Activation of GFRAL+ neurons induces hypothermia and glucoregulatory responses associated with nausea and torpor.

GFRAL-expressing neurons actuate aversion and nausea, are targets for obesity treatment, and may mediate metformin effects by long-term GDF15-GFRAL agonism. Whether GFRAL+ neurons acutely regulate glucose and energy homeostasis is, however, underexplored. Here, we report that cell-specific activation of GFRAL+ neurons using a variety of techniques causes a torpor-like state, including hypothermia, the release of stress hormones, a shift from glucose to lipid oxidation, and impaired insulin sensitivity, glucose tolerance, and skeletal muscle glucose uptake but augmented glucose uptake in visceral fat. Metabolomic analysis of blood and transcriptomics of muscle and fat indicate alterations in ketogenesis, insulin signaling, adipose tissue differentiation and mitogenesis, and energy fluxes. Our findings indicate that acute GFRAL+ neuron activation induces endocrine and gluco- and thermoregulatory responses associated with nausea and torpor. While chronic activation of GFRAL signaling promotes weight loss in obesity, these results show that acute activation of GFRAL+ neurons causes hypothermia and hyperglycemia.

An Approach to Intersectionally Target Mature Enteroendocrine Cells in the Small Intestine of Mice.

Enteroendocrine cells (EECs) constitute only a small proportion of Villin-1 (Vil1)-expressing intestinal epithelial cells (IECs) of the gastrointestinal tract; yet, in sum, they build the largest endocrine organ of the body, with each of them storing and releasing a distinct set of peptides for the control of feeding behavior, glucose metabolism, and gastrointestinal motility. Like all IEC types, EECs are continuously renewed from intestinal stem cells in the crypt base and terminally differentiate into mature subtypes while moving up the crypt-villus axis. Interestingly, EECs adjust their hormonal secretion according to their migration state as EECs receive altering differentiation signals along the crypt-villus axis and thus undergo functional readaptation. Cell-specific targeting of mature EEC subtypes by specific promoters is challenging because the expression of EEC-derived peptides and their precursors is not limited to EECs but are also found in other organs, such as the brain (e.g., Cck and Sst) as well as in the pancreas (e.g., Sst and Gcg). Here, we describe an intersectional genetic approach that enables cell type-specific targeting of functionally distinct EEC subtypes by combining a newly generated Dre-recombinase expressing mouse line (Vil1-2A-DD-Dre) with multiple existing Cre-recombinase mice and mouse strains with rox and loxP sites flanked stop cassettes for transgene expression. We found that transgene expression in triple-transgenic mice is highly specific in I but not D and L cells in the terminal villi of the small intestine. The targeting of EECs only in terminal villi is due to the integration of a defective 2A separating peptide that, combined with low EEC intrinsic Vil1 expression, restricts our Vil1-2A-DD-Dre mouse line and the intersectional genetic approach described here only applicable for the investigation of mature EEC subpopulations.

Vagal pathways for systemic regulation of glucose metabolism.

Maintaining blood glucose at an appropriate physiological level requires precise coordination of multiple organs and tissues. The vagus nerve bidirectionally connects the central nervous system with peripheral organs crucial to glucose mobilization, nutrient storage, and food absorption, thereby presenting a key pathway for the central control of blood glucose levels. However, the precise mechanisms by which vagal populations that target discrete tissues participate in glucoregulation are much less clear. Here we review recent advances unraveling the cellular identity, neuroanatomical organization, and functional contributions of both vagal efferents and vagal afferents in the control of systemic glucose metabolism. We focus on their involvement in relaying glucoregulatory cues from the brain to peripheral tissues, particularly the pancreatic islet, and by sensing and transmitting incoming signals from ingested food to the brain. These recent findings - largely driven by advances in viral approaches, RNA sequencing, and cell-type selective manipulations and tracings - have begun to clarify the precise vagal neuron populations involved in the central coordination of glucose levels, and raise interesting new possibilities for the treatment of glucose metabolism disorders such as diabetes.

Integrative neurocircuits that control metabolism and food intake.

Systemic metabolism has to be constantly adjusted to the variance of food intake and even be prepared for anticipated changes in nutrient availability. Therefore, the brain integrates multiple homeostatic signals with numerous cues that predict future deviations in energy supply. Recently, our understanding of the neural pathways underlying these regulatory principles-as well as their convergence in the hypothalamus as the key coordinator of food intake, energy expenditure, and glucose metabolism-have been revealed. These advances have changed our view of brain-dependent control of metabolic physiology. In this Review, we discuss new concepts about how alterations in these pathways contribute to the development of prevalent metabolic diseases such as obesity and type 2 diabetes mellitus and how this emerging knowledge may provide new targets for their treatment.

Advanced neurobiological tools to interrogate metabolism.

Engineered neurobiological tools for the manipulation of cellular activity, such as chemogenetics and optogenetics, have become a cornerstone of modern neuroscience research. These tools are invaluable for the interrogation of the central control of metabolism as they provide a direct means to establish a causal relationship between brain activity and biological processes at the cellular, tissue and organismal levels. The utility of these methods has grown substantially due to advances in cellular-targeting strategies, alongside improvements in the resolution and potency of such tools. Furthermore, the potential to recapitulate endogenous cellular signalling has been enriched by insights into the molecular signatures and activity dynamics of discrete brain cell types. However, each modulatory tool has a specific set of advantages and limitations; therefore, tool selection and suitability are of paramount importance to optimally interrogate the cellular and circuit-based underpinnings of metabolic outcomes within the organism. Here, we describe the key principles and uses of engineered neurobiological tools. We also highlight inspiring applications and outline critical considerations to be made when using these tools within the field of metabolism research. We contend that the appropriate application of these biotechnological advances will enable the delineation of the central circuitry regulating systemic metabolism with unprecedented potential.

Neural gut-to-brain communication for postprandial control of satiation and glucose metabolism.

The brain is tuned to integrate food-derived signals from the gut, allowing it to accurately adjust behavioral and physiological responses in accordance with nutrient availability. A key element of gut-to-brain communication is the relay of neural cues via peripheral sensory neurons (PSN) which harbor functionally specialized peripheral endings innervating the muscular and mucosal layers of gastrointestinal (GI) tract organs. In this review, we detail the properties of GI tract innervating PSN and describe their roles in regulating satiation and glucose metabolism in response to food consumption. We discuss the complex anatomical organization of vagal and spinal PSN subtypes, their peripheral and central projection patterns, and describe the limitations of unselective lesion and ablation approaches to investigate them. We then highlight the recent identification of molecular markers that allow selective targeting of PSN subtypes that innervate GI tract organs. This has facilitated accurately determining their projections, monitoring their responses to gut stimuli, and manipulating their activity. We contend that these recent developments have significantly improved our understanding of PSN-mediated gut-to-brain communication, which may open new therapeutic windows for the treatment of metabolic disorders, such as obesity and type 2 diabetes.

A synaptic amplifier of hunger for regaining body weight in the hypothalamus.

Restricting caloric intake effectively reduces body weight, but most dieters fail long-term adherence to caloric deficit and eventually regain lost weight. Hypothalamic circuits that control hunger drive critically determine body weight; yet, how weight loss sculpts these circuits to motivate food consumption until lost weight is regained remains unclear. Here, we probe the contribution of synaptic plasticity in discrete excitatory afferents on hunger-promoting AgRP neurons. We reveal a crucial role for activity-dependent, remarkably long-lasting amplification of synaptic activity originating from paraventricular hypothalamus thyrotropin-releasing (PVHTRH) neurons in long-term body weight control. Silencing PVHTRH neurons inhibits the potentiation of excitatory input to AgRP neurons and diminishes concomitant regain of lost weight. Brief stimulation of the pathway is sufficient to enduringly potentiate this glutamatergic hunger synapse and triggers an NMDAR-dependent gaining of body weight that enduringly persists. Identification of this activity-dependent synaptic amplifier provides a previously unrecognized target to combat regain of lost weight.

HypoMap-a unified single-cell gene expression atlas of the murine hypothalamus.

The hypothalamus plays a key role in coordinating fundamental body functions. Despite recent progress in single-cell technologies, a unified catalog and molecular characterization of the heterogeneous cell types and, specifically, neuronal subtypes in this brain region are still lacking. Here, we present an integrated reference atlas, 'HypoMap,' of the murine hypothalamus, consisting of 384,925 cells, with the ability to incorporate new additional experiments. We validate HypoMap by comparing data collected from Smart-Seq+Fluidigm C1 and bulk RNA sequencing of selected neuronal cell types with different degrees of cellular heterogeneity. Finally, via HypoMap, we identify classes of neurons expressing glucagon-like peptide-1 receptor (Glp1r) and prepronociceptin (Pnoc), and validate them using single-molecule in situ hybridization. Collectively, HypoMap provides a unified framework for the systematic functional annotation of murine hypothalamic cell types, and it can serve as an important platform to unravel the functional organization of hypothalamic neurocircuits and to identify druggable targets for treating metabolic disorders.

Technical Note: Modulation of fMRI brainstem responses by transcutaneous vagus nerve stimulation.

Our increasing knowledge about gut-brain interaction is revolutionising the understanding of the links between digestion, mood, health, and even decision making in our everyday lives. In support of this interaction, the vagus nerve is a crucial pathway transmitting diverse gut-derived signals to the brain to monitor of metabolic status, digestive processes, or immune control to adapt behavioural and autonomic responses. Hence, neuromodulation methods targeting the vagus nerve are currently explored as a treatment option in a number of clinical disorders, including diabetes, chronic pain, and depression. The non-invasive variant of vagus nerve stimulation (VNS), transcutaneous auricular VNS (taVNS), has been implicated in both acute and long-lasting effects by modulating afferent vagus nerve target areas in the brain. The physiology of neither of those effects is, however, well understood, and evidence for neuronal response upon taVNS in vagal afferent projection regions in the brainstem and its downstream targets remain to be established. Therefore, to examine time-dependent effects of taVNS on brainstem neuronal responses in healthy human subjects, we applied taVNS during task-free fMRI in a single-blinded crossover design. During fMRI data acquisition, we either stimulated the left earlobe (sham), or the target zone of the auricular branch of the vagus nerve in the outer ear (cymba conchae, verum) for several minutes, both followed by a short 'stimulation OFF' period. Time-dependent effects were assessed by averaging the BOLD response for consecutive 1-minute periods in an ROI-based analysis of the brainstem. We found a significant response to acute taVNS stimulation, relative to the control condition, in downstream targets of vagal afferents, including the nucleus of the solitary tract, the substantia nigra, and the subthalamic nucleus. Most of these brainstem regions remarkably showed increased activity in response to taVNS, and these effect sustained during the post-stimulation period. These data demonstrate that taVNS activates key brainstem regions, and highlight the potential of this approach to modulate vagal afferent signalling. Furthermore, we show that carry-over effects need to be considered when interpreting fMRI data in the context of general vagal neurophysiology and its modulation by taVNS.

Gut-brain communication by distinct sensory neurons differently controls feeding and glucose metabolism.

Sensory neurons relay gut-derived signals to the brain, yet the molecular and functional organization of distinct populations remains unclear. Here, we employed intersectional genetic manipulations to probe the feeding and glucoregulatory function of distinct sensory neurons. We reconstruct the gut innervation patterns of numerous molecularly defined vagal and spinal afferents and identify their downstream brain targets. Bidirectional chemogenetic manipulations, coupled with behavioral and circuit mapping analysis, demonstrated that gut-innervating, glucagon-like peptide 1 receptor (GLP1R)-expressing vagal afferents relay anorexigenic signals to parabrachial nucleus neurons that control meal termination. Moreover, GLP1R vagal afferent activation improves glucose tolerance, and their inhibition elevates blood glucose levels independent of food intake. In contrast, gut-innervating, GPR65-expressing vagal afferent stimulation increases hepatic glucose production and activates parabrachial neurons that control normoglycemia, but they are dispensable for feeding regulation. Thus, distinct gut-innervating sensory neurons differentially control feeding and glucoregulatory neurocircuits and may provide specific targets for metabolic control.

Functionally distinct POMC-expressing neuron subpopulations in hypothalamus revealed by intersectional targeting.

Pro-opiomelanocortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus represent key regulators of metabolic homeostasis. Electrophysiological and single-cell sequencing experiments have revealed a remarkable degree of heterogeneity of these neurons. However, the exact molecular basis and functional consequences of this heterogeneity have not yet been addressed. Here, we have developed new mouse models in which intersectional Cre/Dre-dependent recombination allowed for successful labeling, translational profiling and functional characterization of distinct POMC neurons expressing the leptin receptor (Lepr) and glucagon like peptide 1 receptor (Glp1r). Our experiments reveal that POMCLepr+ and POMCGlp1r+ neurons represent largely nonoverlapping subpopulations with distinct basic electrophysiological properties. They exhibit a specific anatomical distribution within the arcuate nucleus and differentially express receptors for energy-state communicating hormones and neurotransmitters. Finally, we identify a differential ability of these subpopulations to suppress feeding. Collectively, we reveal a notably distinct functional microarchitecture of critical metabolism-regulatory neurons.

The Role of Mediobasal Hypothalamic PACAP in the Control of Body Weight and Metabolism.

Body energy homeostasis results from balancing energy intake and energy expenditure. Central nervous system administration of pituitary adenylate cyclase activating polypeptide (PACAP) dramatically alters metabolic function, but the physiologic mechanism of this neuropeptide remains poorly defined. PACAP is expressed in the mediobasal hypothalamus (MBH), a brain area essential for energy balance. Ventromedial hypothalamic nucleus (VMN) neurons contain, by far, the largest and most dense population of PACAP in the medial hypothalamus. This region is involved in coordinating the sympathetic nervous system in response to metabolic cues in order to re-establish energy homeostasis. Additionally, the metabolic cue of leptin signaling in the VMN regulates PACAP expression. We hypothesized that PACAP may play a role in the various effector systems of energy homeostasis, and tested its role by using VMN-directed, but MBH encompassing, adeno-associated virus (AAVCre) injections to ablate Adcyap1 (gene coding for PACAP) in mice (Adcyap1MBHKO mice). Adcyap1MBHKO mice rapidly gained body weight and adiposity, becoming hyperinsulinemic and hyperglycemic. Adcyap1MBHKO mice exhibited decreased oxygen consumption (VO2), without changes in activity. These effects appear to be due at least in part to brown adipose tissue (BAT) dysfunction, and we show that PACAP-expressing cells in the MBH can stimulate BAT thermogenesis. While we observed disruption of glucose clearance during hyperinsulinemic/euglycemic clamp studies in obese Adcyap1MBHKO mice, these parameters were normal prior to the onset of obesity. Thus, MBH PACAP plays important roles in the regulation of metabolic rate and energy balance through multiple effector systems on multiple time scales, which highlight the diverse set of functions for PACAP in overall energy homeostasis.

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.

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.

NPY mediates the rapid feeding and glucose metabolism regulatory functions of AgRP neurons.

Activation of Agouti-Related Peptide (AgRP)-expressing neurons promotes feeding and insulin resistance. Here, we examine the contribution of neuropeptide Y (NPY)-dependent signaling to the diverse physiological consequences of activating AgRP neurons. NPY-deficient mice fail to rapidly increase food intake during the first hour of either chemo- or optogenetic activation of AgRP neurons, while the delayed increase in feeding is comparable between control and NPY-deficient mice. Acutely stimulating AgRP neurons fails to induce systemic insulin resistance in NPY-deficient mice, while increased locomotor activity upon AgRP neuron stimulation in the absence of food remains unaffected in these animals. Selective re-expression of NPY in AgRP neurons attenuates the reduced feeding response and reverses the protection from insulin resistance upon optogenetic activation of AgRP neurons in NPY-deficient mice. Collectively, these experiments reveal a pivotal role of NPY-dependent signaling in mediating the rapid feeding inducing effect and the acute glucose regulatory function governed by AgRP neurons.

Food Perception Primes Hepatic ER Homeostasis via Melanocortin-Dependent Control of mTOR Activation.

Adaptation of liver to the postprandial state requires coordinated regulation of protein synthesis and folding aligned with changes in lipid metabolism. Here we demonstrate that sensory food perception is sufficient to elicit early activation of hepatic mTOR signaling, Xbp1 splicing, increased expression of ER-stress genes, and phosphatidylcholine synthesis, which translate into a rapid morphological ER remodeling. These responses overlap with those activated during refeeding, where they are maintained and constantly increased upon nutrient supply. Sensory food perception activates POMC neurons in the hypothalamus, optogenetic activation of POMC neurons activates hepatic mTOR signaling and Xbp1 splicing, whereas lack of MC4R expression attenuates these responses to sensory food perception. Chemogenetic POMC-neuron activation promotes sympathetic nerve activity (SNA) subserving the liver, and norepinephrine evokes the same responses in hepatocytes in vitro and in liver in vivo as observed upon sensory food perception. Collectively, our experiments unravel that sensory food perception coordinately primes postprandial liver ER adaption through a melanocortin-SNA-mTOR-Xbp1s axis. VIDEO ABSTRACT.

A hypothalamic circuit for the circadian control of aggression.

'Sundowning' in dementia and Alzheimer's disease is characterized by early-evening agitation and aggression. While such periodicity suggests a circadian origin, whether the circadian clock directly regulates aggressive behavior is unknown. We demonstrate that a daily rhythm in aggression propensity in male mice is gated by GABAergic subparaventricular zone (SPZGABA) neurons, the major postsynaptic targets of the central circadian clock, the suprachiasmatic nucleus. Optogenetic mapping revealed that SPZGABA neurons receive input from vasoactive intestinal polypeptide suprachiasmatic nucleus neurons and innervate neurons in the ventrolateral part of the ventromedial hypothalamus (VMH), which is known to regulate aggression. Additionally, VMH-projecting dorsal SPZ neurons are more active during early day than early night, and acute chemogenetic inhibition of SPZGABA transmission phase-dependently increases aggression. Finally, SPZGABA-recipient central VMH neurons directly innervate ventrolateral VMH neurons, and activation of this intra-VMH circuit drove attack behavior. Altogether, we reveal a functional polysynaptic circuit by which the suprachiasmatic nucleus clock regulates aggression.

Aldosterone-Sensing Neurons in the NTS Exhibit State-Dependent Pacemaker Activity and Drive Sodium Appetite via Synergy with Angiotensin II Signaling.

Sodium deficiency increases angiotensin II (ATII) and aldosterone, which synergistically stimulate sodium retention and consumption. Recently, ATII-responsive neurons in the subfornical organ (SFO) and aldosterone-sensitive neurons in the nucleus of the solitary tract (NTSHSD2 neurons) were shown to drive sodium appetite. Here we investigate the basis for NTSHSD2 neuron activation, identify the circuit by which NTSHSD2 neurons drive appetite, and uncover an interaction between the NTSHSD2 circuit and ATII signaling. NTSHSD2 neurons respond to sodium deficiency with spontaneous pacemaker-like activity-the consequence of "cardiac" HCN and Nav1.5 channels. Remarkably, NTSHSD2 neurons are necessary for sodium appetite, and with concurrent ATII signaling their activity is sufficient to produce rapid consumption. Importantly, NTSHSD2 neurons stimulate appetite via projections to the vlBNST, which is also the effector site for ATII-responsive SFO neurons. The interaction between angiotensin signaling and NTSHSD2 neurons provides a neuronal context for the long-standing "synergy hypothesis" of sodium appetite regulation.

Homeostatic circuits selectively gate food cue responses in insular cortex.

Physiological needs bias perception and attention to relevant sensory cues. This process is 'hijacked' by drug addiction, causing cue-induced cravings and relapse. Similarly, its dysregulation contributes to failed diets, obesity, and eating disorders. Neuroimaging studies in humans have implicated insular cortex in these phenomena. However, it remains unclear how 'cognitive' cortical representations of motivationally relevant cues are biased by subcortical circuits that drive specific motivational states. Here we develop a microprism-based cellular imaging approach to monitor visual cue responses in the insular cortex of behaving mice across hunger states. Insular cortex neurons demonstrate food-cue-biased responses that are abolished during satiety. Unexpectedly, while multiple satiety-related visceral signals converge in insular cortex, chemogenetic activation of hypothalamic 'hunger neurons' (expressing agouti-related peptide (AgRP)) bypasses these signals to restore hunger-like response patterns in insular cortex. Circuit mapping and pathway-specific manipulations uncover a pathway from AgRP neurons to insular cortex via the paraventricular thalamus and basolateral amygdala. These results reveal a neural basis for state-specific biased processing of motivationally relevant cues.