Margarita Curras-Collazo - US grants

9977158

Abstract

This award will be used to acquire an advanced light microscope laboratory that will enable new research projects to be undertaken in the biological sciences at the University of California, Riverside (UCR). This laboratory will consist of an upright confocal scanning laser microscope, an inverted microscope for micromanipulation/injection, and a computer workstation for advanced image data analysis in a mult-user, state-of-the-art facility. These light microscopes will be housed in the currently existing centralized electron microscopy laboratory, thereby creating a Centralized Laboratory for Advanced Microscopy. The light microscopes will service over 30 well established research programs in 12 departments in the College of Natural and Agricultural Sciences. The users in this proposal specifically need this instrumentation for studies of fertilization, oocyte transport, angiogenesis, cell receptors, cell adhesion and dye transfer, cell junctions in neurosecretory cells, molecular motors, ion transport, plant pollination, trafficking of viral proteins in plants, and transcytosis. The equipment will allow Principle Investigators at UCR to extend their current research programsm and will enable initiation of many new projects not currently possible on the UCR campus. The light microscope laboratory has been carefully integrated and designed to be flexible, thereby meeting the broad range of needs of a core facility. The centralized laboratory where the light microscope will be housed is currently staffed by a full-time manager, and the university will provide an additional 50% Staff Research Associate for daily operation and maintainance of the light microscopes and workstation. Training on the light microscopes will be provided in an undergraduate course in Cell Biology, a graduate level class in microscopy, and by one-on-one interaction with the Staff Research Associate. UCR has the highest percentage of minority students of any UC campus and one of the highest nationally. Many students receiving training will be minority students.

Previous studies have shown that NMDA receptors mediate much of the normal synaptic transmission in magnocellular neuroendocrine cells of the supraoptic nucleus of the hypothalamus (SON). Dr. Curras's laboratory has recently shown that osmotic activation of the hypothalamo-neurohypophysial axis produces upregulation of the glycine-binding subunit of the NMDA receptor, NMDAR1. These changes in NMDAR1 expression are specific to the SON and paraventricular nucleus of the hypothalamus (PVN) suggesting that NMDA receptors are also involved in neuroendocrine function.

Recent molecular cloning studies have identified five NMDA receptor subunits: NMDAR1 and NMDAR2A-D. In the adult SON all but the NMDAR2A gene are expressed. Preliminary studies in the investigator's laboratory have detected robust expression of NMDAR2B and NMDAR2D subunit and light expression of NMDAR2A and NMDAR2C subunits. One of the goals of the proposed research is to examine the subunit composition of functional NMDA receptors present on neurons of the SON. Western blot analysis and patch-clamp electrophysiology combined with pharmacological strategies will be used to examine the NMDA receptor structure and function indicative of subunit composition. Since functional NMDA receptors are believed to be composed of NMDAR1 and at least one NMDAR2 subunit, it is proposed to examine expression changes induced by osmotic activation of the glutamate-binding subunit, NMDAR2B.

One major goal of this proposal is to further characterize the subunits that makeup the N-methyl-D-aspartate (NMDA) receptor complex on neuroendocrine cells in the brain. These neurons control body water (osmoregulation) by producing hormones such as vasopressin and oxytocin. These neuroendocrine cells are located in the supraoptic (SON) and paraventricular nucleus (PVN) of the hypothalamus. The composition of NMDAR2 subunits, in particular, is thought to underlie differences in the function of NMDA receptors. The proposed studies will examine the molecular expression of NMDAR2 subunits in neuroendocrine cells using state-of-the-art molecular techniques as well as pharmacological and antisense technology. In addition, the experiments have been designed to examine the functional contribution of NMDAR2 subunits to NMDA receptors on neuroendocrine cells. An examination of the structure of NMDA receptors in the SON and PVN, which may display all five of the NMDA receptor subunits, will provide information on one of the most potentially unique NMDA receptor subtypes in the brain.
One longterm objective of this research is to yield an understanding of the role of NMDA receptor subunits in osmoregulation. To begin to address this issue we will examine the effect of osmotic activation of SON and PVN neuroendocrine cells on their expression of selected NMDA receptor subunits. Our previous research findings indicate that these cells display dramatic changes in NMDAR1 and NMDAR2B subunit levels during dehydration. The proposed experiments will investigate the cellular components showing subunit changes, regional specificity and reversibility upon rehydration. Finally, we will explore the possibility that NMDAR2B-containing receptors on neuroendocrine cells can be biochemically altered and that physiological signals such as dehydration can provide the signal for regulation.
Basic principles revealed by the proposed studies will provide essential information about the properties of NMDA receptor subtypes in the neuroendocrine nuclei of the brain. Effects of osmotic signals on the expression of NMDA receptor subunits may provide clues about the role of NMDA receptors in fluid homeostasis and other neuroendocrine functions. In addition, our findings will contribute new insights into regulatory mechanisms important in central nervous system function.

Vasopressin Neuromodulation in Neuroendocrine Hypothalamus: Receptors, Signal Transduction and Physiological Significance

Neuroendocrine cells (MNCs) located in the supraoptic nucleus (SON) of the brain release the peptides oxytocin (OXY) and vasopressin (VP) that are critical for hydromineral balance, lactation, the birth process and cardiovascular function. VP and OXY get liberated into the bloodstream and target the kidneys, uterus, arteries and mammary glands. Local release of VP and OXY allow MNCs to participate in their autoregulation. It has been previously determined that treatment of SON tissue from adult rats with VP and OXY can decrease the release of the stimulatory chemical glutamate (GLU) that mediates the transfer of information from other brain regions to the SON. During activated states such as dehydration and lactation these signals provoke the release of VP and OXY from MNCs and hence the modulatory roles of these peptides on incoming signals becomes critical. In concert with other effects of local peptide release, this level of control may prevent depletion of hormone supply during periods of prolonged activation and help maintain the efficiency of MNC controlled osmoregulatory and other homeostatic systems that sustain life in land and aquatic vertebrates. The components of this circuit are unknown and the proposed experiments will characterize the receptor subtypes and potential downstream signal transduction cascades involved in VP's actions on GLU release. Levels of GLU and VP in the SON will be correlated in perfusates from SON punches using HPLC and enzyme immunoassay. Other experiments will investigate the source of GLU release and VP's modulatory actions. The physiological significance of the dramatic and potentially important effect of local VP on glutamatergic transmission in rats subjected to dehydration will also be examined. Commensurate with the PI's strong commitment to the development of young scientists, the proposed studies will involve the training of undergraduate, graduate and postdoctoral scientists.