Our long-term objectives are to characterize the hypothalamic cellular and molecular sequelae programmed by ovarian steroids in augmenting the discharge of luteinizing hormone releasing hormone (LHRH), the trigger for the preovulatory surge of pituitary LH secretion and to understand the mechanisms by which an inappropriate steroidal milieu (e.g. estrogen neurotoxicity) disrupts these events. The focus of the current proposal is on the membrane permeant, gaseous nitric oxide (NO) and agmatine (Ag), two newly identified messenger molecules produced by enzymatic conversion of the common precursor amino acid, L-arginine (L-Arg). We propose that (i) in the sequelae of events that initiate and sustain LHRH (LH) surges for several hours, NO and Ag participation is crucial for the genesis and transmission of neural impulses upstream from the neuropeptidergic signals, such as neuropeptide Y (NPY) and galanin that are known to be activated and (ii) unopposed estradiol produces a neurochemical deficit that may be alleviated by L-Arg or progesterone through activation of a common NO- neuropeptidergic signalling pathway. This two-part hypothesis will be tested within the framework of four specific aims. Experiments are designed to elucidate the neurochemical mechanisms underlying the restorative ability of L-Arg (Aim 1), characterize and validate the dynamics of NO-NPY signalling on proestrus and in response to experimentally-induced shifts in opioid-restraint in induction and sustenance of the LH surge (Aim 2), establish the existence of a physiological and anatomical progesterone-NO link for excitation of these surges (Aim 3), and document that L-Arg-induced facilitation of the LH surge also involves hypothalamic Ag, the newly identified imidazoline receptor agonist (Aim 4). The dynamics of NO communication in vivo will be evaluated by microdialysis, antisense deoxynucleotides to constitutive NO synthase mRNA and combined autoradiography and immunocytochemistry; the neurosecretory responses during neuropeptidergic signalling will be studied by measuring gene expression and peptide levels accompanying LH release; Ag participation will be evaluated pharmacologically as well as by analysis of hypothalamic Ag. We anticipate that these investigations will advance our understanding of the timely interactions among various modalities of information processing in the hypothalamic control of ovulation. Adverse reproductive consequence of neurotoxins are well recognized. Environmental estrogens and estrogen-like pollutants (pesticides, chlorinated hydrocarbons, the breakdown products of plastics) as well as improper levels of NO are also known to produce neurotoxicity. This proposal will decipher not only the neurochemical pathway of the estrogen-NO interaction, both under physiological and pharmacological conditions, but also the alleviating influence of progesterone on the neurotoxicity of estrogen-NO on neural processes governing ovulation.
