Alan D. Grinnell - US grants

Twenty-two UCLA faculty members from eight different departments request continued funding for a Training Program in Cellular Neurobiology. This Program has been supported by NIH since 1968 and currently involves 29 predoctoral and 39 postdoctoral trainees, of whom two predoctoral and four postdoctoral positions are funded by the Training Grant. Trainees will be predoctoral students, Ph.D.s or M.D.s, who exhibit unusual potential and commitment to establishing productive careers in neurobiology research, as judged by academic records, examination scores, published research, letters of recommendation, personal interviews and lab experience. Special efforts will be made to recruit under-represented minority candidates to the Program. Trainees spend a minimum of two years learning modern molecular, physiological/optical and structural research techniques and their applications to understanding the biology of excitable cells. Predoctoral students receive a thorough training in rigorous research methodologies superimposed on the requirements of their department. For both pre- and postdoctorals, a rich selection of specialized neurobiology courses is available, as are technique workshops and regular seminars and journal clubs where new technologies, findings, and ideas are freely exchanged. Facilities are excellent and the research environment exciting. The laboratories of the participating faculty occupy approximately 30,000 square feet, most of which is contiguous space or within a few minutes walk. There are widely used common facilities for analytical biochemistry, molecular biology, cell culturing, electron microscopy, and electronics and mechanical shop assistance. A small library/conference room serves as a familiar meeting ground, and UCLA has excellent vivarium and computing facilities, as well as one of the world's great biomedical libraries. The University has traditionally made a strong commitment to neuroscience and their commitment continues.

Frog motor nerve terminals, contrary to common perception, are highly plastic in morphology and function. We have worked out many of the factors that regulate their properties. Further research is proposed in four areas: The first project is concerned with the fact that normal-appearing terminals or parts of terminals are sometimes found to release very little transmitter. As terminals grow and new junctions are added, apparently in response to signals from the muscle, a significant fraction (the distal 20-40%) of long terminal branches, and sometimes whole terminals, release only 5-10% as much as other terminal regions. It seems likely that these seemingly ineffective terminal regions may be functional under conditions we have not examined. We will test several hypotheses for possible functions of "silent" terminal components, and attempt to discover why they release so little transmitter under the conditions tested. A second project is aimed at understanding what causes the large increase in release efficacy of intact terminals in partially denervated muscle. This enhancement of release appears to precede sprouting. We will characterize it, attempt to determine what triggers it, and study possible mechanisms. We will also study the non-quantal "leak" of ACh from motor nerve terminals, using ultrasensitive ACh sensing probes made by patch clamping a small piece of muscle cell membrane containing ACh channels. With these probes, sensitive enough to detect single channel openings, we will study the dependence on quantal release levels, nerve activity, and external ionic environment. Finally, we will study the development of the highly organized innervation pattern of the Xenopus pectoralis muscle, in which single axons selectively innervate one of three types of twitch fibers, are topographically localized to a tightly restricted region of the muscle, and tend to selectively innervate both endplates on fibers. It is probable that this organization is functionally adaptive, and may be refined by use in a way comparable to that of CNS connections.

Frog motor nerve terminals, contrary to common perception, are highly plastic in morphology and function. We have worked out many of the factors that regulate their properties. Further research is proposed in four areas: The first project is concerned with the fact that normal-appearing terminals or parts of terminals are sometimes found to release very little transmitter. As terminals grow and new junctions are added, apparently in response to signals from the muscle, a significant fraction (the distal 20-40%) of long terminal branches, and sometimes whole terminals, release only 5-10% as much as other terminal regions. It seems likely that these seemingly ineffective terminal regions may be functional under conditions we have not examined. We will test several hypotheses for possible functions of "silent" terminal components, and attempt to discover why they release so little transmitter under the conditions tested. A second project is aimed at understanding what causes the large increase in release efficacy of intact terminals in partially denervated muscle. This enhancement of release appears to precede sprouting. We will characterize it, attempt to determine what triggers it, and study possible mechanisms. We will also study the non-quantal "leak" of ACh from motor nerve terminals, using ultrasensitive ACh sensing probes made by patch clamping a small piece of muscle cell membrane containing ACh channels. With these probes, sensitive enough to detect single channel openings, we will study the dependence on quantal release levels, nerve activity, and external ionic environment. Finally, we will study the development of the highly organized innervation pattern of the Xenopus pectoralis muscle, in which single axons selectively innervate one of three types of twitch fibers, are topographically localized to a tightly restricted region of the muscle, and tend to selectively innervate both endplates on fibers. It is probable that this organization is functionally adaptive, and may be refined by use in a way comparable to that of CNS connections.

The function of the nervous system depends on the proper connections between individual and groups of nerve cells. Nerve cells communicate with each other and with muscle cells through the release of chemical transmitters at close contact points called synapse. While much is known about the structure of these "synaptic' connections, far less is understood about how these connections adjust to changes by drugs or endogenous chemical substances like hormones. How the communication between nerve cells is changed or "modulated" in both the short and the long term is important to our understanding of the nervous system under normal conditions and those produced by disease. "Neuromodulation" may also be a consideration in the development of new therapeutic approaches. The chemical transmitters used by nerve cells to signal to each other are released from specialized areas called "active zones." The release process is dependent on the movement of calcium into the nerve cell through channels which are clustered at these active zone regions. Since the release of chemical transmitters is dependent on calcium, any modulation of the movement of calcium through its channels can drastically affect the communication between nerve cells and from nerve cells to muscle. This research project will study calcium movement in frog nerves and in specialized cultured cells which may possess nerve-type calcium channels. Unlike other preparations, these may provide direct access to nerve cell calcium channels in order to study neuromodulation. In this way it is possible to understand the factors which control communication in the central and peripheral nervous systems.

DESCRIPTION: (Applicant's Abstract) This project is aimed at a greater understanding of the mechanisms of regulation of neurotransmitter release, which is critical to an understanding of the function of the nervous system in health and disease. It uses a novel vertebrate preparation: the synapses formed by motoneuron neurites on muscle cells in Xenopus nerve-muscle cultures, in which pre- and postsynaptic processes can be patched-clamped and ionic currents analyzed directly and correlated with quantal neurotransmitter release. Of particular interest is the mechanism whereby Ca++ influx triggers vesicle fusion. Ca++ dynamics at active zones is the subject of many mathematical models, but direct measurements have proved difficult. In the Xenopus synapses, large-conductance Ca++-dependent K+ (KCa) channels are functionally coupled to Ca channels and probably concentrated at active zones. KCa currents will be used to monitor the concentration of Ca++ that occurs near them when Ca channels open during step depolarizations and action potentials. Whole-cell and single channel recordings will be used to calibrate the IKCa as a function of Ca++ concentration and voltage, and the IKCa will be used to describe the changes in Ca++ concentration that occur at or near active zones during different forms of synaptic activity. The changes will be correlated with the levels and timing of transmitter release. These data will be used to construct a realistic mathematical model of the Ca++ dynamics at active zones, which will be further refined by a characterization of the intrinsic buffers and experiments to assess the distance of release sites from Ca channels. We will also investigate the mechanisms of action of peptide fragments of the presynaptic proteins NSF and synapsin, which slow the kinetics of release in squid. We will investigate whether these peptides have the same effect in Xenopus and if they act by desynchronizing fusion events or slowing the kinetics of fusion.

Project Summary: IBN-9722785, Alan D. Grinnell, P.I. Mechanisms of Long-Term Transynaptic Regulation of Neurotransmitter Release" Competitive interactions between synapses innervating the same target cell are critical in the establishment and refinement of connectivity in the nervous system, and in the adjustment of synaptic strength with use or experience. One of the most intriguing forms is known as "Hebbian" plasticity, in which synaptic inputs that are active simultaneously with postsynaptic activity are retained or strengthened, while synapses that are not active at the same time as the postsynaptic cell are suppressed or lost. A particularly clear, accessible instance of such pre-synaptic suppression is that observed in synapses between Xenopus motoneurons and muscle cells in culture. Transmitter release at a synapse is suppressed by manipulations that elevate the calcium concentration in the muscle cell, simulating activation of another synaptic input. The synapse can be protected from suppression if it is active during the time of calcium-induced feedback. The mechanisms of suppression and protection are unknown. We have developed techniques for patch-clamping synaptic terminals and postsynaptic cells simultaneously in this system, allowing us to analyze rigorously the magnitude and time course of ionic currents in the terminal and the associated neurotransmitter release. With a third patch pipette it is possible to introduce reagents into the synaptic terminal through the neuronal cell body. Since calcium influx during a presynaptic action potential is critical to neurotransmitter release, we will first (1) determine whether long-term suppression of synaptic function is associated with a decrease in calcium- influx either by direct modulation of calcium channels or by changes in other ionic currents in the terminal, and what these changes are at the level of single channels. If suppression of release can be explained by changes in ionic currents, we will determine (2) whether the suppression effects are mediated by G-protein associated receptors, and whether this is by a direct ("membrane delimited") or indirect (second messenger mediated) pathway, and (3) whether cyclic nucleotides help mediate the effect. In addition, we will test (4) whether protection by simultaneous synaptic activity is due to prevention of the suppressive influence or to some compensatory change, and (5) what aspect of neuronal activity is responsible for this protection. The Xenopus motoneuron-muscle cell synapses are robust, characteristic in most respects of mature synapses. The Hebbian character of the modulation, the evidence for activity-dependent protection of synapses, the accessibility of the preparation, and above all the ability to monitor and manipulate pre-synaptic events make this an ideal preparation in which to obtain insights about mechanisms of long-term synaptic remodeling and plasticity.