Preeclampsia-Like Features and Partial Lactation Failure in Mice Lacking Cystathionine γ-Lyase-An Animal Model of Cystathioninuria.

Elevated plasma homocysteine levels are considered as a risk factor for cardiovascular diseases as well as preeclampsia-a pregnancy disorder characterized by hypertension and proteinuria. We previously generated mice lacking cystathionine γ-lyase (Cth) as cystathioninuria models and found them to be with cystathioninemia/homocysteinemia. We investigated whether Cth-deficient (Cth-/-) pregnant mice display any features of preeclampsia. Cth-/- females developed normally but showed mild hypertension (~10 mmHg systolic blood pressure elevation) in late pregnancy and mild proteinuria throughout development/pregnancy. Cth-/- dams had normal numbers of pups and exhibited normal maternal behavior except slightly lower breastfeeding activity. However, half of them could not raise their pups owing to defective lactation; they could produce/store the first milk in their mammary glands but not often provide milk to their pups after the first ejection. The serum oxytocin levels and oxytocin receptor expression in the mammary glands were comparable between wild-type and Cth-/- dams, but the contraction responses of mammary gland myoepithelial cells to oxytocin were significantly lower in Cth-/- dams. The contraction responses to oxytocin were lower in uteruses isolated from Cth-/- mice. Our results suggest that elevated homocysteine or other unknown factors in preeclampsia-like Cth-/- dams interfere with oxytocin that regulates milk ejection reflex.

Cerebellar damage with inflammation upregulates oxytocin receptor expression in Bergmann Glia.

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.

Novel 31-kHz calls emitted by female Lewis rats during social isolation and social inequality conditions.

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.

Necessity of integrated genomic analysis to establish a designed knock-in mouse from CRISPR-Cas9-induced mutants.

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.

Oxytocin signaling in the posterior hypothalamus prevents hyperphagic obesity in mice.

Decades of studies have revealed molecular and neural circuit bases for body weight homeostasis. Neural hormone oxytocin (Oxt) has received attention in this context because it is produced by neurons in the paraventricular hypothalamic nucleus (PVH), a known output center of hypothalamic regulation of appetite. Oxt has an anorexigenic effect, as shown in human studies, and can mediate satiety signals in rodents. However, the function of Oxt signaling in the physiological regulation of appetite has remained in question, because whole-body knockout (KO) of Oxt or Oxt receptor (Oxtr) has little effect on food intake. We herein show that acute conditional KO (cKO) of Oxt selectively in the adult PVH, but not in the supraoptic nucleus, markedly increases body weight and food intake, with an elevated level of plasma triglyceride and leptin. Intraperitoneal administration of Oxt rescues the hyperphagic phenotype of the PVH Oxt cKO model. Furthermore, we show that cKO of Oxtr selectively in the posterior hypothalamic regions, especially the arcuate hypothalamic nucleus, a primary center for appetite regulations, phenocopies hyperphagic obesity. Collectively, these data reveal that Oxt signaling in the arcuate nucleus suppresses excessive food intake.

Post-weaning stroking stimuli induce affiliative behavior toward humans and influence brain activity in female rats.

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.

Indispensable role of the oxytocin receptor for allogrooming toward socially distressed cage mates in female mice.

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.

Conditional ablation of vasopressin-synthesizing neurons in transgenic rats.

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.

Roles of fibroblast growth factor 21 in the control of depression-like behaviours after social defeat stress in male rodents.

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 ß-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.

Evidence that oxytocin exerts anxiolytic effects via oxytocin receptor expressed in serotonergic neurons in mice.

The oxytocin receptor has been implicated in the regulation of reproductive physiology as well as social and emotional behaviors. The neurochemical mechanisms by which oxytocin receptor modulates social and emotional behavior remains elusive, in part because of a lack of sensitive and selective antibodies for cellular localization. To more precisely characterize oxytocin receptor-expressing neurons within the brain, we generated an oxytocin receptor-reporter mouse in which part of the oxytocin receptor gene was replaced with Venus cDNA (a variant of yellow fluorescent protein). Examination of the Venus expression revealed that, in the raphe nuclei, about one-half of tryptophan hydroxylase-immunoreactive neurons were positive for Venus, suggesting a potential role for oxytocin in the modulation of serotonin release. Oxytocin infusion facilitated serotonin release within the median raphe nucleus and reduced anxiety-related behavior. Infusion of a 5-HT(2A/2C) receptor antagonist blocked the anxiolytic effect of oxytocin, suggesting that oxytocin receptor activation in serotonergic neurons mediates the anxiolytic effects of oxytocin. This is the first demonstration that oxytocin may regulate serotonin release and exert anxiolytic effects via direct activation of oxytocin receptor expressed in serotonergic neurons of the raphe nuclei. These results also have important implications for psychiatric disorders such as autism and depression in which both the oxytocin and serotonin systems have been implicated.

Gentle stroking stimuli induce affiliative responsiveness to humans in male rats.

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.

Functional Hierarchy of Uterotonics Required for Successful Parturition in Mice.

Parturition is an essential process in placental mammals for giving birth to offspring. However, the molecular machineries of parturition are not fully understood. We investigated whether oxytocin plays a crucial role in the progress of parturition in cooperation with the prostaglandin F2α (PGF2α) receptor. We first examined alterations in the expression of uterine contraction-associated genes in uteri of oxytocin receptor-deficient mice (Oxtr-/-) during parturition. We found that induction of cyclooxygenase (COX)-2 and connexin 43 expression was impaired in Oxtr-/-, whereas that of PGF2α receptor expression was not. We next generated mice with double knockout of genes for the oxytocin receptor/oxytocin and PGF2α receptor (Oxtr-/-;Ptgfr-/- and Oxt-/-;Ptgfr-/-) and evaluated their parturition with Oxtr-/-, Oxt-/-, Ptgfr-/-, and wild-type mice. In Oxtr-/-;Ptgfr-/- and Oxt-/-;Ptgfr-/-, pregnancy rates were similar to those of other genotypes. However, normal parturition was not observed in Oxtr-/-;Ptgfr-/- or Oxt-/-;Ptgfr-/- because of persistent progesterone from the corpus luteum, as observed in Ptgfr-/-. We administered RU486, a progesterone antagonist, to Ptgfr-/-, Oxtr-/-;Ptgfr-/-, and Oxt-/-;Ptgfr-/- on gestation day 19. These mice were able to deliver a living first pup and the parturition onset was similar to that in Ptgfr-/-. Meanwhile, unlike Ptgfr-/-, ∼75% of Oxtr-/-;Ptgfr-/- and Oxt-/-;Ptgfr-/- administered RU486 remained in labor at 24 hours after the onset of parturition. All of the pups that experienced prolonged labor died. We thus revealed that the oxytocin receptor is an upstream regulator of COX-2 and connexin 43 in the uterus during parturition and that both oxytocin/oxytocin receptor and PGF2α receptor are major components for successful parturition.

Increased fibroblast growth factor-21 in chronic kidney disease is a trade-off between survival benefit and blood pressure dysregulation.

Circulating levels of fibroblast growth factor-21 (FGF21) start increasing in patients with chronic kidney disease (CKD) since early stages during the cause of disease progression. FGF21 is a liver-derived hormone that induces responses to stress through acting on hypothalamus to activate the sympathetic nervous system and the hypothalamus-pituitary-adrenal endocrine axis. However, roles that FGF21 plays in pathophysiology of CKD remains elusive. Here we show in mice that FGF21 is required to survive CKD but responsible for blood pressure dysregulation. When introduced with CKD, Fgf21-/- mice died earlier than wild-type mice. Paradoxically, these Fgf21-/- CKD mice escaped several complications observed in wild-type mice, including augmentation of blood pressure elevating response and activation of the sympathetic nervous system during physical activity and increase in serum noradrenalin and corticosterone levels. Supplementation of FGF21 by administration of an FGF21-expressing adeno-associated virus vector recapitulated these complications in wild-type mice and restored the survival period in Fgf21-/- CKD mice. In CKD patients, high serum FGF21 levels are independently associated with decreased baroreceptor sensitivity. Thus, increased FGF21 in CKD can be viewed as a survival response at the sacrifice of blood pressure homeostasis.

Infusion of oxytocin induces successful delivery in prostanoid FP-receptor-deficient mice.

The dramatic increase of oxytocin (OT) receptor (OTR) in the myometrium as well as circulating progesterone withdrawal has been thought to be the most important factor in the induction and accomplishment of parturition since delivery fails in prostaglandin F2alpha receptor (FP) knockout (FP KO) mice. The expression levels of OTR mRNA/protein were not dramatically increased in the near-term uteri of FP KO mice. However, OT-induced myometrial contractions and the concentration-response curves in FP KO in vitro were almost similar to those in wild-type (WT) mice. OT-infusion (0.3 U/day) enabled FP KO mice to experience successful delivery, and furthermore the duration until the onset was hastened by a higher dose of OT (3 U/day). The plasma progesterone levels of FP KO females were maintained at high levels, but decreased during labor by OT-infusion (3 U/day). These results suggest that OT has potentials to induce strong myometrial contractions in uterus with low expression levels of OTR and luteolysis in ovary, which enabled FP KO females to undergo successful delivery.

Oxytocin-Oxytocin Receptor Systems Facilitate Social Defeat Posture in Male Mice.

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.

Mechanical activity of frog esophagus muscle in response to electrical stimulation of intramural nerves.

Microscopic observation of intramural nerves in the frog esophagus, fixed and stained with OsO(4) and ZnI(2), revealed that nerve cell bodies and bundles connecting the nerve cell bodies formed loose and irregular networks. The nerve cell bodies were mostly lying singly in the nerve bundles, with occasional observations of two closely linked nerve cell bodies. Isolated circular and longitudinal segments of esophageal muscle were spontaneously rhythmically contractile, with a frequency of 2.2-3.0 per min. This was not altered by tetrodotoxin (TTX). In longitudinal muscle segments, transmurally applied electrical stimulation produced contractile responses which were not inhibited by atropine or guanethidine, but were reduced in amplitude by TTX, suggesting a nonadrenergic-noncholinergic (NANC) excitatory innervation in the esophagus muscle. In circular muscle segments, transmural application of brief electrical stimulation evoked two types of mechanical response: a biphasic response consisting of an initial relaxation and a following contraction (type I) and a contraction alone (type II). These mechanical responses were not modulated by either atropine or guanethidine. In the type I response, TTX abolished the relaxation component, suggesting that this was produced by non-adrenergic non-cholinergic (NANC) inhibitory nerve excitation. In about half of the type II responses, the amplitude of the contraction was significantly reduced by TTX, suggesting that a part of the contraction was produced by activation of NANC excitatory nerves. Thus, the esophageal smooth muscle of the frog demonstrates myogenic activity, and is innervated by both excitatory and inhibitory NANC nerves.

Activation of Supraoptic Oxytocin Neurons by Secretin Facilitates Social Recognition.

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.

Activation of hypothalamic oxytocin neurons following tactile stimuli in rats.

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.

The medial amygdala-medullary PrRP-synthesizing neuron pathway mediates neuroendocrine responses to contextual conditioned fear in male rodents.

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.

Endothelin-3 expression in the subfornical organ enhances the sensitivity of Na(x), the brain sodium-level sensor, to suppress salt intake.

Salt homeostasis is essential to survival, but brain mechanisms for salt-intake control have not been fully elucidated. Here, we found that the sensitivity of Na(x) channels to [Na(+)](o) is dose-dependently enhanced by endothelin-3 (ET-3). Na(x) channels began to open when [Na(+)](o) exceeded ~150 mM without ET-3, but opened fully at a physiological [Na(+)](o) (135­145 mM) with 1 nM ET-3. Importantly, ET-3 was expressed in the subfornical organ (SFO) along with Nax, and the level was robustly increased by dehydration. Pharmacological experiments revealed that endothelin receptor B (ET(B)R) signaling is involved in this modulation of Na(x) gating through protein kinase C and ERK1/2 activation. ET(B)R agonists increased the firing rate of GABAergic neurons via lactate in the SFO, and an ET(B)R antagonist attenuated salt aversion during dehydration. These results indicate that ET-3 expression in the SFO is tightly coupled with body-fluid homeostasis through modulation of the [Na(+)](o) sensitivity of Na(x).