The synaptic connections between nerve cells define the neural circuits responsible for animal behavior. Changes in behavior can result from changes in the properties of these synapses (synaptic plasticity). Specifically, synapses can be strengthened by increased use and this activity-dependent synapse strengthening plays an important role in the development of the brain, and in learning and memory in the adult. It is difficult to study synaptic plasticity in the mammalian brain due to its enormous complexity. However, the effect of activity upon the development of specific synaptic properties can be studied in simpler organisms where many synapses are identifiable (the same synapse can be located in different animals). The synapses between neurons and muscle fibers in fruit fly (Drosophila) larvae are ideal for these studies since these synapses are identifiable, accessible and amenable to genetic manipulations.
The development and plasticity of processes involved in regulating intracellular calcium concentration ([Ca2+]i) are important for synaptic transmission as well as many other neuronal functions. For instance, [Ca2+]i influences transmitter release, neuronal excitability, gene expression, neuronal growth, neuronal death and learning and memory. Ca2+ plays a particularly crucial role at the presynaptic terminal. Here, Ca2+ enters through voltage-dependent channels producing a brief, localized and large increase in [Ca2+]i at the mouth of the channel, which triggers the release of transmitter. Then Ca2+ equilibrates in the terminal and this residual [Ca2+]i serves multiple functions, such as increasing subsequent transmitter release (synaptic facilitation). The amplitude and duration of the residual [Ca2+]i is largely determined by mechanisms that remove or clear Ca2+. These Ca2+-clearance mechanisms include Ca2+ extrusion from the synaptic terminal by the plasma membrane Ca2+ ATPase (PMCA).
Preliminary studies in Drosophila show that active synaptic terminals develop stronger Ca2+ clearance than inactive ones. This is likely to be an important mechanism in synaptic strengthening since it allows the synapse to limit the increase in[Ca2+]i when activated at high frequencies. The proposed experiments will examine the activity-dependent strengthening of Ca2+ clearance including: defining the Ca2+ clearance mechanism(s) that are strengthened by impulse activity and identifying the molecular signaling pathway(s) involved in linking impulse activity to long-term changes in Ca2+ clearance. In addition, since the PMCA appears to be largely responsible for Ca2+ clearance from these terminals, the expression of this protein will be directly examined. Finally, the consequences of activity-dependent changes in Ca2+ clearance will be explored. In particular, it is hypothesized that changes in Ca2+ clearance will result in metaplasticity; i.e., the changes in Ca2+ clearance will affect subsequent synaptic plasticity, such as synaptic facilitation. This will be directly examined at these synaptic terminals.
These experiments will involve measuring changes in [Ca2+]i at synaptic terminals using fluorescent Ca2+ indicators and sensitive imaging techniques. Specific gene mutations will be used to alter synaptic activity and perturb signaling pathways. Biochemical techniques, immunocytochemistry and confocal microscopy will be used to look at levels of the PMCA.
The findings of these studies will advance our knowledge of activity-dependent changes in synapses and increase our understanding of the basic mechanisms responsible for the development of the nervous system. The synapse is a particularly important locus for experience-dependent changes in brain structure and function leading to adaptive behavioral changes. Thus, these studies will further define the role of experience in specifying neuronal structure and function, and the range of adaptation of the nervous system to altered use. This research will also provide important descriptive information that will be valuable to other Drosophila researchers working on this important model system.
The results of these studies will be widely disseminated through attendance at international meetings and publications in international journals. The research projects will include participation by undergraduate students, including underrepresented minority students. This will provide an entry point for these students to pursue a career in scientific research and teaching. In addition, the proposal will fund the research of graduate students pursuing advanced degrees in the Biological Sciences. Finally, the results of this research will be communicated to students in a Neurobiology lecture course and the experimental techniques will be included in a Neurobiology laboratory course. Overall, the scientific community benefits from the training of future researchers and the general population benefits from the enhancement of science literacy.
