Probes for INS

Chronic pain is under-managed in individuals over 65 years of age due to a dearth of knowledge regarding the impact of age on opioid efficacy in the elderly. We have previously shown that advanced age and sex alter morphine modulation of persistent inflammatory pain (induced by intraplantar administration of Complete Freund's adjuvant (CFA)), such that morphine potency is highest in adult male rats (2mos), with EC50 values 2-fold higher in aged males (18mos) and females regardless of age. Age-induced reductions in morphine potency were accompanied by reduced mu opioid receptor (MOR) expression in the ventrolateral periaqueductal gray (vlPAG), a CNS region critical in pain modulation. The present studies further explore the impact of age on opioid signaling within the PAG. MOR affinity, availability, and G-protein activation were assessed using radioligand binding assays and GTPγS assays in vlPAG tissue from adult and aged, male and female rats collected 72h following CFA administration. Regulation of opioid induced G-protein signaling was assessed using RNAscope to analyze mRNA expression of Regulator of G-Protein Signaling (RGS) proteins RGS4 and RGS9-2. We find that aged males and females (adult and aged) exhibit reduced vlPAG MOR binding potential and reduced G-protein activation efficiency compared to adult males, suggesting age- and sex- differences in MOR machinery drive reduced opioid potency. RNAscope revealed increased expression of RGS4 and RGS9-2 in the vlPAG of aged animals compared to adults, indicating that MOR signaling is subject to greater negative regulation in the aged vlPAG. The observed age-related reductions in vlPAG MOR agonist binding and opioid induced G-protein activation, along with the observed increase in vlPAG RGS expression have significant implications in pain management in the aged population. Our novel findings elucidate several mechanisms mediating reduced morphine potency in aged animals, and identify potential targets to improve pain management in the elderly. R01DA041529-04.

Background A nonsynonymous single nucleotide polymorphism in the neuropeptide S receptor 1 (NPSR1) gene (rs324981) results in isoleucine to asparagine substitution at amino acid 107. In humans, the ancestral variant (NPSR1 I107) is associated with increased anxiety sensitivity and risk of panic disorder, while the human-specific variant (NPSR1 N107) is considered protective against excessive anxiety. In rodents, neurobiological constituents of the NPS system have been analyzed in detail and praised for their anxiolytic-like effects. However, implication for the human situation remains unclear as rodents carry only the ancestral NPSR1 I107 variant. Methods We hypothesized that phenotypic correlates of NPSR1 variants manifest in fear-related circuits in the amygdala. We used CRISPR/Cas9-mediated gene editing to generate a “humanized” mouse strain, where individuals express either NPSR1 I107 or N107. Results Stimulation of NPSR1 evoked excitatory responses in principal neurons of the anterior basal amygdala (aBA) with significant difference in magnitude between genotypes, resulting in synaptic disinhibition of putative extinction neurons in posterior BA in mice expressing the human-specific hypofunctional N107 but not the ancestral I107 variant. N107 mice displayed improved extinction of conditioned fear, which was phenocopied after pharmacological antagonism of NPSR1 in aBA of I107 mice. Differences in fear extinction between male and female mice related to an interaction of Npsr1 genotype and salience of fear training. Conclusions In conclusion, the NPS system regulates extinction circuits in the amygdala depending on Npsr1 genotype, contributing to sex-specific differences in fear extinction and high anxiety sensitivity of individuals bearing the ancestral NPSR1 I107 variant.

Chronic immobilization stress (CIS) results in sex-dependent changes in opioid peptide levels and receptor subcellular distributions within the rat dorsal hippocampus which are paralleled with an inability for males to acquire conditioned place preference (CPP) to oxycodone. Here, RNAScope in situ hybridization was used to determine the expression of hippocampal opioid peptides and receptors in unstressed (US) and CIS estrus female and male adult (~ 14 wk) Sprague Dawley rats. In all groups, dentate granule cells expressed PENK and PDYN; additionally, numerous interneurons expressed PENK. OPRD1 and OPRM1 were primarily expressed in interneurons, and to a lesser extent, in pyramidal and granule cells. OPRK1-was expressed in sparsely distributed interneurons. There were few baseline sex differences: US females compared to US males had more PENK-expressing and fewer OPRD1-expressing granule cells and more OPRM1-expressing CA3b interneurons. Several expression differences emerged after CIS. Both CIS females and males compared to their US counterparts had elevated: 1) PENK-expressing dentate granule cells and interneurons in CA1 and CA2/3a; 2) OPRD1 probe number and cell expression in CA1, CA2/3a and CA3b and the dentate gyrus; and 3) OPRK1-expressing interneurons in the dentate hilus. Also, CIS males compared to US males had elevated: 1) PDYN expression in granule cells; 2) OPRD1 probe and interneuron expression in CA2/3a; 3) OPRM1 in granule cells; and 4) OPRK1 interneuron expression in CA2/3a. The sex-specific changes in hippocampal opioid gene expression may impact network properties and synaptic plasticity processes that may contribute to the attenuation of oxycodone CPP in CIS males. This article is protected by

Anxiety disorders are debilitating psychiatric diseases that affect approximately 16% of the world's population. Although it has been proposed that the central nucleus of the amygdala (CeA) plays a role in anxiety, the molecular and circuit mechanisms through which CeA neurons modulate anxiety-related behaviors are largely uncharacterized. Soluble epoxide hydrolase (sEH) is a key enzyme in the metabolism of polyunsaturated fatty acids, and has been shown to play a role in psychiatric disorders. Here, we reported that sEH was enriched in neurons in the CeA and regulated anxiety-related behaviors in adult male mice. Deletion of sEH in CeA neurons but not astrocytes induced anxiety-like behaviors. Mechanistic studies indicated that sEH was required for maintaining the the excitability of sEH positive neurons (sEHCeA neurons) in the CeA. Using chemogenetic manipulations, we found that sEHCeA neurons bidirectionally regulated anxiety-related behaviors. Notably, we identified that sEHCeA neurons directly projected to the bed nucleus of the stria terminalis (BNST) (sEHCeA-BNST). Optogenetic activation and inhibition of the sEHCeA-BNST pathway produced anxiolytic and anxiogenic effects, respectively. In summary, our studies reveal a set of molecular and circuit mechanisms of sEHCeA neurons underlying anxiety.SIGNIFICANCE STATEMENTsEH, a key enzyme that catalyzes the degradation of EETs, is shown to play a key role in mood disorders. It is well-known that sEH is mostly localized in astrocytes in the prefrontal cortex and regulates depressive-like behaviors. Notably, sEH is also expressed in CeA neurons. While the CeA has been studied for its role in the regulation of anxiety, the molecular and circuit mechanism is quite complex. In the present study, we explored a previously unknown cellular and circuitry mechanism that guides sEHCeA neurons response to anxiety. Our findings reveal a critical role of sEH in the CeA, sEHCeA neurons and CeA-BNST pathway in regulation of anxiety-related behaviors.

Neocortical progenitor cells generate subtypes of excitatory projection neurons in sequential order followed by the generation of astrocytes. The transcription factor zinc finger and BTB domain-containing protein 20 (ZBTB20) has been implicated in regulation of cell specification during neocortical development. Here, we show that ZBTB20 instructs the generation of a subset of callosal projections neurons in cortical layers II/III in mouse. Conditional deletion of Zbtb20 in cortical progenitors, and to a lesser degree in differentiating neurons, leads to an increase in the number of layer IV neurons at the expense of layer II/III neurons. Astrogliogenesis is also affected in the mutants with an increase in the number of a specific subset of astrocytes expressing GFAP. Astrogliogenesis is more severely disrupted by a ZBTB20 protein containing dominant mutations linked to Primrose syndrome, suggesting that ZBTB20 acts in concert with other ZBTB proteins that were also affected by the dominant-negative protein to instruct astrogliogenesis. Overall, our data suggest that ZBTB20 acts both in progenitors and in postmitotic cells to regulate cell fate specification in the mammalian neocortex.

Recent studies have demonstrated that genes related to bitter taste receptors (TAS2Rs) on various chromosomes are expressed in extra-oral organs of various animals. The bitter taste receptor TAS2R14 is conserved among primate species and shows broad ligand sensitivity. Mice have a number of orthologues to primate TAS2R14 located in tandem on chromosome 16; however, their expression patterns are not unique. We characterized the expression of TAS2R14 in various cell types in the intestines of the rhesus macaque and evaluated its role in hormone production in the gut. TAS2R14 expression was examined in the intestines of rhesus macaques, a common non-human primate model, by RT-qPCR and immunohistochemical staining. Mean expression levels of TAS2R14 in the duodenum, ileum, and colon were similar to each other and were lower than those in circumvallate papillae. An immunohistochemical analysis revealed TAS2R14 immunoreactivity in enteroendocrine cells positive for cholecystokinin, serotonin, and the G protein GNAT3. These results suggest that primate TAS2R14 is broadly expressed in the intestine, mainly in enteroendocrine cells, and promotes gut hormone secretion in response to bitter stimuli.

Endometriosis is a complex disease, influenced by genetic factors. Genetic markers associated with endometriosis exist at chromosome 1p36.12 and lead to altered expression of the long intergenic non-coding RNA 339 (LINC00339), however the role of LINC00339 in endometriosis pathophysiology remains unknown. The aim of this work was to characterise the expression patterns of LINC00339 mRNA in endometrium and endometriotic lesions in situ and to determine the functional role of LINC00339 in human endometrium. We employed RNA-sequencing, quantitative RT-PCR and in situ hybridisation to investigate the abundance of LINC00339 transcripts in endometrium and endometrial cell lines and to describe the pattern and localisation of LINC00339 expression in endometrium and endometriotic lesions. LINC00339 mRNA expression was manipulated (overexpressed and silenced) in endometrial stomal cell lines and RNA-sequencing data from overexpression models were analysed using online bioinformatics platforms (STRING and Ingenuity Pathway Analysis) to determine functional processes. We demonstrated the expression of LINC00339 in endometriotic lesions for the first time; we found LINC00339 expression was restricted to the lesion foci and absent in surrounding non-lesion tissue. Furthermore, manipulation of LINC00339 expression in endometrial stromal cell lines significantly impacted the expression of genes involved in immune defense pathways. These studies identify a novel mechanism for LINC00339 activity in endometrium and endometriosis, paving the way for future work, which is essential for understanding the pathogenesis of endometriosis.

Cathelicidins have been reported to inhibit human papillomavirus infection in vitro; however, nothing is known about their activity in vivo. In this study, experimental skin infection with Mus musculus papillomavirus 1 resulted in robust development of cutaneous papillomas in cyclosporine A-treated C57BL/6J mice deficient for the murine cathelicidin-related antimicrobial peptide (CRAMP), in contrast to wild-type controls. Analysis of the underlying mechanisms revealed moderate disruption of virion integrity and lack of interference with viral entry and intracellular trafficking by a synthetic CRAMP peptide. Differences in the immune response to Mus musculus papillomavirus 1 infection were observed between CRAMP-deficient and wild-type mice. These included a stronger reduction in CD4+ and CD8+ T-cell numbers in infected skin, and lack of Mus musculus papillomavirus 1-specific neutralizing antibodies in response to cyclosporine A in the absence of endogenous CRAMP. CRAMP has modest direct anti-papillomaviral effects in vitro, but exerts protective functions against Mus musculus papillomavirus 1 skin infection and disease development in vivo, primarily by modulation of cellular and humoral immunity.

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.

Endocrine disrupting chemicals (EDCs) are raising concerns about adverse effects on fertility in women as they have been shown to disrupt steroidogenesis and ovarian function in animal studies, and they associate to reduced fertility in human cohort studies. However, there is a lack of information regarding mechanisms of action and effects in humans. Our study aims to identify molecular mechanisms of endocrine disruption using two well-known human EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ), via controlled exposure studies in ovarian cell lines and human ovarian tissue culture in vitro. Ovarian cortical tissue slices obtained from tissue collected from Caesarean section (c-section) patients at Karolinska University Hospital was exposed to 10-9 M to 10-5 M KTZ and 10-10 M to 10-6 M DES in vitro for 6 days. Follicle survival and growth were studied using histology, steroid production by liquid-chromatography-mass spectrometry (LC-MS/MS), and tissue viability by cytotoxicity and fibrosis assays. RNA sequencing was performed on primary ovarian cells and ovarian granulosa cell cancer cell lines COV434 and KGN that were exposed for 24 hours to the same concentrations of DES and KTZ as the tissue culture. Selected potential biomarkers were validated using real-time quantitative polymerase chain reaction (RT-qPCR) in the cells, and by in situ RNA hybridization in exposed tissue. Significantly lower non-growing follicle densities (i.e. primordial, intermediary, and primary follicles) were observed in DES 10-10 M group compared to vehicle control. A decrease trend was also observed in DES high dose group and low level KTZ exposed group. On the other hand, slightly higher growing follicle density was shown in high level KTZ exposed group. Levels of pregnenolone and progesterone were significantly reduced in KTZ 10-5 M exposed group. RNA sequencing showed that 445 and 233 differential expressed genes (DEGs) (FDR< 0.1) were affected in DES and KTZ exposed group, respectively, in the cell culture. Gene set variation analysis (GSVA) showed that both DES and KTZ modulated MTORC1 signaling, which was critical for primordial follicle activation and steroidogenesis. We selected stear-oyl-CoA desaturase (SCD), a gene that was shown to involved in cholesterol homeostasis, oocyte maturation and steroidogenesis, for validation as a potential biomarker. Up-regulation of was confirmed in response to KTZ by PCR and RNAscope. In conclusion, DES and KTZ affected folliculogenesis and steroidogenesis in human adult ovarian cortex and SCD may serve as a potential biomarker in response to exposure. Further validation of this potential biomarker may help improve the existing testing guideline and subsequently, contributing to better regulation of chemical in our global market.

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.