 subunit with myorelaxant effects, the 315 α3 subunit with anxiolytic effects, and the α5 subunit with neural plasticity and metabolic 316 effects 57,78,79 . Another study implicated that 2/3-containing GABAA receptors contribute 317 to feeding behavior in a hypothalamic circuit 80 . Therefore, it is important to discover how 318 the GABAA subunit in the hypothalamus is involved in the regulation of feeding. We 

e-g, The food 725 intake of LFD (e), body weight (f) and GTT (g) were performed in Mc4r Cre mice with bilateral 726 injection of either vehicle, AAV9-DIO-Gabra2 cDNA -tdTomato (a2 OE) into the MPO. (n = 8 727 per group; *p < 0.05). h, Real-time qPCR analysis of transcript levels of a2 expressed in MC4R MPO 728 neurons isolated by fluorescence-activated cell sorting in the control and a2 OE mice. (n = 8 per 729 group