Probes for INS

Fibroblasts are highly dynamic cells that play a central role in tissue repair and fibrosis. However, the mechanisms by which they contribute to both physiologic and pathologic states of extracellular matrix deposition and remodeling are just starting to be understood. In this review article, we discuss the current state of knowledge in fibroblast biology and heterogeneity, with a primary focus on the role of fibroblasts in skin wound repair. We also consider emerging techniques in the field, which enable an increasingly nuanced and contextualized understanding of these complex systems, and evaluate limitations of existing methodologies and knowledge. Collectively, this review spotlights a diverse body of research examining an often-overlooked cell type-the fibroblast-and its critical functions in wound repair and beyond.

IgE-mediated anaphylaxis is an acute life-threatening systemic reaction to allergens, including certain foods and venoms. Anaphylaxis is triggered when blood-borne allergens activate IgE-bound perivascular mast cells (MCs) throughout the body, causing an extensive systemic release of MC mediators. Through precipitating vasodilatation and vascular leakage, these mediators are believed to trigger a sharp drop in blood pressure in humans and in core body temperature in animals. We report that the IgE/MC-mediated drop in body temperature in mice associated with anaphylaxis also requires the body's thermoregulatory neural circuit. This circuit is activated when granule-borne chymase from MCs is deposited on proximal TRPV1+ sensory neurons and stimulates them via protease-activated receptor-1. This triggers the activation of the body's thermoregulatory neural network, which rapidly attenuates brown adipose tissue thermogenesis to cause hypothermia. Mice deficient in either chymase or TRPV1 exhibited limited IgE-mediated anaphylaxis, and, in wild-type mice, anaphylaxis could be recapitulated simply by systemically activating TRPV1+ sensory neurons. Thus, in addition to their well-known effects on the vasculature, MC products, especially chymase, promote IgE-mediated anaphylaxis by activating the thermoregulatory neural circuit.

Fibroblasts are the main cell type in the dermis. They are responsible for the synthesis and deposition of structural proteins such as collagen and elastin, which are integrated into the extracellular matrix (ECM). Mouse and human studies using flow cytometry, cell culture, skin reconstitution, and lineage tracing experiments have shown the existence of different subpopulations of fibroblasts, including papillary fibroblasts, reticular fibroblasts, and fibroblasts comprising the dermal papilla at the base of the hair follicle. In recent years, the technological advances in single-cell sequencing have allowed researchers to study the repertoire of cells present in full-thickness skin including the dermis. Multiple groups have confirmed that distinct fibroblast populations can be identified in mouse and human dermis on the basis of differences in the transcriptional profile. Here, we discuss the current state of knowledge regarding dermal fibroblast heterogeneity in healthy mouse and human skin, highlighting the similarities and differences between mouse and human fibroblast subpopulations. We also discuss how fibroblast heterogeneity may provide insights into physiological wound healing and its dysfunction in pathological states such as hypertrophic and keloid scars.

In situ hybridization has been a robust method for detection of mRNA expression in whole-mount samples or tissue sections for more than 50 years. Recent technical advances for in situ hybridization have incorporated oligo-based probes that attain greater tissue penetration and signal amplification steps with restricted localization for visualization of specific mRNAs within single cells. One such method is third-generation in situ hybridization chain reaction (V3HCR). Here, we report an optimized protocol for V3HCR detection of gene expression using sectioned frozen tissues from mouse and human on microscope slides. Our methods and modifications for cryosectioning, tissue fixation, and processing over a three-day V3HCR protocol are detailed along with recommendations for aliquoting and storing V3HCR single-stranded DNA probes and hairpin amplifiers. In addition, we describe a method for blocking background signal from lipofuscin, a highly autofluorescent material that is widespread in human neurons and often complicates imaging efforts. After testing multiple strategies for reduction of lipofuscin, we determined that application of a lipofuscin quencher dye is compatible with V3HCR, in contrast to other methods like cupric sulfate quenching or Sudan Black B blocking that cause V3HCR signal loss. This adaptation enables application of V3HCR for in situ detection of gene expression in human neuronal populations that are otherwise problematic due to lipofuscin autofluorescence.

The blood-brain barrier (BBB) consists of endothelial cells, astrocytic end feet, and pericytes to form a barrier that minimizes the entry of circulating proteins and cells into the brain. However, stroke is known to cause significant damage to the BBB, causing the barrier to become permeable, which allows immune cells and other substances to be extravasated into the brain parenchyma. Preservation of the BBB is associated with better ischemic stroke outcomes; therefore, this synopsis summarizes 3 new studies that aim to characterize specific mechanisms of BBB damage and identify potential therapeutic pathways to preserve barrier integrity. CD36 (cluster of differentiation 36) is a glycoprotein expressed by monocytes and macrophages, as well as by endothelial cells. Previous studies have shown that global knockout of CD36 prevents stroke-induced damage, and Kim et al in 2023 published a study in the Journal of Cerebral Blood Flow and Metabolism titled “Endothelial Cell CD36 Mediates Stroke-Induced Brain Injury via BBB Dysfunction and Monocyte Infiltration in Normal and Obese Conditions,” in which they explore the role of CD36 specifically in endothelial cells. Conditional deletion of CD36 in endothelial cells improved stroke outcomes, as indicated by reduced infarct size and hemispheric swelling. Moreover, this deletion improved survival and motor function. Additionally, CD36 deletion in endothelial cells reduced IgG expression in the brain, indicating improved vascular integrity. There was reduced monocyte infiltration into the brain and reduced MCP-1 (monocyte chemoattractant protein-1) and CCR2 (chemokine receptor type 2) expression in the mice with endothelial cell deletion of CD36. This reduced monocyte trafficking persisted even when normalized for infarct size, suggesting that vascular integrity is maintained independent of cell loss. Intriguingly, endothelial cell-specific deletion of CD36 also made mice resistant to developing an obesity phenotype, providing a potential molecular cause for obesity as a stroke risk factor. In the Proceedings of the National Academy for Science in a publication titled “Myeloid-Derived MIF Drives RIPK1-Mediated Cerebromicrovascular Endothelial Cell Death to Exacerbate Ischemic Brain Injury,” Li et al in 2023 describe how macrophage MIF (migration inhibitory factor) exacerbates endothelial cell death and increases BBB permeability after middle cerebral artery occlusion (MCAo). By treating endothelial cells with MIF and subjecting them to oxygen-glucose deprivation followed by reoxygenation, the authors demonstrated that MIF promotes endothelial cell death specifically by activating RIPK1 (receptor-interacting protein kinase 1). Surgical trauma in both mice and humans increases circulating MIF, and the authors use a perioperative ischemic stroke model to see how this surgically induced increase in MIF affects outcomes after distal MCAo. Two-photon imaging showed that perioperative ischemic stroke mice showed increased adhesion of myeloid cells to ischemic microvascular endothelial cells, and RNAscope analysis showed that MIF expression was increased in microglia surrounding endothelial cells in perioperative ischemic stroke mice. Perioperative ischemic stroke mice also exhibited larger infarct volumes and exacerbated BBB damage. The authors then used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) to delete MIF in myeloid cells, which resulted in reduced expression of necrosis markers phosphorylated RIPK1, phosphorylated RIPK3, and CC3 (cleaved caspase 3) in endothelial cells. Mice with myeloid-deleted MIF showed reduced zonulin-1 loss, a marker of endothelial tight junctions, and sensorimotor deficits after distal MCAo. Peripheral blood mononuclear cells from mice with myeloid-deleted MIF were given after MCAo and resulted in smaller infarct volume and reduced IgG extravasation as compared with mice who were given peripheral blood mononuclear cells from wild types. Endothelial cells were cocultured with peripheral blood mononuclear cells from mice with myeloid-deleted MIF and wild types, and zonulin-1 expression was preserved in endothelial cocultured with peripheral blood mononuclear cells from myeloid-deleted MIF mice. Administration of MIF inhibitor before stroke reduced infarct volume, prevented IgG extravasation, preserved tight junction integrity, and prevented endothelial cell death. Collectively, these data show that myeloid-derived MIF is detrimental to BBB integrity after stroke and deleting this source of MIF can improve outcomes through preserving BBB health. Finally, Li et al in 2023 show in ACS Nano in a publication titled “Inducible Pluripotent Stem Cell-Derived Small Extracellular Vesicles Rejuvenate Senescent Blood-Brain Barrier to Protect Against Ischemic Stroke in Aged Mice” that small extracellular vesicles (sEVs) from induced pluripotent stem cells (iPSCs) are able to restore BBB function in old mice by reversing cellular senescence. Mice treated with iPSC-sEVs showed reduced senescence-associated β-galactosidase, p16, p53, p21, and γ-H2AX (histone family member X), all of which are markers associated with cellular senescence. Pretreatment with iPSC-sEVs before MCAo reduced infarct volume in the aged mice, improved neurological score, and reduced sensorimotor deficits, indicating improved stroke outcomes. Moreover, these mice showed decreased leakage of Evans blue dye into the brain parenchyma and preservation of tight junction proteins, indicating that these sEVs preserve BBB integrity after stroke. Mice treated with sEVs showed decreased immune cell infiltration after stroke and attenuated expression of tumor necrosis factor-α, IL (interleukin)-17, IL-6, and IL-1β, Moreover, sEV treatment reduced ischemia-induced apoptosis of oligodendrocytes and neurons. Reversal of the senescent phenotype of the BBB was tested in vitro, by chemically inducing senescence using D-galactose in endothelial cell cultures and subsequent treatment with iPSC-sEVs. sEV treatment reduced senescence markers and prevented loss of tight junction proteins. Oxygen-glucose deprivation was then used to mimic stroke conditions in these cultures, and sEV treatment preserved angiogenic properties of endothelial cells and reduced dextran leakage in a transwell assay. These experiments affirm the in vivo findings that sEV treatment reverses BBB senescence. The 3 studies summarized in this synopsis show 3 different potential pathways that may serve as a target for preserving BBB function after stroke. Deleting endothelial cell CD36, deleting myeloid-derived MIF, or reversing BBB senescence using iPSC-sEVs resulted in improved vascular integrity and overall better stroke outcomes.

Regional cellular heterogeneity is a fundamental feature of the human neocortex; however, details of this heterogeneity are still undefined. We utilized single-nucleus RNA-sequencing (snRNA-seq) to examine cell-specific transcriptional features in the dorsolateral prefrontal cortex (DLPFC) and the subgenual anterior cingulate cortex (sgACC)-regions implicated in major psychiatric disorders. Droplet-based nuclei-capture and library preparation were performed on replicate samples from eight male donors without history of psychiatric or neurological disorder. Unsupervised clustering identified major neural cell classes. Subsequent iterative clustering of neurons further revealed 20 excitatory and 22 inhibitory subclasses. Inhibitory cells were consistently more abundant in the sgACC and excitatory neuron subclusters exhibited considerable variability across brain regions. Excitatory cell subclasses also exhibited greater within-class transcriptional differences between the two regions. We utilized these molecular definitions to determine which cell classes might be enriched in loci carrying a genetic signal in genome-wide association studies (GWAS) or for differentially expressed genes (DEGs) in mental illness. We found that the heritable signals of psychiatric disorders were enriched in neurons and that while the gene expression changes detected in bulk-RNA-sequencing studies was dominated by glial cells, some alterations could be identified in specific classes of excitatory and inhibitory neurons. Intriguingly, only two excitatory cell classes exhibited concomitant region-specific enrichment for both GWAS loci and transcriptional dysregulation. In sum, by detailing the molecular and cellular diversity of the DLPFC and sgACC, we were able generate hypotheses on regional and cell-specific dysfunctions that may contribute to the development of mental illness.SIGNIFICANCE STATEMENT:Dysfunction of the subgenual anterior cingulate cortex (sgACC) has been implicated in mood disorders, particularly major depressive disorder, and the dorsolateral prefrontal cortex (DLPFC)-a subsection of the Prefrontal cortex involved in executive functioning-has been implicated in schizophrenia. Understanding the cellular composition of these regions is critical to elucidating the neurobiology underlying psychiatric and neurological disorders. We studied cell type diversity of the sgACC and DLPFC of humans with no neuropsychiatric illness utilizing a clustering analysis of single-nuclei RNA-sequencing (snRNA-seq) data. Defining the transcriptomic profile of cellular subpopulations in these cortical regions is a first step to demystifying the cellular and molecular pathways involved in psychiatric disorders.

With the advent of multiplex fluorescence in situ hybridization (FISH) and in situ RNA sequencing technologies, spatial transcriptomics analysis is advancing rapidly, providing spatial location and gene expression information about cells in tissue sections at single cell resolution. Cell type classification of these spatially-resolved cells can be inferred by matching the spatial transcriptomics data to reference atlases derived from single cell RNA-sequencing (scRNA-seq) in which cell types are defined by differences in their gene expression profiles. However, robust cell type matching of the spatially-resolved cells to reference scRNA-seq atlases is challenging due to the intrinsic differences in resolution between the spatial and scRNA-seq data. In this study, we systematically evaluated six computational algorithms for cell type matching across four image-based spatial transcriptomics experimental protocols (MERFISH, smFISH, BaristaSeq, and ExSeq) conducted on the same mouse primary visual cortex (VISp) brain region. We find that many cells are assigned as the same type by multiple cell type matching algorithms and are present in spatial patterns previously reported from scRNA-seq studies in VISp. Furthermore, by combining the results of individual matching strategies into consensus cell type assignments, we see even greater alignment with biological expectations. We present two ensemble meta-analysis strategies used in this study and share the consensus cell type matching results in the Cytosplore Viewer ( https://viewer.cytosplore.org ) for interactive visualization and data exploration. The consensus matching can also guide spatial data analysis using SSAM, allowing segmentation-free cell type assignment.

Rapid growth in aquaculture has resulted in high-density production systems in ecologically and geographically novel conditions in which the emergence of diseases is inevitable. Well-characterized methods for detection and surveillance of infectious diseases are vital for rapid identification, response, and recovery to protect economic and food security. We implemented a proof-of-concept approach for virus detection using a known high-consequence fish pathogen, infectious salmon anemia virus (ISAV), as the archetypal pathogen. In fish infected with ISAV, we integrated histopathology, virus isolation, whole-genome sequencing (WGS), electron microscopy (EM), in situ hybridization (ISH), and reverse transcription real-time PCR (RT-rtPCR). Fresh-frozen and formalin-fixed tissues were collected from virus-infected, control, and sham-infected Atlantic salmon (Salmo salar). Microscopic differences were not evident between uninfected and infected fish. Viral cytopathic effect was observed in cell cultures inoculated with fresh-frozen tissue homogenates from 3 of 3 ISAV-infected and 0 of 4 uninfected or sham-infected fish. The ISAV genome was detected by shotgun metagenomics in RNA extracted from the medium from 3 of 3 inoculated cell cultures, 3 of 3 infected fish, and 0 of 4 uninfected or sham-infected fish, yielding sufficient coverage for de novo assembly. An ISH probe against ISAV revealed ISAV genome in multiple organs, with abundance in renal hematopoietic tissue. Virus was detected by RT-rtPCR in gill, heart, kidney, liver, and spleen. EM and metagenomic WGS from tissues were challenging and unsuccessful. Our proof-of-concept methodology has promise for detection and characterization of unknown aquatic pathogens and also highlights some associated methodology challenges that require additional investigation.

Visceral hypersensitivity (VH) is commonly cited as a major driver of chronic abdominal pain in “functional” gastrointestinal disorders (e.g., irritable bowel syndrome) where persistent and/or recurrent abdominal pain is the primary unifying symptom regardless of any alterations in bowel habits. The complexity of VH is in part influenced by genetic factors and individual differences in gut microbiome composition, yet specific mechanisms that generate VH remain incompletely understood. Correspondingly, current treatments to primarily focus on symptom management rather than targeting physiological mechanisms responsible for generating VH. We have begun to examine the role of genetic susceptibility and microbiome response dynamics in VH development using a preclinical model of intracolonic zymosan (ZYM) administration in which there are strain differences to VH susceptibility. Preliminary data reveals differential susceptibility between ZYM-induced VH in two closely related C57BL/6 sub strains, one from Taconic Biosciences (C57BL/6NTac) and the other from Jackson Laboratory (C57BL/6J). We have identified a VH candidate gene that encodes the arginine-vasopressin receptor 1A (AVPR1A) protein. We have further observed dynamic strain differences in the location and composition of the gut microbiome in response to ZYM corresponding to VH susceptibility. Ongoing studies are focused on teasing apart the potential bidirectional relationship(s) between genetic susceptibility and host-microbiome interactions in the etiology of VH. Identifying underlying mechanisms that drive VH would provide novel targets for pharmacological intervention and decrease reliance on opioids, which are prescribed at a significantly higher rate to patients who report abdominal pain with no accompanying structural disease. Grant support from R21 NS104789/NS/NINDS (KMB), R03 NS096454/NS/NINDS (KMB), Rita Allen Foundation Award in Pain (KMB), P20GM103418 (EEY and KMB), and a K-INBRE recruitment startup package.

There are five cloned muscarinic acetylcholine receptors (M1-M5). Of these, the muscarinic type 5 receptor (M5) is the only one localized to dopamine neurons in the ventral tegmental area and substantia nigra. Unlike M1-M4, the M5 receptor has relatively restricted expression in the brain, making it an attractive therapeutic target. Here we performed an in-depth characterization of M5-dependent potentiation of dopamine transmission in the nucleus accumbens and accompanying exploratory behaviors in male and female mice. We show that M5 receptors potentiate dopamine transmission by acting directly on the terminals within the nucleus accumbens. Using the muscarinic agonist oxotremorine, we revealed a unique concentration-response curve and a sensitivity to repeated forced swim stress or restraint stress exposure. We found that constitutive deletion of M5 receptors reduced exploration of the center of an open field while at the same time impairing normal habituation only in male mice. In addition, M5 deletion reduced exploration of salient stimuli, especially under conditions of high novelty, yet had no effect on hedonia assayed using the sucrose preference test or on stress coping strategy assayed using the forced swim test. We conclude that M5 receptors are critical for both engaging with the environment and updating behavioral output in response to environment cues, specifically in male mice. A cardinal feature of mood and anxiety disorders is withdrawal from the environment. These data indicate that boosting M5 receptor activity may be a useful therapeutic target for ameliorating these symptoms of depression and anxiety.Significance Statement:The basic physiological and behavioral functions of the muscarinic M5 receptor remain understudied. Furthermore, its presence on dopamine neurons, relatively restricted expression in the brain, and recent crystallization make it an attractive target for therapeutic development. Yet, most preclinical studies of M5 receptor function have primarily focused on substance use disorders in male rodents. Here we characterized the role of M5 receptors in potentiating dopamine transmission in the nucleus accumbens, finding impaired functioning after stress exposure. Furthermore, we show that M5 receptors can modulate exploratory behavior in a sex-specific manner, without impacting hedonic behavior. These findings further illustrate the therapeutic potential of the M5 receptor, warranting further research in the context of treating mood disorders.

Semaphorins and Plexins form ligand/receptor pairs that are crucial for a wide range of developmental processes from cell proliferation to axon guidance. The ability of semaphorins to act both as signaling receptors and ligands yields a multitude of responses. Here, we describe a novel role for Semaphorin-6D (Sema6D) and Plexin-A1 in the positioning and targeting of retinogeniculate axons. In Plexin-A1 or Sema6D mutant mice of either sex, the optic tract courses through rather than at the border of the dorsolateral geniculate nucleus (dLGN); and some retinal axons ectopically arborize adjacent and lateral to the optic tract rather than defasciculating and entering the target region. We find that Sema6D and Plexin-A1 act together and in a dose-dependent manner, as the number of the ectopic retinal projections is altered in proportion to the level of Sema6D or Plexin-A1 expression. Moreover, using retinal in utero electroporation of Sema6D or Plexin-A1 shRNA, we show that Sema6D and Plexin-A1 are both required in retinal ganglion cells (RGCs) for axon positioning and targeting. Strikingly, non-electroporated RGC axons also mistarget in the tract region, indicating that Sema6D and Plexin-A1 can act non-cell autonomously, potentially through axon-axon interactions. These data provide novel evidence for a dose-dependent and non-cell autonomous role for Sema6D and Plexin-A1 in retinal axon organization in the optic tract and dLGN.SIGNIFICANCE STATEMENT:Before innervating their central brain targets, retinal ganglion cell (RGC) axons fasciculate in the optic tract then branch and arborize in their target areas. Upon deletion of the guidance molecules Plexin-A1 or Semaphorin-6D, the optic tract becomes disorganized near and extends within the dorsal lateral geniculate nucleus. In addition, some retinal axons form ectopic aggregates within the defasciculated tract. Sema6D and Plexin-A1 act together as a receptor-ligand pair in a dose-dependent manner, and non-cell autonomously, to produce this developmental aberration. Such a phenotype highlights an under-appreciated role for axon guidance molecules in tract cohesion and appropriate defasciculation near and arborization within targets.

Background: The Psychiatric Ratings using Intermediate Stratified Markers (PRISM) project focuses on understanding the biological background behind social deficits, specifically social withdrawal irrespective of diagnosis. Reduced connectional integrity in fiber tracts such as Forceps minor has been indicated in low social individuals as a part of the PRISM 1 project. These fiber tracts are also involved in the Default Mode Network (DMN) and the Social network and they share a common region, the Orbitofrontal Cortex (OFC).This study aims to back-translate the clinical data to preclinical studies and associate social dysfunction in rodents with DMN and particularly OFC. Parvalbumin interneurons are targeted based on their fundamental role in maintaining Excitatory Inhibitory (E/I) balance in brain circuits. Numerous studies indicate behavioral impairment in rodents by increasing excitability of PV+ interneurons. Methods: As an initial step, we characterized the population of projection neurons within OFCs by combining Cholera Toxin subunit B (CTB) as a retrograde tracer and In situ hybridization (ISH) technique (RNAscope). We identified the expression of mRNAs marking glutamatergic (vesicular glutamate transporter [VGLUT]) and GABAergic (vesicular GABA transporter [VGAT]) by using Slc17a7 and Slc32a1 probes. CTB was injected unilaterally in the left OFC (AP=2.68, ML=-0.8, DV=2.2). after 10 days mice were perfused and RNAscope assay was performed using RNAscope™ Multiplex Fluorescent kit (ACDBio™).For inducing hypoactivation of OFC, we introduced an excitatory DREADD (designer receptors exclusively activated by designer drugs) to PV+ interneurons by using a PV-Cre mouse line. Mice were injected either AAV-hSyn-DIO-hM3D(Gq)-mCherry virus (n=12) or AAV-hSyn-DIO-mCherry (n=12) as control virus. As a novel behavioral tool, Radiofrequency identification (RFID)-assisted SocialScan combined with video tracking has been used, which provides a long-term observation of social behaviors. Monitoring the behavior in groups of four was performed for 7 days in total. After two pre-application days, Clozapine-N-oxide (CNO) was injected three times on consecutive days intraperitoneally (5mg/kg) as an activator of hM3D. application days were followed by two post-application days. Mice were perfused and RNAscope was performed to visualize c-fos mRNA expression as neuronal activity marker, and PV expression to validate our virus and mouse line efficacy. Results: ISH results indicated VGLUT1 has the highest expression within projection neurons (81%). 6% are VGAT+ and only 3% are both VGLUT1/VGAT positive neurons. Despite demonstrating the GABAergic projection neurons as a minority, their crucial role as local interneurons to moderate the excitatory neurons is indisputable.In in vivo study, CNO administration induced social dysregulation in DREAAD mice, demonstrated by a reduction in different social parameters (approach, fight, etc.) in terms of duration. During post-application days, DREAAD mice showed significantly higher social interaction in all definedparameters (Social Approach: p=0.0009, unpaired T-test) and locomotion as a non-social parameter (p= 0.0207).Results from ISH support our hypothesis that DREADD activation of PV+ interneurons is followed by high expression of neuronal activity markers in these targeted interneurons. Conclusion: This study indicates that manipulation of PV+ interneurons using artificially engineered activating protein receptors, generates in effect activation of these interneurons, and this manipulation particularly in OFC could cause social dysfunction in mice.