Victor Domingo Ramirez - US grants

This proposal aims to clarify and understand the physiological nature of the effects of hormones on brain dopaminergic neurotransmission. Progesterone (P), prolactin (PRL) and photoperiod can markedly modify the activity of the rat nigrostriatal dopamine (NS-DA) system. P exerts a biphasic action on this system, facilitation followed by inhibition; PRL stimulates the release of DA from male but not from female CS and is capable to evoke grooming and yawning behavior when infused locally within the CS of male rats; and photoperiodic cues appear to exert robust bidirectional influences on the NS-DA system since in vitro CS dopamine activity increases in the morning and decreases in the afternoon in diestrus 1 and 2 rats and these diurnal variations are blocked during the transition from proestrus-estrus. To clarify and better understand the physiological nature of the effect of photoperiod, hormones and their interactions upon the function of the NS-DA system we have chosen three strategies. Strategy I - Push-Pull Cannula (PPC) on-line with High Performance Liquid Chromatography with Electrochemical Detection (HPLC-EC) to estimate the simultaneous in vivo output of a series of neuroactive substances during a 24h period from discrete brain areas of conscious, freely behaving animals. Strategy II - in vitro superfusion to measure the temporal and magnitude of the neuroactive substances released from isolated brain fragments under rigorously controllable experimental conditions. Strategy III - biochemical determinations of cAMP levels and adenylate cyclase activity in specific brain areas. Using these three strategies we will delineate the physiological nature and gain an insight in mechanisms by which P, PRL and photoperiod affect neurotransmission of the NS-DA neuronal system. The significance of these studies reside in the fact that little is known of the action of hormones upon the CS, an extra-hypothalamic structure which bridges neuroendocrine mechanisms with motor, sensory and cognitive functions of the NS-DA system. The combination of hormone and drug infusions locally into the CS of fully awake rats with simultaneous measurements of neurochemical and behavioral responses add a new dimension to the analyses of the NS-DA system. Our studies using in vivo and in vitro methodologies will help to gain a better insight on the biology of hormonal action upon the CS and to aid the understanding of the role of hormones in extrapyramidal motor disfunctions.

The activation of LHRH neural apparatus (a key neuronal system in the control of reproduction) by neural and steroid signals thru a membrane mechanism has become a research topic of great interest in the field of Neuroscience and my laboratory has made contributions to clarify some aspects of such steroid-neural interactions. The present project to continue examining the binding characteristics of a novel ligand introduced by my laboratory, the radio-iodinated albumin conjugated to progesterone in C-11 (the so- called P-11-125I-BSA) to rat hypothalamic membrane fractions and its modification by the hormonal status of the animal will clarify the first step in the action of progesterone at membrane level. The recent demonstration that membranes from female rats but not from male rats have specific binding shows an important sex difference at this level in the CNS and may have important implications. The work of the present project may have clinical implications because it will further our understanding of the effect of steroid hormones in the function of the central nervous system and the role of gender in such actions.//

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of availability data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

9318908 Ramirez The physiological importance of progesterone and its metabolites and their effect on the nervous system has been recognized for a long time. However, the mechanism of action at the molecular level is not clear. Dr. Ramirez will test the hypothesis that progesterone binds with the membrane of nervous tissue and this leads to the control of release of another hormone known as luteinizing hormone-releasing hormone. This work is important because it may lead to a better understanding of the effect of steroid action upon mood and behavior by paving the way for development of new pharmacological agents. ***

DESCRIPTION (Adapted from applicant's abstract): The long-term objectives are to understand the non-genomic actions of E upon the CNS, an issue of relevance since steroid hormones are known to affect a variety of brain functions. We propose, on the grounds of our preliminary work and the work of others, that this steroid can influence cell activity not only by actions on the classical nuclear receptors but also by actions on the plasmalemma of neurons or glia cells through a specific membrane estrogen receptor (mER) leading to changes in intracellular messengers responsible for a particular neurochemical activation. The central hypothesis of this revised project postulates that the rapid stimulation of E upon DA release from rat striatal tissue depends on an interaction between a particular chemical group of the steroid and a specific binding domain in the mER of the corpus striatum (CS) cells. This, depending on the estrous cycle, leads to changes in intracellular messengers ultimately responsible for DA release. The specific aims are: (1) to establish that the rapid effect of E upon in vitro DA release from rat striatal fragments is a membrane mediated event taking place during a particular phase of the rat estrous cycle; (2) to establish that E binds stereospecifically and with high affinity to sites in the plasmalemma fraction from the rat CS and that the specific binding sites for E correspond to mERs and not to "acceptor" molecules; (3) to isolate, purify and chemically characterize the mER from the rat CS and generate polyclonal and monoclonal antibodies against this protein; and (4) to initiate cloning studies to isolate a cDNA clone for E-6-125-IBSA through screening a rat brain lambda ZAP-II library for selective phage plaques producing proteins with affinity for E-6-IBSA. To address these goals, I will take advantage of novel ligands to examine steroid membrane interactions, the so called steroid-BSA complexes. The hormone is covalently bound to bovine serum albumin (BSA), a protein that can either be radioiodinated (for use as a ligand in binding studies) or coupled to an agarose matrix (for use as an affinity chromatography column to isolate and purify the receptor). The work will lead to a better understanding of the role of steroids at the neuronal membrane level and the characterization of a membrane receptor for E may lead to new and useful pharmacological drugs.