Robert S. Zucker - US grants

This project is concerned with the mechanisms for regulating the strength of synaptic transmission. Various forms of synaptic plasticity will be studied, including facilitation, augmentation, post-tetanic potentiation, and long-term potentiation. These processes are involved in synaptic information processing, shaping of motor responses and behaviors, and adaptations of neural circuits to previous experience, and are thought to be essential for higher cognitive functions such as associative learning. Experiments will focus on answering the following specific questions: 1) How does Ca2+ cause synaptic facilitation, augmentation, and potentiation? Presynaptic Ca2+ entering during action potentials may bind to distinct sites to cause phasic exocytosis (the fast release of transmitter immediately following a spike), and other sites to facilitate, augment, and potentiate subsequent release. 2) What is the stoichiometry of Ca2+ action in exocytosis and facilitation? The first process may require high Ca2+ cooperativity, while facilitation may not. These experiments will be done at crayfish neuromuscular junctions and the squid giant synapse. 3) How does depolarization modulate transmitter release? Subthreshold depolarization at the squid giant synapse can enhance spike-evoked release without altering Ca2+ influx; this may reflect a direct Ca2+-independent modification of the exocytotic machinery, or a facilitating or potentiating effect of a rise in [Ca2+]i caused by opening Ca2+ channels. 4) Can genuine long-term potentiation be elicited solely by a rise in postsynaptic [Ca2+]i? A requirement for simultaneous afferent activity and a rise in postsynaptic [Ca2+]i to establish long-term potentiation in mammalian hippocampal CA1 pyramidal cells will be tested. 5) Is the rate- limiting step in Ca2+-evoked peptide secretion different from that for acetylcholine release at co-transmitting synapses? At bullfrog synapses onto sympathetic ganglion neurons, ACh and the peptide LHRH are co- released. Exocytosis of docked vesicles and mobilization of vesicles to release sites may be rate-liming Ca2+-dependent steps in secretion of these respective transmitters. 6) What are the parameters of Ca2+- regulation of growth cone extension? In Helisoma neurons, neurite growth may be enabled by [Ca2+]i in a particular range of intermediate concentration, and directed growth such as turning toward targets may be similarly controlled. These questions will be explored using techniques of electrophysiological recording, fluorescent measurement of [Ca2+]i, and control of [Ca2+]i using photolabile Ca2+ chelators.

This proposal requests funds to establish a Biological Facilities Center for the Study of Cell Morphology and Function. Five major instruments are requested: l. A transmission electron microscope with facilities to section and process specimens at low temperature. It will be used for immunogold localization of a wide variety of antigens and for ultrastructural visualization of cell and cytoskeletal morphology. 2. A high-spatial-resolution low-light fluorescence microscope system to track the dynamics of fluorescently labeled cytoskeletal proteins and the position and shape of labeled cells in developing embryos while minimizing photodynamic damage. 3. A workstation that combines electrophysiological equipment with imaging of fluorescence ratios from fura-2 to map spatial patterns of neuronal activity and intracellular Ca2a in electrically stimulated neural assemblies. 4. A laser-scanning confocal microscope that rejects out-of- focus fluorescence that will enable high-speed optical sectioning of thick or dynamically changing specimens for studying the role of Ca2+ in controlling the cytoskeleton and for measuring local demains of Ca2+ or pH in single neurons, thick ganglia, or heterogeneous epithelia. 5. A Fourier transform nuclear magnetic resonance spectrometer that will permit continuous monitoring of intracellular metabolites, pH, sodium, and hemoglobin polymerization in cell and organelle suspensions such as cyanobacteria, oxyntic cell vesicles, and erythrocytes. This equipment will be used by the forty research groups to be housed in a new Life Sciences building as well as hundreds of graduate students who are involved in ongoing research projects. Expected contributions will be the development of a scanning confocal microscope for high-speed biological observation as well as innovative solutions to current problems in molecular biology.

Synapses are the points of communication between neurons. Alterations in their strength can critically affect the operations and functions of neural circuits in the brain. Many hormones and drugs affect synaptic strength. Sometimes they act by altering presynaptic calcium channels through which calcium ions enter to trigger the secretion of neurotransmitter, the substance one neuron releases in order to communicate with another at a synapse. But often neuromodulators act without affecting calcium channels in ways still not understood. Serotonin is a circulating hormone in crustaceans which strongly enhances transmission at neuromuscular synapses. Serotonin increases transmitter release without altering presynaptic levels of calcium concentration or increasing the influx of calcium into nerve terminals during action potentials. Just how serotonin regulates transmitter release is not known. Long-term facilitation (LTF) is a form of synaptic plasticity observed at crayfish neuromuscular junctions where prolonged neural activity results in an increase in transmitter release per action potential. It is not associated with any change in presynaptic resting calcium level or calcium influx during action potentials, and like serotonin's action, its effects are mediated by the presynaptic biochemical intermediate cyclic adenosine monophosphate (cAMP). It is not known what aspect of the secretory process is altered in LTF. Transmitter is stored in vesicles and released by their fusion with the presynaptic membrane during action potentials. Preliminary results suggest that serotonin acts at crayfish neuromuscular junctions to increase the supply of vesicles available for release. This preparation will be used to test this hypothesis, and also to develop procedures that could be used to assay other drugs that might enhance the size of the vesicle pool. It will also be determined whether vesicle pool size is altered during (LTF). In this project, a membrane labelin g fluorescent dye (FM1-43) will be used to label presynaptic vesicles and determine whether serotonin and LTF increase the number of available vesicles or the probability they are released by action potentials. A slowly developing depression of synaptic transmission due to exhaustion of the vesicles available for release will also be used to estimate the number of vesicles in the releasable pool and whether their number or their probability of release is increased by serotonin and LTF. Other experiments will test for changes in the rate of recovery of released vesicle membrane in serotonin and LTF, and for involvement of cytoskeletal proteins in vesicle availability and release during serotonin action and LTF. Finally, new cAMP-sensitive indicators will be used to measure changes in presynaptic levels of cAMP during serotonin action and LTF.

0075842
Zucker

Neurons communicate by releasing chemical transmitters at synaptic contacts. This project will explore mechanisms by which a hormone, serotonin, regulates synaptic transmission at a neuromuscular junction in crayfish, a preparation that is particularly accessible to modern cell biological probes. Serotonin increases transmitter release by elevating the cyclic adenosine 5'-monophosphate (cAMP) concentration in nerve terminals, which activates a particular presynaptic ion channel referred to as Ih. We will explore the mechanism by which Ih influences transmitter release. We will look for roles for Ca2+ influx through Ih channels and connections to cytoskeletal proteins, because such proteins and Ca2+ are known to influence transmitter release. Ih channels also may mediate the induction of a long-term facilitation (LTF) of synaptic enhancement following presynaptic activity. We will establish whether there are for Ih and cytoskeletal proteins in LTF induction. Serotonergic modulation of transmission involves increasing the size of the presynaptic store of releasable transmitter; we will determine whether LTF works the same way. Finally, we plan to measure the spatio-temporal profile of changes in cAMP concentration accompanying serotonin action and LTF.
Serotonergic regulation of synaptic transmission is a critical locus for control of complex behaviors such as learning and memory. Pharmacological modification of synapses and serotonergic pathways are increasingly used to treat a large variety of neurological and psychiatric disorders. This project provides the basic knowledge essential for the understanding of complex behaviors and the development of therapeutic strategies.

A major research instrumentation grant has been awarded to Dr. Ehud Isacoff at the University of California at Berkeley for the purchase of a 2-photon scanning microscope. The research goal is to probe, non-invasively, by microscopic photometry and imaging, the distribution and structure of macromolecules (RNA, DNA and proteins), and their activity and interactions in living cells. The 2-photon microscope makes it possible to image cells and tissues with high spatial and reasonable temporal resolution, with penetrating excitation, but also with optically confined irradiation, thus limiting photo-damage to cells and photo-destruction of fluorescent tags. The instrument will form a centerpiece of a recently built Berkeley Imaging Center. The Imaging Center will provide access, training and technical support for use of the 2-photon microscope for students, postdocs and faculty in the biological sciences on campus and in the Lawrence Berkeley National Laboratory, and will make the instrument available to academic researchers at other Bay Area campuses.

The research projects conducted with the 2-photon microscope will include; 1) studies on synaptic proteins that have been engineered to be fluorescent and to change their fluorescence upon either activation or interaction with other proteins, 2) optical probes of neural activity to measure the function of neural circuits involved in visual information processing, 3) organic and protein-based indicator dyes to study the roles of calcium ions in synaptic transmission, short- and long-term plasticity, 4) the regulation of other second messenger systems and, 5) fluorescently labeled proteins to study the localization of postsynaptic receptors, and the changes in proteins involved in exocytosis during synaptic transmission.

The aim of the research using the 2-photon microscope is to employ innovative forms of microscopy that can characterize the dynamics of molecular machines in their physiological environments: inside intact cells and tissues.

This is a proposal for a Small Grant for Exploratory Research, to
develop methods and procedures for using fluorescence resonance energy
transfer (FRET) to dynamically measure changes in the structure and
interactions between SNARE proteins during and following neurotransmitter release. SNARE proteins form a complex that docks synaptic vesicles to the plasma membrane and holds them in readiness for secretion by exocytosis triggered by neuronal action potentials. Component proteins will be labeled with fluorescent probes (cyan and yellow fluorescent proteins, CFP and YFP), and FRET between those probes will be used to measure dynamic changes in the SNARE components. The main thrust of the proposal is to develop and perfect the methodology of dynamic FRET detection. As techniques are refined, they will be applied to detecting the assembly and disassembly of SNARE proteins, which should occur before secretion and during recovery of fused vesicles by endocytosis, respectively. This will be accomplished by labeling vesicle associated membrane protein (VAMP) with CFP and SNAP-25 (soluble N-ethylmaleimide-sensitive fusion protein attachment protein of 25 KDaltons) with YFP. A second application involves
the detection of vesicle fusion, accomplished by labeling the extracellular domain of syntaxin with CFP and the intravesicular domain of VAMP with YFP. Procedures will be developed for quantitative evaluation of FRET, and for simultaneous detection of vesicle fusion, by monitoring the FRET-induced change in CFP fluorescence along with the fusion-associated pH-change-induced increase in YFP fluorescence.

This is a novel, high-risk project. Its successful conclusion would be a major methodological breakthrough, enabling the dynamic study of conformational changes among secretory proteins, and allowing the sequencing of molecular events during vesicle priming, fusion, and
recovery. This project will serve as proof of concept for subsequent
regular research proposals to NSF or NIH. Results will be disseminated
broadly by publication and discussion at open meetings. In its broader
impacts, the project will involve the advanced training of a ostdoctoral fellow, contributing to the national scientific talent bank. Undergraduate assistants receive an opportunity for immersion in cutting edge research. Previous NSF support has involved the training of high school teachers of minority students in modern scientific methods (supported by a Research Experience for Teachers supplement to an NSF Research Grant). Finally, development of new techniques strengthens the methodological infrastructure of modern biological research.

Lay Summary

NSF Grant 0413936
Neural and Glial Mechanisms Program
Division of Integrative Biology and Neuroscience
Directorate for Biological Sciences

Synapses are the points of information transfer between neurons and their targets. Synaptic transmission is regulated by neuromodulators and prior activity. This synaptic modifiability greatly increases the complexity of information processing at synapses, it underlies the adaptability of the nervous system to changing circumstances, and it is responsible for its ability to learn. Crayfish neuromuscular junctions (NMJs) are used to study the effects of prior activity and extrinsic modulation. This preparation offers many advantages, including ready availability and minimal cost, easy preparation, and suitability for electrophysiological recording, and physiological and biochemical properties identical to those of mammalian brain synapses
Serotonin is a circulating hormone that regulates the amount of the chemical transmitter (glutamate) released by motor nerve terminals when they are electrically active. The biochemical pathways involved in serotonergic regulation will be explored. In particular, the project will test the hypothesis suggested by preliminary evidence that serotonin operates in part by causing the production of cyclic adenosine 3',5'-monophosphate, which in turn activates an enzyme called exchange protein activated by cyclic AMP (Epac) to modify vesicular proteins involved in making synaptic vesicles containing glutamate available for release. Normally cyclic AMP must also activate another protein (hyperpolarization and cyclic nucleotide-activated ion channels or HCNCs) to enhance transmission, but following extensive electrical activity, HCNCs are not needed for serotonin to have an effect. The hypothesis that under these circumstances serotonin acts only via the Epac pathway will be tested. It is also planned to extend these experiments to fruit fly (Drosophila), because this would allow genetic manipulations not feasible with crayfish. A final aim of this project is to understand the mechanisms responsible for differences between phasic motor neurons, which are very effective in releasing a lot of glutamate to single action potentials, versus tonic synapses, which are unresponsive to single action potentials but show tremendous synaptic facilitation in releasing glutamate to a train of action potentials. Biophysical experiments using photosensitive chemical probes of molecular function ("caged calcium" chelators) will be used to distinguish these synapses. The project will involve training junior scientists for future careers in teaching and research, with an emphasis on recruiting women and minority participants as has been done in the past.

DESCRIPTION (provided by applicant): The specific goals of this project are: 1) Development of methods and procedures for detecting conformational changes in SNARE proteins during and after secretion. This is a developmental/exploratory proposal for a project whose chief goal is the development of fluorescence resonance transfer (FRET) as a dynamic measure of changes in the structure and binding of SNARE proteins during and following the release of transmitter in neurons. Cultured hippocampal cells will be transfected with green fluorescent protein (GFP)-Iabeled presynaptic SNARE proteins, and procedures will be developed and perfected to detect changes in the spatial relationships and interactions between these proteins during and following secretion that will be triggered by depolarization. 2) Application of FRET to detecting SNARE complex assembly and disassembly. The N-termini of SNAP-25 and VAMP become closely associated during SNARE complex formation, and dissociate when SNARE complexes are disassembled. These changes will be monitored by FRET following secretion of the readily releasable pool of vesicles, when new vesicles dock and SNARE complexes assemble, and when SNARE complexes of previously exocytosed vesicles are disassembled prior to endocytosis. 3) Application of FRET to detection SNARE complex reorientation following vesicle fusion. The C-termini of VAMP and syntaxin come together on the external surface of the plasma membrane on vesicle fusion. We will attempt to detect this reorientation during secretion, and measure its lifetime before vesicle recovery by endocytosis. These are novel applications of the FRET technique to studies of synaptic transmission, and require solution of numerous technical obstacles. Success will open wide possibilities for the study of dynamic changes in protein structure during a variety of cell activities, and pave the way for the study of protein structural alterations in diseased tissue.

Synapses are the functional points of interaction between neurons, where information in one neuron is passed onto and processed by another. At some synapses, action potentials cause secretion of transmitter with a high efficacy, and a train of action potentials rapidly depletes the readily releasable pool of vesicles resulting in the diminishing responses of synaptic depression. At other synapses, release to single action potentials can be rare, while the probability of release facilitates in a train of action potentials. This project will explore why some synapses are strong, but weaken on use, while others are initially weak, but become stronger on use.

Crayfish neuromuscular junctions are functionally similar to mammalian central nervous system synapses while offering significant advantages. At leg extensor muscles, phasic motor neuron synapses transmit strongly and show depression, while tonic motor neuron synapses initially transmit extremely weakly but facilitate tremendously. Previous work showed both synapses to be structurally similar, to admit similar amounts of Ca2+ per action potential, and to have similar numbers of vesicles available for release to a train of action potentials. Phasic and tonic synapses differ dramatically in their secretory responses to steps of presynaptic calcium concentration ([Ca2+]i) evoked by photolysis of photosensitive calcium buffers (liberation of "caged calcium"). A computational model was constructed in which the differences between phasic and tonic synapses are due to vesicles at tonic synapses being less likely to be "primed" and ready for immediate release, while [Ca2+]i accumulating in an action potential train primes vesicles for later release. The goal of this project is to test this model, and revise or refine it as necessary, to gain a more complete understanding of this crucial form of regulation of synaptic strength and plasticity.

The project will involve the training of junior scientists to assume independent posts in research and university teaching, the teaching of specialized techniques to graduate students, exposure of undergraduates to and involvement in the scientific research process, and the training of secondary teachers in the scientific method and research procedures. Minority and women students and scientists have been and will continue to be recruited as participants. Collaborations with domestic and foreign scientists are proposed. Active participation in national and international scientific forums will be pursued. In addition, specialized techniques of broad use in biology will be refined.

DESCRIPTION (provided by applicant): The synapse is the point of functional contact between neurons, where information is communicated between cells. Each electrical impulse in the presynaptic neuron leads to the secretion of chemical neurotransmitter, which is packaged at the presynaptic active zone in small membrane-bound vesicles. Fusion of the vesicles with the plasma membrane results in liberation of transmitter molecules, which bind postsynaptically to receptor channels and signal the postsynaptic cell of the presence of presynaptic activity. Vesicle fusion is accomplished by a molecular machine consisting of several components. Vesicles are docked at the membrane by the SNARE complex of vesicular membrane protein VAMP (vesicle associated membrane protein) and the plasma membrane proteins syntaxin and SNAP-25 (soluble NSF-attachment protein of 25 kD). Presynaptic impulses open calcium channels to admit calcium to the cytoplasm, where it binds to the vesicular protein synaptotagmin, which interacts with SNAREs and the plasma membrane to initiate fusion. SNAREs must assemble before arrival of action potentials in order for vesicles to be primed for release. After fusion, SNAREs must disassemble before the vesicle membrane is recovered by endocytosis and recycled for reuse. Some models of vesicle fusion involve the tightening of the SNARE complex as an early step in fusion. The timing of these processes is unknown, and they have never been measured directly. This project will use fluorescence resonance energy transfer (FRET) interactions between fluorescent tags on vesicle and plasma membrane components of SNAREs to characterize their assembly, disassembly, and conformational changes on fusion. Specifically, cerulean-SNAP-25B and citrine-VAMP-2 N-terminal interactions, and VAMP-2-cerulean and syntaxin-1-citrine C-terminal interactions, will be studied in cultured rat hippocampal neurons under field stimulation. FRET will be measured by enhanced exciter emission or by reduced donor emission on donor excitation, and by two-photon fluorescence lifetime imaging microscopy (FLIM). The dispersion and re-aggregation of SNAP-25 following secretion will also be analyzed. This project will advance the specific goal of the NIH Roadmap of developing innovative tools to study interactions between individual proteins within single cells. Understanding the molecular mechanisms of synaptic function provides an essential foundation for the development of rational and effective therapeutic interventions to treat synaptic dysfunctions underlying numerous neurological disorders. Understanding the molecular machinery of synaptic transmission is a specific priority of the NINDS Strategic Plan in furtherance of the NINDS primary mission of reducing the burden of neurological disease.

DESCRIPTION (provided by applicant): This proposal requests a new training grant in physical biosciences, to replace the expiring non-renewable NSF IGERT grant in Physical Biosciences. The program will be administered by the interdisciplinary Graduate Group in Biophysics. Its 35 faculty mentors are drawn from the departments of Bioengineering, Chemical Engineering, Chemistry, Integrative Biology, Molecular and Cell Biology, Physics, Psychology, Public Health, and Vision Science. The 12 trainees may matriculate in any participating department, but must have a research focus in one of five research areas in biomedical imaging and bioengineering in which this campus excels, namely: 1) single molecule / single cell mechanics and dynamics, 2) molecular microscopy and optical-electrical probes, 3) brain imaging and systems neuroscience, 4) computational biology, and 5) biomechanics and animal locomotion. The program attracts physical scientists to the quantitative study of biological problems, and trains them flexibly and broadly for emerging disciplines, making use of the extraordinary research facilities of UCB and the adjacent Lawrence Berkeley National Laboratory, with whom many of the faculty are associated. No other training grant on campus supports these areas. The program is characterized by intensive advising, tracking, and counseling, and individualized curricula designed to provide breadth and depth appropriate to each trainee's interests and background. Initially, trainees take graduate level survey and comprehensive courses, followed by specialized laboratory and focus courses, directed reading and oral presentation courses, and graduate seminars. They attend an annual biophysics retreat, laboratory meetings, and journal clubs, receive teaching experience, and perform original dissertation research in the final years. The expiring IGERT program has attracted outstanding students and provided core support for the first two years of study, supplemented with Graduate Student Instructor stipends, plus UCB, individual NRSA, and private foundation scholarships that support additional trainees. Minority applicants have been welcome and are succeeding in the program. This grant will allow UCB to continue in its tradition of training the best biomedical researchers who provide the scientific foundation for the improvement of modern medical practice and extending the health and well being of the American public.