All forms of life rely on biochemical processes and these processes are either accelerated or inhibited according to the concentration of protons (pH) in their immediate vicinity. The pH changes that occur within nerve cells are therefore predicted to have a profound influence on the ability of nerve cells to transfer and store information. However, it is very difficult to measure or control pH in the nerve cells of live animals, and as a result the influence of pH changes on the nervous system, is difficult to test. Here, the investigators propose to use optical techniques, combined with genetically-encoded proteins, to measure and control the pH of live nerve cells in fruit flies (Drosophila melanogaster). Fluorescent proteins will be used to measure pH, and light-activated proton pumps will be used to control pH. When deployed in the same cell, these proteins will provide the means for establishing dynamic control of pH. These tools will be invaluable for investigating the extent to which the pH fluctuates in nerve cells during naturally occurring activity, and for testing the role of these pH changes in maintaining the capacity of the nerve cells to transfer and store information. Further, the development of these tools for fruit fly nerve cells will facilitate their application in other fields of the biological sciences, from mycology and plant physiology to renal physiology and oncology. Transgenic animals and plasmids generated through this project will be made available directly to the scientific research community. Information regarding the performance and application of the tools will be made available through publications in scientific journals and presentations at scientific meetings. This project will directly provide research training for an MD/PhD graduate student and training opportunities for PhD graduate students, undergraduate students, high-school students and K-12 teachers.
All biochemical reactions are sensitive to the acid-base balance of their environment - also referred to as the pH of their environment. Although pH does change during nerve activity, the pH sensitivities of many fundamental neurophysiological processes have yet to be elucidated. The main reason for this lack of progress is that tools to simultaneously control and measure pH in cellular processes and organelles have not been developed. Such tools would provide the means to test critical hypotheses, not just in the field of neurophysiology, but also in other fields of the biological sciences, such as mycology, plant physiology, renal physiology and oncology. Broadly, our goal was to develop tools to optically measure and control pH in various cellular compartments in the nervous system in vivo. For the purpose of pH measurement we created several transgenic fruit fly (Drosophila) lines that allow the expression of genetically-encoded fluorescent pH indicators in the cytosol of nerve endings. Data collected from live fruit fly larvae using these tools revealed that the pH within nerve endings acidifies to a far greater degree than was previously appreciated. These finding were published in the Journal of Physiology. The fruit flies are now freely available to other researchers. Data regarding nerve activity dependent acidification of mitochondria, the organelles that power communication between nerves, have also been collected. These data, which are very informative about how nerves manage their energy supply, are being prepared for publication. These fly lines will also be made freely available. For the purpose of pH control we created transgenic fruit fly lines that allow the expression of genetically-encoded proton-selective channels and pumps in nerve cell membranes. These channels and pumps are light responsive and provide the means to control the pH of the nerve cell using light (they are optogenetic tools). We continue to refine the design and performance of these tools along with the hardware and software programs that will allow us to integrate rapid pH measurement and control in various cellular compartments. This project provided for the involvement and development of a number of graduate, undergraduate and high school students. Most directly it provided research training for an MD PhD graduate student (who successfully defended their dissertation in July 2013), and a high-school student in their first and second years of an immersive biomedical research education and college preparatory program.
