This project will use a combination of analytical, computational and experimental techniques to gain a deeper understanding of the synaptic release of neurotransmitter, termed exocytosis. It will address such open questions as the precise sequence of steps linking calcium ion entry into the cell to exocytosis, the involvement of individual calcium channels in neurotransmitter release, and the mechanisms of transient calcium-dependent facilitation of synaptic response, presumably caused by the accumulation of calcium at the exocytosis site. More specifically, this project will focus on the impact of intrinsic and externally applied calcium binding substances termed calcium buffers on exocytosis and its facilitation, using mathematical and computational modeling of calcium diffusion inside the cell. The importance of calcium buffers stems from the fact that they absorb more than 95% of calcium ions entering the cell; further, the sensitivity of exocytosis and facilitation to applied buffers provides one of the most widely used methods to probe the intrinsic calcium sensitivity of these processes. The first specific goal of this project is to reveal the effect of such buffers on the cooperativity of individual calcium channels in triggering exocytosis of a single vesicle, and to re-examine the so-called calcium current cooperativity measurements that probe the arrangement of channels at the release site. The second specific goal is to examine the proposed non-local property of the buffer saturation phenomenon, whereby the transient whole-terminal depletion of free buffer by calcium influx through one group of channels may cause subsequent opening of the same or another group of channels to produce a greater calcium elevation. Further, the competition between two calcium buffers in their regulation of intracellular calcium will be explored. Finally, this project will examine the possible functional consequences of synaptic facilitation for the dynamics of neural circuits.
Neurotransmitter release at chemical synapses (exocytosis) represents the most common form of communication between two neurons in any biological neural system, including the mammalian cortex, and the knowledge of its mechanisms is indispensable for a full understanding of inter-neuronal interactions and neural information processing. Further, the regulation of neurotransmitter release and binding to receptors represents the main pharmacological treatment method in many neurological and psychiatric pathologies. The importance of this project stems from its combined use of advanced computational tools and experimental physiological techniques in gaining deeper understanding of the neurotransmitter release process, known to depend on the action of calcium ions inside the cell. This project will focus on the precise sequence of exocytosis steps starting with the entry of calcium through cell membrane calcium channels, their subsequent accumulation and binding to intracellular calcium-binding substances, and ending with the calcium-triggered release of neurotransmitter-filled vesicles into the synapse. This investigation should also lead to a better understanding of other vital physiological processes controlled by calcium ions, from gene expression regulation to muscle cell contraction in the heart. Further, this project will involve the use and further development of a publicly accessible computational modeling tool called CalC ("Calcium Calculator"), designed by the Principle Investigator for the modeling of three-dimensional calcium ion diffusion inside the cell (www.calciumcalculator.org). This will contribute to the infrastructure for computational modeling in the biological sciences and will also serve as a useful training instrument in the fields of cell neurophysiology and biophysics. All modeling results obtained in the course of this project will be made publicly available through the on-line model database, ensuring the most rapid and effective dissemination of the obtained results. Finally, this project will create student training opportunities in the highly interdisciplinary fields of mathematical biology, biophysics and computational neuroscience, including the training of students from under-represented ethnic groups, since this work will be conducted in an institution with one of the most ethnically diverse student bodies in the country (NJIT).
Using advanced computational modeling techniques, this project explored the time course of calcium ion diffusion inside neuronal cells, validating the results of the modeling work through collaboration with experimental neurophysiologists. Understanding of cell calcium ion dynamics is an important goal since calcium controls practically all vital physiological processes, from cell fertilization and gene expression to cell growth and muscle contraction. This project focused on a very fundamental calcium-dependent cell process termed vesicle exocytosis, whereby several calcium ions bind to certain control proteins, triggering the fusion of tiny intracellular container organelles called vesicles with the outer cell membrane, and allowing the contents of a vesicle to be released into the extracellular space. In neuronal synapses connecting together pairs of neurons, exocytosis (fusion) of neurotransmitter-filled vesicles causes their neurotransmitter cargo to be released into the narrow synaptic gap separating two neurons, and thereby allowing activity of one neuron to influence the activity of another neuron. Although the general mechanisms of vesicle exocytosis have been revealed in recent decades, the precise sequence of steps and the amount of calcium required for vesicle fusion are still poorly understood. One difficulty is that calcium inside a cell is not evenly distributed but is highly localized, entering through membrane-spanning proteins called calcium ion channels. Calcium elevation remains constrained within the small region surrounding calcium channels, in part through the action of calcium-binding intracellular molecules termed calcium buffers, which limit the spread of calcium away from the channel. Another difficulty is that calcium concentration is very hard to measure experimentally, since such measurement requires adding extra buffers that emit light upon calcium binding, thereby distorting the very signal they are supposed to measure. For this reason computational modeling has played a central role in the understanding of calcium-regulated processes. This project continued and extended this modeling approach, providing new results that help to tackle the following concrete questions: 1) To what degree do individual calcium ion channels cooperate in triggering exocytosis of a single neurotransmitter-containing vesicle? Is an opening of a single calcium ion channel enough to trigger exocytosis of a vesicle? This project rigorously re-examined the experimental protocol used to answer this question, which involves a partial pharmacological block of synaptic calcium channels. The results of this work greatly help in the correct interpretation of such channel block experiments, allowing to infer the arrangement of vesicles and channels on small spatial scales. 2) How do the properties of calcium buffers influence the time course of calcium ion diffusion at a synaptic terminal? This project is one of the first to explore the impact of more realistic calcium buffers with multiple calcium binding sites, as compared to previous modeling efforts that focused for simplicity on buffers that can only bind a single calcium ion, even though such simple buffers are rare in biology. 3) How does synaptic transmission strength change in response to calcium ion accumulation inside a cell? Changes in the reliability of synaptic transmission caused by changes in synaptic calcium concentration have very important consequences for the activity of any real neuronal circuit, creating a memory effect whereby the transmission efficiency changes depending on the previous history of synaptic activation. This project elucidated the mechanisms of such calcium-dependent changes in synaptic properties in a well-studied invertebrate neuronal circuit. 4) How is the activity of a neural circuit affected by the properties of synapses connecting these cells? One of the results of this project was to show that neurons connected by strong inhibitory synapses display a surprisingly rich activity repertoire compared to neuronal circuits coupled by weak synapses. This project also contributed to the computational modeling infrastructure in the broader area of cell biophysics and physiology through the continued development of the cell calcium diffusion modeling software tool called CalC ("Calcium Calculator"). CalC is publically available at the website maintained by the PI of this project (www.calciumcalculator.org), and is used by several laboratories throughout the word, not only in the study of synaptic function, but also in the modeling of calcium-dependent contraction of cardiac myocytes. The results of this project should also shed light on other calcium-dependent cell processes, for instance hormone release by endocrine cells and immune system’scytotoxic T-cell activation, both of which relie on calcium-dependent vesicle exocytosis. Finally, this project contributed to the creation of student training opportunities in the highly interdisciplinary fields of mathematical biology, biophysics and computational neuroscience. Four graduate students were directly involved in different specific aims of this project, of which two are women, helping to alleviate the gender gap in science and technology careers. One of the two female graduate students has already completed her Ph.D. degree based on her work on this project, and is pursuing a career in mathematics education, contributing to the development of human resources in science and math education.
