Oral Supplementation with Maca Improves Social Recognition Deficits in the Valproic Acid Animal Model of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a congenital, lifelong neurodevelopmental disorder whose main symptom is impaired social communication and interaction. However, no drug can treat social deficits in patients with ASD, and treatments to alleviate social behavioral deficits are sorely needed. Here, we examined the effect of oral supplementation of maca (Lepidium meyenii) on social deficits of in utero-exposed valproic acid (VPA) mice, widely used as an ASD model. Although maca is widely consumed as a fertility enhancer and aphrodisiac, it possesses multiple beneficial activities. Additionally, it benefits learning and memory in experimental animal models. Therefore, the effect of maca supplementation on the social behavioral deficit of VPA mice was assessed using a social interaction test, a three-stage open field test, and a five-trial social memory test. The oral supplementation of maca attenuated social interaction behavior deficit and social memory impairment. The number of c-Fos-positive cells and the percentage of c-Fos-positive oxytocin neurons increased in supraoptic and paraventricular neurons of maca-treated VPA mice. These results reveal for the first time that maca is beneficial to social memory and that it restores social recognition impairments by augmenting the oxytocinergic neuronal pathways, which play an essential role in diverse social behaviors.

Early-onset of social communication and locomotion activity in F2 pups of a valproic acid-induced mouse model of autism.

Autism spectrum disorder (ASD) is a heterogeneously pervasive developmental disorder that usually occurs before 3 years old. Animal models of psychiatric disorders are essential for elucidating the underlying preclinical neural mechanisms. Mice that are prenatally exposed to valproic acid (VPA, F1) are widely used as an ASD model. Epigenetics has recently been suggested as a contributor to ASD etiology with the hypothesis that epigenetic marks can be transgenerationally inherited. Previous studies have indicated that autism-like behavioral phenotypes detected in F1 VPA mice transgenetically appear in F2 and F3 generations. However, studies on the autism-like behavioral phenotypes during the early postnatal days in subsequent generations are scarce. Here, the behavioral deficit on postnatal day 5 of the F2 generation was examined to assess the onset of ASD symptoms. Communication disorders were examined by analyzing maternal separation-induced ultrasonic vocalizations (USVs). Although the duration and frequency of USVs were not significantly altered, the emission rate was significantly lower in F2 VPA pups. Furthermore, the locomotive activity with or without littermates was altered in F2 VPA pups. The data of the current study suggest that social deficit and impaired locomotion are inherited by the subsequent generation and were apparent on early postnatal day 5.

Oxytocin Dynamics in the Body and Brain Regulated by the Receptor for Advanced Glycation End-Products, CD38, CD157, and Nicotinamide Riboside.

Investigating the neurocircuit and synaptic sites of action of oxytocin (OT) in the brain is critical to the role of OT in social memory and behavior. To the same degree, it is important to understand how OT is transported to the brain from the peripheral circulation. To date, of these, many studies provide evidence that CD38, CD157, and receptor for advanced glycation end-products (RAGE) act as regulators of OT concentrations in the brain and blood. It has been shown that RAGE facilitates the uptake of OT in mother's milk from the digestive tract to the cell surface of intestinal epithelial cells to the body fluid and subsequently into circulation in male mice. RAGE has been shown to recruit circulatory OT into the brain from blood at the endothelial cell surface of neurovascular units. Therefore, it can be said that extracellular OT concentrations in the brain (hypothalamus) could be determined by the transport of OT by RAGE from the circulation and release of OT from oxytocinergic neurons by CD38 and CD157 in mice. In addition, it has recently been found that gavage application of a precursor of nicotinamide adenine dinucleotide, nicotinamide riboside, for 12 days can increase brain OT in mice. Here, we review the evaluation of the new concept that RAGE is involved in the regulation of OT dynamics at the interface between the brain, blood, and intestine in the living body, mainly by summarizing our recent results due to the limited number of publications on related topics. And we also review other possible routes of OT recruitment to the brain.

Oral Supplementation with L-Carnosine Attenuates Social Recognition Deficits in CD157KO Mice via Oxytocin Release.

The outcomes of supplementation with L-carnosine have been investigated in clinical trials in children with autism spectrum disorder (ASD). However, reports on the effects of L-carnosine in humans have been inconsistent, and the efficacy of L-carnosine supplementation for improving ASD symptoms has yet to be investigated in animal studies. Here, we examined the effects of oral supplementation with L-carnosine on social deficits in CD157KO mice, a murine model of ASD. Social deficits in CD157KO mice were assessed using a three-chamber social approach test. Oral supplementation with L-carnosine attenuated social behavioral deficits. The number of c-Fos-positive oxytocin neurons in the supraoptic nucleus and paraventricular nucleus was increased with L-carnosine supplementation in CD157KO mice after the three-chamber social approach test. We observed an increase in the number of c-Fos-positive neurons in the basolateral amygdala, a brain region involved in social behavior. Although the expression of oxytocin and oxytocin receptors in the hypothalamus was not altered by L-carnosine supplementation, the concentration of oxytocin in cerebrospinal fluid was increased in CD157KO mice by L-carnosine supplementation. These results suggest that L-carnosine supplementation restores social recognition impairments by augmenting the level of released oxytocin. Thus, we could imply the possibility of a safe nutritional intervention for at least some types of ASD in the human population.

Oxytocin administration modulates the complex type of ultrasonic vocalisation of mice pups prenatally exposed to valproic acid.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by communication disability with no curative treatment. Maternal separation-induced ultrasonic vocalisation (USV) was widely used to assess communication disability between pups and dams. Particularly, USV calls in many genetically modified ASD model mice were altered. Previously, we demonstrated that mice pups exposed to valproic acid in utero (VPA pups) showed decreased number of USV calls on postnatal day 11 and were rescued by subcutaneous injection of oxytocin. However, the qualitative change of USV calls by oxytocin has not been evaluated in VPA pups. In the present study, we examined the duration of oxytocin effect and analysed the altered pattern of USV calls using VPA pups. The oxytocin administration increased the total number of USV calls and the effect persisted up to 120 min in VPA pups. The pattern analysis revealed that the increase in the number of complex calls also persisted up to 120 min. These results suggested that oxytocin had a prolonged effect on USV calls, mainly on complex calls, in VPA pup, showing that oxytocin could recover their social modality to respond to maternal separation.

Oxytocin ameliorates impaired social behavior in a Chd8 haploinsufficiency mouse model of autism.

BACKGROUND: Autism spectrum disorder (ASD) is characterized by the core symptoms of impaired social interactions. Increasing evidence suggests that ASD has a strong genetic link with mutations in chromodomain helicase DNA binding protein 8 (CHD8), a gene encoding a chromatin remodeler. It has previously been shown that Chd8 haplodeficient male mice manifest ASD-like behavioral characteristics such as anxiety and altered social behavior. Along with that, oxytocin (OT) is one of the main neuropeptides involved in social behavior. Administration of OT has shown improvement of social behavior in genetic animal models of ASD. The present study was undertaken to further explore behavioral abnormalities of Chd8 haplodeficient mice of both sexes, their link with OT, and possible effects of OT administration. First, we performed a battery of behavioral tests on wild-type and Chd8+/∆SL female and male mice. Next, we measured plasma OT levels and finally studied the effects of intraperitoneal OT injection on observed behavioral deficits. RESULTS: We showed general anxiety phenotype in Chd8+/∆SL mice regardless of sex, the depressive phenotype in Chd8+/∆SL female mice only and bidirectional social deficit in female and male mice. We observed decreased level of OT in Chd+/∆SL mice, possibly driven by males. Mice injected by OT demonstrated recovery of social behavior, while reduced anxiety was observed only in male mice. CONCLUSIONS: Here, we demonstrated that abnormal social behaviors were observed in both male and female Chd8+/∆SL mice. The ability of peripheral OT administration to affect such behaviors along with altered plasma OT levels indicated a possible link between Chd8 + /∆SL and OT in the pathogenesis of ASD as well as the possible usefulness of OT as a therapeutic tool for ASD patients with CHD8 mutations.

Transport of oxytocin to the brain after peripheral administration by membrane-bound or soluble forms of receptors for advanced glycation end-products.

Oxytocin (OT) is a neuropeptide hormone. Single and repetitive administration of OT increases social interaction and maternal behaviour in humans and mammals. Recently, it was found that the receptor for advanced glycation end-products (RAGE) is an OT-binding protein and plays a critical role in the uptake of OT to the brain after peripheral OT administration. Here, we address some unanswered questions on RAGE-dependent OT transport. First, we found that, after intranasal OT administration, the OT concentration increased in the extracellular space of the medial prefrontal cortex (mPFC) of wild-type male mice, as measured by push-pull microperfusion. No increase of OT in the mPFC was observed in RAGE knockout male mice. Second, in a reconstituted in vitro blood-brain barrier system, inclusion of the soluble form of RAGE (endogenous secretory RAGE [esRAGE]), an alternative splicing variant, in the luminal (blood) side had no effect on the transport of OT to the abluminal (brain) chamber. Third, OT concentrations in the cerebrospinal fluid after i.p. OT injection were slightly higher in male mice overexpressing esRAGE (esRAGE transgenic) compared to those in wild-type male mice, although this did not reach statistical significance. Although more extensive confirmation is necessary because of the small number of experiments in the present study, the reported data support the hypothesis that RAGE may be involved in the transport of OT to the mPFC from the circulation. These results suggest that the soluble form of RAGE in the plasma does not function as a decoy in vitro.

Nicotinamide riboside supplementation corrects deficits in oxytocin, sociability and anxiety of CD157 mutants in a mouse model of autism spectrum disorder.

Oxytocin (OT) is a critical molecule for social recognition and memory that mediates social and emotional behaviours. In addition, OT acts as an anxiolytic factor and is released during stress. Based on the activity of CD38 as an enzyme that produces the calcium-mobilizing second messenger cyclic ADP-ribose (cADPR), CD157, a sister protein of CD38, has been considered a candidate mediator for the production and release of OT and its social engagement and anti-anxiety functions. However, the limited expression of CD157 in the adult mouse brain undermined confidence that CD157 is an authentic and/or actionable molecular participant in OT-dependent social behaviour. Here, we show that CD157 knockout mice have low levels of circulating OT in cerebrospinal fluid, which can be corrected by the oral administration of nicotinamide riboside, a recently discovered vitamin precursor of nicotinamide adenine dinucleotide (NAD). NAD is the substrate for the CD157- and CD38-dependent production of cADPR. Nicotinamide riboside corrects social deficits and fearful and anxiety-like behaviours in CD157 knockout males. These results suggest that elevating NAD levels with nicotinamide riboside may allow animals with cADPR- and OT-forming deficits to overcome these deficits and function more normally.

A monoclonal antibody raised against a synthetic oxytocin peptide stains mouse hypothalamic neurones.

A monoclonal antibody against oxytocin was generated in 7a5 hybridoma cells derived from myeloma cells and lymphocytes from the spleen of mice immunised with a synthetic oxytocin peptide. The 7a5 monoclonal antibody bound with oxytocin in enzyme-linked immunosorbent assays. 7a5 cell growth medium was diluted up to 5000-fold and used for immunohistochemistry. First, to test the specificity of the 7a5 antibody against oxytocin, we stained brain tissues of oxytocin knockout mice, comprising mice in which the first exon of the oxytocin-neurophysin gene is deleted. No 7a5 immunoreactivity was detected in the paraventricular nucleus (PVN) of the hypothalamus of oxytocin knockout mice; however, this area was strongly stained with the anti-vasopressin polyclonal antibody, HM07. Tissue preparations of the wild-type mouse PVN and supraoptic nucleus (SON) displayed 7a5 immunoreactivity that was indistinguishable from the staining produced with an anti-oxytocin polyclonal antibody, HM06. The immunoreactivity of HM06 in the PVN was similar to that of an anti-oxytocin monoclonal antibody, PS38. We then examined the cross-reactivity of 7a5 with arginine vasopressin. The majority of cell soma and processes stained by 7a5 were not co-stained with the vasopressin antibody in SON and PVN regions. Furthermore, the suprachiasmatic nucleus was stained by the vasopressin antibody but not by 7a5. These results demonstrate that 7a5 is a new anti-oxytocin monoclonal antibody recognising oxytocin and not vasopressin; therefore, 7a5 can be used to investigate the role of oxytocin in the brain.

Vascular RAGE transports oxytocin into the brain to elicit its maternal bonding behaviour in mice.

Oxytocin sets the stage for childbirth by initiating uterine contractions, lactation and maternal bonding behaviours. Mice lacking secreted oxcytocin (Oxt -/-, Cd38 -/-) or its receptor (Oxtr -/-) fail to nurture. Normal maternal behaviour is restored by peripheral oxcytocin replacement in Oxt -/- and Cd38 -/-, but not Oxtr -/- mice, implying that circulating oxcytocin crosses the blood-brain barrier. Exogenous oxcytocin also has behavioural effects in humans. However, circulating polypeptides are typically excluded from the brain. We show that oxcytocin is transported into the brain by receptor for advanced glycation end-products (RAGE) on brain capillary endothelial cells. The increases in oxcytocin in the brain which follow exogenous administration are lost in Ager -/- male mice lacking RAGE, and behaviours characteristic to abnormalities in oxcytocin signalling are recapitulated in Ager -/- mice, including deficits in maternal bonding and hyperactivity. Our findings show that RAGE-mediated transport is critical to the behavioural actions of oxcytocin associated with parenting and social bonding.

Sex Differences in Salivary Oxytocin and Cortisol Concentration Changes during Cooking in a Small Group.

Background: Oxytocin (OT), a neuropeptide, has positive effects on social and emotional processes during group activities. Because cooking is an integrated process in the cognitive, physical, and socio-emotional areas, cooking in a group is reported to improve emotion and cognition. However, evidence for efficacy in group cooking has not been well established at the biological level. Methods: To address this shortcoming, we first measured salivary levels of OT and cortisol (CORT), a biomarker of psychological stress, before and after group cooking for approximately 1 h by people who know each other in healthy married or unmarried men and women. We then compared the initial OT and CORT concentrations with those during individual non-cooking activities in isolation. Results: Baseline OT concentrations before group and non-group sessions did not significantly differ and OT levels increased after both types of activity in men and women. In men, however, the percentage changes of OT levels in the first over the second saliva samples were significantly small during cooking compared with those in individual activities. In women, however, such a difference was not observed. In contrast, the mean salivary CORT concentrations after group cooking were significantly decreased from the baseline level in both sexes, though such decreases were not significant after individual activity sessions. The sex-specific differences were marital-status independent. Conclusion: These results indicate that OT and CORT concentrations after two activity sessions by a familiar group changed in opposite directions in a sex-specific manner. This suggests that, because cooking is experience-based, we need to consider the sex-specific features of group cooking if we apply it for intervention.

Salivary Oxytocin Concentration Changes during a Group Drumming Intervention for Maltreated School Children.

Many emotionally-disturbed children who have been maltreated and are legally separated from their parents or primary caregivers live in group homes and receive compulsory education. Such institutions provide various special intervention programs. Taiko-ensou, a Japanese style of group drumming, is one such program because playing drums in a group may improve children's emotional well-being. However, evidence for its efficacy has not been well established at the biological level. In this study, we measured salivary levels of oxytocin (OT), a neuropeptide associated with social memory and communication, in three conditions (recital, practice, and free sessions) in four classes of school-aged children. Following the sessions, OT concentrations showed changes in various degrees and directions (no change, increases, or decreases). The mean OT concentration changes after each session increased, ranging from 112% to 165%. Plasma OT concentrations were equally sensitive to drum playing in school-aged boys and girls. However, the difference between practice and free play sessions was only significant among elementary school boys aged 8-12 years. The results suggest that younger boys are most responsive to this type of educational music intervention.

Intestinal transepithelial permeability of oxytocin into the blood is dependent on the receptor for advanced glycation end products in mice.

Plasma oxytocin (OT) originates from secretion from the pituitary gland into the circulation and from absorption of OT in mother's milk into the blood via intestinal permeability. However, the molecular mechanism underlying the absorption of OT remains unclear. Here, we report that plasma OT concentrations increased within 10 min after oral delivery in postnatal day 1-7 mice. However, in Receptors for Advanced Glycation End Products (RAGE) knockout mice after postnatal day 3, an identical OT increase was not observed. In adult mice, plasma OT was also increased in a RAGE-dependent manner after oral delivery or direct administration into the intestinal tract. Mass spectrometry evaluated that OT was absorbed intact. RAGE was abundant in the intestinal epithelial cells in both suckling pups and adults. These data highlight that OT is transmitted via a receptor-mediated process with RAGE and suggest that oral OT supplementation may be advantageous in OT drug development.

Communication Impairment in Ultrasonic Vocal Repertoire during the Suckling Period of Cd157 Knockout Mice: Transient Improvement by Oxytocin.

Communication consists of social interaction, recognition, and information transmission. Communication ability is the most affected component in children with autism spectrum disorder (ASD). Recently, we reported that the CD157/BST1 gene is associated with ASD, and that CD157 knockout (Cd157-/-) mice display severe impairments in social behavior that are improved by oxytocin (OXT) treatment. Here, we sought to determine whether Cd157-/- mice can be used as a suitable model for communication deficits by measuring ultrasonic vocalizations (USVs), especially in the early developmental stage. Call number produced in pups due to isolation from dams was higher at postnatal day (PND) 3 in knockout pups than wild-type mice, but was lower at PNDs 7 and 10. Pups of both genotypes had similarly limited voice repertoires at PND 3. Later on, at PNDs 7 and 10, while wild-type pups emitted USVs consisting of six different syllable types, knockout pups vocalized with only two types. This developmental impairment in USV emission was rescued within 30 min by intraperitoneal OXT treatment, but quickly returned to control levels after 120 min, showing a transient effect of OXT. USV impairment was partially observed in Cd157+/- heterozygous mice, but not in Cd157-/- adult male mice examined while under courtship. These results demonstrate that CD157 gene deletion results in social communication insufficiencies, and suggests that CD157 is likely involved in acoustic communication. This unique OXT-sensitive developmental delay in Cd157-/- pups may be a useful model of communicative interaction impairment in ASD.

Cyclic ADP-Ribose and Heat Regulate Oxytocin Release via CD38 and TRPM2 in the Hypothalamus during Social or Psychological Stress in Mice.

Hypothalamic oxytocin (OT) is released into the brain by cyclic ADP-ribose (cADPR) with or without depolarizing stimulation. Previously, we showed that the intracellular free calcium concentration ([Ca(2+)]i) that seems to trigger OT release can be elevated by ß-NAD(+), cADPR, and ADP in mouse oxytocinergic neurons. As these ß-NAD(+) metabolites activate warm-sensitive TRPM2 cation channels, when the incubation temperature is increased, the [Ca(2+)]i in hypothalamic neurons is elevated. However, it has not been determined whether OT release is facilitated by heat in vitro or hyperthermia in vivo in combination with cADPR. Furthermore, it has not been examined whether CD38 and TRPM2 exert their functions on OT release during stress or stress-induced hyperthermia in relation to the anxiolytic roles and social behaviors of OT under stress conditions. Here, we report that OT release from the isolated hypothalami of male mice in culture was enhanced by extracellular application of cADPR or increasing the incubation temperature from 35°C to 38.5°C, and simultaneous stimulation showed a greater effect. This release was inhibited by a cADPR-dependent ryanodine receptor inhibitor and a nonspecific TRPM2 inhibitor. The facilitated release by heat and cADPR was suppressed in the hypothalamus isolated from CD38 knockout mice and CD38- or TRPM2-knockdown mice. In the course of these experiments, we noted that OT release differed markedly between individual mice under stress with group housing. That is, when male mice received cage-switch stress and eliminated due to their social subclass, significantly higher levels of OT release were found in subordinates compared with ordinates. In mice exposed to anxiety stress in an open field, the cerebrospinal fluid (CSF) OT level increased transiently at 5 min after exposure, and the rectal temperature also increased from 36.6°C to 37.8°C. OT levels in the CSF of mice with lipopolysaccharide-induced fever (+0.8°C) were higher than those of control mice. The TRPM2 mRNA levels and immunoreactivities increased in the subordinate group with cage-switch stress. These results showed that cADPR/CD38 and heat/TRPM2 are co-regulators of OT secretion and suggested that CD38 and TRPM2 are potential therapeutic targets for OT release in psychiatric diseases caused by social stress.

Salivary oxytocin concentrations in seven boys with autism spectrum disorder received massage from their mothers: a pilot study.

Seven male children with autism spectrum disorder (ASD), aged 8-12 years, attending special education classrooms for ASD and disabled children, were assigned to receive touch therapy. Their mothers were instructed to provide gentle touch in the massage style of the International Liddle Kidz Association. The mothers gave massages to their child for 20 min every day over a period of 3 months, followed by no massage for 4 months. To assess the biological effects of such touch therapy, saliva was collected before and 20 min after a single session of massage for 20 min from the children and mothers every 3 weeks during the massage period and every 4 weeks during the non-massage period, when they visited a community meeting room. Salivary oxytocin levels were measured using an enzyme immunoassay kit. During the period of massage therapy, the children and mothers exhibited higher oxytocin concentrations compared to those during the non-massage period. The changes in oxytocin levels before and after a single massage session were not significantly changed in children and mothers. The results suggested that the ASD children (massage receivers) and their mothers (massage givers) show touch therapy-dependent changes in salivary oxytocin concentrations.

Anxiety- and depression-like behavior in mice lacking the CD157/BST1 gene, a risk factor for Parkinson's disease.

CD157, known as bone marrow stromal cell antigen-1, is a glycosylphosphatidylinositol-anchored ADP-ribosyl cyclase that supports the survival and function of B-lymphocytes and hematopoietic or intestinal stem cells. Although CD157/Bst1 is a risk locus in Parkinson's disease (PD), little is known about the function of CD157 in the nervous system and contribution to PD progression. Here, we show that no apparent motor dysfunction was observed in young knockout (CD157 (-/-)) male mice under less aging-related effects on behaviors. CD157 (-/-) mice exhibited anxiety-related and depression-like behaviors compared with wild-type mice. These behaviors were rescued through treatment with anti-psychiatric drugs and oxytocin. CD157 was weakly expressed in the amygdala and c-Fos immunoreactivity in the amygdala was less evident in CD157 (-/-) mice than in wild-type mice. These results demonstrate for the first time that CD157 plays a role as a neuro-regulator and suggest a potential role in pre-motor symptoms in PD.