Spatial, temporal, and context-dependent features of GPCR-mediated protein kinase A activity

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The spatial specificity, temporal dynamics, and context dependence of neuromodulator-induced intracellular signals are essential to explain neuromodulator function. However, although the identity of many signaling molecules downstream of neuromodulator receptors are known, the nature and functions of these features are poorly understood. The long-term goal is to uncover the cellular and subcellular specificity, the temporal dynamics, and the context-dependence of neuromodulator-induced intracellular signals. The overall objective here is to determine the features and synaptic functions of acetylcholine (ACh)-mediated protein kinase A (PKA) activity in the hippocampus. The central hypothesis is that ACh regulates PKA with spatial, temporal, and context-dependent specificity that is essential to synaptic plasticity. The rationale for this project came from multiples lines of evidence. First, Gαq-coupled muscarinic ACh receptors (mAChRs) elevate PKA activity. Second, PKA activity demonstrates rich spatial, temporal, and context-dependent features. Third, perturbations of the spread and duration of PKA activity alter cellular and behavior functions, illustrating the importance of its spatiotemporal dynamics. Finally, mAChRs and PKA are both powerful regulators of synaptic plasticity. The central hypothesis will be tested in both acute hippocampal slices and head-fixed mice, with three specific aims to determine the subcellular compartments (Aim 1), the temporal dynamics (Aim 2), and the context- dependence (Aim 3) of PKA activation by ACh and the roles of these features for synaptic plasticity. To determine the features of mAChR-mediated PKA activity, optogenetics will be used to induce ACh release, and ACh level and PKA activity will be measured with novel biosensors and two-photon fluorescence lifetime imaging microscopy (2pFLIM). To determine the contribution of these features to synaptic plasticity, subcellular compartment-targeted, light-activated actuators will be used to perturb PKA activity with spatial and temporal precision, and electrophysiology will be used to measure synaptic transmission. The proposed research is innovative because conceptually, it goes beyond the identity of molecules to revealing their actions, goes beyond static snapshots to revealing signaling dynamics, and goes beyond knowing the involvement of a signal to revealing their contributions. Methodologically, the research employs cutting-edge technology to induce neuromodulator release, and to measure and perturb intracellular signals with spatial and temporal precision – these approaches will find widespread application in cellular signaling beyond neuromodulator research. The proposed research is significant because it will offer explanatory power on how features, and not just identity of intracellular signals, shape cellular physiology and behavior. These results will reveal new principles of neuromodulator action, and provide insights into how molecular mechanisms general behaviorally relevant features. In the long run, these results will help design better therapies that target the relevant features in neurological and psychiatric disorders.