Satya Kalra - US grants

In this proposal we plan to test the hypothesis that progesterone-induced increments in LHRH levels in the estrogen-primed ovariectomized rats result from increased influx of excitatory signals which, in turn, activate the appearance of new LHRH in the median-eminence. Electrical stimulation of appropriate site(s) in the diencephalon of freely-moving rats and analysis of labelled precursor incorporation into LHRH by high performance liquid chromatography (HPLC) will be the major new methods of investigation. In another related project the site(s) of impairment--pre or post-adrenergic receptors--which may be responsible for blockade of the pre-ovulatory LH surge will be examined in progesterone-blocked ovariectomized rats and middle-aged spontaneous persistent estrous rats. The effects of naturally occurring neuroactive agents--neurotransmitters and prostaglandins--infused intermittently into the third ventricle of freely-moving rats and their effects on LH secretion will be analyzed. The application of these three new approaches will clarify our understanding of the action of progesterone on the brain.

Recent evidence suggests that a complex neural circuitry, the components of which may communicate with each other by diverse neurochemical signals, participates in initiation and sustenance of preovulatory LHRH and LH hypersecretion. This proposal focuses on contributions of the peptidergic components of this neural circuitry. The precise modes of involvement of an excitatory component - neuropeptide Y (NPY), and an inhibitory component - endogenous opioid peptides (EOP) in the preovulatory LH discharge will be studied. NPY participation will be examined by studying the dynamic changes in hypothalamic NPY levels and release rates preceding and following the LH surge. Whether NPY acts independently or in concert with excitatory adrenergic transmitters or whether adrenergic transmitters mediate NPY excitation of LHRH and LH release will be the subject of intensive investigations. Curtailment of the inhibitory influence of EOP as an early neurochemical event responsible for triggering the excitatory neural chain on proestrus, will be documented by analysis of beta-endorphin (betaE) release in association with the LH surge. Experiments are designed to test whether EOP exert a "braking" influence on basal episodic LH secretion during the estrous cycle and to ascertain the roles played by NPY and adrenergic transmitters in mediating EOP effects on LH release. The release of NPY, betaE, LHRH and neurotransmitters will be assessed in vivo by push-pull cannulate and in vitro by hypothalamic perifusion methods. In vivo experiments will utilize chronic intraventricular cannulae for continuous or episodic delivery of test materials and blood sampling by intrajugular cannulae in freely-moving rats. In some experiments, neuropeptides will be measured in microdissected brain sites. Neuropeptides and LH will be measured by RIA and catecholamines by radioenzymatic assays and HPLC.

Clinically, eating disorders (obesity, anorexia nervosa, dieting) are associated with subnormal or impaired reproductive function. Recent evidence shows that Neuropeptide Y (NPY), neuropeptide K (NPK) and endogenous opioid peptides (EOP) control food intake as well as reproduction. Although produced in the same neuronal population(s) these neuropeptides are differentially released at specific hypothalamic sites to modulate these two basic functions. Consequently, an understanding of the factors that regulate neuropeptide production in the perikarya and release from remote nerve terminals at the site(s) of action is a new challenge. In this proposal, we plan to address these issues in the control of ovulation, especially in the preovulatory LH surge (PLS). In the rat increased episodic secretion of LH releasing hormone (LHRH) during the critical period on the afternoon of proestrus causes the discharge of PLS. Although it is well known that the neural clock (NC) entrained to the daily photoperiod activates LHRH secretion during the critical period, the origin and route of transmission of neural signals to LHRH neurons have not been delineated. In this context we propose to test the hypothesis that the NC restrains the inhibitory peptidergic circuits leading to activation of excitatory circuits which cause LHRH hypersecretion. Therefore, our focus will be on how the NC and inhibitory circuits, viz. EOP and NPK and the excitatory circuit, viz. NPY and adrenergic systems, operate under the direction of ovarian steroids. The operation of these circuits will be examined locally in the hypothalamus by studying the inter- and intraneuronal secretory events. In addition, the hypothesis that NPK is a potent inhibitory system that mediates the negative feedback effects of gonadal steroids on LH release will be tested. Interneuronal communication will be studied by analyses of peptide release in vivo and in vitro and the intraneuronal events will be assessed by elucidating steroidal effects on Pre-Pro NPY mRNA and peptide levels in microdissected sites. The connections between these circuits will be analyzed by employing pharmacological, surgical and electrolytic lesioning approaches to interrupt the progression of signals. Major techniques are RIA of neuropeptides, LH and ovarian steroids, anterior pituitary dispersed cell cultures, combined autoradiography and immunocytochemistry, neuropeptide output analysis by push-pull cannulae in vivo and hypothalamic perfusion and incubation in vitro. Catecholamines will be measured by HPLC and radioenzymatic assays.

DESCRIPTION (Scanned from the Applicant's Description): With one out of every two adult Americans rated as medically obese, obesity is the number one health problem in the United States. Since it is a major risk factor for heart disease, diabetes, stroke, hypertension, and morbidity, the treatment of obesity and associated diseases entails enormous medical costs. The major objective of this proposal is (1) to examine whether viral vector mediated delivery of weight-reducing signals, such as leptin, is a viable therapy (gene therapy), alternative to the pharmacologic approach, to control body weight (BW) gain for extended periods in normal male and female rats and in rodents with obesity due to environmental (diet-induced) and genetic factors. We believe that leptin acts by (1) augmenting energy expenditure (thermogenesis) and/or curbing appetite and (2) that the beneficial effects of leptin gene therapy are manifest at sites that enhance energy expenditure and modify hypothalamic appetite regulating signals such as the orexigenic signals, NPY, AgrP and GABA, and the anorexigenic melanocortin signal alpha-MSH, and intracellular signal transduction sequalae involving STAT3 and SOCS-3. We have successfully generated a recombinant adeno-associated virus (rAAV) vector to efficiently transfer the naturally occurring body weight reducing peptide, leptin (rAAV-lep). Aim 1: Examine the long-term (one-year) efficacy of rAAV-lep to reduce BW gain when delivered intracerebroventricular (icv) or systemically in out-bred Sprague-Dawley (SD) rats and obese rats maintained on a high-energy (HE) diet. Aim 2: Evaluate the efficacy of icv and peripheral rAAV-lep in those genetic models of obesity that (1) lack leptin (ob/ob mice) and (2) display resistance to peripheral and not central leptin (New Zealand obese mice); and (3) lack NPY (-/-) and display obesity when maintained on HE diet. Finally, we will also characterize the underlying mechanism(s) if excessive ectopic leptin itself induces leptin ineffectiveness (resistance) in these experiments. The causal mechanisms responsible for phenotype changes will be identified by evaluation of hypothalamic leptin expression and signaling through analysis of gene expression, and peptide levels, signal transduction sequalae (p-STAT3 and SOCS-3) and metabolic indices (body temperature, 24h urinary NE activity, oxygen consumption, UCP1 mRNA, and blood leptin, insulin glucose, and corticosterone levels). The outcome of these investigations will provide fundamental information on the broader potential of using gene delivery of naturally occurring anorectic molecules for BW control and their mechanism of action. This new knowledge will be applied to the ultimate goal of site-directed gene delivery aimed at the newly identified vulnerable loci in hypothalamic signaling to curb overeating and abnormal weight gain.