Postnatal expression of CD38 in astrocytes regulates synapse formation and adult social memory.

Social behavior is essential for health, survival, and reproduction of animals; however, the role of astrocytes in social behavior remains largely unknown. The transmembrane protein CD38, which acts both as a receptor and ADP-ribosyl cyclase to produce cyclic ADP-ribose (cADPR) regulates social behaviors by promoting oxytocin release from hypothalamic neurons. CD38 is also abundantly expressed in astrocytes in the postnatal brain and is important for astroglial development. Here, we demonstrate that the astroglial-expressed CD38 plays an important role in social behavior during development. Selective deletion of CD38 in postnatal astrocytes, but not in adult astrocytes, impairs social memory without any other behavioral abnormalities. Morphological analysis shows that depletion of astroglial CD38 in the postnatal brain interferes with synapse formation in the medial prefrontal cortex (mPFC) and hippocampus. Moreover, astroglial CD38 expression promotes synaptogenesis of excitatory neurons by increasing the level of extracellular SPARCL1 (also known as Hevin), a synaptogenic protein. The release of SPARCL1 from astrocytes is regulated by CD38/cADPR/calcium signaling. These data demonstrate a novel developmental role of astrocytes in neural circuit formation and regulation of social behavior in adults.

Two oxytocin analogs, N-(p-fluorobenzyl) glycine and N-(3-hydroxypropyl) glycine, induce uterine contractions ex vivo in ways that differ from that of oxytocin.

Contraction of the uterus is critical for parturient processes. Insufficient uterine tone, resulting in atony, can potentiate postpartum hemorrhage; thus, it is a major risk factor and is the main cause of maternity-related deaths worldwide. Oxytocin (OT) is recommended for use in combination with other uterotonics for cases of refractory uterine atony. However, as the effect of OT dose on uterine contraction and control of blood loss during cesarean delivery for labor arrest are highly associated with side effects, small amounts of uterotonics may be used to elicit rapid and superior uterine contraction. We have previously synthesized OT analogs 2 and 5, prolines at the 7th positions of which were replaced with N-(p-fluorobenzyl) glycine [thus, compound 2 is now called fluorobenzyl (FBOT)] or N-(3-hydroxypropyl) glycine [compound 5 is now called hydroxypropyl (HPOT)], which exhibited highly potent binding affinities for human OT receptors in vitro. In this study, we measured the ex vivo effects of FBOT and HPOT on contractions of uteri isolated from human cesarean delivery samples and virgin female mice. We evaluated the potency and efficacy of the analogs on uterine contraction, additivity with OT, and the ability to overcome the effects of atosiban, an OT antagonist. In human samples, the potency rank judged by the calculated EC50 (pM) was as follows: HPOT (189) > FBOT (556) > OT (5,340) > carbetocin (12,090). The calculated Emax was 86% for FBOT and 75% for HPOT (100%). Recovery from atosiban inhibition after HPOT treatment was as potent as that after OT treatment. HPOT showed additivity with OT. FBOT (56 pM) was found to be the strongest agonist in virgin mouse uterus. HPOT and FBOT demonstrated high potency and partial agonist efficacy in the human uterus. These results suggested that HPOT and FBOT are highly uterotonic for the human uterus and performed better than OT, indicating that they may prevent postpartum hemorrhage.

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.

An improved sample extraction method reveals that plasma receptor for advanced glycation end-products (RAGE) modulates circulating free oxytocin in mice.

The receptor for advanced glycation end-products (RAGE) binds oxytocin (OT) and transports it from the blood to the brain. As RAGE's OT-binding capacity was lost in RAGE knockout (KO) mice, we predicted that circulating concentrations of unbound (free) OT should be elevated compared to wild-type (WT) mice. However, this hypothesis has not yet been investigated. Unfortunately, the evaluation of the dynamics of circulating free and bound plasma OT is unclear in immunoassays, in part because of interference from plasma proteins. A radioimmunoassay (RIA) is considered the gold standard method for overcoming this issue, but is more challenging to implement; thus, commercially available enzyme-linked immunosorbent assays (ELISAs) are more commonly used. Here, we developed a pre-treatment method to remove the interference-causing components from plasma before performing ELISA. The acetonitrile protein precipitation (PPT) approach was reliable, with fewer steps needed to measure free OT concentrations than by solid-phase extraction of plasma samples. PPT-extracted plasma samples yielded higher concentrations of OT in RAGE KO mice than in WT mice using ELISA. After peripheral OT injection, free OT plasma levels spiked immediately then rapidly declined in WT mice, but remained high in KO mice. These results suggest that plasma samples with PPT pre-treatment appear to be superior and that circulating soluble RAGE can most likely serve as a buffer for plasma OT, which indicates a novel physiological function of RAGE.

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.

Distinct physical condition and social behavior phenotypes of CD157 and CD38 knockout mice during aging.

The ability of CD38 and CD157 to utilize nicotinamide adenine dinucleotide (NAD) has received much attention because the aging-induced elevation of CD38 expression plays a role in the senescence-related decline in NAD levels. Therefore, it is of interest to examine and compare the effects of age-associated changes on the general health and brain function impairment of Cd157 and Cd38 knockout (CD157 KO and CD38 KO) mice. The body weight and behaviors were measured in 8-week-old (young adult) or 12-month-old (middle-aged) male mice of both KO strains. The locomotor activity, anxiety-like behavior, and social behavior of the mice were measured in the open field and three-chamber tests. The middle-aged CD157 KO male mice gained more body weight than young adult KO mice, while little or no body weight gain was observed in the middle-aged CD38 KO mice. Middle-aged CD157 KO mice displayed increased anxiety-like behavior and decreased sociability and interaction compared with young adult KO mice. Middle-aged CD38 KO mice showed less anxiety and hyperactivity than CD157 KO mice, similar to young adult CD38 KO mice. The results reveal marked age-dependent changes in male CD157 KO mice but not in male CD38 KO mice. We discuss the distinct differences in aging effects from the perspective of inhibition of NAD metabolism in CD157 and CD38 KO mice, which may contribute to differential behavioral changes during aging.

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.

CD38, CD157, and RAGE as Molecular Determinants for Social Behavior.

Recent studies provide evidence to support that cluster of differentiation 38 (CD38) and CD157 meaningfully act in the brain as neuroregulators. They primarily affect social behaviors. Social behaviors are impaired in Cd38 and Cd157 knockout mice. Single-nucleotide polymorphisms of the CD38 and CD157/BST1 genes are associated with multiple neurological and psychiatric conditions, including autism spectrum disorder, Parkinson's disease, and schizophrenia. In addition, both antigens are related to infectious and immunoregulational processes. The most important clues to demonstrate how these molecules play a role in the brain are oxytocin (OT) and the OT system. OT is axo-dendritically secreted into the brain from OT-containing neurons and causes activation of OT receptors mainly on hypothalamic neurons. Here, we overview the CD38/CD157-dependent OT release mechanism as the initiation step for social behavior. The receptor for advanced glycation end-products (RAGE) is a newly identified molecule as an OT binding protein and serves as a transporter of OT to the brain, crossing over the blood-brain barrier, resulting in the regulation of brain OT levels. We point out new roles of CD38 and CD157 during neuronal development and aging in relation to nicotinamide adenine dinucleotide+ levels in embryonic and adult nervous systems. Finally, we discuss how CD38, CD157, and RAGE are crucial for social recognition and behavior in daily life.

Synthesis of oxytocin derivatives lipidated via a carbonate or carbamate linkage as a long-acting therapeutic agent for social impairment-like behaviors.

In the course of our studies of hydrophobic oxytocin (OT) analogues, we newly synthesized lipidated OT (LOT-4a-c and LOT-5a-c), in which a long alkyl chain (C14-C16) is conjugated via a carbonate or carbamate linkage at the Tyr-2 phenolic hydroxy group and a palmitoyl group at the terminal amino group of Cys-1. These LOTs did not activate OT and vasopressin receptors. Among the LOTs, however, LOT-4c, having a C16-chain via a carbonate linkage at the phenolic hydroxyl group of the Tyr-2, showed very long-lasting action for the recovery of impaired social behavior in CD38 knockout mice, a rodent model of autistic phenotypes, whereas the effect of OT itself rapidly diminished. These results indicate that LOT-4c may serve as a potential prodrug in mice.

Development of a Highly Potent Analogue and a Long-Acting Analogue of Oxytocin for the Treatment of Social Impairment-Like Behaviors.

The nonapeptide hormone oxytocin (OT) has pivotal brain roles in social recognition and interaction and is thus a promising therapeutic drug for social deficits. Because of its peptide structure, however, OT is rapidly eliminated from the bloodstream, which decreases its potential therapeutic effects in the brain. We found that newly synthesized OT analogues in which the Pro7 of OT was replaced with N-( p-fluorobenzyl)glycine (2) or N-(3-hydroxypropyl)glycine (5) exhibited highly potent binding affinities for OT receptors and Ca2+ mobilization effects by selectively activating OT receptors over vasopressin receptors in HEK cells, where 2 was identified as a superagonist ( EMax = 131%) for OT receptors. Furthermore, the two OT analogues had a remarkably long-acting effect, up to 16-24 h, on recovery from impaired social behaviors in two strains of CD38 knockout mice that exhibit autism spectrum disorder-like social behavioral deficits, whereas the effect of OT itself rapidly diminished.

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.

Effects of Three Lipidated Oxytocin Analogs on Behavioral Deficits in CD38 Knockout Mice.

Oxytocin (OT) is a nonapeptide that plays an important role in social behavior. Nasal administration of OT has been shown to improve trust in healthy humans and social interaction in autistic subjects. As is consistent with the nature of a peptide, OT has some unfavorable characteristics: it has a short half-life in plasma and shows poor permeability across the blood-brain barrier. Analogs with long-lasting effects may overcome these drawbacks. To this end, we have synthesized three analogs: lipo-oxytocin-1 (LOT-1), in which two palmitoyl groups are conjugated to the cysteine and tyrosine residues, lipo-oxytocin-2 (LOT-2) and lipo-oxytocin-3 (LOT-3), which include one palmitoyl group conjugated at the cysteine or tyrosine residue, respectively. The following behavioral deficits were observed in CD38 knockout (CD38-/-) mice: a lack of paternal nurturing in CD38-/- sires, decreased ability for social recognition, and decreased sucrose consumption. OT demonstrated the ability to recover these disturbances to the level of wild-type mice for 30 min after injection. LOT-2 and LOT-3 partially recovered the behaviors for a short period. Conversely, LOT-1 restored the behavioral parameters, not for 30 min, but for 24 h. These data suggest that the lipidation of OT has some therapeutic benefits, and LOT-1 would be most useful because of its long-last activity.

Structure-specific effects of lipidated oxytocin analogs on intracellular calcium levels, parental behavior, and oxytocin concentrations in the plasma and cerebrospinal fluid in mice.

Oxytocin (OT) is a neuroendocrine nonapeptide that plays an important role in social memory and behavior. Nasal administration of OT has been shown to improve trust in healthy humans and social interaction in autistic subjects in some clinical trials. As a central nervous system (CNS) drug, however, OT has two unfavorable characteristics: OT is short-acting and shows poor permeability across the blood-brain barrier, because it exists in charged form in the plasma and has short half-life. To overcome these drawbacks, an analog with long-lasting effects is required. We previously synthesized the analog, lipo-oxytocin-1 (LOT-1), in which two palmitoyl groups are conjugated to the cysteine and tyrosine residues. In this study, we synthesized and evaluated the analogs lipo-oxytocin-2 (LOT-2) and lipo-oxytocin-3 (LOT-3), which feature the conjugation of one palmitoyl group at the cysteine and tyrosine residues, respectively. In human embryonic kidney-293 cells overexpressing human OT receptors, these three LOTs demonstrated comparably weak effects on the elevation of intracellular free calcium concentrations after OT receptor activation, compared to the effects of OT. The three LOTs and OT exhibited different time-dependent effects on recovery from impaired pup retrieval behavior in sires of CD38-knockout mice. Sires treated with LOT-1 showed the strongest effect, whereas others had no or little effects at 24 h after injection. These results indicated that LOTs have structure-specific agonistic effects, and suggest that lipidation of OT might have therapeutic benefits for social impairment.

Lipo-oxytocin-1, a Novel Oxytocin Analog Conjugated with Two Palmitoyl Groups, Has Long-Lasting Effects on Anxiety-Related Behavior and Social Avoidance in CD157 Knockout Mice.

Oxytocin (OT) is a nonapeptide hormone that is secreted into the brain and blood circulation. OT has not only classical neurohormonal roles in uterine contraction and milk ejection during the reproductive phase in females, but has also been shown to have new pivotal neuromodulatory roles in social recognition and interaction in both genders. A single administration of OT through nasal spray increases mutual recognition and trust in healthy subjects and psychiatric patients, suggesting that OT is a potential therapeutic drug for autism spectrum disorders, schizophrenia, and some other psychiatric disorders. Although the mechanism is not well understood, it is likely that OT can be transported into the brain where it activates OT receptors to exert its function in the brain. However, the amount transported into the brain may be low. To ensure equivalent effects, an OT analog with long-lasting and effective blood-brain barrier penetration properties would be beneficial for use as a therapeutic drug. Here, we designed and synthesized a new oxytocin analog, lipo-oxytocin-1 (LOT-1), in which two palmitoyl groups are conjugated at the amino group of the cysteine9 residue and the phenolic hydroxyl group of the tyrosine8 residue of the OT molecule. To determine whether LOT-1 actually has an effect on the central nervous system, we examined its effects in a CD157 knockout model mouse of the non-motor psychiatric symptoms of Parkinson's disease. Similar to OT, this analog rescued anxiety-like behavior and social avoidance in the open field test with the social target in a central arena 30 min after intraperitoneal injection in CD157 knockout mice. When examined 24 h after injection, the mice treated with LOT-1 displayed more recovery than those given OT. The results suggest that LOT-1 has a functional advantage in recovery of social behavioral impairment, such as those caused by neurodegenerative diseases, autism spectrum disorders, and schizophrenia.

CD38 in the nucleus accumbens and oxytocin are related to paternal behavior in mice.

BACKGROUND: Mammalian sires participate in infant care. We previously demonstrated that sires of a strain of nonmonogamous laboratory mice initiate parental retrieval behavior in response to olfactory and auditory signals from the dam during isolation in a new environment. This behavior is rapidly lost in the absence of such signals when the sires are caged alone. The neural circuitry and hormones that control paternal behavior are not well-understood. CD38, a membrane glycoprotein, catalyzes synthesis of cyclic ADP-ribose and facilitates oxytocin (OT) secretion due to cyclic ADP-ribose-dependent increases in cytosolic free calcium concentrations in oxytocinergic neurons in the hypothalamus. In this paper, we studied CD38 in the nucleus accumbens (NAcc) and the role of OT on paternal pup retrieval behavior using CD38 knockout (CD38-/-) mice of the ICR strain. RESULTS: CD38-/- sires failed to retrieve when they were reunited with their pups after isolation together with the mate dams, but not with pup, in a novel cage for 10 min. CD38-/- sires treated with a single subcutaneous injection of OT exhibited recovery in the retrieval events when caged with CD38-/- dams treated with OT. We introduced human CD38 in the NAcc of CD38-/- sires using a lentiviral infection technique and examined the effects of local expression of CD38. Pairs of knockout dams treated with OT and sires expressing CD38 in the NAcc showed more retrieval (83% of wild-type sire levels). Complete recovery of retrieval was obtained in sires with the expression of CD38 in the NAcc in combination with OT administration. Other paternal behaviors, including pup grooming, crouching and huddling, were also more common in CD38-/- sires with CD38 expression in the NAcc compared with those in CD38-/- sires without CD38 expression in the NAcc. CONCLUSIONS: CD38 in the NAcc and OT are critical in paternal behavior.

Displays of paternal mouse pup retrieval following communicative interaction with maternal mates.

Compared with the knowledge of maternal care, much less is known about the factors required for paternal parental care. Here we report that new sires of laboratory mice, though not spontaneously parental, can be induced to show maternal-like parental care (pup retrieval) using signals from dams separated from their pups. During this interaction, the maternal mates emit 38-kHz ultrasonic vocalizations to their male partners, which are equivalent to vocalizations that occur following pheromone stimulation. Without these signals or in the absence of maternal mates, the sires do not retrieve their pups within 5 min. These results show that, in mice, the maternal parent communicates to the paternal parent to encourage pup care. This new paradigm may be useful in the analysis of the parental brain during paternal care induced by interactive communication.