Probes for INS

Oxytocin is an endogenous neuropeptide hormone that influences social behaviour and bonding in mammals. Variations in oxytocin receptor (OXTR) expression may play a role in the social deficits seen in autism spectrum disorder. Previous studies from our laboratory found a dense population of OXTR in the human substantia nigra (SN), a basal ganglia structure in the midbrain that is important in both movement and reward pathways. Here, we explore whether differences in OXTR can be identified in the dopaminergic SN pars compacta of individuals with autism. Postmortem human brain tissue specimens were processed for OXTR autoradiography from four groups: males with autism, females with autism, typically developing (TD) males and TD females. We found that females with autism had significantly lower levels of OXTR than the other groups. To examine potential gene expression differences, we performed in situ hybridization in adjacent slides to visualize and quantify OXTR mRNA as well as mRNA for tyrosine hydroxylase. We found no differences in mRNA levels for either gene across the four groups. These results suggest that a dysregulation in local OXTR protein translation or increased OXTR internalization/recycling may contribute to the differences in social symptoms seen in females with autism. This article is part of the theme issue 'Interplays between oxytocin and other neuromodulators in shaping complex social behaviours'.

Diabetes leads to intestinal barrier dysfunction. 5-Hydroxytryptamine receptor 4 (5-HT4R) is distributed in the colonic mucosa, but little is known about the role of 5-HT4R activation in diabetes-evoked colonic barrier dysfunction. This study investigates whether activation of 5-HT4Rs on goblet cells (GCs) protects the colon from commensal bacterial translocation in diabetic mice. Expression of 5-HT4R detected inside the colonic epithelium by RNAscope in situ hybridization was further observed within the mucin 2 (MUC2)-immunoreactive GCs. In diabetic mice, neither 5-HT4R transcription nor protein levels were altered compared with those in nondiabetic mice. Bacterial translocation was characterized by 16S rRNA RNAscope in situ hybridization and manifested in both crypts and lamina propria of the colon in diabetic mice. Moreover, mucin production and MUC2 expression were significantly decreased in diabetic mice. Furthermore, the loss of mitochondrial cristae of GCs and the down-regulation of mitofilin, the core protein maintaining mitochondrial homeostasis, were observed in diabetic mice. However, long-term treatment with 5-HT4R agonist in diabetic mice not only prevented bacterial penetration of the whole colonic mucosa but also promoted mucin production and MUC2 expression. Markedly, 5-HT4R agonist also restored the mitochondrial cristae of GCs and up-regulated mitofilin. However, co-administration of 5-HT4R antagonist abolished the effects of 5-HT4R agonist on diabetic mice. These findings indicate that 5-HT4R in colonic mucosa is an effective target for the treatment of diabetes-induced colonic mucous barrier dysfunction.

The neuropeptide oxytocin (Oxt) plays important roles in modulating social behaviors. Oxt receptor (Oxtr) is abundantly expressed in the brain and its relationship to socio-behavioral controls has been extensively studied using mouse brains. Several genetic tools to visualize and/or manipulate Oxtr-expressing cells, such as fluorescent reporters and Cre recombinase drivers, have been generated by ES-cell based gene targeting or bacterial artificial chromosome (BAC) transgenesis. However, these mouse lines displayed some differences in their Oxtr expression profiles probably because of the complex context and integrity of their genomic configurations in each line. Here, we apply our sophisticated genome-editing techniques to the Oxtr locus, systematically generating a series of knock-in mouse lines, in which its endogenous transcriptional regulations are intactly preserved and evaluate their expression profiles to ensure the reliability of our new tools. We employ the epitope tagging strategy, with which C-terminally fused tags can be detected by highly specific antibodies, to successfully visualize the Oxtr protein distribution on the neural membrane with super-resolution imaging for the first time. By using T2A self-cleaving peptide sequences, we also induce proper expressions of tdTomato reporter, codon-improved Cre recombinase (iCre), and spatiotemporally inducible Cre-ERT2 in Oxtr-expressing neurons. Electrophysiological recordings from tdTomato-positive cells in the reporter mice support the validity of our tool design. Retro-orbital injections of AAV-PHP.eB vector into the Cre line further enabled visualization of recombinase activities in the appropriate brain regions. Moreover, the first-time Cre-ERT2 line drives Cre-mediated recombination in a spatiotemporally controlled manner on tamoxifen (TMX) administration. These tools thus provide an excellent resource for future functional studies in Oxt-responsive neurons and should prove of broad interest in the field.

The classical motor center cerebellum is one of the most consistent structures of abnormality in autism spectrum disorders (ASD), and neuropeptide oxytocin is increasingly explored as a potential pharmacotherapy for ASD. However, whether oxytocin targets the cerebellum for therapeutic effects remains unclear. Here, we report a localization of oxytocin receptor (OXTR) in Purkinje cells (PCs) of cerebellar lobule Crus I, which is functionally connected with ASD-implicated circuits. OXTR activation neither affects firing activities, intrinsic excitability, and synaptic transmission of normal PCs nor improves abnormal intrinsic excitability and synaptic transmission of PCs in maternal immune activation (MIA) mouse model of autism. Furthermore, blockage of OXTR in Crus I in wild-type mice does not induce autistic-like social, stereotypic, cognitive, and anxiety-like behaviors. These results suggest that oxytocin signaling in Crus I PCs seems to be uninvolved in ASD pathophysiology, and contribute to understanding of targets and mechanisms of oxytocin in ASD treatment.

Hyperactivity of pyramidal neurons (PNs) in CA1 is an early event in Alzheimer’s disease (AD). However, factors accounting for the hyperactivity of CA1 PNs remain to be completely investigated. In the present study, we found that the serotonergic signaling was abnormal in the hippocampus of hAPP-J20 mice. Interestingly, chemogenetic activation of serotonin (5-hydroxytryptamine, 5-HT) neurons in the median raphe nucleus (MRN) attenuated the activity of CA1 PNs in hAPP-J20 mice by regulating the intrinsic properties or inhibitory synaptic transmission of CA1 PNs through 5-HT3aR and/or 5-HT1aR. Furthermore, activating MRN 5-HT neurons improved memory in hAPP-J20 mice and this effect was mediated by 5-HT3aR and 5-HT1aR. Direct activation of 5-HT3aR and 5-HT1aR with their selective agonists also improved memory of hAPP-J20 mice. Together, we identified the impaired 5-HT/5-HT3aR and/or 5-HT/5-HT1aR signaling as new pathways contributing to the hyperexcitability of CA1 PNs and the impaired cognition in hAPP-J20 mice.

The nonapeptide system modulates numerous social behaviors through oxytocin and vasopressin activation of the oxytocin receptor (OXTR) and vasopressin receptor (AVPR1A) in the brain. OXTRs and AVPR1As are widely distributed throughout the brain and binding densities exhibit substantial variation within and across species. Although OXTR and AVPR1A binding distributions have been mapped for several rodents, this system has yet to be characterized in the spiny mouse (Acomys cahirinus). Here we conducted receptor autoradiography and in situ hybridization to map distributions of OXTR and AVPR1A binding and Oxtr and Avpr1a mRNA expression throughout the basal forebrain and midbrain of male and female spiny mice. We found that nonapeptide receptor mRNA is diffuse throughout the forebrain and midbrain and does not always align with OXTR and AVPR1A binding. Analyses of sex differences in brain regions involved in social behavior and reward revealed that males exhibit higher OXTR binding densities in the lateral septum, bed nucleus of the stria terminalis, and anterior hypothalamus. However, no association with gonadal sex was observed for AVPR1A binding. Hierarchical clustering analysis further revealed that co-expression patterns of OXTR and AVPR1A binding across brain regions involved in social behavior and reward differ between males and females. These findings provide mapping distributions and sex differences in nonapeptide receptors in spiny mice. Spiny mice are an excellent organism for studying grouping behaviors such as cooperation and prosociality, and the nonapeptide receptor mapping here can inform the study of nonapeptide-mediated behavior in a highly social, large group-living rodent.

Oxytocin regulates social behavior via direct modulation of neurons, regulation of neural network activity, and interaction with other neurotransmitter systems. The behavioral effects of oxytocin signaling are determined by the species-specific distribution of brain oxytocin receptors. The socially monogamous prairie vole has been a useful model organism for elucidating the role of oxytocin in social behaviors, including pair bonding, response to social loss, and consoling. However, there has been no comprehensive mapping of oxytocin receptor-expressing cells throughout the prairie vole brain. Here, we employed a highly sensitive in situ hybridization, RNAscope, to construct an exhaustive, brain-wide map of oxytocin receptor mRNA-expressing cells. We found that oxytocin receptor mRNA expression was widespread and diffused throughout the brain, with specific areas displaying a particularly robust expression. Comparing receptor binding with mRNA revealed that regions of the hippocampus and substantia nigra contained oxytocin receptor protein but lacked mRNA, indicating that oxytocin receptors can be transported to distal neuronal processes, consistent with presynaptic oxytocin receptor functions. In the nucleus accumbens, a region involved in oxytocin-dependent social bonding, oxytocin receptor mRNA expression was detected in both the D1 and D2 dopamine receptor-expressing subtypes of cells. Furthermore, natural genetic polymorphisms robustly influenced oxytocin receptor expression in both D1 and D2 receptor cell types in the nucleus accumbens. Collectively, our findings further elucidate the extent to which oxytocin signaling is capable of influencing brain-wide neural activity, responses to social stimuli, and social behavior. KEY POINTS: Oxytocin receptor mRNA is diffusely expressed throughout the brain, with strong expression concentrated in certain areas involved in social behavior. Oxytocin receptor mRNA expression and protein localization are misaligned in some areas, indicating that the receptor protein may be transported to distal processes. In the nucleus accumbens, oxytocin receptors are expressed on cells expressing both D1 and D2 dopamine receptor subtypes, and the majority of variation in oxytocin receptor expression between animals is attributable to polymorphisms in the oxytocin receptor gene.

Seasonal changes in food intake and adiposity in many animal species are triggered by changes in the photoperiod. These latter changes are faithfully transduced into a biochemical signal by melatonin secreted by the pineal gland. Seasonal variations, encoded by melatonin, are integrated by third ventricular tanycytes of the mediobasal hypothalamus through the detection of the thyroid-stimulating hormone (TSH) released from the pars tuberalis. The mediobasal hypothalamus is a critical brain region that maintains energy homeostasis by acting as an interface between the neural networks of the central nervous system and the periphery to control metabolic functions, including ingestive behavior, energy homeostasis, and reproduction. Among the cells involved in the regulation of energy balance and the blood-hypothalamus barrier (BHB) plasticity are tanycytes. Increasing evidence suggests that anterior pituitary hormones, specifically TSH, traditionally considered to have unitary functions in targeting single endocrine sites, display actions on multiple somatic tissues and central neurons. Notably, modulation of tanycytic TSH receptors seems critical for BHB plasticity in relation to energy homeostasis, but this needs to be proven.