Probes for INS

Background Selective serotonin reuptake inhibitors such as fluoxetine have a limited treatment efficacy. The mechanism by which some patients respond to fluoxetine while others do not remains poorly understood, limiting treatment effectiveness. We have found the opioid system to be involved in the responsiveness to fluoxetine treatment in a mouse model for anxiety- and depressive-like behavior. Methods We analyzed gene expression changes in the dentate gyrus of mice chronically treated with corticosterone and fluoxetine. After identifying a subset of genes of interest, we studied their expression patterns in relation to treatment responsiveness. We further characterized their expression through in situ hybridization and the analysis of a single-cell RNA-Seq data set. Finally, we behaviorally tested mu and delta opioid receptor knockout mice in the Novelty Suppressed Feeding test and the Forced Swim Test after chronic corticosterone and fluoxetine treatment. Results Chronic fluoxetine treatment upregulates proenkephalin expression in the dentate gyrus, and this upregulation is associated with treatment responsiveness. The expression of several of the most significantly upregulated genes, including proenkephalin, is localized to an anatomically and transcriptionally specialized subgroup of mature granule cells in the dentate gyrus. We have also found that the delta opioid receptor contributes to some, but not all, of the behavioral effects of fluoxetine. Conclusions These data indicate that the opioid system is involved in the antidepressant effects of fluoxetine, and this effect may be mediated through the upregulation of proenkephalin in a subpopulation of mature granule cells.

Green light exposure has been shown to reduce pain in animal models. Here, we report a vision-associated enkephalinergic neural circuit responsible for green light-mediated analgesia. Full-field green light exposure at an intensity of 10 lux produced analgesic effects in healthy mice and in a model of arthrosis. Ablation of cone photoreceptors completely inhibited the analgesic effect, whereas rod ablation only partially reduced pain relief. The analgesic effect was not modulated by the ablation of intrinsically photosensitive retinal ganglion cells (ipRGCs), which are atypical photoreceptors that control various nonvisual effects of light. Inhibition of the retino-ventrolateral geniculate nucleus (vLGN) pathway completely abolished the analgesic effects. Activation of this pathway reduced nociceptive behavioral responses; such activation was blocked by the inhibition of proenkephalin (Penk)-positive neurons in the vLGN (vLGNPenk). Moreover, green light analgesia was prevented by knockdown of Penk in the vLGN or by ablation of vLGNPenk neurons. In addition, activation of the projections from vLGNPenk neurons to the dorsal raphe nucleus (DRN) was sufficient to suppress nociceptive behaviors, whereas its inhibition abolished the green light analgesia. Our findings indicate that cone-dominated retinal inputs mediated green light analgesia through the vLGNPenk-DRN pathway and suggest that this signaling pathway could be exploited for reducing pain.

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/β-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.

IntroductionExact measurement of renal function is essential for the treatment of patients. Elevated serum-creatinine levels, while established are influenced by other parameters and show a significant time-lag. This drives the search for novel biomarkers of renal function and injury. Beside Lipocalin-2 and kidney-injury-molecule-1(KIM-1), the endogenous opioid precursor proenkephalin-A(Penk) has recently emerged as a promising marker for renal function. But the cellular origin and regulation of Penk outside the brain has not yet been investigated in depth.Materials and MethodsThis study characterizes the cellular origin of Penk expression with high resolution in-situ hybridization in two models of renal fibrosis in mice and human tissue.ResultsInterstitial cells are the main expression site for renal Penk. This classifies Penk as biomarker for interstitial damage as opposed to tubular damage markers like Lipocalin-2 and KIM-1. Furthermore, our data indicate that renal Penk expression is not regulated by classical profibrotic pathways.DiscussionThis study characterizes changing Penk expression in the kidneys. The similarity of Penk expression across species gives rise to further investigations into the function of Penk in healthy and injured kidneys.ConclusionPenk is a promising biomarker for interstitial renal damage that warrants further studies to utilize its predictive potential.

The central nucleus of the amygdala (CeA) is a key hub integrating sensory inputs and modulating behavioural outputs. The CeA is a complex structure with discrete subdivisions, high peptidergic heterogeneity and broad CNS afferent and efferent projections. While several neuropeptide systems within the CeA have been examined in detail, less is known about CeA preproenkephalin (ppENK) cells. Here, we used a recently developed transgenic Penk-Cre mouse line to advance our understanding of the efferent and afferent connectivity of ppENK in the CeA. First, to determine the fidelity of Cre expression in Penk-Cre transgenic mice, we conducted RNAscope in the CeA of Penk-Cre mice. Our analysis revealed that 96.6% of CeA Cre+ neurons co-expressed pENK mRNA, and 99.7% of CeA pENK+ neurons co-expressed Cre mRNA, indicating faithful recapitulation of Cre expression in CeA ppENK-expressing cells, supporting the fidelity of the Penk-Cre reporter mouse. Anterograde tracing of CeAPenk cells showed strong efferent projections to the extended amygdala, midbrain and hindbrain PBN and NTS. Retrograde tracing of Penk afferents to the CeA were more restricted, with primary innervation originating within the amygdala complex and bed nucleus of the stria terminalis, and minor innervation from the parabrachial nucleus and nucleus of the solitary tract. Together, our data provide a comprehensive map of ENKergic efferent and afferent connectivity of the CeA in Penk-Cre mice. Further, we highlight both the utility and limitations of the Penk-Cre mice to study the function of CeA, PBN and NTS ppENK cells.

In adults, hepatocytes are mainly replenished from the existing progenitor pools of hepatocytes and cholangiocytes during chronic liver injury. However, it is unclear whether other cell types in addition to classical hepatocytes and cholangiocytes contribute to hepatocyte regeneration after chronic liver injuries. Here, we identified a new biphenotypic cell population that contributes to hepatocyte regeneration during chronic liver injuries. We found that a cell population expressed Gli1 and EpCAM (EpCAM+Gli1+), which was further characterized with both epithelial and mesenchymal identities by single-cell RNA sequencing. Genetic lineage tracing using dual recombinases revealed that Gli1+ nonhepatocyte cell population could generate hepatocytes after chronic liver injury. EpCAM+Gli1+ cells exhibited a greater capacity for organoid formation with functional hepatocytes in vitro and liver regeneration upon transplantation in vivo. Collectively, these findings demonstrate that EpCAM+Gli1+ cells can serve as a new source of liver progenitor cells and contribute to liver repair and regeneration.

Barrier epithelia depend upon resident stem cells for homeostasis, defense, and repair. Epithelial stem cells of small and large intestines (ISCs) respond to their local microenvironments (niches) to fulfill a continuous demand for tissue turnover. The complexity of these niches and underlying communication pathways are not fully known. Here, we report a lymphatic network at the intestinal crypt base that intimately associates with ISCs. Employing in vivo loss of function and lymphatic:organoid cocultures, we show that crypt lymphatics maintain ISCs and inhibit their precocious differentiation. Pairing single-cell and spatial transcriptomics, we apply BayesPrism to deconvolve expression within spatial features and develop SpaceFold to robustly map the niche at high resolution, exposing lymphatics as a central signaling hub for the crypt in general and ISCs in particular. We identify WNT-signaling factors (WNT2, R-SPONDIN-3) and a hitherto unappreciated extracellular matrix protein, REELIN, as crypt lymphatic signals that directly govern the regenerative potential of ISCs.