Probes for INS

There are sex differences in microglia, which can maintain sex-related gene expression and functional differences in the absence of circulating sex steroids. The angiotensin type 2 (AT2) receptors mediate anti-inflammatory actions in different tissues, including brain. In mice, we performed RT-PCR analysis of microglia isolated from adult brains and RNA scope in situ hybridization from males, females, ovariectomized females, orchiectomized males and brain masculinized females. We also compared wild type and AT2 knockout mice. The expression of AT2 receptors in microglial cells showed sex differences with much higher AT2 mRNA expression in females than in males, and this was not dependent on circulating gonadal hormones, as observed using ovariectomized females, brain masculinized females and orchiectomized males. These results suggest genomic reasons, possibly related to sex chromosome complement, for sex differences in AT2 expression in microglia, as the AT2 receptor gene is located in the X chromosome. Furthermore, sex differences in expression of AT2 receptors were associated to sex differences in microglial expression of key anti-inflammatory cytokines such as interleukin-10 and pro-inflammatory cytokines such as interleukin-1β and interleukin-6. In conclusion, sex differences in microglial AT2 receptor expression appear as a major factor contributing to sex differences in the neuroinflammatory responses beyond the effects of circulating steroids.

Background Pain is the most common cause of disability in IBD. What causes inter-individual variability in chronic pain after successful treatment of inflammation remains elusive. We have shown that activation of TRPV1+ colonic nociceptors is essential for the establishment of persistent pain in DSS colitis. Nociceptor development coincides with microbial colonization, while early life dysbiosis can lead to visceral hypersensitivity in adulthood. Whether the microbiota dictates nociceptor development and pain susceptibility remains unknown. Here we test the hypothesis that the microbiota programs nociceptor specification during early development, rendering them more susceptible to sensitization later in life. We have identified the aryl hydrocarbon receptor (AHR) that senses bacterial-derived metabolites as a candidate target that orchestrates transcriptional regulation in nociceptors. Aims We investigated the developmental regulation of nociceptors by the microbiome and how it influences pain sensitivity. We will determine the effects of AHR activation on nociceptor lineage and function as well as the long term impact of AHR signaling on pain sensitivity. Methods We have developed a germ-free (GF) TRPV1-GFP reporter mouse that was used to phenotype and visualise TRPV1+ nociceptors in the absence of a microbiota. We will isolate TRPV1+ neurons by FACS to identify genes that are under the control of the microbiota and to characterise the phosphoproteome of TRPV1+ nociceptors in GF conditions. Finally, we will investigate the role of AHR signaling in nociceptors both acutely and during development. Results We showed a reduction in thermal pain threshold and a reduction in capsaicin test responses in GF mice. The number and size of DRG neurons was unchanged in GF mice. Examination of molecular markers for peptidergic (CGRP) and non-peptidergic (IB4) neurons did not show a difference. Finally, there was no difference in the expression of TRPV1, suggesting post-translational modification of the channel. In cultured DRG neurons, we found a decrease in capsaicin induced action potentials and a decrease in the amplitude of the capsaicin response in GF mice. Using RNAscope, we showed that TRPV1+ neurons express AHR. Conclusions Our results highlight the importance of bacterial composition in regulating the development of nociceptors and pain sensitivity in adulthood. Furthermore, we are the first to demonstrate the expression of AHR in sensory neurons. These findings point to a role of the microbiota in programming nociceptors during development. My work will advance our understanding of the role of commensal bacteria in regulating pain and could lead to recommendations for the treatment of neonates in early life to reduce their risk of developing chronic pain later in life. Funding Agencies CAG, CIHR

One negative emotional state from morphine protracted abstinence is anxiety which can drive craving and relapse risk in opioid addicts. Although the orexinergic system has been reported to be important in mediating emotion processing and addiction, the role of orexinergic system in anxiety from drug protracted abstinence remains elusive. In this study, by using behavioral test, western blot, electrophysiology and virus-mediated regulation of orexin receptor 1 (OX1R), we found that: (1) Intraperitoneal and intra-VTA administration of a selective OX1R antagonist SB334867 alleviated anxiety-like behaviors in open field test (OFT) but not in elevated plus maze test (EPM) in morphine protracted abstinent male mice. (2) OX1R expression in the VTA was upregulated by morphine withdrawal. (3) Virus-mediated knockdown of OX1R in the VTA prevented morphine abstinence-induced anxiety-like behaviors and virus-mediated overexpression of OX1R in the VTA was sufficient to produce anxiety-like behaviors in male mice. (4) The VTA neuronal activity was increased significantly induced by morphine protracted abstinence, which was mediated by OX1R. (5) OX1R was widely distributed in the neuronal soma and processes of dopaminergic and non-dopaminergic neurons in the VTA. The findings revealed that the OX1R mediates morphine abstinence-induced anxiety-like behaviors and the VTA plays a critical role in this effect.

Cannabinoid CB2 receptors (CB2R) are importantly involved in drug reward and addiction. However, the cellular mechanisms underlying CB2R action remain unclear. We have previously reported that cocaine self-administration upregulates CB2R expression in midbrain dopamine (DA) neurons. In the present study, we investigated whether cocaine or heroin also alters CB2R expression in striatal medium-spiny neurons that express dopamine D1 or D2 receptors (D1-MSNs, D2-MSNs) and microglia. Due to the concern of CB2R antibody specificity, we developed three mouse CB2-specific probes to detect CB2R mRNA using quantitative RT-PCR and RNAscope in situ hybridization (ISH) assays. We found that a single injection of cocaine failed to alter, while repeated cocaine injections or self-administration dose-dependently upregulated CB2R gene expression in both brain (cortex and striatum) and periphery (spleen). In contrast, repeated administration of heroin produced a dose-dependent reduction in striatal CB2 mRNA expression. RNAscope ISH assays detected CB2R mRNA in striatal D1- and D2-MSNs, not in microglia. We then used transgenic CX3CR1eGFP/+ microglia reporter mice and D1- or D2-Cre-RiboTag mice to purify striatal microglia or ribosome-associated mRNAs from CX3CR1eGFP/+, D1-MSNs, or D2-MSNs, respectively. We found that CB2R upregulation occurred mainly in D1-MSNs, not in D2-MSNs or microglia, in the nucleus accumbens rather than the dorsal striatum. These findings indicate that repeated cocaine exposure may upregulate CB2R expression in both brain and spleen, with regional and cell type-specific profiles. In the striatum, CB2R upregulation occurs mainly in D1-MSNs in the nucleus accumbens. Given the important role of D1-MSNs in brain reward function, the present findings provide new insight into mechanisms by which brain CB2Rs modulate cocaine action.

Nociceptive markers in mice have been identified in two distinct peptidergic and nonpeptidergic neurons in the dorsal root ganglion (DRG) and distributed in different laminae of the dorsal horn of the spinal cord. Recently, however, a study in humans showed a significant overlapping in these two populations. In this study, we investigated the distribution of various nociceptive markers in the lumbar DRG and spinal cord of the dromedary camel. Immunohistochemical data showed a remarkable percentage of total neurons in the DRG expressed IB4 binding (54.5%), calcitonin gene-related peptide (CGRP; 49.5%), transient receptor potential vanilloid 1 (TRPV1; 48.2%), and nitric oxide synthase (NOS; 30.6%). The co-localization data showed that 89.6% and 74.0% of CGRP- and TRPV1-labeled neurons, respectively, were IB4 positive. In addition, 61.6% and 84.2% of TRPV1- and NOS-immunoreactive neurons, respectively, were also co-localized with CGRP. The distribution of IB4, CGRP, TRPV1, substance P, and NOS immunoreactivities in the spinal cord were observed in lamina I and outer lamina II (IIo). Quantitative data showed that 82.4% of IB4-positive nerve terminals in laminae I and IIo were co-localized with CGRP, and 86.0% of CGRP-labeled terminals were co-localized with IB4. Similarly, 85.1% of NOS-labeled nerve terminals were co-localized with CGRP. No neuropeptide Y (NPY) or cholecystokinin (CCK) immunoreactivities were detected in the DRG, and no co-localization between IB4, NPY, and CCK were observed in the spinal cord. Our results demonstrate marked convergence of nociceptive markers in the primary afferent neurons in camels, which is similar to humans rather than the mouse. The data also emphasizes the importance of interspecies differences when selecting ideal animal models for studying nociception and treating chronic pain.

Significance Molecules regulated by neuronal activity are necessary for circuits to adapt to changing inputs. Specific classical major histocompatibility class I (MHCI) molecules play roles in circuit and synaptic plasticity, but the function of most members of this family remains unexplored in brain. Here, we show that a nonclassical MHCI molecule, Qa-1 (H2-T23), is expressed in a subset of excitatory neurons and regulated by visually driven activity in the cerebral cortex. Moreover, CD94/NKG2 heterodimers, cognate receptors for Qa-1, are expressed in microglia. A functional interaction between Qa-1 and CD94/NKG2 is necessary for regulating the magnitude of ocular dominance plasticity during the critical period in the visual cortex, implying an interaction in which activity-dependent changes in neurons may be monitored by microglia.

Recent contradictory data has renewed discussion regarding the existence of adult hippocampal neurogenesis (AHN) in humans, i.e., the continued production of new neurons in the brain after birth. The present review revisits the debate of AHN in humans from a historical point of view in the face of contradictory evidence, analyzing the methods employed to investigate this phenomenon. Thus, to date, of the 57 studies performed in humans that we reviewed, 84% (48) concluded in favor of the presence of newborn neurons in the human adult hippocampus. Besides quality of the tissue (such as postmortem intervals below 26hours as well as tissue conservation and fixation), considerations for assessing and quantify AHN in the human brain require the use of stereology and toxicological analyses of clinical data of the patient.

Hypothalamic-pituitary-adrenal (HPA) axis dynamics are disrupted by opioids and may be involved in substance abuse; this persists during withdrawal and abstinence and is associated with co-morbid sleep disruption leading to vulnerability to relapse. We hypothesized that chronic sleep restriction (SR) alters the HPA axis diurnal rhythm and the sexually dimorphic response to acute stressor during opioid abstinence. We developed a rat model to evaluate the effect of persistent sleep loss during opioid abstinence on HPA axis dynamics in male and female rats. Plasma ACTH and corticosterone were measured diurnally and in response to acute restraint stress in rats Before (control) compared to During subsequent opioid abstinence without or with SR. Abstinence, regardless of sleep state, led to an increase in plasma ACTH and corticosterone in the morning in males. There was a tendency for higher PM plasma ACTH during abstinence in SR males (p = 0.076). ACTH and corticosterone responses to restraint were reduced in male SR rats whereas there was a failure to achieve the post-restraint nadir in female SR rats. There was no effect of the treatments or interventions on adrenal weight normalized to body weight. SR resulted in a dramatic increase in hypothalamic PVN AVP mRNA and plasma copeptin in male but not female rats. This corresponded to the attenuation of the HPA axis stress response in SR males during opioid abstinence. We have identified a potentially unique, sexually dimorphic role for magnocellular vasopressin in the control of the HPA axis during opioid abstinence and sleep restriction.

Growth hormone (GH) acts in several hypothalamic neuronal populations to modulate metabolism and the autoregulation of GH secretion via negative-feedback loops. However, few studies have investigated whether GH receptor (GHR) expression in specific neuronal populations is required for the homeostatic control of GH secretion and energy homeostasis. In the present study, we investigated the consequences of the specific GHR ablation in GABAergic (VGAT-expressing) or glutamatergic (VGLUT2-expressing) cells. GHR ablation in GABAergic neurons led to increased GH secretion, lean mass, and body growth in male and female mice. VGAT-specific GHR knockout (KO) male mice also showed increased serum insulin-like growth factor-1, hypothalamic Ghrh, and hepatic Igf1 messenger RNA levels. In contrast, normal GH secretion, but reduced lean body mass, was observed in mice carrying GHR ablation in glutamatergic neurons. GHR ablation in GABAergic cells increased weight loss and led to decreased blood glucose levels during food restriction, whereas VGLUT2-specific GHR KO mice showed blunted feeding response to 2-deoxy-D-glucose both in males and females, and increased relative food intake, oxygen consumption, and serum leptin levels in male mice. Of note, VGLUT2-cre female mice, independently of GHR ablation, exhibited a previously unreported phenotype of mild reduction in body weight without further metabolic alterations. The autoregulation of GH secretion via negative-feedback loops requires GHR expression in GABAergic cells. Furthermore, GHR ablation in GABAergic and glutamatergic neuronal populations leads to distinct metabolic alterations. These findings contribute to the understanding of the neuronal populations responsible for mediating the neuroendocrine and metabolic effects of GH.

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.

It is the centenary of the discovery of oligodendrocytes and we are increasingly aware of their importance in the functioning of the brain in development, adult learning, normal ageing and in disease across the life course, even in those diseases classically thought of as neuronal. This has sparked more interest in oligodendroglia for potential therapeutics for many neurodegenerative/neurodevelopmental diseases due to their more tractable nature as a renewable cell in the central nervous system. However, oligodendroglia are not all the same. Even from the first description, differences in morphology were described between the cells. With advancing techniques to describe these differences in human tissue, the complexity of oligodendroglia is being discovered, indicating apparent functional differences which may be of critical importance in determining vulnerability and response to disease, and targeting of potential therapeutics. It is timely to review the progress we have made in discovering and understanding oligodendroglial heterogeneity in health and neuropathology.