All organisms' ability to successfully live and function within their own habitat depends on cell-to-cell communication that is necessary for the most basic reflexes to the highest order thought processes. This cellular communication by neurons occurs by specialized signals that rapidly allow a chemical transmitter to be released from one neuronal cell and have an effect on an adjacent neuronal cell. It is not understood how these signals can be modulated to cause more or less chemical transmitter to be released, or to release one type of chemical transmitter preferentially over another. Previous work has established that two chemical transmitters, neuropeptide Y (NPY) and catecholamines (CAs), that are packaged into the same small vesicles undergo differential release. That is, NPY requires a longer stimulation duration and/or a higher frequency of stimulation than required to release CAs from the same vesicles. How this differential regulation of chemical transmitter release occurs is not known. The aims of this research will test the hypothesis that different members of a family of proteins, called synaptotagmins (syt), sense and control calcium-dependent release of the chemical transmitters from the vesicles. This project will utilize a combination of electrophysiology, molecular biology and biochemistry techniques in a model secretory cell system to study how these proteins may regulate release of NPY compared to the CAs. The objectives of this study are 1) to determine what types of stimuli are required for differential release, 2) to determine whether a Ca2+-dependent , or 3) a Ca2+-independent syt protein regulates differential release of chemical transmitter. Intellectually, this project will establish whether one protein that functions to sense Ca2+ and trigger secretion of transmitter can also preferentially determine the release of one particular transmitter over another.
BROADER IMPACT: These results will have broad application to understanding how one type of vesicle can preferentially release transmitter from the same population of vesicles, a basic mechanism that underlies neuronal communication. Results from this project will provide the basis for future research directions to understand how transmitter is regulated in release not only from secretory cells and neurons, but also from brain tissues. Education initiatives will involve students at all levels. The PI will continue to a) participate in an outreach science program for middle school inner city girls, b) participate in a highly competitive high school student research program (STARS), c) involve undergraduate students in research from simple lab techniques to Honors research theses, and d) train graduate students pursuing their PhDs to enter the academic arena. Students at all levels will be introduced to the disciplines of biophysics, molecular biology and chemistry as a combined approach to research. Current efforts to recruit female and minority researchers will be continued and expanded. All generated cell lines will be freely distributed to other researchers. To disseminate findings from this project, students will continue to present results at national conferences, and publish their work in peer-reviewed journals. As an outreach program for high school and undergraduate students, as well as a tool for all researchers to use, the students will provide lay descriptions of their projects and their scientific protocols on the laboratory's website. This research program will provide a platform for integrating research with education for students, scientists, and the lay community.
In this project, we have pursued three major research questions. The first research question involves differences in release mechanisms for different types of neurotransmitters, neuropeptide Y, a large peptide that is packaged into large vesicles, and a small soluble transmitter family of catecholamines that are packaged in both large and small vesicles. We have found that the peptides require longer periods of stimulation and higher amounts of stimulation, whether it is higher electrical stimulation or higher concentrations of activating chemicals, to evoke the same release as the soluble small transmitters. We have performed these experiments in both culture models of neurons and acutely isolated neuronal cultures, utilizing multiple types of population and single cell methods for measuring the released neurotransmitters. The second research project included specifically targeting a protein made by neurons that regulate the release mechanisms of neurotransmitter from the membrane enclosed vesicles, called synaptotagmin I. We have found that the large vesicles are more dependent on synaptotagmin I, whereas the family of soluble catecholamines are less dependent for release on synaptotagmin I. When we measured release with specific amounts of synaptotagmin I present on the vesicles, from 0 – 100% of control levels of synaptotagmin I protein, we found that synaptotagmin I is required for the peptide release from the large vesicles, but not the soluble transmitters from the small vesicles. We have postulated that the release mechanism of synaptotagmin I acting to release vesicles is needed to open the fusion pore formed when the vesicle membranes and the cellular membranes fuse. Therefore, another protein can function in this capacity than only synaptotagmin I for the small vesicles, but not for the large vesicles. The third and last research project involved reducing a second synaptotagmin that is postulated to work in tandem, and possibly substitute for, synaptotagmin I and that is also found on the vesicle membranes, synaptotagmin IV. We found that when both proteins are removed from vesicles, there is a larger effect on inhibiting release of vesicles than with either one alone. We have also increased expression of synaptotagmin IV by upregulating expression of this protein by chemical treatment as well as overexpressing a plasmid that expresses the synaptotagmin IV protein. We have measured the results of increased expression levels of synaptotagmin IV in both control cells, and from cells that have had synaptotagmin I removed from the vesicle membranes to determine the effects that overexpression of synaptotagmin IV has on catecholamine release from individual cells. We find that synaptotagmin IV does not function redundantly for synaptotagmin I. We have published these results in peer-reviewed journals. Many students including high school, undergraduate, summer research students, graduate student, and medical students, were trained in various stages of their academic careers while working on these projects.
