Probes for INS

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.

Skeletal muscle myofibers can be separated into functionally distinct cell types that differ in gene and protein expression. Current single cell expression data is generally based upon single nucleus RNA, rather than whole myofiber material. We examined if a whole-cell flow sorting approach could be applied to perform single cell RNA-seq (scRNA-seq) in a single muscle type. We performed deep, whole cell, scRNA-seq on intact and fragmented skeletal myofibers from the mouse fast-twitch flexor digitorum brevis muscle utilizing a flow-gated method of large cell isolation. We performed deep sequencing of 763 intact and fragmented myofibers. Quality control metrics across the different gates indicated only 171 of these cells were optimal, with a median read count of 239,252 and an average of 12,098 transcripts per cell. scRNA-seq identified three clusters of myofibers (a slow/fast 2A cluster and two fast 2X clusters). Comparison to a public skeletal nuclear RNA-seq dataset demonstrated a diversity in transcript abundance by method. RISH validated multiple genes across fast and slow twitch skeletal muscle types. This study introduces and validates a method to isolate intact skeletal muscle myofibers to generate deep expression patterns and expands the known repertoire of fiber-type-specific genes.

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.

The selection of goal-directed behaviors is supported by neural circuits located within the frontal cortex. Frontal cortical afferents arise from multiple brain areas, yet the cell-type-specific targeting of these inputs is unclear. Here, we use monosynaptic retrograde rabies mapping to examine the distribution of afferent neurons targeting distinct classes of local inhibitory interneurons and excitatory projection neurons in mouse infralimbic frontal cortex. Interneurons expressing parvalbumin, somatostatin, or vasoactive intestinal peptide receive a large proportion of inputs from the hippocampus, while interneurons expressing neuron-derived neurotrophic factor receive a large proportion of inputs from thalamic regions. A similar dichotomy is present among the four different excitatory projection neurons. These results show a prominent bias among long-range hippocampal and thalamic afferent systems in their targeting to specific sets of frontal cortical neurons. Moreover, they suggest the presence of two distinct local microcircuits that control how different inputs govern frontal cortical information processing.

As a significant cause of malignancy mortality, gastric carcinoma (GC) has been well documented to be an often-fatal diagnosis. Despite the limitations of effective therapy, immunotherapy has emerged as a promising therapeutic approach capable of killing cancer cells via the immune system. The current study was conducted to investigate the effect of cytokine C-C motif chemokine ligand 21 (CCL21) on GC progression through the metastasis-associated lung adenocarcinoma transcript 1/serine arginine-rich splicing factor 1/mammalian target of rapamycin (MALAT1/SRSF1/mTOR) axis. Bioinformatics analysis was conducted to identify the key genes associated with GC and to subsequently predict their downstream genes. The effect of CCL21, MALAT1, and SRSF1 on the malignant phenotypes and epithelial-mesenchymal transition (EMT) of SGC-7901 and MGC-803 cells in-vitro and the tumorigenesis of SGC-7901 and MGC-803 cells in-vivo were assessed by expression determination and plasmid transfection. Additionally, RNA pull-down and RNA binding protein immunoprecipitation experiments were performed to determine the MALAT1-microRNA-202-3p (miR-203-3p) interaction and miR-202-3p-SRSF1 interaction followed by the analysis of their effect on the mTOR pathway. CCL21 was identified as a key GC immune gene. Overexpressed CCL21, MALAT1, and SRSF1 along with poorly expressed miR-202-3p were identified in the GC cells. CCL21 induced the MALAT1 expression in a time- and dose-dependent manner. Functionally, MALAT1 targeted miR-202-3p but upregulated SRSF1 and activated mTOR. Crucially, evidence was obtained indicating that CCL21 promoted both the malignant phenotypes and EMT of SGC-7901 and MGC-803 cells in-vitro and the tumorigenesis of SGC-7901 and MGC-803 cells in-vivo by increasing the MALAT1-induced upregulation of SRSF1. Taken together, the key observations of our study provide evidence that CCL21 enhances the progression of GC via the MALAT1/SRSF1/mTOR axis, providing a novel therapeutic target for the treatment of GC.

Obesity is a global pandemic that is causally linked to many life-threatening diseases. Apart from some rare genetic conditions, the biological drivers of overeating and reduced activity are unclear. Here, we show that neurotensin-expressing neurons in the mouse interstitial nucleus of the posterior limb of the anterior commissure (IPAC), a nucleus of the central extended amygdala, encode dietary preference for unhealthy energy-dense foods. Optogenetic activation of IPACNts neurons promotes obesogenic behaviors, such as hedonic eating, and modulates food preference. Conversely, acute inhibition of IPACNts neurons reduces feeding and decreases hedonic eating. Chronic inactivation of IPACNts neurons recapitulates these effects, reduces preference for sweet, non-caloric tastants and, furthermore, enhances locomotion and energy expenditure; as a result, mice display long-term weight loss and improved metabolic health and are protected from obesity. Thus, the activity of a single neuronal population bidirectionally regulates energy homeostasis. Our findings could lead to new therapeutic strategies to prevent and treat obesity.

Behavior arises from concerted activity throughout the brain. Consequently, a major focus of modern neuroscience is defining the physiology and behavioral roles of projection neurons linking different brain areas. Single-cell RNA sequencing has facilitated these efforts by revealing molecular determinants of cellular physiology and markers that enable genetically targeted perturbations such as optogenetics, but existing methods for sequencing defined projection populations are low throughput, painstaking, and costly. We developed a straightforward, multiplexed approach, virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq). VECTORseq repurposes commercial retrogradely infecting viruses typically used to express functional transgenes (e.g., recombinases and fluorescent proteins) by treating viral transgene mRNA as barcodes within single-cell datasets. VECTORseq is compatible with different viral families, resolves multiple populations with different projection targets in one sequencing run, and identifies cortical and subcortical excitatory and inhibitory projection populations. Our study provides a roadmap for high-throughput identification of neuronal subtypes based on connectivity.