Probes for INS

Rhythmic inspiratory activity is generated in the preBötzinger complex (preBötC), a neuronal network located bilaterally in the ventrolateral medulla. Cholinergic neurotransmission affects respiratory rhythmogenic neurons and inhibitory glycinergic neurons in the preBötC. Acetylcholine has been extensively investigated given that cholinergic fibers and receptors are present and functional in the preBötC, are important in sleep/wake cycling, and modulate inspiratory frequency through its action on preBötC neurons. Despite its role in modulating inspiratory rhythm, the source of acetylcholine input to the preBötC is not known. In the present study, we used retrograde and anterograde viral tracing approaches in transgenic mice expressing Cre-recombinase driven by the choline acetyltransferase promoter to identify the source of cholinergic inputs to the preBötC. Surprisingly, we observed very few, if any, cholinergic projections originating from the laterodorsal and pedunculopontine tegmental nuclei (LDT/PPT), two main cholinergic, state-dependent systems long hypothesized as the main source of cholinergic inputs to the preBötC. On the contrary, we identified glutamatergic and GABAergic/glycinergic neurons in the PPT/LDT that send projections to the preBötC. Although these neurons contribute minimally to the direct cholinergic modulation of preBötC neurons, they could be involved in state-dependent regulation of breathing. Our data also suggest that the source of cholinergic inputs to the preBötC appears to originate from cholinergic neurons in neighboring regions of the medulla, the intermediate reticular formation, the lateral paragigantocellularis, and the nucleus of the solitary tract.

Brain-derived neurotrophic factor (BDNF) signals through its cognate receptor tropomyosin receptor kinase B (TrkB) to promote the function of several classes of inhibitory interneurons. We previously reported that loss of BDNF-TrkB signaling in cortistatin (Cort)-expressing interneurons leads to behavioral hyperactivity and spontaneous seizures in mice. We performed bulk RNA sequencing (RNA-seq) from the cortex of mice with disruption of BDNF-TrkB signaling in cortistatin interneurons, and identified differential expression of genes important for excitatory neuron function. Using translating ribosome affinity purification and RNA-seq, we define a molecular profile for Cort-expressing inhibitory neurons and subsequently compare the translatome of normal and TrkB-depleted Cort neurons, revealing alterations in calcium signaling and axon development. Several of the genes enriched in Cort neurons and differentially expressed in TrkB-depleted neurons are also implicated in autism and epilepsy. Our findings highlight TrkB-dependent molecular pathways as critical for the maturation of inhibitory interneurons and support the hypothesis that loss of BDNF signaling in Cort interneurons leads to altered excitatory/inhibitory balance

Gastrin-releasing peptide (GRP) functions as a neurotransmitter for non-histaminergic itch, but its site of action (sensory neurons vs spinal cord) remains controversial. To determine the role of GRP in sensory neurons, we generated a floxed Grp mouse line. We found that conditional knockout of Grp in sensory neurons results in attenuated non-histaminergic itch, without impairing histamine-induced itch. Using a Grp-Cre knock-in mouse line, we show that the upper epidermis of the skin is exclusively innervated by GRP fibers, whose activation via optogeneics and chemogenetics in the skin evokes itch- but not pain-related scratching or wiping behaviors. In contrast, intersectional genetic ablation of spinal Grp neurons does not affect itch nor pain transmission, demonstrating that spinal Grp neurons are dispensable for itch transmission. These data indicate that GRP is a neuropeptide in sensory neurons for non-histaminergic itch, and GRP sensory neurons are dedicated to itch transmission

Sarcopenia and frailty are urgent socio-economic problems worldwide. Here we demonstrate a functional connection between the lateral hypothalamus (LH) and skeletal muscle through Slc12a8, a recently identified nicotinamide mononucleotide transporter, and its relationship to sarcopenia and frailty. Slc12a8-expressing cells are mainly localized in the LH. LH-specific knockdown of Slc12a8 in young mice decreases activity-dependent energy and carbohydrate expenditure and skeletal muscle functions, including muscle mass, muscle force, intramuscular glycolysis, and protein synthesis. LH-specific Slc12a8 knockdown also decreases sympathetic nerve signals at neuromuscular junctions and β2-adrenergic receptors in skeletal muscle, indicating the importance of the LH-sympathetic nerve-β2-adrenergic receptor axis. LH-specific overexpression of Slc12a8 in aged mice significantly ameliorates age-associated decreases in energy expenditure and skeletal muscle functions. Our results highlight an important role of Slc12a8 in the LH for regulation of whole-body metabolism and skeletal muscle functions and provide insights into the pathogenesis of sarcopenia and frailty during aging.

Synaptic zinc is a neuromodulator that shapes synaptic transmission and sensory processing. The maintenance of synaptic zinc is dependent on the vesicular zinc transporter, ZnT3. Hence, the ZnT3 knockout mouse has been a key tool for studying the mechanisms and functions of synaptic zinc. However, the use of this constitutive knockout mouse has notable limitations, including developmental, compensatory, and brain and cell type specificity issues. To overcome these limitations, we developed and characterized a dual recombinase transgenic mouse, which combines the Cre and Dre recombinase systems. This mouse allows for tamoxifen-inducible Cre-dependent expression of exogenous genes or knockout of floxed genes in ZnT3-expressing neurons and DreO-dependent region and cell type-specific conditional ZnT3 knockout in adult mice. Using this system, we reveal a neuromodulatory mechanism whereby zinc release from thalamic neurons modulates N-methyl-d-aspartate receptor activity in layer 5 pyramidal tract neurons, unmasking previously unknown features of cortical neuromodulation.

Rodents perceive pheromones via vomeronasal receptors encoded by highly evolutionarily dynamic Vr and Fpr gene superfamilies. We report here that high numbers of V1r pseudogenes are scattered in mammalian genomes, contrasting with the clustered organization of functional V1r and Fpr genes. We also found that V1r pseudogenes are more likely to be expressed when located in a functional V1r gene cluster than when isolated. To explore the potential regulatory role played by the association of functional vomeronasal receptor genes with their clusters, we dissociated the mouse Fpr-rs3 from its native cluster via transgenesis. Singular and specific transgenic Fpr-rs3 transcription was observed in young vomeronasal neurons but was only transient. Our study of natural and artificial dispersed gene duplications uncovers the existence of transcription-stabilizing elements not coupled to vomeronasal gene units but rather associated with vomeronasal gene clusters and thus explains the evolutionary conserved clustered organization of functional vomeronasal genes.

Motor control of the striated esophagus originates in the nucleus ambiguus (nAmb), a vagal motor nucleus that also contains upper airway motor neurons and parasympathetic preganglionic neurons for the heart and lungs. We disambiguate nAmb neurons based on their genome-wide expression profiles, efferent circuitry, and ability to control esophageal muscles. Our single-cell RNA sequencing analysis predicts three molecularly distinct nAmb neuron subtypes and annotates them by subtype-specific marker genes: Crhr2, Vipr2, and Adcyap1. Mapping the axon projections of the nAmb neuron subtypes reveals that Crhr2nAmb neurons innervate the esophagus, raising the possibility that they control esophageal muscle function. Accordingly, focal optogenetic stimulation of cholinergic Crhr2+ fibers in the esophagus results in contractions. Activating Crhr2nAmb neurons has no effect on heart rate, a key parasympathetic function of the nAmb, whereas activating all of the nAmb neurons robustly suppresses heart rate. Together, these results reveal a genetically defined circuit for motor control of the esophagus.

Drugs of abuse induce persistent remodeling of reward circuit function, a process thought to underlie the emergence of drug craving and relapse to drug use. However, how circuit-specific, drug-induced molecular and cellular plasticity can have distributed effects on the mesolimbic dopamine reward system to facilitate relapse to drug use is not fully elucidated. Here, we demonstrate that dopamine receptor D3 (DRD3)-dependent plasticity in the ventral pallidum (VP) drives potentiation of dopamine release in the nucleus accumbens during relapse to cocaine seeking after abstinence. We show that two distinct VP DRD3+ neuronal populations projecting to either the lateral habenula (LHb) or the ventral tegmental area (VTA) display different patterns of activity during drug seeking following abstinence from cocaine self-administration and that selective suppression of elevated activity or DRD3 signaling in the LHb-projecting population reduces drug seeking. Together, our results uncover how circuit-specific DRD3-mediated plasticity contributes to the process of drug relapse.

Erythropoietin (Epo) is produced by a subpopulation of resident fibroblasts in the healthy kidney. We have previously demonstrated that, during kidney fibrosis, kidney fibroblasts including Epo-producing cells transdifferentiate into myofibroblasts and lose their Epo-producing ability. However, it remains unclear whether Epo-producing cells survive and transform into myofibroblasts during fibrosis because previous studies did not specifically label Epo-producing cells in pathophysiological conditions. Here, we generated EpoCreERT2/+ mice, a novel mouse strain that enables labeling of Epo-producing cells at desired time points and examined the behaviors of Epo-producing cells under pathophysiological conditions. Lineage -labeled cells that were producing Epo when labeled were found to be a small subpopulation of fibroblasts located in the interstitium of the kidney, and their number increased during phlebotomy-induced anemia. Around half of lineage-labeled cells expressed Epo mRNA, and this percentage was maintained even 16 weeks after recombination, supporting the idea that a distinct subpopulation of cells with Epo-producing ability makes Epo repeatedly. During fibrosis caused by ureteral obstruction, EpoCreERT2/+ -labeled cells were found to transdifferentiate into myofibroblasts with concomitant loss of Epo-producing ability, and their numbers and the proportion among resident fibroblasts increased during fibrosis, indicating their high proliferative capacity. Finally, we confirmed that EpoCreERT2/+-labeled cells that lost their Epo-producing ability during fibrosis regained their ability after kidney repair due to relief of the ureteral obstruction. Thus, our analyses have revealed previously unappreciated characteristic behaviors of Epo-producing cells, which had not been clearly distinguished from those of resident fibroblasts.