Synaptic transmission plays a key role in the information processing mechanism of the nervous system. One major focus of our research is the basic mechanisms underlying synaptic transmission. Our research is characterized by the use of identified neurons and muscle fibers and the integrated system composed of those identified cells. In this way, the data obtained from the component cells can be applied directly to the analysis of the system's function. We use a multidisciplinary approach that combines physiological, immunocytochemical, electron microscopical and genetic techniques. More specifically, our research is characterized by the use of conditional single-gene mutants of Drosophila that reversibly affect a particular step of the transmission process. One such mutant is shibire ts, in which the process of endocytosis is blocked when the temperature is raised to 29°C but is normal at 19°C. Since exocytosis is not affected by the mutation, transmitter release at the synapse proceeds normally at 29°C until depletion of synaptic vesicles occurs as a result of the blockage of recycling of synaptic vesicle membrane. Thus, the number of synaptic vesicles in the synapse can be experimentally regulated. Another mutant is cha ts, in which the activity of choline acetyltransferase, the enzyme involved in the synthesis of acetylcholine, is reversibly blocked at 29°C. Using this mutant, the amount of transmitter in cholinergic synapses can be experimentally regulated.
By using these two conditional mutants, which allow us to manipulate two key properties of the transmission process (i.e., transmitter substance and the synaptic vesicles), we are able to ask questions that cannot be addressed in any other way. For example, by using the shi mutant to precisely regulate the number of vesicles in the synapse, we are able to observe the relationship between the number of vesicles at a synapse and the e.j.p. amplitude of that synapse. Also, by depleting the synapse at 29°C using shi and then allowing recycling to begin in a synchronized fashion by lowering the temperature to 19°C, we were able to observe the various steps in the recycling pathway in a sequential manner. Observing vesicle recycling following depletion at 29°C has led to the important discovery that two distinct recycling pathways coexist in the terminal. One pathway emanates from sites away from the active zone and involves collared pits and/or cisternae, while a second, much faster pathway emanates directly from the active zone membrane adjacent to the dense body and involves direct pinch-off of vesicles from the plasma membrane. These two pathways appear to replenish two distinct vesicle populations--a small active zone population and a larger, nonactive zone population dispersed throughout the terminal cytoplasm. We have further demonstrated that the active zone pathway is Ca2+-dependent, while the nonactive zone pathway is not. Using differences in the time of recovery of vesicles from the depleted state of these two pathways, we have been able to determine the individual contribution of each of them to spontaneous and evoked release.