Probes for INS

The central extended amygdala (CEA) and ventral pallidum (VP) are involved in diverse motivated behaviors based on rodent models. These structures are conserved, but expanded, in higher primates including human. Corticotropin releasing factor (CRF), a canonical 'stress molecule' associated with the CEA and VP circuitry across species, is dynamically regulated by stress and drugs of abuse and misuse. CRF's effects on circuits critically depend on its colocation with primary 'fast' transmitters, making this crucial for understanding circuit effects. We surveyed the distribution and colocalization of CRF-, VGluT2- (vesicular glutamate transporter 2) and VGAT- (vesicular GABA transporter) mRNA in specific subregions of the CEA and VP in young male monkeys. Although CRF-containing neurons were clustered in the lateral central bed nucleus (BSTLcn), the majority were broadly dispersed throughout other CEA subregions, and the VP. CRF/VGAT-only neurons were highest in the BSTLcn, lateral central amygdala nucleus (CeLcn), and medial central amygdala nucleus (CeM) (74%, 73%, and 85%, respectively). In contrast, lower percentages of CRF/VGAT only neurons populated the sublenticular extended amygdala (SLEAc), ventrolateral bed nucleus (BSTLP), and VP (53%, 54%, 17%, respectively), which had higher complements of CRF/VGAT/VGluT2 labeled neurons (33%, 29%, 67%, respectively). Thus, the majority of CRF-neurons at the 'poles' (BSTLcn and CeLcn/CeM) of the CEA are inhibitory, while the 'extended' BSTLP and SLEAc subregions, and neighboring VP, have a more complex profile with admixtures of 'multiplexed' excitatory CRF neurons. CRF's colocalization with its various fast transmitters is likely circuit-specific, and relevant for understanding CRF actions on specific target sites.SIGNIFICANCE STATEMENT:The central extended amygdala (CEA) and ventral pallidum (VP) regulate multiple motivated behaviors through differential downstream projections. The stress neuropeptide corticotropin releasing factor (CRF) is enriched in the CEA, and is thought to 'set the gain' through modulatory effects on co-expressed primary transmitters. Using protein and transcript assays in monkey, we found that CRF neurons are broadly and diffusely distributed in CEA and VP. CRF mRNA+ neurons colocalize with VGAT (GABA) and VGluT2 (glutamate) mRNAs in different proportions depending on subregion. CRF mRNA was also co-expressed in a subpopulation of VGAT/VGluT2 mRNA ('multiplexed') cells which were most prominent in the VP and 'pallidal'-like parts of the CEA. Heterogeneous CRF and fast transmitter co-expression across CEA/VP subregions implies circuit-specific effects.

26RFa (pyroglutamilated RFamide peptide [QRFP]) is a biologically active peptide that regulates glucose homeostasis by acting as an incretin and by increasing insulin sensitivity at the periphery. 26RFa is also produced by a neuronal population localised in the hypothalamus. In this study we investigated whether 26RFa neurons are involved in the hypothalamic regulation of glucose homeostasis.26Rfa+/+, 26Rfa-/- and insulin-deficient male C57Bl/6J mice were used in this study. Mice received an acute intracerebroventricular (i.c.v.) injection of 26RFa, insulin or the 26RFa receptor (GPR103) antagonist 25e and were subjected to IPGTTs, insulin tolerance tests, acute glucose-stimulated insulin secretion tests and pyruvate tolerance tests (PTTs). Secretion of 26RFa by hypothalamic explants after incubation with glucose, leptin or insulin was assessed. Expression and quantification of the genes encoding 26RFa, agouti-related protein, the insulin receptor and GPR103 were evaluated by quantitative reverse transcription PCR and RNAscope in situ hybridisation.Our data indicate that i.c.v.-injected 26RFa induces a robust antihyperglycaemic effect associated with an increase in insulin production by the pancreatic islets. In addition, we found that insulin strongly stimulates 26Rfa expression and secretion by the hypothalamus. RNAscope experiments revealed that neurons expressing 26Rfa are mainly localised in the lateral hypothalamic area, that they co-express the gene encoding the insulin receptor and that insulin induces the expression of 26Rfa in these neurons. Concurrently, the central antihyperglycaemic effect of insulin is abolished in the presence of a GPR103 antagonist and in 26RFa-deficient mice. Finally, our data indicate that the hypothalamic 26RFa neurons are not involved in the central inhibitory effect of insulin on hepatic glucose production, but mediate the central effects of the hormone on its own peripheral production.We have identified a novel mechanism in the hypothalamic regulation of glucose homeostasis, the 26RFa/GPR103 system, and we provide evidence that this neuronal peptidergic system is a key relay for the central regulation of glucose metabolism by insulin.

Restoring the control of food intake is the key to obesity management and prevention. The arcuate nucleus (ARC) of the hypothalamus is extensively being studied as a potential anti-obesity target. Animal studies showed that neuropeptide FF (NPFF) reduces food intake by its action in neuropeptide Y (NPY) neurons of the hypothalamic ARC, but the detailed mode of action observed in human neurons is missing, due to the lack of a human-neuron-based model for pharmacology testing. Here, we validated and utilized a human-neural-stem-cell-based (hNSC) model of ARC to test the effects of NPFF on cellular pathways and neuronal activity. We found that in the human neurons, decreased cAMP levels by NPFF resulted in a reduced rate of cytoplasmic calcium oscillations, indicating an inhibition of ARC NPY neurons. This suggests the therapeutic potential of NPFFR2 in obesity. In addition, we demonstrate the use of human-stem-cell-derived neurons in pharmacological applications and the potential of this model to address functional aspects of human hypothalamic neurons.

Obesity and type 2 diabetes are associated with cognitive dysfunction. Because the hypothalamus is implicated in energy balance control and memory disorders, we hypothesized that specific neurons in this brain region are at the interface of metabolism and cognition. Acute obesogenic diet administration in mice impaired recognition memory due to defective production of the neurosteroid precursor pregnenolone in the hypothalamus. Genetic interference with pregnenolone synthesis by Star deletion in hypothalamic POMC, but not AgRP neurons, deteriorated recognition memory independently of metabolic disturbances. Our data suggest that pregnenolone's effects on cognitive function were mediated via an autocrine mechanism on POMC neurons, influencing hippocampal long-term potentiation. The relevance of central pregnenolone on cognition was also confirmed in metabolically unhealthy patients with obesity. Our data reveal an unsuspected role for POMC neuron-derived neurosteroids in cognition. These results provide the basis for a framework to investigate new facets of POMC neuron biology with implications for cognitive disorders.

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

Leptin acts on hypothalamic neurons expressing agouti-related protein (AgRP) or pro-opiomelanocortin (POMC) to suppress appetite and increase energy expenditure, but the intracellular mechanisms that modulate central leptin signalling are not fully understood. Here we show that growth factor receptor-bound protein 10 (Grb10), an adaptor protein that binds to the insulin receptor and negatively regulates its signalling pathway, can interact with the leptin receptor and enhance leptin signalling. Ablation of Grb10 in AgRP neurons promotes weight gain, while overexpression of Grb10 in AgRP neurons reduces body weight in male and female mice. In parallel, deletion or overexpression of Grb10 in POMC neurons exacerbates or attenuates diet-induced obesity, respectively. Consistent with its role in leptin signalling, Grb10 in AgRP and POMC neurons enhances the anorexic and weight-reducing actions of leptin. Grb10 also exaggerates the inhibitory effects of leptin on AgRP neurons via ATP-sensitive potassium channel-mediated currents while facilitating the excitatory drive of leptin on POMC neurons through transient receptor potential channels. Our study identifies Grb10 as a potent leptin sensitizer that contributes to the maintenance of energy homeostasis by enhancing the response of AgRP and POMC neurons to leptin.

Enhanced appetite occurs as a means of behavioral thermoregulation at low temperature. Neural circuitry mediating this crosstalk between behavioral thermoregulation and energy homeostasis remains to be elucidated. We find that the hypothalamic orexigenic agouti-related neuropeptide (AgRP) neurons in the arcuate nucleus (ARC) are profoundly activated by cold exposure. The calcium signals in ARCAgRP neurons display an immediate-response pattern in response to cold stimulation. Cold-responsive neurons in the medial preoptic area (mPOA) make excitatory synapses onto ARCAgRP neurons. Inhibition of either ARCAgRP neurons or ARC-projecting mPOA neurons attenuates cold-evoked feeding, while activation of the mPOA-to-ARC projection increases food intake. These findings reveal an mPOA-ARCAgRP neural pathway that modulates cold-evoked feeding behavior.

The availability of Cre-based mouse lines for visualizing and targeting populations of hormone-sensitive cells has helped identify the neural circuitry driving hormone effects. However, these mice have limitations and may not even be available. For instance, the development of the first ghrelin receptor (Ghsr)-IRES-Cre model paved the way for using the Cre-lox system to identify and selectively manipulate ghrelin-responsive populations. The insertion of the IRES-Cre cassette, however, interfered with Ghsr expression, resulting in defective GHSR signaling and a pronounced phenotype in the homozygotes. As an alternative strategy to target ghrelin-responsive cells, we hereby utilize TRAP2 (targeted recombination in active populations) mice in which it is possible to gain genetic access to ghrelin-activated populations. In TRAP2 mice crossed with a reporter strain, we visualized ghrelin-activated cells and found, as expected, much activation in the arcuate nucleus (Arc). We then stimulated this population using a chemogenetic approach and found that this was sufficient to induce an orexigenic response of similar magnitude to that induced by peripheral ghrelin injection. The stimulation of this population also impacted food choice. Thus, the TRAPing of hormone-activated neurons (here exemplified by ghrelin-activated pathways) provides a complimentary/alternative technique to visualize, access and control discrete pathways, linking hormone action to circuit function.

Environmental cues recalling palatable foods motivate eating beyond metabolic need, yet the timing of this response and whether it can develop towards a less palatable but readily available food remain elusive. Increasing evidence indicates that external stimuli in the olfactory modality communicate with the major hub in the feeding neurocircuitry, namely the hypothalamic arcuate nucleus (Arc), but the neural substrates involved have been only partially uncovered. By means of a home-cage hidden palatable food paradigm, aiming to mimic ubiquitous exposure to olfactory food cues in Western societies, we investigated whether the latter could drive the overeating of plain chow in non-food-deprived male rats and explored the neural mechanisms involved, including the possible engagement of the orexigenic ghrelin system. The olfactory detection of a familiar, palatable food impacted upon meal patterns, by increasing meal frequency, to cause the persistent overconsumption of chow. In line with the orexigenic response observed, sensing the palatable food in the environment stimulated food-seeking and risk-taking behavior, which are intrinsic components of food acquisition, and caused active ghrelin release. Our results suggest that olfactory food cues recruited intermingled populations of cells embedded within the feeding circuitry within the Arc, including, notably, those containing the ghrelin receptor. These data demonstrate the leverage of ubiquitous food cues, not only for palatable food searching, but also to powerfully drive food consumption in ways that resonate with heightened hunger, for which the orexigenic ghrelin system is implicated.

Obesity is associated with the activation of cellular responses, such as endoplasmic reticulum (ER) stress. Here, we show that leptin-deficient ob/ob mice display elevated hypothalamic ER stress as early as postnatal day 10, i.e., prior to the development of obesity in this mouse model. Neonatal treatment of ob/ob mice with the ER stress-relieving drug tauroursodeoxycholic acid (TUDCA) causes long-term amelioration of body weight, food intake, glucose homeostasis, and pro-opiomelanocortin (POMC) projections. Cells exposed to ER stress often activate autophagy. Accordingly, we report that in vitro induction of ER stress and neonatal leptin deficiency in vivo activate hypothalamic autophagy-related genes. Furthermore, genetic deletion of autophagy in pro-opiomelanocortin neurons of ob/ob mice worsens their glucose homeostasis, adiposity, hyperphagia, and POMC neuronal projections, all of which are ameliorated with neonatal TUDCA treatment. Together, our data highlight the importance of early life ER stress-autophagy pathway in influencing hypothalamic circuits and metabolic regulation