Glenn I. Hatton - US grants

The work proposed here will investigate the roles played in the osmodetection process by the neurons and glial cells in and around three magnocellular nuclei paraventricular and supraoptic nuclei and the nucleus circularis all of the hypothalamus. In addition, osmosensitive cells, in other nearby locations, that may be presynaptic to the neurons of the three magnocellular nuclei will be investigated. The approaches to be used in these studies include light and electron microscopy, electrophysiology of brain slices and immunocytochemistry.

The long-range goal of our research is to understand the mechanisms involved in the control of hormone secretion from the magnocellular neuroendocrine cells (MNCs) whose cell bodies reside predominantly in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus. Since the patterns of electrical activity are dominant factors in the process of neurosecretion by these cells, our immediate goals for the research proposed here are to investigate some of the factors which may influence or determine their activity patterns. In particular we wish to focus on investigating membrane properties, synaptic inputs and non-synaptic interactions, all of which probably play roles in controlling or modulating the process of neurosecretion in this system. In order to accomplish these objectives, we will record intracellular from PVN and perifornical neurons and from both neurons and glial cells in the area of the SON in slices of rat hypothalamus. In some instances the electrodes will be filled with the fluorescent dye, Lucifer Yellow (LY), and in others with horseradish peroxidase (HRP) in order to identify the recorded cell and trace its processes. Cells marked with LY will undergo either subsequent immunocytochemical analysis of hormone content (oxytocin, vasopressin or enkephalin) or electron microscopic (EM) analysis to demonstrate gap junctions after rendering the LY electron dense. HRP-injected cells and their processes will be analyzed at both the light and EM levels in order to determine the collateral branching and axonal terminations of these neurons. Studies of putative transmitters will also be carried out. Thus, we will be able to determine the physiological, morphological and hormonal characteristics of cells in this brain area, as well as to learn more about the local circuitry in which these cell groups apparently participate.

The work proposed here will investigate the roles played in the osmodetection process by the neurons and glial cells in and around three magnocellular nuclei paraventricular and supraoptic nuclei and the nucleus circularis all of the hypothalamus. In addition, osmosensitive cells, in other nearby locations, that may be presynaptic to the neurons of the three magnocellular nuclei will be investigated. The approaches to be used in these studies include light and electron microscopy, electrophysiology of brain slices and immunocytochemistry.

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.

DESCRIPTION (applicant's abstract): The long term objectives of this research are: 1) to understand the functioning and control of the mammalian magnocellular hypothalamo-neurohypophysial system (mHNS) as it senses and responds to changes in the animal's physiological state, including fluctuations in both the internal and external milieu. Since this oxytocin- and vasopressin-producing system is ubiquitously expressed in mammalian brains, what we learn should have general applicability and importance in health and disease. 2) to identify the various cell types, neural and non-neural, involved in the homeostatic roles played by the mHNS, and to understand the processes and mechanisms by which these cell types interact. Consisting chiefly of the supraoptic and paraventricular nuclei of the hypothalamus and their main axonal terminations in the pituitary neural lobe, the mHNS has achieved model system status because it is well characterized physiologically (e.g., in water, blood pressure and temperature regulation, parturition and lactation/nursing) and has revealed much in investigations at many levels of analysis. So well known are the peripheral effects of mHNS outputs that even findings obtained in biophysical and molecular biological investigations using brain slices or cell cultures can most often be meaningfully related to functioning of the intact system. The work proposed here includes studies of the mHNS using microscopic, immunocytochemical, biochemical and physiological approaches as converging operations aimed at uncovering fundamental mechanisms of CNS function. Specific aims for the requested period of support are the following: Aim 1. To further analyze the functional consequences of morphological and chemical changes that accompany physiological activation of the mHNS. Specific experiments are directed at determining mechanisms involved in glial influences on neurohypophysial peptide release, and at the roles played by calcium binding proteins. Aim 2. To investigate the newly discovered cellular network that appears to link the meningeal, vascular and parenchymal glial compartments, and that has the potential to be a signaling system in the hypothalamus and perhaps elsewhere in the brain. Specific experiments address questions of the cell types involved, their modes of intercellular communication with cells of homotypic and heterotypic nature, and their signaling capacity.

A grant has been awarded to Dr. James G. Baldwin at The University of California, Riverside (UCR) for a new transmission electron microscope (TEM). The research and teaching needs of this growing campus and scheduling problems on the existing microscope will be met by adding this new TEM. The new microscope is highly complementary with the existing TEM and both instruments will be housed and administered together in the campuswide Central Facility for Advanced Microscopy and Microanalysis (CFAMM) administered within the College of Natural and Agricultural Sciences (CNAGS).

This instrument provides functionality needed by experts, yet simplicity appreciated by students and less experienced microscopists. The new microscope will be scheduled for projects that leverage its ease-of-use for diverse TEM work done by many biology researchers and students that do not require high accelerating voltage nor analytical capabilities. Flexibility and scheduling conflicts will be further addressed by equipping the microscope with an electronically controlled rotating stage (required by many Cell, Molecular and Developmental Biology [CMDB]) investigators, e.g. those using stereo pairs) and digital image capture required for most CMDB scientists and crucial for cost effective printing, storage, 3D reconstruction, and easy interfacing with the adjacent Center for Visual Computing (CVC) with which it will be linked by an existing fiber optics system. A new vacuum evaporator is also awarded and is vital for sample preparation for the electron microscope.

UCR is the most rapidly growing UC campus, with the highest representation of minority students in the UC system and among the highest nationally. New faculty hires and new laboratory construction are underway to accommodate a 100% increase by 2010, with particular growth in life sciences. The new TEM is essential for research and teaching programs to keep pace with the rapidly expanding interdepartmental program in CMDB and to address a worsening scheduling crisis on the only existing modern TEM on campus and shared by both life and materials scientists. For a significant group of Federally-funded UCR researchers in the life sciences, TEM is an indispensable tool, complementary to a range of additional techniques for acquiring structural information at the cellular and sub-cellular level. These include special applications in cell biology that are relevant to basic science as well as agriculture, biomedicine, pathogenesis, mode of action of antibiotics, and exploring character changes in evolution. UCR's existing CFAMM, interfaced with the CVC, provides a unique context for the new TEM dedicated for life science/CMDB research and training. The new TEM will be the primary graduate and undergraduate TEM teaching tool for seven existing courses, and the PIs of this proposal will collaborate to develop a new training course specific to TEM techniques centered around the new instrument at the CFAMM.