The spatiotemporal patterns of second messenger signals and interactions among pathways are central to the integration and processing of synaptic information and to regulating the strength of synaptic connections. The general goal of this proposal is to understand the role of calcium (Ca) signaling in the synaptic function of cerebellar Purkinje neurons. Our focus is on the properties and function of Ca signals produced by inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), which mediate Ca release from intracellular stores. Our hypothesis is that the Ca release pathways function as integrators of synaptic activity, and that this property is the cellular basis for associativity and synapse specificity during the induction of a form of synaptic plasticity known as cerebellar long-term depression (LTD). My previous studies established that Ca release from intracellular stores via the IP3-sensitive pathway in Purkinje neurons is centrally involved in the induction of LTD and that Ca release by IP3 receptors is dynamically regulated by both IP3 and cytosolic Ca in a way that suggests this pathway acts as a coincidence detector for the integration of synaptic signals. Other studies have also implicated nitric oxide (NO) as another key determinant of LTD. The studies proposed here have two primary objectives. The first goal is to understand how the spatial and temporal dynamics of Ca release in Purkinje cell dendrites are regulated by the interplay between Ca signaling pathways and other second messenger cascades involved in LTD. The second goal is to determine the functional consequences of these interactions for the induction of LTD. For these experiments, we will use a combination of high-speed confocal microscopic Ca measurements, electrophysiology, localized photolysis of caged compounds, and pharmacological manipulations of signaling pathways in acute cerebellar slices. We first test the idea that amplification of synaptic Ca signals by IP3Rs and RyRs mediates associativity between synaptic inputs during the induction of LTD. We then examine the relationship between Ca release and NO in the induction and spatial spread of LTD, with particular focus on a model in which NO regulates LTD by enhancing the sensitivity of IP3Rs and RyRs. The results of these studies should clarify how synaptic signaling pathways contribute to the induction of LTD, and give insight into fundamental mechanisms by which biochemical computation regulates communication between neurons and shapes the signal processing capabilities of individual neurons. This information is important for understanding the control of motor function by the cerebellum and the cerebellar dysfunction that underlies ataxia and other motor problems.
