Kazumi Narita

Variation in the Expression of Orexin and Orexin Receptors in the Rat Hypothalamus During the Estrous Cycle, Pregnancy, Parturition, and Lactation

The widespread distribution of mRNA encoding orexin-1 (OX1R) and -2 receptors (OX2R) in the central nervous system suggests that orexin may be involved in multiple functional pathways. Central administration of orexin stimulates feeding and also affects ovarian steroid-dependent luteinizing hormone secretion, suggesting involvement of orexin in the regulation of reproductive function. To investigate a possible role for orexin in reproductive function, we examined variations in prepro-OX, OX1R, and OX2R mRNA levels in the female rat hypothalamus during the estrous cycle, pregnancy, parturition, and lactation using competitive reverse transcription-polymerase chain reaction. During the estrous cycle, only OX1R mRNA expression during late proestrus was significantly higher than that at metestrus. The prepro-OX and OX1R mRNA levels on d 1 of lactation were significantly higher than that during late pregnancy and lactation. Immunohistochemistry revealed the presence of orexin-A immunoreactive cells and the OX1R subtype in the lateral hypothalamic area as well as the magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nuclei, respectively, in pregnant and lactating rats. These results suggest a role for orexin in reproduction that may be involved in regulating physiological function in early lactation through important binding sites in hypothalamic PVN and SON.

Excitation of oxytocin cells in the hypothalamic supraoptic nucleus by electrical stimulation of the dorsal penile nerve and tactile stimulation of the penis in the rat.

In urethane-anaesthetized male rats, electrical stimulation of the dorsal penile nerve (DPN) excited 29 of 48 (60%) oxytocin cells in the contralateral supraoptic nucleus, whereas only 5 of 28 (18%) vasopressin cells were excited by the stimulation. The stimulus applied to the ipsilateral DPN to the recorded neurone also excited a similar proportion of oxytocin cells (25 of 43; 58%). Tactile stimulation of the glans penis excited 7 of 12 (58%) oxytocin cells, whereas the same stimulation excited only 3 of 16 (19%) vasopressin cells. The results suggest that sensory information arising from the penis preferentially excites oxytocin cells in the supraoptic nucleus.

Leptin affects the electrical activity of neurones in the hypothalamic supraoptic nucleus

The present experiments were undertaken to examine whether leptin affects the electrical activity of neurones in the supraoptic nucleus (SON) by using brain slice preparation of male Wistar and obese Zucker rats. Bath application of leptin (10(-8) - 10(-12) M) induced mainly inhibitory response in SON neurones of Wistar rats, although a minority showed excitation. These effects were observed in both continuously and phasically active cells. The inhibitory effect of leptin still persisted in low Ca(2+), high Mg(2+) medium. Bath application of tolbutamide, which is known to inhibit ATP-sensitive potassium channel activity, did not reverse the inhibitory effect of leptin on SON neurones. The effect of bath application of leptin was also tested in SON neurones of obese Zucker rats. Although leptin still affected the electrical activity of some SON neurones of Zucker rats, the proportion of unaffected neurones was significantly higher than in Wistar rats. The results suggest that leptin may inhibit the secretion of both oxytocin and vasopressin by inhibiting the electrical activity of neurones in the SON via direct action. This inhibitory effect of leptin may be exerted through mechanisms other than activation of ATP-sensitive potassium channels.

Orexin‐A Immunoreactivity and Prepro‐Orexin mRNA Expression in Hyperphagic Rats Induced By Hypothalamic Lesions and Lactation

Orexins are endogenous neuropeptides that potently facilitate appetite and food consumption. In the present study, we examined orexin immunoreactivity and prepro‐orexin mRNA expression in the lateral hypothalamus by immunohistochemistry and competitive reverse transcription‐polymerase chain reaction (RT‐PCR) methods in different models of hyperphagia in rats. Hyperphagia was induced by lesions of either the ventromedial hypothalamus (VMHL) or the paraventricular nucleus (PVNL), and we also compared lactating rats to nonlactating controls. Both VMHL and PVNL increased food intake and body weight compared to shams. On day 7 post lesion, serum leptin and insulin concentrations exhibited 3.2‐ and 2.8‐fold increases in VMHL rats, and nonsignificant 1.8‐ and 1.8‐fold increases in PVNL rats; there were significant decreases (48% and 33%) in lactating rats on day 12 postpartum compared to controls, respectively. Serum glucose concentrations were not significantly changed compared to controls in these rats. Quantification by image analysis suggests that VMHL significantly decreased the number and mean staining intensity of orexin‐A immunoreactive neurones compared to those in the sham‐lesioned group; while PVNL did not change orexin‐A immunoreactivity. Competitive RT‐PCR analysis showed that VMHL significantly decreased the prepro‐orexin mRNA expression compared to those in the sham‐lesioned group, and PVNL did not change it. Lactating rats on days 11–12 of lactation had significantly greater number and mean staining intensity of orexin‐A immunoreactive neurones, prepro‐orexin mRNA expression food intake and body weight than nonlactating postpartum rats. Thus, changes in orexin‐A immunoreactivity and prepro‐orexin mRNA expression were not consistent between the hyperphagia models. These results suggest that the hyperphagia from VMHL or PVNL and lactating rats differ in their involvement of orexin‐A, and the change in circulating leptin and insulin concentrations may be involved in the change of orexin‐A immunoreactivity in these rats.

Rat Uterine Oxytocin Receptor and Estrogen Receptor α and β mRNA Levels are Regulated by Estrogen Through Multiple Estrogen Receptors

Abstract Estrogen action is mediated through several types of receptors (ERs), such as ERα, ERβ and putative membrane ERs. Oxytocin receptor (OTR) and ER expression levels in the rat uterus are regulated by estrogen; however, which types of ERs are involved has not been elucidated. This study examined OTR, ERα and ERβ levels in ovariectomized rats treated with 17β-estradiol (E2), an ERα agonist (PPT), an ERβ agonist (DPN) or estren (Es). E2 and PPT increased OTR mRNA levels and decreased ERα and ERβ mRNA levels 3 and 6 h posttreatment. DPN decreased ERα and ERβ mRNA levels at 3 and 6 h, while OTR mRNA levels increased at 3 h and decreased at 6 h. OTR mRNA levels increased 3 h after the Es treatment and then declined until 6 h. ERα and ERβ mRNA levels decreased by 3 h and remained low until 6 h posttreatment with Es. The ER antagonist ICI182,780 (ICI) suppressed the increases in OTR mRNA levels induced 3 h after the Es treatment. However, ICI and tamoxifen (Tam) had no significant effect on ERα and ERβ mRNA levels in the Es-treated or vehicle-treated group. In intact rats, proestrus-associated increases in OTR mRNA levels were antagonized by both ICI and Tam. However, decreases in ERα and ERβ mRNA levels were not antagonized by Tam and ICI, respectively. Therefore, uterine OTR gene expression is upregulated by estrogen through the classical nuclear (or non-nuclear) ERs, ERα and ERβ, while the levels of these ERs are downregulated by estrogen through multiple pathways including Es-sensitive nonclassical ERs.

Prolactin releasing peptides modulate background firing rate and milk-ejection related burst of oxytocin cells in the supraoptic nucleus

The hypothalamic dorsomedial nucleus is suggested to be a final relay site for the afferent pathway of milk-ejection reflex. Existence of prolactin releasing peptide-immunoreactive cells in the dorsomedial nucleus and synaptic contact of prolactin releasing peptide-immunoreactive terminals with oxytocin cells was reported. Experiments were done to test the effect of prolactin releasing peptide on the electrical activity of oxytocin cells in the supraoptic nucleus. In rat brain slice preparations, oxytocin cells were unresponsive to the peptide. In lactating rats, although lateral ventricular injection of prolactin releasing peptide (20 nmol) was ineffective, a hundred nanomoles of the peptide increased basal activity and amplitude of milk-ejection related burst firing of oxytocin cells. Cells responded to lateral ventricular injection of peptides were unresponsive to direct application of peptides by pressure ejection from the recording electrode. These results suggest that prolactin releasing peptide may modulate electrical activity of oxytocin cells not through its direct action on oxytocin cells but through its action on area other than supraoptic nucleus.

Dietary Deficiency of Essential Amino Acids Rapidly Induces Cessation of the Rat Estrous Cycle

Reproductive functions are regulated by the sophisticated coordination between the neuronal and endocrine systems and are sustained by a proper nutritional environment. Female reproductive function is vulnerable to effects from dietary restrictions, suggesting a transient adaptation that prioritizes individual survival over reproduction until a possible future opportunity for satiation. This adaptation could also partially explain the existence of amenorrhea in women with anorexia nervosa. Because amino acid nutritional conditions other than caloric restriction uniquely alters amino acid metabolism and affect the hormonal levels of organisms, we hypothesized that the supply of essential amino acids in the diet plays a pivotal role in the maintenance of the female reproductive system. To test this hypothesis, we examined ovulatory cyclicity in female rats under diets that were deficient in threonine, lysine, tryptophan, methionine or valine. Ovulatory cyclicity was monitored by daily cytological evaluations of vaginal smears. After continuous feeding of the deficient diet, a persistent diestrus or anovulatory state was induced most quickly by the valine-deficient diet and most slowly by the lysine-deficient diet. A decline in the systemic insulin-like growth factor 1 level was associated with a dietary amino acid deficiency. Furthermore, a paired group of rats that were fed an isocaloric diet with balanced amino acids maintained normal estrous cyclicity. These disturbances of the estrous cycle by amino acid deficiency were quickly reversed by the consumption of a normal diet. The continuous anovulatory state in this study is not attributable to a decrease in caloric intake but to an imbalance in the dietary amino acid composition. With a shortage of well-balanced amino acid sources, reproduction becomes risky for both the mother and the fetus. It could be viewed as an adaptation to the diet, diverting resources away from reproduction and reallocating them to survival until well-balanced amino acid sources are found.

The ventromedial hypothalamus oxytocin induces locomotor behavior regulated by estrogen.

Our previous studies demonstrated that excitation of neurons in the rat ventromedial hypothalamus (VMH) induced locomotor activity. An oxytocin receptor (Oxtr) exists in the VMH and plays a role in regulating sexual behavior. However, the role of Oxtr in the VMH in locomotor activity is not clear. In this study we examined the roles of oxytocin in the VMH in running behavior, and also investigated the involvement of estrogen in this behavioral change. Microinjection of oxytocin into the VMH induced a dose-dependent increase in the running behavior in male rats. The oxytocin-induced running activity was inhibited by simultaneous injection of Oxtr-antagonist, (d(CH2)5(1), Try(Me)(2), Orn(8))-oxytocin. Oxytocin injection also induced running behavior in ovariectomized (OVX) female rats. Pretreatment of the OVX rats with estrogen augmented the oxytocin-induced running activity twofold, and increased the Oxtr mRNA in the VMH threefold. During the estrus cycle locomotor activity spontaneously increased in the dark period of proestrus. The Oxtr mRNA was up-regulated in the proestrus afternoon. Blockade of oxytocin neurotransmission by its antagonist before the onset of the dark period of proestrus decreased the following nocturnal locomotor activity. These findings demonstrate that Oxtr in the VMH is involved in the induction of running behavior and that estrogen facilitates this effect by means of Oxtr up-regulation, suggesting the involvement of oxytocin in the locomotor activity of proestrus female rats.