Oxytocin neurons enable social transmission of maternal behaviour.

Maternal care, including by non-biological parents, is important for offspring survival1-8. Oxytocin1,2,9-15, which is released by the hypothalamic paraventricular nucleus (PVN), is a critical maternal hormone. In mice, oxytocin enables neuroplasticity in the auditory cortex for maternal recognition of pup distress15. However, it is unclear how initial parental experience promotes hypothalamic signalling and cortical plasticity for reliable maternal care. Here we continuously monitored the behaviour of female virgin mice co-housed with an experienced mother and litter. This documentary approach was synchronized with neural recordings from the virgin PVN, including oxytocin neurons. These cells were activated as virgins were enlisted in maternal care by experienced mothers, who shepherded virgins into the nest and demonstrated pup retrieval. Virgins visually observed maternal retrieval, which activated PVN oxytocin neurons and promoted alloparenting. Thus rodents can acquire maternal behaviour by social transmission, providing a mechanism for adapting the brains of adult caregivers to infant needs via endogenous oxytocin.

Systemic Co-Administration of Low-Dose Oxytocin and Glucagon-Like Peptide 1 Additively Decreases Food Intake and Body Weight.

INTRODUCTION: GLP-1 receptor agonists are the number one drug prescribed for the treatment of obesity and type 2 diabetes. These drugs are not, however, without side effects, and in an effort to maximize therapeutic effect while minimizing adverse effects, gut hormone co-agonists received considerable attention as new drug targets in the fight against obesity. Numerous previous reports identified the neuropeptide oxytocin (OXT) as a promising anti-obesity drug. The aims of this study were to evaluate OXT as a possible co-agonist for GLP-1 and examine the effects of its co-administration on food intake (FI) and body weight (BW) in mice. METHODS: FI and c-Fos levels were measured in the feeding centers of the brain in response to an intraperitoneal injection of saline, OXT, GLP-1, or OXT/GLP-1. The action potential frequency and cytosolic Ca2+ ([Ca2+]i) in response to OXT, GLP-1, or OXT/GLP-1 were measured in ex vivo paraventricular nucleus (PVN) neuronal cultures. Finally, FI and BW changes were compared in diet-induced obese mice treated with saline, OXT, GLP-1, or OXT/GLP-1 for 13 days. RESULTS: Single injection of OXT/GLP-1 additively decreased FI and increased c-Fos expression specifically in the PVN and supraoptic nucleus. Seventy percent of GLP-1 receptor-positive neurons in the PVN also expressed OXT receptors, and OXT/GLP-1 co-administration dramatically increased firing and [Ca2+]i in the PVN OXT neurons. The chronic OXT/GLP-1 co-administration decreased BW without changing FI. CONCLUSION: Chronic OXT/GLP-1 co-administration decreases BW, possibly via the activation of PVN OXT neurons. OXT might be a promising candidate as an incretin co-agonist in obesity treatment.

Oxytocin receptor-expressing neurons in the medial preoptic area are essential for lactation, whereas those in the lateral septum are not critical for maternal behavior.

INTRODUCTION: In nurturing systems, the oxytocin (Oxt)-oxytocin receptor (Oxtr) system is important for parturition, and essential for lactation and parental behavior. Among the nerve nuclei that express Oxtr, the lateral septal nucleus (LS) and medial preoptic area (MPOA) are representative regions that control maternal behavior. METHODS: We investigated the role of Oxtr- and Oxtr-expressing neurons, located in the LS and MPOA, in regulating maternal behavior by regulating Oxtr expression in a region-specific manner using recombinant mice and adeno-associated viruses. We quantified the prolactin (Prl) concentrations in the pituitary gland and plasma when Oxtr expression in the MPOA was reduced. RESULTS: The endogenous Oxtr gene in the neurons of the LS did not seem to play an essential role in maternal behavior. Conversely, decreased Oxtr expression in the MPOA increased the frequency of pups being left outside the nest and reduced their survival rate. Deletion of Oxtr in MPOA neurons prevented elevation of Prl levels in plasma and pituitary at postpartum day 2. DISCUSSION/CONCLUSION: Oxtr-expressing neurons in the MPOA are involved in the postpartum production of Prl. We confirmed the essential functions of Oxtr-expressing neurons and the Oxtr gene itself in the MPOA for the sustainability of maternal behavior, which involved Oxtr-dependent induction of Prl.

Effects of oxytocin on responses to nociceptive and non-nociceptive stimulation in the upper central nervous system.

Oxytocin is known as a social bonding hormone, but it also functions as an anxiolytic or analgesic neurotransmitter. When oxytocin regulates pain or anxiousness centrally as a neurotransmitter, it is secreted by neurons and directly projected to targeted regions. Although the function of oxytocin at the spinal level is well studied, its effects at the supraspinal level are poorly understood. We aimed to investigate the effect of oxytocin at the supraspinal level in vivo using C57BL/6J (wild-type [WT]), oxytocin-deficient (Oxt-/-), oxytocin receptor-deficient (Oxtr-/-), and oxytocin receptor-Venus (OxtrVenus/+) mice lines. Response thresholds in Oxtr-/- mice in Hargreaves and von-Frey tests were significantly lower than those in WT mice, whereas open field and light/dark tests showed no significant differences. Moreover, response thresholds in Oxt-/- mice were raised to those in WT mice after oxytocin administration. Following the Hargreaves test, we observed the co-localisation of c-fos with Venus or the oxytocin receptor in the periaqueductal gray (PAG), medial amygdala (MeA), and nucleus accumbens (NAc) regions in OxtrVenus/+ mice. Furthermore, in the PAG, MeA, and NAc regions, the co-localisation of oxytocin with c-fos and gamma-aminobutyric acid was much stronger in Oxtr-/- mice than in WT mice. However, following von-Frey test, the same findings were observed only in the MeA and NAc regions. Our results suggest that oxytocin exerts its analgesic effect on painful stimulation via the PAG region and a self-protective effect on unpleasant stimulation via the MeA and NAc regions.

Single administration of resveratrol improves social behavior in adult mouse models of autism spectrum disorder.

Resveratrol (RSV) is a natural polyphenol present in grapes, the skin of peanuts, and several other plants with many health benefits. Autism spectrum disorder (ASD) is a neurodevelopmental disorder that may be linked to neural and synaptic development impairments. The present study aimed to analyze the preventive effects of RSV on the development of ASD-like behavior, using oxytocin receptor gene knockout (Oxtr-KO) and valproic acid-induced ASD (VPA-ASD) model mice. Genetic deficiencies in Oxtr are suggested to be involved in ASD etiology. Twenty-four hours after a single RSV injection to the Oxtr-KO mice, the social impairments caused by OXTR deficiency were ameliorated. RSV also improved social impairments in the VPA-ASD mice. Administration of RSV up-regulated silent information regulator 1 (Sirt1) gene and early growth response factor 3 (Egr3) gene expressions in the amygdala of the Oxtr-KO mice. Our data suggest that RSV may have therapeutic effects on ASD with multiple targets.

Oxytocin induced labor causes region and sex-specific transient oligodendrocyte cell death in neonatal mouse brain.

AIM: Previous reports showed associations between oxytocin induced labor and mental disorders in offspring. However, those reports are restricted in epidemiological analyses and its mechanism remains unclear. In this study, we hypothesized that induced labor directly causes brain damage in newborns and results in the development of mental disorders. Therefore we aimed to investigate this hypothesis with animal model. METHODS: The animal model of induced labor was established by subcutaneous oxytocin administration to term-pregnant C57BL/6J mice. We investigated the neonatal brain damage with evaluating immediate early gene expression (c-Fos, c-Jun and JunB) by quantitative polymerase reaction and TdT-mediated dUTP nick end labeling staining. To investigate the injured brain cell types, we performed double-immunostaining with TdT-mediated dUTP nick end labeling staining and each brain component specific protein, such as Oligo2, NeuN, GFAP and Iba1. RESULTS: Brain damage during induced labor led to cell death in specific brain regions, which are implicated in mental disorders, in only male offspring at P0. Furthermore, oligodendrocyte precursors were selectively vulnerable compared to the other cell types. This oligodendrocyte-specific impairment during the perinatal period led to an increased numbers of Olig2-positive cells at P5. Expression levels of oxytocin and Oxtr in the fetal brain were not affected by the oxytocin administered to mothers during induced labor. CONCLUSION: Oligodendrocyte cell death in specific brain regions, which was unrelated to the oxytocin itself, was caused by induced labor in only male offspring. This may be an underlying mechanism explaining the human epidemiological data suggesting an association between induced labor and mental disorders.

Relay of peripheral oxytocin to central oxytocin neurons via vagal afferents for regulating feeding.

Oxytocin (Oxt), a neurohormone synthesized in the neurons of hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus induces milk-ejection and uterine contraction and regulates social behavior, stress responses, memory and food intake. Peripheral (intraperitoneal and subcutaneous) infusion of Oxt decreases food intake and body weight in obese animals via mechanisms involving vagal afferent nerves and in obese subjects when administered nasally. Peripherally injected and intracerebroventricularly injected Oxt inhibit food intake to similar extent and with similar time course. Thus, peripheral Oxt mimics the effects of central Oxt, however, underlying mechanisms are unclear. In the present study we explored whether intraperitoneal Oxt activates Oxt neurons in PVN via vagal afferents and whether this pathway is linked to inhibition of feeding. We here show that intraperitoneal Oxt injection induces c-Fos expression in PVN largely in Oxt neurons and inhibits food intake, and these effects are blunted by subdiaphragmatic vagotomy. The intraperitoneal Oxt-induced inhibition of food intake was blunted in Oxt KO mice, by intracerebroventricular injection of Oxt receptor antagonist, and by vagotomy. These results demonstrate that intraperitoneal Oxt injection activates PVN Oxt neurons via vagal afferent nerves, thereby inhibiting food intake. This vagal afferents-mediated Oxt's peripheral-to-central coupling may serve to promote satiety and possibly a series of neural functions of Oxt and to treat their disorders.

Oxytocin receptor knockout prairie voles generated by CRISPR/Cas9 editing show reduced preference for social novelty and exaggerated repetitive behaviors.

Behavioral neuroendocrinology has benefited tremendously from the use of a wide range of model organisms that are ideally suited for particular questions. However, in recent years the ability to manipulate the genomes of laboratory strains of mice has led to rapid advances in our understanding of the role of specific genes, circuits and neural populations in regulating behavior. While genome manipulation in mice has been a boon for behavioral neuroscience, the intensive focus on the mouse restricts the diversity in behavioral questions that can be investigated using state-of-the-art techniques. The CRISPR/Cas9 system has great potential for efficiently generating mutants in non-traditional animal models and consequently to reinvigorate comparative behavioral neuroendocrinology. Here we describe the efficient generation of oxytocin receptor (Oxtr) mutant prairie voles (Microtus ochrogaster) using the CRISPR/Cas9 system, and describe initial behavioral phenotyping focusing on behaviors relevant to autism. Oxtr mutant male voles show no disruption in pup ultrasonic vocalization, anxiety as measured by the open field test, alloparental behavior, or sociability in the three chamber test. Mutants did however show a modest elevation in repetitive behavior in the marble burying test, and an impairment in preference for social novelty. The ability to efficiently generate targeted mutations in the prairie vole genome will greatly expand the utility of this model organism for discovering the genetic and circuit mechanisms underlying complex social behaviors, and serves as a proof of principle for expanding this approach to other non-traditional model organisms.

LGR4 is essential for R-spondin1-mediated suppression of food intake via pro-opiomelanocortin.

Leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) suppresses food intake after its activation by binding of its ligands, R-spondins. We investigated the mechanism of food intake suppression by R-spondin1 in a region-specific Lgr4 gene knockout (LGR4 cKO) mouse model, generated by deletion of the Lgr4 gene in arcuate nucleus (ARC) using Lgr4fx/fx mice combined with infection of an AAV-Cre vector. After R-spondin1 administration, LGR4 cKO mice didn't exhibit a suppressed appetite, compared to that in control mice, which received a vehicle. In ARC of LGR4 cKO mice, Pomc mRNA expression was reduced, leading to suppressed food intake. On the other hand, neurons-specific LGR4 KO mice exhibited no differences in Pomc expression, and no structural differences were observed in the ARC of mutant mice. These results suggest that LGR4 is an essential part of the mechanism, inducing Pomc gene expression with R-spondin1 in ARC neurons in mice, thereby regulating feeding behavior. Abbreviations: LGR4: Leucine-rich repeat-containing G-protein coupled receptor 4; RSPOs: roof plate-specific spondins; ARC: arcuate nucleus; AAV: adeno associated virus; POMC: pro-opiomelanocortin; CART: cocaine and amphetamine-regulated transcript; NPY: neuropeptide Y; AgRP: agouti-related peptide; Axin2: axis inhibition protein 2; Lef1: lymphoid enhancer binding factor 1; ccnd1: cyclin D1.

Generation of Oxtr cDNA(HA)-Ires-Cre Mice for Gene Expression in an Oxytocin Receptor Specific Manner.

The neurohypophysial hormone oxytocin (OXT) and its receptor (OXTR) have critical roles in the regulation of pro-social behaviors, including social recognition, pair bonding, parental behavior, and stress-related responses. Supporting this hypothesis, a portion of patients suffering from autism spectrum disorder have mutations, such as single nucleotide polymorphisms, or epigenetic modifications in their OXTR gene. We previously reported that OXTR-deficient mice exhibit pervasive social deficits, indicating the critical role of OXTR in social behaviors. In the present study, we generated Oxtr cDNA(HA)-Ires-Cre knock-in mice, expressing both OXTR and Cre recombinase under the control of the endogenous Oxtr promoter. Knock-in cassette of Oxtr cDNA(HA)-Ires-Cre consisted of Oxtr cDNA tagged with the hemagglutinin epitope at the 3' end (Oxtr cDNA(HA)), internal ribosomal entry site (Ires), and Cre. Cre was expressed in the uterus, mammary gland, kidney, and brain of Oxtr cDNA(HA)-Ires-Cre knock-in mice. Furthermore, the distribution of Cre in the brain was similar to that observed in Oxtr-Venus fluorescent protein expressing mice (Oxtr-Venus), another animal model previously generated by our group. Social behavior of Oxtr cDNA(HA)-Ires-Cre knock-in mice was similar to that of wild-type animals. We demonstrated that this construct is expressed in OXTR-expressing neurons specifically after an infection with the recombinant adeno-associated virus carrying the flip-excision switch vector. Using this system, we showed the transport of the wheat-germ agglutinin tracing molecule from the OXTR-expressing neurons to the innervated neurons in knock-in mice. This study might contribute to the monosynaptic analysis of neuronal circuits and to the optogenetic analysis of neurons expressing OXTR.

Prenatal minocycline treatment alters synaptic protein expression, and rescues reduced mother call rate in oxytocin receptor-knockout mice.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired communication, difficulty in companionship, repetitive behaviors and restricted interests. Recent studies have shown amelioration of ASD symptoms by intranasal administration of oxytocin and demonstrated the association of polymorphisms in the oxytocin receptor (Oxtr) gene with ASD patients. Deficient pruning of synapses by microglial cells in the brain has been proposed as potential mechanism of ASD. Other researchers have shown specific activation of microglial cells in brain regions related to sociality in patients with ASD. Although the roles of Oxtr and microglia in ASD are in the spotlight, the relationship between them remains to be elucidated. In this study, we found abnormal activation of microglial cells and a reduction of postsynaptic density protein PSD95 expression in the Oxtr-deficient brain. Moreover, pharmacological inhibition of microglia during development can alter the expression of PSD95 and ameliorate abnormal mother-infant communication in Oxtr-deficient mice. Our results suggest that microglial abnormality is a potential mechanism of the development of Oxt/Oxtr mediated ASD-like phenotypes.

Transgenic expression of Telomerase reverse transcriptase (Tert) improves cell proliferation of primary cells and enhances reprogramming efficiency into the induced pluripotent stem cell.

The enzymatic activity of telomerase is important for the extension of the telomere repeat sequence and overcoming cellular senescence. We generated a conditional transgenic mouse line, carrying the telomerase reverse transcriptase (Tert) expression cassette, controlled by the Cre-loxP-mediated recombination. In our study, Cre recombinase expression efficiently activated Tert expression, resulting in its increased enzymatic activity, which extended the period of cellular proliferation until the keratinocytes entered senescence. This suggests that transgenic Tert expression is effective in enhancing primary cell proliferation. Notably, Tert expression increased colony formation of induced pluripotent stem (iPS) cells after the introduction of four reprogramming factors, Oct-4, klf4, SOX-2, and c-Myc into the transgenic fibroblasts. To the best of our knowledge, this is the first study to show that the transgenic Tert expression enhances reprogramming efficiency of iPS cells, which indicates a critical role for Tert in the reprogramming process.

In vitro culture and in vitro fertilization techniques for prairie voles (Microtus ochrogaster).

Prairie vole (Microtus ochrogaster) is a highly social animal and is a commonly used animal model for neuropsychopharmacological and psychiatric studies. To date, only a few reports on the development of transgenic prairie voles which was primarily due to the suboptimal development of assisted reproductive technology (ART) in prairie voles. Limitations in ART further hinder the development of genetically modified prairie voles such as the application of conventional gene targeting technologies using embryonic stem (ES) or induced pluripotent stem (iPS) cells to generate chimeric prairie voles. Moreover, recent advancement in genome-editing tools such as transcription activator-like effector nuclease (TALEN) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas technology provide an unprecedented opportunity to create gene targeting animal model and the development of ART in prairie voles is necessary for future development of novel transgenic prairie vole model. We have established efficient method for in vitro embryo culture and sperm cryopreservation with high fertilization rate. In G-1 PLUS and G-2 PLUS sequential culture condition, 81.0% (# of Blastocysts/total n) of one-cell embryos developed to blastocysts. In contrary, no embryos were developed to blastocyst stage in KSOM medium (0/total # of embryos in culture). In vitro fertilization rate using fresh and frozen-thawed sperm was 32.6% and 29.3%, respectively. This is the first report of IVF using cryopreserved prairie vole sperm. We employed mouse IVF methods in prairie voles and optimize culture conditions using human G-1/G-2 PLUS sequential culture method that resulted in high embryonic development rate. The development in vole reproductive technology will facilitate the generation of transgenic voles in the future.

Lgr4 controls specialization of female gonads in mice.

Leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is a type of membrane receptor with a seven-transmembrane structure. LGR4 is homologous to gonadotropin receptors, such as follicle-stimulating hormone receptor (Fshr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr). Recently, it has been reported that Lgr4 is a membrane receptor for R-spondin ligands, which mediate Wnt/beta-catenin signaling. Defects of R-spondin homolog (Rspo1) and wingless-type MMTV integration site family, member 4 (Wnt4) cause masculinization of female gonads. We observed that Lgr4(-/-) female mice show abnormal development of the Wolffian ducts and somatic cells similar to that in the male gonads. Lgr4(-/-) female mice exhibited masculinization similar to that observed in Rspo1-deficient mice. In Lgr4(-/-) ovarian somatic cells, the expression levels of lymphoid enhancer-binding factor 1 (Lefl) and Axin2 (Axin2), which are target genes of Wnt/beta-catenin signaling, were lower than they were in wild-type mice. This study suggests that Lgr4 is critical for ovarian somatic cell specialization via the cooperative signaling of Rspo1 and Wnt/beta-catenin.

Lgr4 is required for endometrial receptivity acquired through ovarian hormone signaling.

Previously, using the Keratin5-Cre transgenic mouse model we reported that female Lgr4-conditional KO mice (Lgr4(K5 KO)) showed subfertility with defective stromal decidualization due to abnormal development of the uterine gland. However, the impact of the LGR4 defect on luminal epithelial cells was not investigated in the previous report. Here, we focused on the receptive state of the luminal epithelium in Lgr4(K5 KO) mice that received ovarian hormone treatment. In Lgr4(K5 KO) mice, progesterone failed to inhibit the luminal epithelial cell proliferation. Immunohistochemical and qRT-PCR analyses revealed down-regulated progesterone signaling in the uterus of Lgr4(K5 KO) mice. These results demonstrated that LGR4 is essential for the acquisition of endometrial receptivity through ovarian hormone signaling.

In need of a specific antibody against the oxytocin receptor for neuropsychiatric research: A KO validation study.

Antibodies are one of the most utilized tools in biomedical research. However, few of them are rigorously evaluated, as there are no accepted guidelines or standardized methods for determining their validity before commercialization. Often, an antibody is considered validated if it detects a band by Western blot of the expected molecular weight and, in some cases, if blocking peptides result in loss of staining. Neither of these approaches are unquestionable proof of target specificity. Since the oxytocin receptor has recently become a popular target in neuropsychiatric research, the need for specific antibodies to be used in brain has arisen. In this work, we have tested the specificity of six commercially available oxytocin receptor antibodies, indicated by the manufacturers to be suitable for Western blot and with an available image showing the correct size band (45-55 KDa). Antibodies were first tested by Western blot in brain lysates of wild-type and oxytocin receptor knockout mice. Uterus tissue was also tested as control for putative differential tissue specificity. In brain, the six tested antibodies lacked target specificity, as both wild-type and receptor knockout samples resulted in a similar staining pattern, including the expected 45-55 KDa band. Five of the six antibodies detected a selective band in uterus (which disappeared in knockout tissue). These five specific antibodies were also tested for immunohistochemistry in uterus, where only one was specific. However, when the uterine-specific antibody was tested in brain tissue, it lacked specificity. In conclusion, none of the six tested commercial antibodies are suitable to detect oxytocin receptor in brain by either Western blot or immunohistochemistry, although some do specifically detect it in uterus. The present work highlights the need to develop standardized antibody validation methods, including a proper negative control, in order to grant quality and reproducibility of the generated data.

Traditional Japanese medicine Kamikihito ameliorates sucrose preference, chronic inflammation and obesity induced by a high fat diet in middle-aged mice.

The high prevalence of obesity has become a pressing global public health problem and there exists a strong association between increased BMI and mortality at a BMI of 25 kg/m2 or higher. The prevalence of obesity is higher among middle-aged adults than among younger groups and the combination of aging and obesity exacerbate systemic inflammation. Increased inflammatory cytokines such as interleukin 6 and tumor necrosis factor alpha (TNFα) are hallmarks of obesity, and promote the secretion of hepatic C-reactive protein (CRP) which further induces systematic inflammation. The neuropeptide oxytocin has been shown to have anti-obesity and anti-inflammation effects, and also suppress sweet-tasting carbohydrate consumption in mammals. Previously, we have shown that the Japanese herbal medicine Kamikihito (KKT), which is used to treat neuropsychological stress disorders in Japan, functions as an oxytocin receptors agonist. In the present study, we further investigated the effect of KKT on body weight (BW), food intake, inflammation, and sweet preferences in middle-aged obese mice. KKT oral administration for 12 days decreased the expression of pro-inflammatory cytokines in the liver, and the plasma CRP and TNFα levels in obese mice. The effect of KKT administration was found to be different between male and female mice. In the absence of sucrose, KKT administration decreased food intake only in male mice. However, while having access to a 30% sucrose solution, both BW and food intake was decreased by KKT administration in male and female mice; but sucrose intake was decreased in female mice alone. In addition, KKT administration decreased sucrose intake in oxytocin deficient lean mice, but not in the WT lean mice. The present study demonstrates that KKT ameliorates chronic inflammation, which is strongly associated with aging and obesity, and decreases food intake in male mice as well as sucrose intake in female mice; in an oxytocin receptor dependent manner.

Impairment of pachytene spermatogenesis in Dmrt7 deficient mice, possibly causing meiotic arrest.

Although Dmrt7 has been reported to be essential for male spermatogenesis, the molecular mechanism underlying pachytene spermatogenesis by Dmrt7 is not known. In the present study, by detailed analysis of Dmrt7 protein distribution in spermatocytes in the first wave of spermatogenesis, we clarified the profile of Dmrt7 expression and localization in pachytene spermatogenesis. Dmrt7-deficient spermatocytes were arrested in the pachytene stage, followed by apoptosis. We analyzed to determine whether every event in the spermatogenesis at the Dmrt7-deficient mice progressed normally, because in several gene knockout mice with spermatogenic arrest described in the previous reports impairments of these events often appeared. Mutant mice showed normal synapsis and XY body formation, while impairment of meiotic sex chromosome inactivation (MSCI), decreased expression of backup genes, and increased expression of retrotransposons indicated incomplete meiotic recombination.

Identification of oxytocin expression in human and murine microglia.

BACKGROUND: Oxytocin is a neuropeptide synthesized in the hypothalamus. In addition to its role in parturition and lactation, oxytocin mediates social behavior and pair bonding. The possibility of using oxytocin to modify behavior in neurodevelopmental disorders, such as autism spectrum disorder, is of clinical interest. Microglia are tissue-resident macrophages with roles in neurogenesis, synapse pruning, and immunological mediation of brain homeostasis. Recently, oxytocin was found to attenuate microglial secretion of proinflammatory cytokines, but the source of this oxytocin was not established. This prompted us to investigate whether microglia themselves were the source. METHODS: We examined oxytocin expression in human and murine brain tissue in both sexes using immunohistochemistry. Oxytocin mRNA expression and secretion were examined in isolated murine microglia from wild type and oxytocin-knockout mice. Also, secretion of oxytocin and cytokines was measured in cultured microglia (MG6) stimulated with lipopolysaccharide (LPS). RESULTS: We identified oxytocin expression in microglia of human brain tissue, cultured microglia (MG6), and primary murine microglia. Furthermore, LPS stimulation increased oxytocin mRNA expression in primary murine microglia and MG6 cells, and oxytocin secretion as well. A positive correlation between oxytocin and IL-1ß, IL-10 secretion emerged, respectively. CONCLUSION: This may be the first demonstration of oxytocin expression in microglia. Functionally, oxytocin might regulate inflammatory cytokine release from microglia in a paracrine/autocrine manner.

SxtA localizes to chloroplasts and changes to its 3'UTR may reduce toxin biosynthesis in non-toxic Alexandrium catenella (Group I)✰.

SxtA is the enzyme that catalyses the first step of saxitoxin biosynthesis. We developed an immunofluorescent method to detect SxtA using antibodies against SxtA peptides. Confocal microscopy revealed the presence of abundant, sub-cellularly localized signal in cells of toxic species and its absence in non-toxic species. Co-localization of SxtA with Rubisco II and ultra-structural observation by transmission electron microscopy strongly suggested the association of SxtA with chloroplasts. We also characterized a non-toxic sub-clone of Alexandrium catenella (Group I) to elucidate the mutation responsible for its loss of toxicity. Although sxtA4 gene copy number was indistinguishable in toxic and non-toxic sub-clones, mRNA and protein expression were significantly reduced in the non-toxic sub-clone and we uncovered sequence variation at the 3' untranslated region (3'UTR) of sxtA4 mRNA. We propose that differences in the sxtA4 mRNA 3'UTR lead to down-regulation of STX biosynthesis post-transcriptionally, thereby explaining the differences in toxicity amongst different A. catenella (Group I) sub-clones.