Probes for VGAT

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.

Despite notable genetic influences, obesity mainly results from the overconsumption of food, which arises from the interplay of physiological, cognitive and environmental factors. In patients with obesity, eating is determined more by external cues than by internal physiological needs. However, how environmental context drives non-homeostatic feeding is elusive. Here, we identify a population of somatostatin (TNSST) neurons in the mouse hypothalamic tuberal nucleus that are preferentially activated by palatable food. Activation of TNSST neurons enabled a context to drive non-homeostatic feeding in sated mice and required inputs from the subiculum. Pairing a context with palatable food greatly potentiated synaptic transmission between the subiculum and TNSST neurons and drove non-homeostatic feeding that could be selectively suppressed by inhibiting TNSST neurons or the subiculum but not other major orexigenic neurons. These results reveal how palatable food, through a specific hypothalamic circuit, empowers environmental context to drive non-homeostatic feeding.

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.

Spinal astrocytes contribute to chronic itch via sensitization of itch-specific neurons expressing gastrin-releasing peptide receptor (GRPR). However, whether microglia-neuron interactions contribute to itch remains unclear. In this study, we aimed to explore how microglia interact with GRPR+ neurons and promote chronic itch.RNA sequencing, quantitative real-time PCR, western blot, immunohistochemistry, RNAscope ISH, pharmacologic and genetic approaches were performed to examine the roles of spinal NLRP3 (The NOD-like receptor family, pyrin-containing domain 3) inflammasome activation and IL-1β-IL1R1 signaling in chronic itch. Grpr-eGFP and Grpr KO mice were used to investigate microglia-GRPR+ neuron interactions.We observed NLRP3 inflammasome activation and IL-1β production in spinal microglia under chronic itch conditions. Blockade of microglial activation and the NLRP3/caspase-1/IL-1β axis attenuated chronic itch and neuronal activation. Type 1 IL-1 receptor (IL-1R1) was expressed in GRPR+ neurons, which are essential for the development of chronic itch. Our studies also find that IL-1β+ microglia are localized in close proximity to GRPR+ neurons. Consistently, intrathecal injection of IL1R1 antagonist or exogenous IL-1β indicate that the IL-1β-IL-1R1 signaling pathway enhanced the activation of GRPR+ neurons. Furthermore, our results demonstrate that the microglial NLRP3/caspase-1/IL-1β axis contributes to several different chronic itches triggered by small molecules and protein allergens from the environment and drugs.Our findings reveal a previously unknown mechanism in which microglia enhances the activation of GRPR+ neurons through the NLRP3/caspase-1/IL-1β/IL1R1 axis. These results will provide new insights into the pathophysiology of pruritus and novel therapeutic strategies for patients with chronic itch.

Aguti-related protein (AGRP) neurons in the arcuate nucleus of the hypothalamus (ARC), which co-express neuropeptide Y (NPY), are key regulators of feeding and energy homeostasis. However, the precise role NPY has within these neurons and the specific pathways that it control are still unclear. In this article, we aimed to determine what aspects of feeding behaviour and energy homeostasis are controlled by NPY originating from AGRP neurons and which Y-receptor pathways are utilised to fulfil this function.Novel conditional Agrpcre/+;Npylox/lox knockout mice were generated and comprehensively phenotyped, both under standard chow as well as high-fat-diet conditions. Designer receptor exclusively activated by designer drugs (DREADD) technology was used to assess the altered responses on feeding and energy homeostasis control in the absence of NPY in these neurons. Rescue experiments utilising Npy1r- and Npy2r-selective NPY ligands were performed to assess which component of the energy homeostasis control is dependent by which specific Y-receptor pathway.We show that the specific deletion of Npy only in AGRP neurons leads to a paradoxical mild obese phenotype associated with reduced locomotion and energy expenditure and increased feeding and Respiratory Quotient (RQ) that remain elevated under a positive energy balance. The activation of Npy-deficient AGRP neurons via DREADD's is still able to drive feeding, yet with a delayed onset. Additionally, Clozapine-N-oxide (CNO) treatment reduces locomotion without impacting on energy expenditure. Rescue experiments re-introducing Npy1r- and Npy2r-selective NPY ligands revealed that the increased feeding and RQ are mostly driven by Npy1r, whereas energy expenditure and locomotion are controlled by Npy2r signalling.Together, these results demonstrate that NPY originating from AGRP neurons is not only critical to initiate but also for continuously driving feeding, and we for the first time identify which Y-receptor controls which pathway.