高柳 友紀

BACKGROUND: The flexibility to adjust actions and attitudes in response to varying social situations is a fundamental aspect of adaptive social behavior. Adaptive social behaviors influence an individual's vulnerability to social stress. While oxytocin has been proposed to facilitate active coping behaviors during social stress, the exact mechanisms remain unknown. METHODS: By using a social defeat stress paradigm in male mice, we identified the distribution of oxytocin receptor (OXTR)-expressing neurons in the ventrolateral part of the ventromedial hypothalamus (vlVMH) that are activated during stress by detection of c-Fos protein expression. We then investigated the role of vlVMH OXTR-expressing neurons in social defeat stress responses by chemogenetic methods or deletion of local OXTRs. The social defeat posture was measured for quantification of adaptive social behavior during repeated social stress. RESULTS: Social defeat stress activated OXTR-expressing neurons rather than estrogen type 1-expressing neurons in the rostral vlVMH. OXTR-expressing neurons in the vlVMH were glutamatergic. Chemogenetic activation of vlVMH OXTR-expressing neurons facilitated exhibition of the social defeat posture during exposure to social stress, while local OXTR deletion suppressed it. In contrast, over-activation of vlVMH-OXTR neurons induced generalized social avoidance after exposure to chronic social defeat stress. Neural circuits for the social defeat posture centered on OXTR-expressing neurons were identified by viral tracers and c-Fos mapping. CONCLUSIONS: VlVMH OXTR-expressing neurons are a functionally unique population of neurons that promote an active coping behavior during social stress, but their excessive and repetitive activation under chronic social stress impairs subsequent social behavior.

Rodent ultrasonic vocalisations can be used to assess social behaviour and have attracted increasing attention. Rats emit 50-kHz and 22-kHz calls during appetitive and aversive states, respectively. These calls induce behavioural and neural responses in the receiver by transmitting the internal states of the rats, thus serving communicative functions. Recently, we discovered that female Lewis rats emit 31-kHz calls under social isolation and inequality conditions; however, the biological significance of 31-kHz calls remains unknown. In the present study, we conducted three playback experiments to examine the behavioural effects of 31-kHz calls. In the first experiment, Lewis female rats were exposed to four types of sound: 22-kHz, 50-kHz, 31-kHz calls, and environmental noise. As a result, rats stayed significantly longer in the area with a sound-producing speaker, regardless of the sound type, than in the silent speaker area. The duration spent around the sound-producing speaker was particularly extended during the 50-kHz or 31-kHz call playback, compared to the environmental noise or 22-kHz call playback. In the second experiment, rats were exposed to refined versions of sound stimuli that were synthesised to preserve prominent frequency components while removing background noise from original calls. Rats significantly preferred to stay around the speaker for the synthesised 50-kHz and 31-kHz sounds, but not for the synthesised 22-kHz sound. However, in the third experiment, additional 31-kHz sound synthesised from calls emitted by a different rat did not elicit a significant preference for the source side. These results suggest that the rats paid attention to the 31-kHz call, although it is plausible that acoustic variability in the 31-kHz USV may affect their approach behaviour.

The cerebellum plays an important role in cognitive and social functioning. Childhood damage in the cerebellum increases the risk of autism spectrum disorder. Cerebellar inflammation induces social avoidance in mice. Oxytocin regulates social relationship and expression pattern of the oxytocin receptor in the brain is related to social behaviors. However, the expression patterns of the oxytocin receptor in the cerebellum remain controversial. Here, we report that the expression patterns of the oxytocin receptor in the cerebellum are highly variable among knock-in transgenic lines. We used Oxtr-Cre knock-in mice combined with a fluorescent reporter line and found that oxytocin receptor expression in Bergmann glia was more variable than that in Purkinje cells. We found that physical damage with inflammation induced the selective upregulation of the oxytocin receptor in Bergmann glia. Our findings indicate high variability in oxytocin receptor expression in the cerebellum and suggest that the oxytocin receptor can affect neural processing in pathological conditions, such as inflammation.

BACKGROUND: Fabry disease (FD) is an inherited lysosomal storage disease caused by deficiency of α-galactosidase A (α-Gal A) encoded by the GLA gene. The symptoms of FD occur as a result of the accumulation of globotriaosylceramide (Gb3), comprising a substrate of α-Gal A, in the organs. Adeno-associated virus (AAV)-mediated gene therapy is a promising treatment for FD. METHODS: α-Gal A knockout (GLAko) mice were injected intravenously with AAV2 (1 × 1011 viral genomes [vg]) or AAV9 (1 × 1011 or 2 × 1012 vg) vectors carrying human GLA (AAV-hGLA), and plasma, brain, heart, liver and kidney were tested for α-Gal A activity. The vector genome copy numbers (VGCNs) and Gb3 content in each organ were also examined. RESULTS: The plasma α-Gal A enzymatic activity was three-fold higher in the AAV9 2 × 1012 vg group than wild-type (WT) controls, which was maintained for up to 8 weeks after injection. In the AAV9 2 × 1012 vg group, the level of α-Gal A expression was high in the heart and liver, intermediate in the kidney, and low in the brain. VGCNs in the all organs of the AAV9 2 × 1012 vg group significantly increased compared to the phosphate-buffered-saline (PBS) group. Although Gb3 in the heart, liver and kidney of the AAV9 2 × 1012 vg was reduced compared to PBS group and AAV2 group, and the amount of Gb3 in the brain was not reduced. CONCLUSIONS: Systemic injection of AAV9-hGLA resulted in α-Gal A expression and Gb3 reduction in the organs of GLAko mice. To expect a higher expression of α-Gal A in the brain, the injection dosage, administration route and the timing of injection should be reconsidered.

Whether commonly used experimental animals show aversion toward inequality of social rewards, as humans do remains unknown. We examined whether rats emitted the 22-kHz distress calls under social reward inequality. Rats showed affiliative behavior for a specific human who repeatedly stroked and tickled them. When experimenter stroked another rat in front of them and during social isolation, these rats emitted novel calls with acoustic characteristics different from those of calls emitted under physical stress, namely air-puff. Under inequality conditions, rats emitted calls with higher frequency (∼31 kHz) and shorter duration (<0.5 s) than those emitted when receiving air-puff. However, with an affiliative human in front of them, the number of novel calls was lower and rats emitted 50-kHz calls, indicative of the appetitive state. These results indicate that rats distinguish between conditions of social reward inequality and the presence of an experimenter, and emit novel 31-kHz calls.

The CRISPR-Cas9 method for generation of knock-in mutations in rodent embryos yields many F0 generation candidates that may have the designed mutations. The first task for selection of promising F0 generations is to analyze genomic DNA which likely contains a mixture of designed and unexpected mutations. In our study, while generating Prlhr-Venus knock-in reporter mice, we found that genomic rearrangements near the targeted knock-in allele, tandem multicopies at a target allele locus, and mosaic genotypes for two different knock-in alleles occurred in addition to the designed knock-in mutation in the F0 generation. Conventional PCR and genomic sequencing were not able to detect mosaicism nor discriminate between the designed one-copy knock-in mutant and a multicopy-inserted mutant. However, by using a combination of Southern blotting and the next-generation sequencing-based RAISING method, these mutants were successfully detected in the F0 generation. In the F1 and F2 generations, droplet digital PCR assisted in establishing the strain, although a multicopy was falsely detected as one copy by analysis of the F0 generation. Thus, the combination of these methods allowed us to select promising F0 generations and facilitated establishment of the designed strain. We emphasize that focusing only on positive evidence of knock-in can lead to erroneous selection of undesirable strains.

During obesity, tissue macrophages increase in number and become proinflammatory, thereby contributing to metabolic dysfunction. Lipoprotein lipase (LPL), which hydrolyzes triglyceride in lipoproteins, is secreted by macrophages. However, the role of macrophage-derived LPL in adipose tissue remodeling and lipoprotein metabolism is largely unknown. To clarify these issues, we crossed leptin-deficient Lepob/ob mice with mice lacking the Lpl gene in myeloid cells (Lplm-/m-) to generate Lplm-/m-;Lepob/ob mice. We found the weight of perigonadal white adipose tissue (WAT) was increased in Lplm-/m-;Lepob/ob mice compared with Lepob/ob mice due to substantial accumulation of both adipose tissue macrophages and collagen that surrounded necrotic adipocytes. In the fibrotic epidydimal WAT of Lplm-/m-;Lepob/ob mice, we observed an increase in collagen VI and high mobility group box 1, while α-smooth muscle cell actin, a marker of myofibroblasts, was almost undetectable, suggesting that the adipocytes were the major source of the collagens. Furthermore, the adipose tissue macrophages from Lplm-/m-;Lepob/ob mice showed increased expression of genes related to fibrosis and inflammation. In addition, we determined Lplm-/m-;Lepob/ob mice were more hypertriglyceridemic than Lepob/ob mice. Lplm-/m-;Lepob/ob mice also showed slower weight gain than Lepob/ob mice, which was primarily due to reduced food intake. In conclusion, we discovered that the loss of myeloid Lpl led to extensive fibrosis of perigonadal WAT and hypertriglyceridemia. In addition to illustrating an important role of macrophage LPL in regulation of circulating triglyceride levels, these data show that macrophage LPL protects against fibrosis in obese adipose tissues.

Oxytocin has been revealed to work for anxiety suppression and anti-stress as well as for psychosocial behavior and reproductive functions. Oxytocin neurons are activated by various stressful stimuli. The oxytocin receptor is widely distributed within the brain, and oxytocin that is released or diffused affects behavioral and neuroendocrine stress responses. On the other hand, there has been an increasing number of reports on the role of oxytocin in allostasis and resilience. It has been shown that oxytocin maintains homeostasis, shifts the set point for adaptation to a changing environment (allostasis) and contributes to recovery from the shifted set point by inducing active coping responses to stressful stimuli (resilience). Recent studies have suggested that oxytocin is also involved in stress-related disorders, and it has been shown in clinical trials that oxytocin provides therapeutic benefits for patients diagnosed with stress-related disorders. This review includes the latest information on the role of oxytocin in stress responses and adaptation.

Vasopressin-synthesizing neurons are located in several brain regions, including the hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON) and suprachiasmatic nucleus (SCN). Vasopressin has been shown to have various functions in the brain, including social recognition memory, stress responses, emotional behaviors and circadian rhythms. The precise physiological functions of vasopressin-synthesizing neurons in specific brain regions remain to be clarified. Conditional ablation of local vasopressin-synthesizing neurons may be a useful tool for investigation of the functions of vasopressin neurons in the regions. In the present study, we characterized a transgenic rat line that expresses a mutated human diphtheria toxin receptor under control of the vasopressin gene promoter. Under a condition of salt loading, which activates the vasopressin gene in the hypothalamic PVN and SON, transgenic rats were i.c.v. injected with diphtheria toxin. Intracerebroventricular administration of diphtheria toxin after salt loading depleted vasopressin-immunoreactive cells in the hypothalamic PVN and SON, but not in the SCN. The number of oxytocin-immunoreactive cells in the hypothalamus was not significantly changed. The rats that received i.c.v. diphtheria toxin after salt loading showed polydipsia and polyuria, which were rescued by peripheral administration of 1-deamino-8-d-arginine vasopressin via an osmotic mini-pump. Intrahypothalamic administration of diphtheria toxin in transgenic rats under a normal hydration condition reduced the number of vasopressin-immunoreactive neurons, but not the number of oxytocin-immunoreactive neurons. The transgenic rat model can be used for selective ablation of vasopressin-synthesizing neurons and may be useful for clarifying roles of vasopressin neurons at least in the hypothalamic PVN and SON in the rat.

Early-life experience influences social and emotional behaviour in adulthood. Affiliative tactile stimuli in early life facilitate the development of social and emotional behaviour, whereas early-life adverse stimuli have been shown to increase the risk of various diseases in later life. On the other hand, oxytocin has been shown to have organizational actions during early-life stages. However, the detailed mechanisms of the effects of early-life experience and oxytocin remain unclear. Here, we review the effects of affiliative tactile stimuli during the neonatal period and neonatal oxytocin treatment on the activity of the oxytocin-oxytocin receptor system and social or emotional behaviour in adulthood. Both affiliative tactile stimuli and early-life adverse stimuli in the neonatal period acutely activate the oxytocin-oxytocin receptor system in the brain but modulate social behaviour and anxiety-related behaviour apparently in an opposite direction in adulthood. Accumulating evidence suggests that affiliative tactile stimuli and exogenous application of oxytocin in early-life stages induce higher activity of the oxytocin-oxytocin receptor system in adulthood, although the effects are dependent on experimental procedures, sex, dosages and brain regions examined. On the other hand, early-life stressful stimuli appear to induce reduced activity of the oxytocin-oxytocin receptor system, possibly leading to adverse actions in adulthood. It is possible that activation of a specific oxytocin system can induce beneficial actions against early-life maltreatments and thus could be used for the treatment of developmental psychiatric disorders.

Fibroblast growth factor 21 (FGF21) modulates energy metabolism and neuroendocrine stress responses. FGF21 synthesis is increased after environmental or metabolic challenges. Detailed roles of FGF21 in the control of behavioural disturbances under stressful conditions remain to be clarified. Here, we examined the roles of FGF21 in the control of behavioural changes after social defeat stress in male rodents. Central administration of FGF21 increased the number of tyrosine hydroxylase-positive catecholaminergic cells expressing c-Fos protein, an activity marker of neurones, in the nucleus tractus solitarius and area postrema. Double in situ hybridisation showed that some catecholaminergic neurones in the dorsal medulla oblongata expressed β-Klotho, an essential co-receptor for FGF21, in male mice. Social defeat stress increased FGF21 concentrations in the plasma of male mice. FGF21-deficient male mice showed social avoidance in a social avoidance test with C57BL/6J mice (background strain of FGF21-deficient mice) and augmented immobility behaviour in a forced swimming test after social defeat stress. On the other hand, overexpression of FGF21 by adeno-associated virus vectors did not significantly change behaviours either in wild-type male mice or FGF21-deficient male mice. The present data are consistent with the view that endogenous FGF21, possibly during the developmental period, has an inhibitory action on stress-induced depression-like behaviour in male rodents.

Niemann-Pick disease type C1 (NPC1) is a fatal congenital neurodegenerative disorder caused by mutations in the NPC1 gene, which is involved in cholesterol transport in lysosomes. Broad clinical manifestations of NPC1 include liver failure, pulmonary disorder, neurological deficits, and psychiatric symptoms. The main cause of death in NPC1 patients involves central nervous system (CNS) dysfunction; there is no essential treatment. We generated a tyrosine-mutant adeno-associated virus (AAV) 9/3 vector that expresses human NPC1 under a CMV promotor (AAV-CMV-hNPC1) and injected it into the left lateral ventricle (5 μl) and cisterna magna (10 μl) of Npc1 homo-knockout (Npc1^-/-) mice. Each mouse received total 1.35×10¹¹ vector genome on day 4 or 5 of life. AAV-treated Npc1^-/- mice (n=11) had an average survival of >28 weeks, while all saline-treated Npc1^-/- mice (n=11) and untreated Npc1^-/- mice (n=6) died within 16 weeks. Saline-treated and untreated Npc1^-/- mice lost body weight from seven weeks until death. However, the average body weight of AAV-treated Npc1^-/- mice increased until 15 weeks. AAV-treated Npc1^-/- mice also showed a significant improvement in Rotarod test performance. A pathological analysis at 11 weeks showed that cerebellar Purkinje cells were preserved in AAV-treated Npc1^-/- mice. In contrast, untreated Npc1^-/- mice showed an almost total loss of cerebellar Purkinje cells. Combined injection into both the lateral ventricle and cisterna magna achieved broader delivery of the vector to the CNS, leading to better outcomes than noted in previous reports with injection into the lateral ventricles or veins alone. In AAV-treated Npc1^-/- mice, vector genome DNA was detected widely in the CNS and liver. Human NPC1 RNA was detected in the brain, liver, lung, and heart. Accumulated unesterified cholesterol in liver was reduced in the AAV-treated Npc1^-/- mice. Our results suggest the feasibility of gene therapy for patients with NPC1.

Long-term and mild confinement or isolation in an enclosed environment can occur in situations such as disasters, specific political, economic or social events, nuclear shelters, seabed exploration, polar expeditions, and space travel. To investigate the effects of stress caused by long-term confinement in an enclosed environment in mammals, we divided 8-week-old C57BL/6J mice into four groups that were housed in a closed environment with a narrow metabolic cage (stress group), normal metabolic cage (control group), conventional cage (conventional group) or conventional cage with wire mesh floor (wire mesh group). The phenotypes of the mice were examined for four weeks, followed by behavioral tests. Weight gain suppression was observed in the stress group. Continuous analysis of these mice every two minutes for four weeks using an implanted measuring device showed a significantly decreased amount of spontaneous activity and subcutaneous temperature in the stress group. After housing in each environment for four weeks, the behavioral tests of mice in the stress group also revealed a shorter latency to fall off in the rotarod test and shorter stride length and interstep distance in the footprint test. Interestingly, the lower spontaneous activity of mice in the stress group was rescued by housing in conventional cages. These results suggest a temporary effect of long-term confinement in an enclosed environment as a chronic and mild stress on homeostasis in mammals.

Social contact reduces stress responses in social animals. Mice have been shown to show allogrooming behaviour toward distressed conspecifics. However, the precise neuronal mechanisms underlying allogrooming behaviour remain unclear. In the present study, we examined whether mice show allogrooming behaviour towards distressed conspecifics in a social defeat model and we also determined whether oxytocin receptor-expressing neurons were activated during allogrooming by examining the expression of c-Fos protein, a marker of neurone activation. Mice showed allogrooming behaviour toward socially defeated conspecifics. After allogrooming behaviour, the percentages of oxytocin receptor-expressing neurones expressing c-Fos protein were significantly increased in the anterior olfactory nucleus, cingulate cortex, insular cortex, lateral septum and medial amygdala of female mice, suggesting that oxytocin receptor-expressing neurones in these areas were activated during allogrooming behaviour toward distressed conspecifics. The duration of allogrooming was correlated with the percentages of oxytocin receptor-expressing neurones expressing c-Fos protein in the anterior olfactory nucleus, insular cortex, lateral septum and medial amygdala. In oxytocin receptor-deficient mice, allogrooming behaviour toward socially defeated cage mates was markedly reduced in female mice but not in male mice, indicating the importance of the oxytocin receptor for allogrooming behaviour in female mice toward distressed conspecifics. The results suggest that the oxytocin receptor, possibly in the anterior olfactory nucleus, insular cortex, lateral septum and/or medial amygdala, facilitates allogrooming behaviour toward socially distressed familiar conspecifics in female mice.

Gentle touch contributes to affiliative interactions. We investigated the effects of gentle stroking in female rats on the development of affiliative behaviors toward humans and we exploratively examined brain regions in which activity was influenced by stroking. Rats that had received stroking stimuli repeatedly after weaning emitted 50-kHz calls, an index of positive emotion, and showed affiliative behaviors toward the experimenter. Hypothalamic paraventricular oxytocin neurons were activated in the rats after stroking. The septohypothalamic nucleus (SHy) in the post-weaningly stroked rats showed decreased activity in response to stroking stimuli compared with that in the non-stroked control group. There were negative correlations of neural activity in hypothalamic regions including the SHy with the number of 50-kHz calls. These findings revealed that post-weaning stroking induces an affiliative relationship between female rats and humans, possibly via activation of oxytocin neurons and suppression of the activity of hypothalamic neurons.

Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), statins, which are used to prevent cardiovascular diseases, are associated with a modest increase in the risk of new-onset diabetes. To investigate the role of HMGCR in the development of β-cells and glucose homeostasis, we deleted Hmgcr in a β-cell-specific manner by using the Cre-loxP technique. Mice lacking Hmgcr in β-cells (β-KO) exhibited hypoinsulinemic hyperglycemia as early as postnatal day 9 (P9) due to decreases in both β-cell mass and insulin secretion. Ki67-positive cells were reduced in β-KO mice at P9; thus, β-cell mass reduction was caused by proliferation disorder immediately after birth. The mRNA expression of neurogenin3 (Ngn3), which is transiently expressed in endocrine progenitors of the embryonic pancreas, was maintained despite a striking reduction in the expression of β-cell-associated genes, such as insulin, pancreatic and duodenal homeobox 1 (Pdx1), and MAF BZIP transcription factor A (Mafa) in the islets from β-KO mice. Histological analyses revealed dysmorphic islets with markedly reduced numbers of β-cells, some of which were also positive for glucagon. In conclusion, HMGCR plays critical roles not only in insulin secretion but also in the development of β-cells in mice.

Gentle tactile stimuli have been shown to play an important role in the establishment and maintenance of affiliative social interactions. Oxytocin has also been shown to have similar actions. We investigated the effects of gentle stroking on affiliative relationships between humans and rats and the effects of gentle stroking on activation of oxytocin neurons. Male rats received 5-min stroking stimuli from an experimenter every other day for 4 weeks between 3 and 6 weeks of age (S3-6 group), for 4 weeks between 7 and 10 weeks of age (S7-10 group), or for 8 weeks between 3 and 10 weeks of age (S3-10 group). Control rats did not receive stroking stimuli. Rats in the S7-10 and S3-10 groups emitted 50-kHz calls, an index of positive emotion, more frequently during stroking stimuli. Rats in the S3-6, S7-10, and S3-10 groups showed affiliative behaviors toward the experimenter. Oxytocin neurons in the hypothalamic paraventricular nucleus of rats in the S3-6, S7-10, and S3-10 groups were activated following stroking stimuli. These findings revealed that post-weaning repeated stroking stimuli induce an affiliative relationship between rats and humans and activation of oxytocin neurons.

Oxytocin neurones in the hypothalamus are activated by stressful stimuli and food intake. The oxytocin receptor is located in various brain regions, including the sensory information-processing cerebral cortex; the cognitive information-processing prefrontal cortex; reward-related regions such as the ventral tegmental areas, nucleus accumbens and raphe nucleus; stress-related areas such as the amygdala, hippocampus, ventrolateral part of the ventromedial hypothalamus and ventrolateral periaqueductal gray; homeostasis-controlling hypothalamus; and the dorsal motor complex controlling intestinal functions. Oxytocin affects behavioural and neuroendocrine stress responses and terminates food intake by acting on the metabolic or nutritional homeostasis system, modulating emotional processing, reducing reward values of food intake, and facilitating sensory and cognitive processing via multiple brain regions. Oxytocin also plays a role in interactive actions between stress and food intake and contributes to adaptive active coping behaviours.

Social stress has deteriorating effects on various psychiatric diseases. In animal models, exposure to socially dominant conspecifics (i.e., social defeat stress) evokes a species-specific defeat posture via unknown mechanisms. Oxytocin neurons have been shown to be activated by stressful stimuli and to have prosocial and anxiolytic actions. The roles of oxytocin during social defeat stress remain unclear. Expression of c-Fos, a marker of neuronal activation, in oxytocin neurons and in oxytocin receptor‒expressing neurons was investigated in mice. The projection of oxytocin neurons was examined with an anterograde viral tracer, which induces selective expression of membrane-targeted palmitoylated green fluorescent protein in oxytocin neurons. Defensive behaviors during double exposure to social defeat stress in oxytocin receptor‒deficient mice were analyzed. After social defeat stress, expression of c-Fos protein was increased in oxytocin neurons of the bed nucleus of the stria terminalis, supraoptic nucleus, and paraventricular hypothalamic nucleus. Expression of c-Fos protein was also increased in oxytocin receptor‒expressing neurons of brain regions, including the ventrolateral part of the ventromedial hypothalamus and ventrolateral periaqueductal gray. Projecting fibers from paraventricular hypothalamic oxytocin neurons were found in the ventrolateral part of the ventromedial hypothalamus and in the ventrolateral periaqueductal gray. Oxytocin receptor‒deficient mice showed reduced defeat posture during the second social defeat stress. These findings suggest that social defeat stress activates oxytocin-oxytocin receptor systems, and the findings are consistent with the view that activation of the oxytocin receptor in brain regions, including the ventrolateral part of the ventromedial hypothalamus and the ventrolateral periaqueductal gray, facilitates social defeat posture.

BACKGROUND: Social recognition underlies social behavior in animals, and patients with psychiatric disorders associated with social deficits show abnormalities in social recognition. Oxytocin is implicated in social behavior and has received attention as an effective treatment for sociobehavioral deficits. Secretin receptor-deficient mice show deficits in social behavior. The relationship between oxytocin and secretin concerning social behavior remains to be determined. METHODS: Expression of c-Fos in oxytocin neurons and release of oxytocin from their dendrites after secretin application were investigated. Social recognition was examined after intracerebroventricular or local injection of secretin, oxytocin, or an oxytocin receptor antagonist in rats, oxytocin receptor-deficient mice, and secretin receptor-deficient mice. Electron and light microscopic immunohistochemical analysis was also performed to determine whether oxytocin neurons extend their dendrites into the medial amygdala. RESULTS: Supraoptic oxytocin neurons expressed the secretin receptor. Secretin activated supraoptic oxytocin neurons and facilitated oxytocin release from dendrites. Secretin increased acquisition of social recognition in an oxytocin receptor-dependent manner. Local application of secretin into the supraoptic nucleus facilitated social recognition, and this facilitation was blocked by an oxytocin receptor antagonist injected into, but not outside of, the medial amygdala. In the medial amygdala, dendrite-like thick oxytocin processes were found to extend from the supraoptic nucleus. Furthermore, oxytocin treatment restored deficits of social recognition in secretin receptor-deficient mice. CONCLUSIONS: The results of our study demonstrate that secretin-induced dendritic oxytocin release from supraoptic neurons enhances social recognition. The newly defined secretin-oxytocin system may lead to a possible treatment for social deficits.

Gentle touching or stroking has anxiolytic actions and contributes to the establishment of an intimate relationship between individuals. Oxytocin administration also has anxiolytic actions and facilitates social behaviors. In this study, we examined effects of stroking stimuli on activation of oxytocin neurons and emission of 50-kHz ultrasonic vocalizations, an index of positive emotion, in rats. The number of oxytocin neurons expressing Fos protein was increased in the hypothalamus, especially in the dorsal zone of the medial parvicellular part of the paraventricular nucleus. The number of 50-kHz ultrasonic vocalizations was also increased. These findings suggest that pleasant sensory stimuli activate hypothalamic oxytocin neurons.

Oxytocin plays an essential role in milk ejection and parturition in mammals. Oxytocin has also been shown to be involved in the control of various behaviors, including anxiety-related behaviors, food intake and affiliative behaviors.<br>We previously showed that noxious stimuli or stimuli previously paired with noxious stimuli (conditioned fear stimuli) activate hypothalamic oxytocin neurons via activation of brainstem catecholaminergic/prolactin-releasing peptide (PrRP)-positive neurons. Oxytocin neurons are activated not only by noxious stimuli but also by non-noxious touch stimuli. Social contact has been suggested to activate oxytocin neurons. Non-noxious tactile stimuli induce 50-kHz ultrasonic vocalization, an index of positive states in rats, and activate hypothalamic oxytocin neurons, suggesting that pleasant tactile stimuli activate oxytocin neurons.<br>Physiological roles of oxytocin released during noxious or non-noxious tactile stimuli remain to be clarified. Noxious stimuli increase anxiety-related behavior, while pleasant sensory stimuli have pro-social actions. We have shown that endogenous oxytocin reduces anxiety-related behaviors, induces a decrease in amounts of food intake per meal, and facilitates social recognition via distinct neural pathways. Roles of oxytocin released during sensory stimuli may be dependent upon the sensory stimuli used, and oxytocin may contribute to the prevention of overreactions to noxious stimuli or mediate pro-social or anxiolytic actions of pleasant tactile stimuli.

Fear responses play evolutionarily beneficial roles, although excessive fear memory can induce inappropriate fear expression observed in posttraumatic stress disorder, panic disorder, and phobia. To understand the neural machineries that underlie these disorders, it is important to clarify the neural pathways of fear responses. Contextual conditioned fear induces freezing behavior and neuroendocrine responses. Considerable evidence indicates that the central amygdala plays an essential role in expression of freezing behavior after contextual conditioned fear. On the other hand, mechanisms of neuroendocrine responses remain to be clarified. The medial amygdala (MeA), which is activated after contextual conditioned fear, was lesioned bilaterally by infusion of N-methyl-d-aspartate after training of fear conditioning. Plasma oxytocin, ACTH, and prolactin concentrations were significantly increased after contextual conditioned fear in sham-lesioned rats. In MeA-lesioned rats, these neuroendocrine responses but not freezing behavior were significantly impaired compared with those in sham-lesioned rats. In contrast, the magnitudes of neuroendocrine responses after exposure to novel environmental stimuli were not significantly different in MeA-lesioned rats and sham-lesioned rats. Contextual conditioned fear activated prolactin-releasing peptide (PrRP)-synthesizing neurons in the medulla oblongata. In MeA-lesioned rats, the percentage of PrRP-synthesizing neurons activated after contextual conditioned fear was significantly decreased. Furthermore, neuroendocrine responses after contextual conditioned fear disappeared in PrRP-deficient mice. Our findings suggest that the MeA-medullary PrRP-synthesizing neuron pathway plays an important role in neuroendocrine responses to contextual conditioned fear.

RFamide-related peptides (RFRP-1 and RFRP-3) are localised in neurones of the dorsomedial hypothalamus in rats. The dorsomedial hypothalamus plays an essential role in neuroendocrine and behavioural stress responses. In the present study, we examined the role of RFRP in the control of neuroendocrine and behavioural responses in rats. Stressful stimuli increased expression of Fos protein in RFRP-immunoreactive neurones of the dorsomedial hypothalamus, suggesting that stressful stimuli activate RFRP neurones. Intracerebroventricular injection of RFRPs increased the expression of Fos protein in oxytocin neurones in the hypothalamus and plasma concentrations of adrenocorticotrophic hormone and oxytocin. The hypothalamic paraventricular and supraoptic nuclei expressed mRNA of GPR147, the putative RFRP receptor, and application of RFRPs to isolated supraoptic nuclei facilitated oxytocin release, suggesting that RFRPs activate oxytocin neurones directly. Furthermore, the administration of RFRPs induced anxiety-related behaviour in rats in open-field tests. All these data taken together suggest that RFRPs play a role in the control of neuroendocrine and behavioural stress responses in rats.

Subsequent to the isolation of the first recognized RFamide neuropeptide, FMRFamide, from the clam, a large number of these peptides have been identified. There are now five groups of RFamide peptides identified in mammals. RFamide peptides show diversity with respect to their N-terminal sequence and biological activity. RFamide peptides have been implicated in a variety of roles, including energy metabolism, stress and pain modulation, as well as effects in the neuroendocrine and cardiovascular systems. In the present minireview, we focus on prolactin-releasing peptide (PrRP) and RFamide related peptide (RFRP) with respect to their roles in the control of energy metabolism and stress responses. Both food intake and stressful stimuli activate PrRP neurons. The administration of PrRP affects energy metabolism and neuroendocrine systems. PrRP-deficient or PrRP receptor-deficient mice show abnormal energy metabolism and/or stress responses. On the other hand, RFRP neurons are activated by stressful stimuli and the administration of RFRP induces neuroendocrine and behavioral stress responses. Taken together, these data suggests that PrRP and RFRP neurons play a role in the control of energy metabolism and/or stress responses.

Food intake is controlled by signals from the brainstem that mediate signals from the gut. Prolactin-releasing peptide (PrRP) neurons are localized in the brainstem. In the present study, we investigated whether food intake activates PrRP neurons. We examined expression of phospho-CREB in PrRP neurons after food intake. The percentage of phospho-CREB-positive PrRP neurons in the nucleus of tractus solitarii was increased by food intake, suggesting that food intake activates PrRP neurons in the nucleus tractus solitarii. To further study the role of endogenous PrRP in food intake, we generated PrRP-deficient mice. PrRP-deficient mice developed late onset obestity associated with metabolic disorders due to hyperphagia but not to energy expenditure, and showed an attenuated response to the peripheral satiety signal, cholecystokinin. Blockade of endogenous PrRP signaling by a central injection of monoclonal anti-PrRP neutralizing antibodies increased food intake, reflecting an increase in meal size. Furthermore, we demonstrated that leptin-induced reduction in food intake and body weight was impaired in PrRP-deficient mice. All these data suggest that PrRP relays satiety signals within the brain and that disturbance of this system can result in obesity and associated metabolic disorders. [J Physiol Sci. 2008;58 Suppl:S106]