Stuart H. Thompson - US grants

Electrical activity of nerve cells is determined by ion channels found within the cell membrane. The electrical excitability of individual nerve cells depends upon the types of ion channels expressed, the relative and absolute densities, and the spatial distributions of ion channels in different regions of the cell. These factors have a functional significance and any modification to these factors could be a cause of neuronal plasticity. The molluscan nervous system provides an important model for the study of neural circuits and neuronal plasticity. This research project will investigate the mechanisms molluscan neurons use to process information in the nervous system. Electrophysiological methods of voltage and patch clamping will be utilized to study the biophysics of membrane ion channels and to map the spatial distribution of ion channels in different areas of the cell. Biophysical properties that are modulated by activity, such as the inactivation gating of calcium and potassium, will be examined to help encode the time history of neuronal activity. Video fluorescence microscopy will be used to study the spatial patterning of intracellular calcium signals and the nature of the cytoskeleton. Results of this study will contribute to the understanding of neuronal information processing and the specializations responsible for the functional individuality of neurons.

The activation of muscarinic acetylcholine receptors initiates a complex physiological response that involves a number of second messenger systems. We are studying muscarinic signal transduction in the murine neuroblastoma cell line N1E-115 and in CHO cells with the goal of understanding how the various second messenger signals are coordinated. Our experiments are designed to examine interactions between intermediates in the signal transduction process by manipulating the pathways at specific points by applying exogenous metabolites or pharmacologically altering the activity of key enzymes. We will be concentrating on the Ca signals due to influx release, the cGMP & AA signals, and changes in ionic currents. Our methods include video imaging of fluorescent indicators, voltage clamp, release of caged metabolites, microinjection, and permeabilization. Muscarinic agonists, and other ligands, initiate (Ca)i oscillations in N1E-115 cells. Experiments are proposed to investigate the mechanisms responsible for these temporally and spatially complicated Ca signals. A key experiment involves attempts to phase shift the oscillation with brief pulses of Ca, IP3, cGMP, and IP4 using voltage clamp, microinjection and photolysis of caged compounds. The goal is to identify rate limiting steps in the processes leading to oscillation. We found that Ca influx is required for stimulating cGMP production and that Ca release will not substitute for influx. Our hypothesis is that agonist elicits two Ca signals that are compartmentalized and have different effects on metabolism. Voltage clamp experiments will be done to study the modulation of Ca influx by agonist. N1E-115 cells express receptors for several transmitters and we found that the transmitters interact and produce a cross-facilitation effect that we call recruitment. We will study the mechanism of interaction between the messenger pathways activated by different transmitters. One hypothesis is that cGMP production is important for recruitment. We will also be studying signal transduction in CHO cell lines separately transfected with the m1, m2, m3, and m4 gene sequences. These lines provide powerful new preparations for studying the signaling pathways engaged by muscarinic agonists. The results of our studies should be generally applicable to the broad class of receptors whose effects are mediated through G proteins.

This facilities project at the Hopkins Marine Station, Stanford University, involves the construction of a versatile sea water laboratory/culture facility. The project is motivated by the recognition that marine organisms provide unique model systems for biomedical, ecological, and evolution research and the importance of these systems is continually expanding. The laboratory will be an enclosed structure suitable for culturing marine specimens, maintaining cloned species, containing transgenic organisms, and for conduction experiments under controlled conditions. The building is single storied with a 50X40 ft. footprint and it is sited adjacent to existing seawater delivery and outlet pipes. It will be of masonry construction with tile roof and cement slab floor. The interior space is divided into two rooms by a central wall and connecting doors. The interior design is very flexible in order to accommodate new experiments or containment needs as they arise. Special equipment such as water chillers, sterilization equipment, tank racks, video monitoring will be the responsibility of the individual laboratories using the flexible space. The new laboratory/culture facility will enhance the current research program at the Hopkins Marine Station and open exciting new research opportunities.

9416210 Stuart Thompson Calcium ions hold a central position in the hierarchy of events that regulate both the metabolism and the unique functional attributes of neurons in the nervous system. A delicate interplay exits between the entry of calcium into the cells through ion channels in the membrane and the removal of calcium from the cells by ion pumps. When this balance is disturbed for longer than a few minutes, the result is nerve cell death. Consequently, there is great interest among researchers, clinicians and pharmaceutical manufacturers in understanding the mechanisms that control calcium levels in neurons. This conference will bring together researchers, educators, graduate students and drug company representatives to discuss the latest findings in this research area. The focus is on a neuly discovered pathway that directly links the metabolic state of the neuron to the control of the calcium entry pathway. This pathway is changing our view of metabolic regulation and neuronal function. This meeting will bring together the key persons involved in this research around the world.

9514421 Thompson Understanding the functioning of messenger molecules inside cells is critical for a complete explanation of how nerve cells communicate. Activation of molecular receptors on the surface of nerve cells by neurotransmitter molecules unleashes a cascade of events, which is orchestrated by the intracellular messengers. The key messenger is the concentration of calcium ions, which controls the production of the gaseous neurotransmitter, nitric oxide (NO). Nitric oxide, in turn, stimulates the production of the cyclic nucleotide messenger, cGMP. Calcium ions, NO, and cGMP together regulate numerous physiological events, including synaptic transmission and the opening of membrane ion channels. The focus of this research project is on the dynamics of calcium ion concentrations, which are affected by calcium release from intracellular storage compartments and by calcium ion inflow through the cell membrane. More specifically, the experiments are concerned with the internal signaling pathway that is activated by muscarinic cholinergic receptors in cultured cells. Techniques employed include the voltage clamp and calcium imaging; these are combined with modern molecular techniques to examine in detail rate-limiting steps in the activation of calcium release, for calcium influx, and in the production of cGMP. It is expected that these experiments will greatly advance our understanding of fundamental cellular mechanisms that regulate signaling within nerve cells.

Abstract Thompson

A confocal laser scanning imaging system will be used by faculty, graduate students, undergraduates and visitors to Stanford University's Hopkins Marine Station. The equipment includes a Zeiss Axiovert SI00 inverted microscope and a Noran Oz confocal scanning system.This equipment will: I) Enhance ongoing faculty research, 2) Promote new research activities, 3) Promote the training of graduate and post-doctoral students in modern methods of optical microscopy in a way that will enhance their research and career
development, and 4) Strengthen ongoing efforts to permit undergraduate
students access to the lasted research techniques.
The confocal imaging system will be used by four principle investigators
to address a number of cell biological questions using methods ranging from
ion imaging, sophisticated fluorescence microscopy, high resolution
Nomarski DIC, to morphometry. One group is investigating the spatial and
temporal aspects of second messenger signaling in neurons following the
activation of surface receptors by neurotransmitters, including the
dynamics of Ca2+ and NO signals. A second group is investigating the
development and biophysical properties of motor systems in the squid using
neuroanatomical and immunocytochemical methods. A third study focuses on
the specializations of fish heater organs and cardiac cells using
immunocytochemistry and antibodies specific for different isoforms of Ca
pumps and myosin ATPase. The fourth group uses fluorescence imaging
techniques to investigate the time course and spatial pattern of NO and
Ca2+ during fertilization of the sea urchin egg.
The principle investigators are addressing questions at the leading edge
of their disciplines. This shared confocal microscopy center adds
substantially to their ability to further their science, enhances their
teaching efforts, and increases collaboration with Stanford colleagues and
visitors from other institutions. This is in keeping with the role of the
Hopkins Marine Station as a regional center for marine biology research.