Function of gastrin-releasing peptide receptors in ocular itch transmission in the mouse trigeminal sensory system.

The prevalence of allergic conjunctivitis in itchy eyes has increased constantly worldwide owing to environmental pollution. Currently, anti-allergic and antihistaminic eye drops are used; however, there are many unknown aspects about the neural circuits that transmit itchy eyes. We focused on the gastrin-releasing peptide (GRP) and GRP receptor (GRPR), which are reportedly involved in itch transmission in the spinal somatosensory system, to determine whether the GRP system is involved in itch neurotransmission of the eyes in the trigeminal sensory system. First, the instillation of itch mediators, such as histamine (His) and non-histaminergic itch mediator chloroquine (CQ), exhibited concentration-dependent high levels of eye scratching behavior, with a significant sex differences observed in the case of His. Histological analysis revealed that His and CQ significantly increased the neural activity of GRPR-expressing neurons in the caudal part of the spinal trigeminal nucleus of the medulla oblongata in GRPR transgenic mice. We administered a GRPR antagonist or bombesin-saporin to ablate GRPR-expressing neurons, followed by His or CQ instillation, and observed a decrease in CQ-induced eye-scratching behavior in the toxin experiments. Intracisternal administration of neuromedin C (NMC), a GRPR agonist, resulted in dose-dependent excessive facial scratching behavior, despite the absence of an itch stimulus on the face. To our knowledge, this is the first study to demonstrate that non-histaminergic itchy eyes were transmitted centrally via GRPR-expressing neurons in the trigeminal sensory system, and that NMC in the medulla oblongata evoked facial itching.

Chronic corticosterone exposure evokes itch hypersensitivity and sexual dysfunction in male rats: Relationship between the two distinct gastrin-releasing peptide systems in the spinal cord.

In today's society, people are subjected to many social stressors, and excessive chronic stress causes functional disruption of the neuroendocrine system and many diseases. Although the exacerbation of atopic dermatitis with symptoms of itching and erectile dysfunction is induced by chronic stress, the details of the mechanisms are unknown. Here, we examined the effects of chronic stress on itch sensation and male sexual function at the behavioral and molecular levels, focusing on two distinct gastrin-releasing peptide (GRP) systems that independently regulate itch transmission, i.e., the somatosensory GRP system, and male sexual function, i.e., the lumbosacral autonomic GRP system, in the spinal cord. In a rat model of chronic stress induced by chronic corticosterone (CORT) administration, we observed increased plasma CORT concentrations, decreased body weight, and increased anxiety-like behavior, similar to that observed in humans. Chronic CORT exposure induced hypersensitivity to itch and increased the Grp mRNA level in the spinal somatosensory system, but there was no change in pain or tactile sensitivity. Antagonists of the somatosensory GRP receptor, an itch-specific mediator, suppressed itch hypersensitivity induced by chronic CORT exposure. In contrast, chronic CORT exposure decreased male sexual behavior, ejaculated semen volume, vesicular gland weight, and plasma testosterone levels. However, there were no effects on the expression of Grp mRNA or protein in the lumbosacral autonomic GRP system, which regulates male sexual function. In summary, chronic stress model rats showed itch hypersensitivity and impaired sexual function in males, and the involvement of the spinal GRP systems was apparent in itch hypersensitivity.

Footedness for scratching itchy eyes in rodents.

The neural bases of itchy eye transmission remain unclear compared with those involved in body itch. Here, we show in rodents that the gastrin-releasing peptide receptor (GRPR) of the trigeminal sensory system is involved in the transmission of itchy eyes. Interestingly, we further demonstrate a difference in scratching behaviour between the left and right hindfeet in rodents; histamine instillation into the conjunctival sac of both eyes revealed right-foot biased laterality in the scratching movements. Unilateral histamine instillation specifically induced neural activation in the ipsilateral sensory pathway, with no significant difference between the activations following left- and right-eye instillations. Thus, the behavioural laterality is presumably due to right-foot preference in rodents. Genetically modified rats with specific depletion of Grpr-expressing neurons in the trigeminal sensory nucleus caudalis of the medulla oblongata exhibited fewer and shorter histamine-induced scratching movements than controls and eliminated the footedness. These results taken together indicate that the Grpr-expressing neurons are required for the transmission of itch sensation from the eyes, but that foot preference is generated centrally. These findings could open up a new field of research on the mechanisms of the laterality in vertebrates and also offer new potential therapeutic approaches to refractory pruritic eye disorders.

Nanobody-based RFP-dependent Cre recombinase for selective anterograde tracing in RFP-expressing transgenic animals.

Transgenic animals expressing fluorescent proteins are widely used to label specific cells and proteins. By using a split Cre recombinase fused with mCherry-binding nanobodies or designed ankyrin repeat proteins, we created Cre recombinase dependent on red fluorescent protein (RFP) (Cre-DOR). Functional binding units for monomeric RFPs are different from those for polymeric RFPs. We confirmed selective target RFP-dependent gene expression in the mouse cerebral cortex using stereotaxic injection of adeno-associated virus vectors. In estrogen receptor-beta (Esr2)-mRFP1 mice and gastrin-releasing peptide receptor (Grpr)-mRFP1 rats, we confirmed that Cre-DOR can be used for selective tracing of the neural projection from RFP-expressing specific neurons. Cellular localization of RFPs affects recombination efficiency of Cre-DOR, and light and chemical-induced nuclear translocation of an RFP-fused protein can modulate Cre-DOR efficiency. Our results provide a method for manipulating gene expression in specific cells expressing RFPs and expand the repertory of nanobody-based genetic tools.

Inhibition of itch by neurokinin 1 receptor (Tacr1) -expressing ON cells in the rostral ventromedial medulla in mice.

The rostral ventromedial medulla (RVM) is important in descending modulation of spinal nociceptive transmission, but it is unclear if the RVM also modulates spinal pruriceptive transmission. RVM ON cells are activated by noxious algesic and pruritic stimuli and are pronociceptive. Many RVM-spinal projection neurons express the neurokinin-1 receptor (Tacr1), and ON-cells are excited by local administration of substance P (SP). We hypothesized that Tacr1-expressing RVM ON cells exert an inhibitory effect on itch opposite to their pronociceptive action. Intramedullary microinjection of SP significantly potentiated RVM ON cells and reduced pruritogen-evoked scratching while producing mild mechanical sensitization. Chemogenetic activation of RVM Tacr1-expressing RVM neurons also reduced acute pruritogen-evoked scratching. Optotagging experiments confirmed RVM Tacr1-expressing neurons to be ON cells. We conclude that Tacr1-expressing ON cells in RVM play a significant role in the modulation of pruriceptive transmission.

Characterization of the expression of gastrin-releasing peptide and its receptor in the trigeminal and spinal somatosensory systems of Japanese macaque monkeys: Insight into humans.

Gastrin-releasing peptide (GRP) and its receptor (GRPR) have been identified as itch mediators in the spinal and trigeminal somatosensory systems in rodents. In primates, there are few reports of GRP/GRPR expression or function in the spinal sensory system and virtually nothing is known in the trigeminal system. The aim of the present study was to characterize GRP and GRPR in the trigeminal and spinal somatosensory system of Japanese macaque monkeys (Macaca fuscata). cDNA encoding GRP was isolated from the macaque dorsal root ganglion (DRG) and exhibited an amino acid sequence that was highly conserved among mammals and especially in primates. Immunohistochemical analysis demonstrated that GRP was expressed mainly in the small-sized trigeminal ganglion and DRG in adult macaque monkeys. Densely stained GRP-immunoreactive (ir) fibers were observed in superficial layers of the spinal trigeminal nucleus caudalis (Sp5C) and the spinal cord. In contrast, GRP-ir fibers were rarely observed in the principal sensory trigeminal nucleus and oral and interpolar divisions of the spinal trigeminal nucleus. cDNA cloning, in situ hybridization, and Western blot revealed substantial expression of GRPR mRNA and GRPR protein in the macaque spinal dorsal horn and Sp5C. Our Western ligand blot and ligand derivative stain for GRPR revealed that GRP directly bound in the macaque Sp5C and spinal dorsal horn as reported in rodents. Finally, GRP-ir fibers were also detected in the human spinal dorsal horn. The spinal and trigeminal itch neural circuits labeled with GRP and GRPR appear to function also in primates.

A spinal microglia population involved in remitting and relapsing neuropathic pain.

Neuropathic pain is often caused by injury and diseases that affect the somatosensory system. Although pain development has been well studied, pain recovery mechanisms remain largely unknown. Here, we found that CD11c-expressing spinal microglia appear after the development of behavioral pain hypersensitivity following nerve injury. Nerve-injured mice with spinal CD11c+ microglial depletion failed to recover spontaneously from this hypersensitivity. CD11c+ microglia expressed insulin-like growth factor-1 (IGF1), and interference with IGF1 signaling recapitulated the impairment in pain recovery. In pain-recovered mice, the depletion of CD11c+ microglia or the interruption of IGF1 signaling resulted in a relapse in pain hypersensitivity. Our findings reveal a mechanism for the remission and recurrence of neuropathic pain, providing potential targets for therapeutic strategies.

Estrogens influence female itch sensitivity via the spinal gastrin-releasing peptide receptor neurons.

There are sex differences in somatosensory sensitivity. Circulating estrogens appear to have a pronociceptive effect that explains why females are reported to be more sensitive to pain than males. Although itch symptoms develop during pregnancy in many women, the underlying mechanism of female-specific pruritus is unknown. Here, we demonstrate that estradiol, but not progesterone, enhances histamine-evoked scratching behavior indicative of itch in female rats. Estradiol increased the expression of the spinal itch mediator, gastrin-releasing peptide (GRP), and increased the histamine-evoked activity of itch-processing neurons that express the GRP receptor (GRPR) in the spinal dorsal horn. The enhancement of itch behavior by estradiol was suppressed by intrathecal administration of a GRPR blocker. In vivo electrophysiological analysis showed that estradiol increased the histamine-evoked firing frequency and prolonged the response of spinal GRP-sensitive neurons in female rats. On the other hand, estradiol did not affect the threshold of noxious thermal pain and decreased touch sensitivity, indicating that estradiol separately affects itch, pain, and touch modalities. Thus, estrogens selectively enhance histamine-evoked itch in females via the spinal GRP/GRPR system. This may explain why itch sensation varies with estrogen levels and provides a basis for treating itch in females by targeting GRPR.

The gastrin-releasing peptide/bombesin system revisited by a reverse-evolutionary study considering Xenopus.

Bombesin is a putative antibacterial peptide isolated from the skin of the frog, Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (Xenopus tropicalis), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand the derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in Xenopus. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of Xenopus. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the 'gut-brain peptide' systems.

Variation of pro-vasopressin processing in parvocellular and magnocellular neurons in the paraventricular nucleus of the hypothalamus: Evidence from the vasopressin-related glycopeptide copeptin.

Arginine vasopressin (AVP) is synthesized in parvocellular- and magnocellular neuroendocrine neurons in the paraventricular nucleus (PVN) of the hypothalamus. Whereas magnocellular AVP neurons project primarily to the posterior pituitary, parvocellular AVP neurons project to the median eminence (ME) and to extrahypothalamic areas. The AVP gene encodes pre-pro-AVP that comprises the signal peptide, AVP, neurophysin (NPII), and a copeptin glycopeptide. In the present study, we used an N-terminal copeptin antiserum to examine copeptin expression in magnocellular and parvocellular neurons in the hypothalamus in the mouse, rat, and macaque monkey. Although magnocellular NPII-expressing neurons exhibited strong N-terminal copeptin immunoreactivity in all three species, a great majority (~90%) of parvocellular neurons that expressed NPII was devoid of copeptin immunoreactivity in the mouse, and in approximately half (~53%) of them in the rat, whereas in monkey hypothalamus, virtually all NPII-immunoreactive parvocellular neurons contained strong copeptin immunoreactivity. Immunoelectron microscopy in the mouse clearly showed copeptin-immunoreactivity co-localized with NPII-immunoreactivity in neurosecretory vesicles in the internal layer of the ME and posterior pituitary, but not in the external layer of the ME. Intracerebroventricular administration of a prohormone convertase inhibitor, hexa-d-arginine amide resulted in a marked reduction of copeptin-immunoreactivity in the NPII-immunoreactive magnocellular PVN neurons in the mouse, suggesting that low protease activity and incomplete processing of pro-AVP could explain the disproportionally low levels of N-terminal copeptin expression in rodent AVP (NPII)-expressing parvocellular neurons. Physiologic and phylogenetic aspects of copeptin expression among neuroendocrine neurons require further exploration.

Oxytocin Influences Male Sexual Activity via Non-synaptic Axonal Release in the Spinal Cord.

Oxytocinergic neurons in the paraventricular nucleus of the hypothalamus that project to extrahypothalamic brain areas and the lumbar spinal cord play an important role in the control of erectile function and male sexual behavior in mammals. The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord is an important component of the neural circuits that control penile reflexes in rats, circuits that are commonly referred to as the "spinal ejaculation generator (SEG)." We have examined the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system in rats. Here, we show that SEG/GRP neurons express oxytocin receptors and are activated by oxytocin during male sexual behavior. Intrathecal injection of oxytocin receptor antagonist not only attenuates ejaculation but also affects pre-ejaculatory behavior during normal sexual activity. Electron microscopy of potassium-stimulated acute slices of the lumbar cord showed that oxytocin-neurophysin-immunoreactivity was detected in large numbers of neurosecretory dense-cored vesicles, many of which are located close to the plasmalemma of axonal varicosities in which no electron-lucent microvesicles or synaptic membrane thickenings were visible. These results suggested that, in rats, release of oxytocin in the lumbar spinal cord is not limited to conventional synapses but occurs by exocytosis of the dense-cored vesicles from axonal varicosities and acts by diffusion-a localized volume transmission-to reach oxytocin receptors on GRP neurons and facilitate male sexual function.

Detection and Characterization of Estrogen Receptor Beta Expression in the Brain with Newly Developed Transgenic Mice.

Two types of nuclear estrogen receptors, ERα and ERß, have been shown to be differentially involved in the regulation of various types of behaviors. Due to a lack of tools for identifying ERß expression, detailed anatomical distribution and neurochemical characteristics of ERß expressing cells and cellular co-expression with ERα remain unclear. We have generated transgenic mice ERß-RFPtg, in which RFP was inserted downstream of ERß BAC promotor. We verified RFP signals as ERß by confirming: (1) high ERß mRNA levels in RFP-expressing cells collected by fluorescence-activated cell sorting; and (2) co-localization of ERß mRNA and RFP proteins in the paraventricular nucleus (PVN). Strong ERß-RFP signals were found in the PVN, medial preoptic area (MPOA), bed nucleus of the stria terminalis, medial amygdala (MeA), and dorsal raphe nucleus (DRN). In the MPOA and MeA, three types of cell populations were identified; those expressing both ERα and ERß, and those expressing exclusively either ERα or ERß. The majority of PVN and DRN cells expressed only ERß-RFP. Further, ERß-RFP positive cells co-expressed oxytocin in the PVN, and tryptophan hydroxylase 2 and progesterone receptors in the DRN. In the MeA, some ERß-RFP positive cells co-expressed oxytocin receptors. These findings collectively suggest that ERß-RFPtg mice can be a powerful tool for future studies on ERß function in the estrogenic regulation of social behaviors.

The Amphibious Mudskipper: A Unique Model Bridging the Gap of Central Actions of Osmoregulatory Hormones Between Terrestrial and Aquatic Vertebrates.

Body fluid regulation, or osmoregulation, continues to be a major topic in comparative physiology, and teleost fishes have been the subject of intensive research. Great progress has been made in understanding the osmoregulatory mechanisms including drinking behavior in teleosts and mammals. Mudskipper gobies can bridge the gap from aquatic to terrestrial habitats by their amphibious behavior, but the studies are yet emerging. In this review, we introduce this unique teleost as a model to study osmoregulatory behaviors, particularly amphibious behaviors regulated by the central action of hormones. Regarding drinking behavior of mammals, a thirst sensation is aroused by angiotensin II (Ang II) through direct actions on the forebrain circumventricular structures, which predominantly motivates them to search for water and take it into the mouth for drinking. By contrast, aquatic teleosts can drink water that is constantly present in their mouth only by reflex swallowing, and Ang II induces swallowing by acting on the hindbrain circumventricular organ without inducing thirst. In mudskippers, however, through the loss of buccal water by swallowing, which appears to induce buccal drying on land, Ang II motivates these fishes to move to water for drinking. Thus, mudskippers revealed a unique thirst regulation by sensory detection in the buccal cavity. In addition, the neurohypophysial hormones, isotocin (IT) and vasotocin (VT), promote migration to water via IT receptors in mudskippers. VT is also dipsogenic and the neurons in the forebrain may mediate their thirst. VT regulates social behaviors as well as osmoregulation. The VT-induced migration appears to be a submissive response of subordinate mudskippers to escape from competitive and dehydrating land. Together with implications of VT in aggression, mudskippers may bridge the multiple functions of neurohypophysial hormones. Interestingly, cortisol, an important hormone for seawater adaptation and stress response in teleosts, also stimulates the migration toward water, mediated possibly via the mineralocorticoid receptor. The corticosteroid system that is responsive to external stressors can accelerate emergence of migration to alternative habitats. In this review, we suggest this unique teleost as an important model to deepen insights into the behavioral roles of these hormones in relation to osmoregulation.

Effects of Sex Steroids on the Spinal Gastrin-Releasing Peptide System Controlling Male Sexual Function in Rats.

The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord controls male sexual function in rats. In contrast, in female rats, GRP neurons could scarcely be detected around puberty when circulating ovarian steroid hormones such as estradiol and progesterone levels are increasing. However, little information is available on feminizing or demasculinizing effects of ovarian steroids on the central nervous system in female puberty and adulthood. In this study, to visualize the spinal GRP neurons in vivo, we generated a GRP-promoter-Venus transgenic (Tg) rat line and studied the effects of the sex steroid hormones on GRP expression in the rat lumbar cord by examining the Venus fluorescence. In these Tg rats, the sexually dimorphic spinal GRP neurons controlling male sexual function were clearly labeled with Venus fluorescence. As expected, Venus fluorescence in the male lumbar cord was markedly decreased after castration and restored by chronic androgen replacement. Furthermore, androgen-induced Venus expression in the spinal cord of adult Tg males was significantly attenuated by chronic treatment with progesterone but not with estradiol. A luciferase assay using a human GRP-promoter construct showed that androgens enhance the spinal GRP system, and more strikingly, that progesterone acts to inhibit the GRP system via an androgen receptor-mediated mechanism. These results demonstrate that circulating androgens may play an important role in the spinal GRP system controlling male sexual function not only in rats but also in humans and that progesterone could be an important feminizing factor in the spinal GRP system in females during pubertal development.

Decrease in neuronal spine density in the postpartum period in the amygdala and bed nucleus of the stria terminalis in rat.

In pregnancy and the postpartum period, many women have emotional instability and some suffer from depression. The ovarian steroid hormone milieu is markedly changed during these periods, and this hormonal change may be an important cause of peripartum emotional instability. The amygdala is a central region of emotion, and the bed nucleus of the stria terminalis (BNST), which is considered to be the extended amygdala, is also involved in the emotional response. The amygdala and BNST are well characterized as target brain regions for ovarian steroid hormones, and this suggests that the functional response of neurons in these regions to hormonal fluctuation is affected in the peripartum period. In this study, we investigated the neuronal morphology in the central (CeA) and basolateral (BLA) nucleus of the amygdala and BNST on gestational days 15 (G15) (mid-gestation) and 20 (G20) (late gestation) and 4days after delivery (P4) (early postpartum) in rat. Golgi staining showed that the dendritic spine density, and particularly the number of mature mushroom-type spines, in the CeA, BLA and BNST was significantly decreased at P4, compared with G15 and G20 and with virgin females in the estrous phase in the normal estrous cycle (Est). Interestingly, the presence of pups after delivery influenced the spine density in the BNST. The density was significantly decreased with pup presence compared with pup absence at P4, and compared with G15, G20 and Est. These results provide fundamental insights into the neuronal basis underlying emotional instability during pregnancy and postpartum.

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.

Identification of the sexually dimorphic gastrin-releasing peptide system in the lumbosacral spinal cord that controls male reproductive function in the mouse and Asian house musk shrew (Suncus murinus).

Several regions of the brain and spinal cord control male reproductive function. We previously demonstrated that the gastrin-releasing peptide (GRP) system, located in the lumbosacral spinal cord of rats, controls spinal centers to promote penile reflexes during male copulatory behavior. However, little information exists on the male-specific spinal GRP system in animals other than rats. The objective of this study was to examine the functional generality of the spinal GRP system in mammals using the Asian house musk shrew (Suncus murinus; suncus named as the laboratory strain), a specialized placental mammal model. Mice are also used for a representative model of small laboratory animals. We first isolated complementary DNA encoding GRP in suncus. Phylogenetic analysis revealed that suncus preproGRP was clustered to an independent branch. Reverse transcription-PCR showed that GRP and its receptor mRNAs were both expressed in the lumbar spinal cord of suncus and mice. Immunohistochemistry for GRP demonstrated that the sexually dimorphic GRP system and male-specific expression/distribution patterns of GRP in the lumbosacral spinal cord in suncus are similar to those of mice. In suncus, we further found that most GRP-expressing neurons in males also express androgen receptors, suggesting that this male-dominant system in suncus is also androgen-dependent. Taken together, these results indicate that the sexually dimorphic spinal GRP system exists not only in mice but also in suncus, suggesting that this system is a conserved property in mammals. J. Comp. Neurol. 525:1586-1598, 2017. © 2016 Wiley Periodicals, Inc.

Expression of Sex Steroid Hormone Receptors in Vagal Motor Neurons Innervating the Trachea and Esophagus in Mouse.

The medullary vagal motor nuclei, the nucleus ambiguus (NA) and dorsal motor nucleus of the vagus (DMV), innervate the respiratory and gastrointestinal tracts. We conducted immunohistochemical analysis of expression of the androgen receptor (AR) and estrogen receptor α (ERα), in relation to innervation of the trachea and esophagus via vagal motor nuclei in mice. AR and ERα were expressed in the rostral NA and in part of the DMV. Tracing experiments using cholera toxin B subunit demonstrated that neurons of vagal motor nuclei that innervate the trachea and esophagus express AR and ERα. There was no difference in expression of sex steroid hormone receptors between trachea- and esophagus-innervating neurons. These results suggest that sex steroid hormones may act on vagal motor nuclei via their receptors, thereby regulating functions of the trachea and esophagus.

Postnatal development of the gastrin-releasing peptide system in the lumbosacral spinal cord controlling male reproductive function in rats.

A sexually dimorphic spinal gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord, which projects to the lower spinal centers, controls erection and ejaculation in rats. However, little is known about the postnatal development of this system. In this study, we therefore examined the postnatal development of the male-dominant spinal GRP system and its sexual differentiation in rats using immunohistochemistry. Our results show that male-dominant expression of GRP is prominent from the onset of puberty and that sexually dimorphism persists into adulthood. These results suggest that androgen surge during male puberty plays an important role in the development and maintenance of the male-specific GRP function in the rat spinal cord.

Perinatal testosterone exposure is critical for the development of the male-specific sexually dimorphic gastrin-releasing peptide system in the lumbosacral spinal cord that mediates erection and ejaculation.

BACKGROUND: In rats, a sexually dimorphic spinal gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord projects to spinal centers that control erection and ejaculation. This system controls the sexual function of adult males in an androgen-dependent manner. In the present study, we assessed the influence of androgen exposure on the spinal GRP system during a critical period of the development of sexual dimorphism. METHODS: Immunohistochemistry was used to determine if the development of the spinal GRP system is regulated by the perinatal androgen surge. We first analyzed the responses of neonates administered with anti-androgen flutamide. To remove endogenous androgens, rats were castrated at birth. Further, neonatal females were administered androgens during a critical period to evaluate the development of the male-specific spinal GRP system. RESULTS: Treatment of neonates with flutamide on postnatal days 0 and 1 attenuated the spinal GRP system during adulthood. Castrating male rats at birth resulted in a decrease in the number of GRP neurons and the intensity of neuronal GRP in the spinal cord during adulthood despite testosterone supplementation during puberty. This effect was prevented if the rats were treated with testosterone propionate immediately after castration. Moreover, treating female rats with androgens on the day of birth and the next day, masculinized the spinal GRP system during adulthood, which resembled the masculinized phenotype of adult males and induced a hypermasculine appearance. CONCLUSIONS: The perinatal androgen surge plays a key role in masculinization of the spinal GRP system that controls male sexual behavior. Further, the present study provides potentially new approaches to treat sexual disorders of males.