Janet A. Amico - US grants

In this laboratory, a new protein which is antigenically similar to oxytocin (OT) and arginine vasotocin (AVT) has been found in human plasma and is termed "the novel OT-like peptide." This novel peptide rises in human plasma in response to administration of estrogen (acute and chronic); rises when endogenous levels of estrogen increase (mid-cycle and pregnancy); and is also found in high concentrations in the plasma of patients with chronic renal failure. This novel peptide has heretofore not been described in humans. Reverse phase HPLC separates this material from OT. Two peaks of immunoreactivity, one eluting before and one after OT, are found when plasma from individuals given estrogen, plasma from pregnant women, and plasma from patients with renal failure are chromatographed. All conditions in which the OT-like peptide have been increased have also been characterized by high levels of the estrogen-stimulated neurophysin (ESN), suggesting that ESN and the OT-like peptide are released together and perhaps synthesized and/or are stored together. The quantity of this material in human plasma is less than 10 pg/ml and the identity of this substance is dependent on isolating large amounts of the material. The quantity of plasma needed to recover 100 ng of a salt-free protein - the minimum required for amino acid analysis using the most sensitive of methods - would be in excess of 10 liters. Therefore, a body fluid (as urine from an individual given estrogen or pregnant women or the used dialysate from chronic renal failure patients) and/or a tissue source of the material are being sought and methods of extraction of the peptide, concentration of body fluid and tissue extracts, HPLC isolation and amino acid analysis form one focus of this grant. Because the peptide bears structural similarities to OT, concentrated extracts of the peptide will be tested for oxytocic activity by performing bio- and radioreceptor assays and the release of the OT-like peptide in response to stimuli known to promote release of oxytocin will be tested. By so doing we will learn whether the OT-like peptide and OT are regulated differentially or in parallel and in the process also learn more about the physiology of oxytocin, a hormone about which very little is known.

Oxytocin(OT) is a nonapeptide that functions as both a hormone and a neuropeptide during certain reproductive processes and behaviors. OT is essential for milk ejection during lactation, enhances uterine contractility at parturition and induces maternal behavior in the rat. Maternal behavior in most mammals is stimulated by the hormonal milieu of late pregnancy. In the rat, rising levels of estrogen (E) superimposed on the late-term decline in progesterone (P), stimulate maternal behavior. The ovarian steroid exposure which optimally stimulate maternal behavior (declining P in the setting of E priming) also enhances levels of oxytocin (OT) mRNA and OT peptide in the paraventricular and supraoptic nuclei (PVN and SON) of the hypothalamus, which are the sites of synthesis of over 95% of the OT formed within the CNS. Progesterone plays a key role in the regulation of both maternal behavior and OT expression by mechanisms which are not well understood. P can affect neuronal transmission by binding to its cognate nuclear receptor (PR), which acts as a hormone- dependent transcription factor, or by binding to the membrane bound GABAA receptor complex. We hypothesize that changes in OT expression in the PVN and SON of the steroid-treated and pregnant rat are regulated by P-mediated plasticity in subunits of the GABAA receptor and/or by alterations in concentrations of neurosteroids. Increased OT expression within magnocellular and/or parvocellular neurons of the PVN may in turn facilitate the onset of maternal behavior. The specific aims of this proposal are as follows:1-administer allopregnanolone, or its agonist, ganaxolone, or inhibitors of allopregnanolone formation to the steroid-treated ovariectomized rat and measure changes in OT mRNA and OT peptide in the PVN and SON of the hypothalamus; 2-administer allopregnanolone or its agonist, ganaxolone, or block formation of allopregnanolone and measure indices of maternal behavior, hypothalamic OT mRNA and OT peptide in rats receiving a steroid treatment known to induce maternal behaviors and; 3-measure by in situ hybridization the relative abundance of OT mRNA and GABAA receptor subunit mRNAs and measure by in vitro receptor autoradiography 3[H]muscimol GABAA receptor binding in the hypothalami of the steroid- treated or allopregnanolone (ganaxolone)- treated ovariectomized rat and the pregnant or lactating rat; 4 - administer an antisense oligonucleotide to OT or an OT antagonist i.c.v. to the steroid-treated ovariectomized rat or the pregnant rat and measure indices of maternal behavior and levels of hypothalamic OT mRNA and OT peptide.

DESCRIPTION (provided by applicant): In the female laboratory rodent, oxytocin (OT) is believed to be anxiolytic and to dampen the response of the hypothalamic-pituitary- adrenal axis to stress. Psychogenic stress or anxiety activates limbic regions of the rat or mouse brain, such as the amygdaloid nuclei and hippocampus. These regions project to the hypothalamic paraventricular nucleus (PVN), a major site of OT production in the brain. The PVN in turn innervates the limbic system. Brain pathways that are activated by psychogenic stressors or anxiety contain OT immunoreactivity (ir) and/or OT receptors (OTR). Preliminary studies in this proposal indicate that female OT deficient mice release more corticosterone following a psychogenic stress, have a greater stress-induced hyperthermic response to novel environment, and display more anxiety-related behavior than wild type mice. The enhanced anxiety in this genotype is accompanied by enhanced expression of Fos immunoreactivity (ir), a marker of neuronal activation, in the medial amygdala of null versus wild type mice. We hypothesize that central OT is necessary for attenuation of stress and anxiety. If OT pathways are absent, then limbic activation (as measured by Fos ir) will be altered and responses to psychogenic stress (e.g., corticosterone release or stress-induced hyperthermia) will be heightened. The specific aims of this proposal are as follows: 1- Measure plasma corticosterone concentrations in OT null and wild type mice before and following either psychogenic or physical stressors and score anxiety-related behavior during tests of anxiety. Peripheral and brain indicators of the stress response and anxiety-related behavior will be measured to ascertain potential genotypic differences in compensatory homeostatic mechanisms. 2- Immunocytochemical localization of the immediate-early gene protein product, Fos, will be used to identify neurons activated after stress or anxiety testing in wild type and OT null mice. Single- and dual-labeling methods will be used to map the distribution and neurochemical phenotypes of activated neurons in the brain with a particular focus on neurons in the limbic system, which are believed to mediate psychogenic stress and anxiety-related behaviors. 3- Administer exogenous OT into the brain of OT null and wild type mice and measure anxiety-related behavior or corticosterone following stressful stimuli; and 4- Measure brain OT receptors by autoradiography pre and post anxiety and stress testing in wild type versus OT null mice.