Probes for VGAT

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.

Despite accumulating evidence in rodents, the functional role of neuromedin B (NMB) in regulating somatosensory systems in primate spinal cord is unknown. We aimed to compare the expression patterns of NMB and its receptor (NMBR) and the behavioral effects of intrathecal (i.t.) NMB with gastrin-releasing peptide (GRP) on itch or pain in non-human primates (NHPs). We used six adult rhesus monkeys. The mRNA or protein expressions of NMB, GRP, and their receptors were evaluated by quantitative reverse transcription polymerase chain reaction, immunohistochemistry, or in situ hybridization. We determined the behavioral effects of NMB or GRP via acute thermal nociception, capsaicin-induced thermal allodynia, and itch scratching response assays. NMB expression levels were greater than those of GRP in the dorsal root ganglia and spinal dorsal horn. Conversely, NMBR expression was significantly lower than GRP receptor (GRPR). I.t. NMB elicited only mild scratching responses, whereas GRP caused robust scratching responses. GRP- and NMB-elicited scratching responses were attenuated by GRPR (RC-3095) and NMBR (PD168368) antagonists, respectively. Moreover, i.t. NMB and GRP did not induce thermal hypersensitivity and GRPR and NMBR antagonists did not affect peripherally elicited thermal allodynia. Consistently, NMBR expression was low in both itch- and pain-responsive neurons in the spinal dorsal horn. Spinal NMB-NMBR system plays a minimal functional role in the neurotransmission of itch and pain in primates. Unlike the functional significance of the GRP-GRPR system in itch, drugs targeting the spinal NMB-NMBR system may not effectively alleviate non-NMBR-mediated itch.