Ryoichi Teruyama, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): My research interest has been focused on the physiological adaptation of animals to their different reproductive states. The oxytocin (OT) neuron in the supraoptic nucleus (SON) of the hypothalamus is one of the best models to study such adaptation, because the importance of OT during parturition and lactation are well documented. The reports of neuronal activity and morphological responses of OT neurons to lactation are abundant as well. However, the mechanisms underlying the physiological adaptation of OT neurons to lactation are only sparsely discussed. OT neurons in the supraoptic nucleus are known to undergo a remarkable change in neuronal activity in response to the increased hormonal demand that occurs during parturition and lactation. OT neurons display a short, high frequency burst proceeding each milk ejection (Poulain & Wakerley, 1982). The bursting activity is synchronized among all OT neurons (Belin et al., 1984) and results in a bolus release of OT into the blood stream, necessary for uterine contraction or contraction of myoepithelial cells in the mammary glands. This pulsatility is believed to maximize the biological effects of OT and allows the neurons to recover from secretory fatigue (Bicknell, 1988). Because of their significance in controlling hormone secretion, mechanisms involve in the regulation of firing rate and pattern in OT neurons are the subject of great interest. The pattern of electrical activity of neurons generally results from the interaction of intrinsic ionic mechanisms and synaptic activity. The intrinsic membrane properties of OT neuron, the spike after-potentials (after hyperpolarizing potentials (AHP) and depolarization after-potential (DAP)), appear to change in favor of the short bursting activity of OT neuron necessary during parturition and lactation. The ionic conductances mediating these properties are, at least partly, involving in the regulation of the firing rate and pattern that ultimately control the release of hormone. Therefore, this application is to elucidate the mechanisms responsible for the changes in physiological properties of OT neurons in response to lactation. The specific aims of the application are to determine 1) what specific component of spike after-potentials (AHPs and DAP) is altered during lactation; 2) whether the responsive component is responsive to changes in the gonadal steroids. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The neurohypophysial hormones vasopressin (VP) and oxytocin (OT) are synthesized in the magnocellular neurons (MNCs) located within the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus, and released from the neurohypophysis into the general circulation in response to physiological demands. The secretion of VP increases in response to hyperosmolality, hypovolemia, and hypotension, and produces antidiuretic and pressor effects (Sladek 2000). In addition to the well known effects of OT during parturition and lactation, plasma OT increases in response to hypernatremia (Huang et al., 1995) and induces natriuresis (Conrad et al., 1986; Huang et al., 1994). The non-voltage-dependent, amiloride-sensitive Epithelial Na+ channels (ENaCs) are present in kidney and are known to contribute to Na+ and water homeostasis (Benos et al., 1995). In humans, most of the known genetic causes of hypertension are due to defects in ENaC itself or its regulation, which results in abnormal increases in renal Na+ reabsorption (Dahlberg et al., 2007; Lifton 1996; Mune et al., 1995; Shimkets et al., 1994; Zhou et al., 2007). Interestingly, both messengers and proteins for all three ENaC subunits (1, 2, and 3) have been demonstrated in the cardiovascular regulatory centers of the rat brain including the MNCs in the SON and PVN (Amin et al., 2005). Intracerebroventricular injections of the ENaC blocker, the amiloride analogue benzamil, significantly attenuated the hypertension in animal models with salt-dependent forms of hypertension (Gomez-Sanchez and Gomez-Sanchez 1995; Nishimura et al., 1998). In addition, a known target for altered ENaC expression, the mineralocorticoid receptor (MR), is present in MNCs (Amin et al., 2005). These findings suggest central ENaC inhibition may be a potential new target in the treatment of cardiovascular disease (Teiwes and Toto 2007). Despite these findings, the functional significance of ENaCs and their regulation by MRs in MNCs is completely unknown. Therefore, the overall objective of this research project is to characterize the functional significance of ENaCs in MNCs. We hypothesize that ENaCs affect the firing patterns of VP and OT neurons that ultimately affect the secretion of these hormones, and abnormal expression/regulation of ENaCs in these neurons contributes, at least partly, to abnormal secretion of VP and/or OT in salt-sensitive individuals. To address this hypothesis, we will employ whole-cell patch clamp technique combined with single-cell RT-PCR and immunocytochemisry to determine: 1) the presence and electrophysiological characteristics of ENaC-mediated current in VP and OT neurons; 2) whether abnormal expression/regulation of ENaCs in VP and OT neurons is observed in an animal model of the salt-sensitive rat. PUBLIC HEALTH RELEVANCE Epithelial sodium channels which present in kidney and which play an important role in development of hypertension in human, have been also found in the cardiovascular regulatory centers of the brain vasopressin (VP) and oxytocin (OT) neurons in the hypothalamus. While the brain ENaC may be a potential new target in the treatment of cardiovascular disease, the functional significance of ENaCs in VP and OT neurons is unknown. The research in this proposal will elucidate this critical mechanism, and will increase our ability to manage hypertension. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Salt-sensitive individuals have blood pressure that is unusually sensitive to salt intake (1). Salt-sensitivity increases the risk of death whether or nota person has high blood pressure. Furthermore, salt-sensitive persons are likely to develop high blood pressure as they age (2). Because salt sensitivity is common in the U.S., it is of significan public health concern. Current interventional approaches counter only the peripheral effects of salt sensitive hypertension, and the results are often unsatisfactory. Therefore, additional therapies that treat the cause of the disorder are needed. Although the mechanism of salt sensitivity is not well understood, a growing body of evidence suggests that it is caused by, at least partly, an abnormal regulation of the epithelial Na+ channels (ENaCs) in the brain. Both messengers and proteins for all three ENaC subunits were demonstrated in the rat brain including in vasopressin (VP) and oxytocin (OT) synthesizing magnocellular cells (MNCs) in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei (3). In addition, a known target for altered ENaC expression, the mineralocorticoid receptor (MR), is present in MNCs (3). VP and OT are released from the neurohypophysis into the general circulation. The secretion of VP increases in response to hyperosmolality, hypovolemia, and hypotension, and produces antidiuretic and pressor effects (4). Plasma OT increases in response to hypernatremia (5) and induces natriuresis (6, 7). Because, intracerebroventricular infusion of the ENaC blockers significantly attenuated the hypertension in animal models with salt-sensitive hypertension (8, 9), these findings strongly suggest that ENaC in MNCs play a significant role in the development of salt-sensitive hypertension. However, the role of ENaCs and their regulation in the brain is not well understood. Therefore, the overall objective of this proposed research is to characterize the functional significance of ENaCs in MNCs. Our recent study demonstrated that ENaC is a Na+-leak current modulating membrane potential and affecting the frequency action potentials evoked in MNCs (18). This implies that modulation of ENaC activity is a powerful means to modulate hormone secretion according to physiological demands. Based on results from my preliminary study, I hypothesize that dietary Na+ intake affects ENaCs activity that alters the patterns of action potentials in VP and OT MNCs which ultimately affect the secretion of these hormones. Therefore, abnormal regulation of ENaCs in these neurons contributes to the development of salt-sensitive hypertension. To address this hypothesis, we will employ the whole-cell patch clamp technique combined with immunocytochemistry, semi-quantitative RT-PCR and immunoblotting to determine: 1) how dietary salt intake affects both ENaC activity and the neuronal activity in MNCs; 2) the regulatory roles of the mineralocorticoid aldosterone and VP on ENaC expression in MNCs; and 3) the activation mechanisms of ENaCs in MNCs. Results from this proposed project will provide critical information concerning central ENaC inhibition as a potential new target in the treatment of cardiovascular disease (10).

Project Summary / Abstract The purpose of this research is to characterize the autoregulatory mechanism of the hypothalamic oxytocin (OT) system on the systemic release of OT during reproductive states. OT is synthesized by magnocellular cells (MNCs) in the supraoptic (SON) and paraventricular nuclei. OT is released into the general circulation to induce uterine contraction during parturition and contraction of mammary glands during milk ejection. The release of OT is, therefore, essential for birth and normal growth of offspring. The systemic release of OT is largely regulated by the somato-dendritic release of OT that is elicited by physiological stimuli, such as parturition and suckling. The somato-dendritic release OT on OT MNCs is thought to be mediated by the OT receptor (OTR) on OT MNCs themselves via an autocrine-paracrine mechanism. Despite this commonly accepted notion, our preliminary study using OTR reporter mice found no OTR in OT MNCs in the SON; however, OTR was found in non-OT cells in the perinuclear zone (PNZ), the area immediately dorsal to the SON. Moreover, these OTR cells have processes projecting into the SON. Based upon reports in the literature and our preliminary results, we hypothesize that: 1) OTR cells in the PNZ are GABAergic interneurons for OT MNCs in the SON; and 2) GABA inputs from OTR cells become stimulatory due to increase in intracellular chloride concentration in OT MNCs in lactating females. We will use an integrative multidisciplinary approach that includes electrophysiology combined with chemogenetic manipulation of neural activity, neural tracing, immunocytochemistry on transgenic mouse models to explicitly test our hypotheses. The findings from these studies will provide new information concerning the regulation of OT release and the neural adaptations that are necessary for proper parturition and lactation.

Project Summary / Abstract The neurohypophysial hormone, oxytocin, is known for its critical role in female reproductive physiology, such as uterine contraction during labor and milk ejection while nursing. Oxytocin is also released in the brain and modulates many aspects of social behaviors, including social recognition, maternal behavior and pair bonding. Oxytocin influences social behaviors by binding to the oxytocin receptor (OXTR) located in various parts of the brain. In recent years, the oxytocin system in the brain has received tremendous attention as a potential pharmacological target for the treatment of many psychiatric disorders, such as anxiety, autism spectrum disorders, and postpartum depression. Despite the importance, the cellular characterization, connectivity, and regulation of OXTR expressing neurons in the brain is still largely unknown. We recently discovered a group of estrogen-dependent OXTR neurons that is exclusively present in the anteroventral periventricular nucleus (AVPV) in females, but not in males. The overall long-term objective of our project is to elucidate the behavioral significance and regulatory mechanisms of OXTR neurons in the AVPV. Because the AVPV is known to regulate parental behavior in a sex-specific manner, we hypothesize that oxytocin exerts parental behavior via OXTR neurons in the AVPV. To address this hypothesis, we will employ a "Designer Receptors Exclusively Activate by Designer Drug" (DREADD)-based approach to specifically manipulate activity of OXTR neurons in the AVPV in vivo and in vitro. DREADDs are mutated G-protein coupled receptors that are exclusively activated by the pharmacologically inert ligand clozapine-N-oxide (CNO) at nanomolar potency. Both the stimulatory and inhibitory DREADD will be introduced specifically to OXTR neurons using Cre-recombinase-dependent viral vectors. Neural activity of the DREADD-expressing OXTR neurons will be manipulated by CNO. In Aim 1, the effect of inhibition/activation of OXTR neurons in the AVPV on maternal behavior will be examined. In Aim 2, anatomical and functional connectivity of OXTR neurons in the AVPV will be examined using the Channelrhodopsin-assisted circuit mapping (CRACM) technique combined with electrophysiology and Ca++ imaging. The proposed studies will elucidate the sex-specific oxytocin neural circuitry system that regulates sex- specific social behaviors. The findings from this project will provide useful insight into sex-specific pharmacological interventions that may likely treat sex typical psychiatric disorders, such as postpartum depression (PPD).