Probes for INS

Gene expression analyses by molecular histology are a crucial step in understanding gene function in any model organism. In the teleost _Astyanax mexicanus_, here we describe in detail the method we have developed to perform double fluorescent in situ hybridization on whole-mount samples. As an illustration, in the result section, we present an analysis of the expression patterns of four mRNAs expressed in the hypothalamus of the surface and cave morphs of _A. mexicanus_ at 3.5 days postfertilization, three neuropeptides (_npy_, _pomca_, _agrp_) and one transcription factor (_isl1_). Confocal imaging after fluorescent in situ hybridization allows counting cells in distinct but closely related hypothalamic areas. The step-by-step protocol and the comprehensive table of reagents presented here will allow researchers to analyze gene expression in different structures and at various stages, from embryos to older larvae.

Although the immunogenicity of formalin-fixed paraffin-embedded tissue sections can decrease during storage and transport, the exact mechanism of antigenic loss and how to prevent it are not clear. Herein, we investigated changes in the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), E-cadherin, and Ki-67 in human breast tissue microarray (TMA) tissue sections stored for up to 3 months in dry and wet conditions. The positive rates of ER and PR expression were minimally changed after 3 months of storage, but the Allred scores of ER and PR stored in humid conditions decreased remarkably in comparison to fresh-cut tissue. The HER-2 antigenicity and RNA integrity of breast TMA sections stored in dry conditions diminished gradually with storage time, whereas the immunoreactivity and RNA quality of HER-2 in humid conditions decreased sharply as storage length increased. The area and intensity of E-cadherin staining in tissue sections stored in dry conditions did not change significantly and were minimally changed after 3 months, respectively. In contrast, the area and intensity of E-cadherin staining in tissue sections stored in humid conditions decreased significantly as storage length increased. Finally, the Ki-67 labeling index of tissue sections stored for 3 months in dry (9% decrease) and wet (31.9% decrease) conditions was decreased in comparison to fresh sections. In conclusion, these results indicate that water is a crucial factor for protein and RNA degradation in stored tissue sections, and detailed guidelines are required in the clinic.

Radiochemical in situ hybridization enables detection of gene expression in small areas of the brain, such as the developing pineal gland in rodents. The method combines determination of spatial and temporal gene expression profiles with semiquantitative analyses. We here describe the procedure of radiochemical in situ hybridization on the developing rat pineal gland ranging from preparation of fetal tissue for in situ hybridization to principles of quantification.

Single-cell technology has become an indispensable tool in cardiovascular research since its first introduction in 2009. Here, we highlight the recent remarkable progress in using single-cell technology to study transcriptomic and epigenetic heterogeneity in cardiac disease and development. We then introduce the key concepts in single-cell multi-omics modalities that apply to cardiovascular research. Lastly, we discuss some of the trending concepts in single-cell technology that are expected to propel cardiovascular research to the next phase of single-cell research.

Epidermal keratinocytes express various purinergic 2 receptors that play an essential role in cell growth, differentiation, and proliferation. In the conditions of injury, concentrations of extracellular adenosine triphosphate (ATP) may dramatically increase due to cell damage and inflammatory processes. In this situation activation of purinergic signaling in keratinocytes could act as a double-edged sword contributing to skin regeneration or cell apoptosis. As the role of keratinocytes in transducing and modulating nociceptive stimuli has been increasingly appreciated in recent years, the aim of the present study was to evaluate whether peripheral nerve injury affects purinergic signaling in keratinocytes. Spared nerve injury (SNI), a classical model of peripheral neuropathic pain, was induced in mice. The injury was induced by sparing of the tibial nerve, and ligation and cut of the sural and common peroneal nerves. Keratinocytes were isolated and cultured on Days 2-4 post-injury and ATP-mediated calcium responses in keratinocytes were examined by confocal imaging. On average, the number of keratinocytes that responded to ATP with an increase in intracellular calcium gradient as well as the magnitude of the peak response was not significantly different between sham and SNI groups. However, significantly less delay in ATP-induced increase in intracellular calcium concentration was observed in keratinocytes in SNI group compared to sham. Selective pharmacological inhibition of keratinocyte response to ATP indicated a major role of P2 × 4 receptors in the modulation of calcium homeostasis in SNI. Our results indicate that epidermal purinergic signaling undergoes dramatic changes following peripheral nerve injury that may contribute to injury-induced mechanical hypersensitivity.

Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide-deficiency phenotypes with either role is first to assess potential phenotypes in so-called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well-known constellation of behavioral and metabolic phenotypes associated with constitutive PACAP knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP-null phenotype from wild-type in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in wild-type mice that do not occur in knock-out mice (aPRGs). Comparing constitutive PACAP knock-out mice to a variety of temporally and regionally specific PACAP knock-outs, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP-expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knock-out mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress.

Localization of mRNAs at the front of migrating cells is a widely used mechanism that functionally supports efficient cell movement. It is observed in single cells on two-dimensional surfaces, as well as in multicellular three-dimensional (3D) structures and in tissue in vivo. 3D multicellular cultures can reveal how the topology of the extracellular matrix and cell-cell contacts influence subcellular mRNA distributions. Here we describe a method for mRNA imaging in an inducible system of collective cancer cell invasion. MDA-MB-231 cancer cell spheroids are embedded in Matrigel, induced to invade, and processed to image mRNAs with single-molecule sensitivity. An analysis algorithm is used to quantify and compare mRNA distributions at the front of invasive leader cells. The approach can be easily adapted and applied to analyze RNA distributions in additional settings where cells polarize along a linear axis.

Chronic stress is a well-known risk factor for the development of hypertension. However, the underlying mechanisms remain unclear. Corticotropin-releasing hormone (CRH) neurons in the central nucleus of the amygdala (CeA) are involved in the autonomic responses to chronic stress. Here, we determined the role of CeA-CRH neurons in chronic stress-induced hypertension.Borderline hypertensive rats (BHRs) and Wistar-Kyoto (WKY) rats were subjected to chronic unpredictable stress (CUS). Firing activity and M-currents of CeA-CRH neurons were assessed, and a CRH-Cre-directed chemogenetic approach was used to suppress CeA-CRH neurons. CUS induced a sustained elevation of arterial blood pressure (ABP) and heart rate (HR) in BHRs, while in WKY rats, CUS-induced increases in ABP and HR quickly returned to baseline levels after CUS ended. CeA-CRH neurons displayed significantly higher firing activities in CUS-treated BHRs than unstressed BHRs. Selectively suppressing CeA-CRH neurons by chemogenetic approach attenuated CUS-induced hypertension and decreased elevated sympathetic outflow in CUS-treated BHRs. Also, CUS significantly decreased protein and mRNA levels of Kv7.2 and Kv7.3 channels in the CeA of BHRs. M-currents in CeA-CRH neurons were significantly decreased in CUS-treated BHRs compared with unstressed BHRs. Blocking Kv7 channel with its blocker XE-991 increased the excitability of CeA-CRH neurons in unstressed BHRs but not in CUS-treated BHRs. Microinjection of XE-991 into the CeA increased sympathetic outflow and ABP in unstressed BHRs but not in CUS-treated BHRs.CeA-CRH neurons are required for chronic stress-induced sustained hypertension. The hyperactivity of CeA-CRH neurons may be due to impaired Kv7 channel activity, which represents a new mechanism involved in chronic stress-induced hypertension.We found that hyperactivity of CRH neurons in the CeA, likely due to diminished Kv7 channel activity, play a major role in the development of chronic stress-induced hypertension. Our study suggests that CRH neurons in the brain may be targeted for treating chronic stress-induced hypertension. Thus, increasing Kv7 channel activity or overexpressing Kv7 channels in the CeA may reduce stress-induced hypertension. Further studies are needed to delineate how chronic stress diminishes Kv7 channel activity in the brain.

Infant avoidance and aggression are promoted by activation of the Urocortin-3 expressing neurons of the perifornical area of hypothalamus (PeFAUcn3) in male and female mice. PeFAUcn3 neurons have been implicated in stress, and stress is known to reduce maternal behavior. We asked how chronic restraint stress (CRS) affects infant-directed behavior in virgin and lactating females and what role PeFAUcn3 neurons play in this process. Here we show that infant-directed behavior increases activity in the PeFAUcn3 neurons in virgin and lactating females. Chemogenetic inhibition of PeFAUcn3 neurons facilitates pup retrieval in virgin females. CRS reduces pup retrieval in virgin females and increases activity of PeFAUcn3 neurons, while CRS does not affect maternal behavior in lactating females. Inhibition of PeFAUcn3 neurons blocks stress-induced deficits in pup-directed behavior in virgin females. Together, these data illustrate the critical role for PeFAUcn3 neuronal activity in mediating the impact of chronic stress on female infant-directed behavior.

Somatosensory neurons are highly heterogeneous with distinct types of neural cells responding to specific stimuli. However, the distribution and roles of cell-type-specific long intergenic noncoding RNAs (lincRNAs) in somatosensory neurons remain largely unexplored. Here, by utilizing droplet-based single-cell RNA-seq (scRNA-seq) and full-length Smart-seq2, we show that lincRNAs, but not coding mRNAs, are enriched in specific types of mouse somatosensory neurons. Profiling of lincRNAs from single neurons located in dorsal root ganglia (DRG) identifies 200 lincRNAs localized in specific types or subtypes of somatosensory neurons. Among them, the conserved cell-type-specific lincRNA CLAP associates with pruritus and is abundantly expressed in somatostatin (SST)-positive neurons. CLAP knockdown reduces histamine-induced Ca2+ influx in cultured SST-positive neurons and in vivo reduces histamine-induced scratching in mice. In vivo knockdown of CLAP also decreases the expression of neuron-type-specific and itch-related genes in somatosensory neurons, and this partially depends on the RNA binding protein MSI2. Our data reveal a cell-type-specific landscape of lincRNAs and a function for CLAP in somatosensory neurons in sensory transmission.

Microglia are widely distributed in the central nervous system (CNS), where they aid in the maintenance of neuronal function and perform key auxiliary roles in phagocytosis, neural repair, immunological control, and nutrition delivery. Microglia in the undamaged spinal cord is in a stable state and serve as immune monitors. In the event of spinal cord injury (SCI), severe changes in the microenvironment and glial scar formation lead to axonal regeneration failure. Microglia participates in a series of pathophysiological processes and behave both positive and negative consequences during this period. A deep understanding of the characteristics and functions of microglia can better identify therapeutic targets for SCI. Technological innovations such as single-cell RNA sequencing (Sc-RNAseq) have led to new advances in the study of microglia heterogeneity throughout the lifespan. Here,We review the updated studies searching for heterogeneity of microglia from the developmental and pathological state, survey the activity and function of microglia in SCI and explore the recent therapeutic strategies targeting microglia in the CNS injury.

Seasonal changes in day length (photoperiod) affect numerous physiological functions. The suprachiasmatic nucleus (SCN)-paraventricular nucleus (PVN) axis plays a key role in processing photoperiod-related information. Seasonal variations in SCN and PVN neurotransmitter expression have been observed in humans and animal models. However, the molecular mechanisms by which the SCN-PVN network responds to altered photoperiod is unknown. Here, we show in mice that neuromedin S (NMS) and vasoactive intestinal polypeptide (VIP) neurons in the SCN display photoperiod-induced neurotransmitter plasticity. In vivo recording of calcium dynamics revealed that NMS neurons alter PVN network activity in response to winter-like photoperiod. Chronic manipulation of NMS neurons is sufficient to induce neurotransmitter switching in PVN neurons and affects locomotor activity. Our findings reveal previously unidentified molecular adaptations of the SCN-PVN network in response to seasonality and the role for NMS neurons in adjusting hypothalamic function to day length via a coordinated multisynaptic neurotransmitter switching affecting behavior.