Probes for VGAT

Opioid withdrawal signs, such as hyperalgesia, are manifestations of opioid use disorder that may contribute to opioid seeking and taking. We have previously identified an association between dorsal raphe (DR) neurons and the expression of hyperalgesia during spontaneous heroin withdrawal. Here, we found that chemogenetic inhibition of DR neurons decreased hyperalgesia during spontaneous heroin withdrawal in male and female C57/B6 mice. By neuroanatomy, we identified three major subtypes of DR neurons expressing μ-opioid receptors (MOR) that were activated in hyperalgesia during spontaneous withdrawal, those expressing vesicular GABA transporter (VGaT), glutamate transporter 3 (VGluT3), or co-expressing VGluT3 and tryptophan hydroxylase (TPH). In contrast, we identified a small population of DR-MOR neurons expressing solely TPH, which were not activated in hyperalgesia during spontaneous withdrawal. Collectively, these findings indicate a role of the DR in hyperalgesia during spontaneous heroin withdrawal mediated, in part, by the activation of local MOR-GABAergic, MOR-glutamatergic and MOR-co-releasing glutamatergic-serotonergic neurons. We found that  specific chemogenetic inhibition of DR-VGaT neurons blocked hyperalgesia during spontaneous heroin withdrawal in male and female mice. Collectively, these findings indicate that DR-GABAergic neurons play a role in the expression of hyperalgesia during spontaneous heroin withdrawal.

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.

Diverse neurons in the parabrachial nucleus (PB) communicate with widespread brain regions. Despite evidence linking them to a variety of homeostatic functions, it remains difficult to determine which PB neurons influence which functions because their subpopulations intermingle extensively. An improved framework for identifying these intermingled subpopulations would help advance our understanding of neural circuit functions linked to this region. Here, we present the foundation of a developmental-genetic ontology that classifies PB neurons based on their intrinsic, molecular features. By combining transcription factor labeling with Cre fate-mapping, we find that the PB is a blend of two, developmentally distinct macropopulations of glutamatergic neurons. Neurons in the first macropopulation express Lmx1b (and, to a lesser extent, Lmx1a) and are mutually exclusive with those in a second macropopulation, which derive from precursors expressing Atoh1. This second, Atoh1-derived macropopulation includes many Foxp2-expressing neurons, but Foxp2 also identifies a subset of Lmx1b-expressing neurons in the Kölliker-Fuse nucleus (KF) and a population of GABAergic neurons ventrolateral to the PB ("caudal KF"). Immediately ventral to the PB, Phox2b-expressing glutamatergic neurons (some coexpressing Lmx1b) occupy the KF, supratrigeminal nucleus, and reticular formation. We show that this molecular framework organizes subsidiary patterns of adult gene expression (including Satb2, Calca, Grp, and Pdyn) and predicts output projections to the amygdala (Lmx1b), hypothalamus (Atoh1), and hindbrain (Phox2b/Lmx1b). Using this molecular ontology to organize, interpret, and communicate PB-related information could accelerate the translation of experimental findings from animal models to human patients.