Synaptic transmission is regulated by calcium-dependent plasticity signals in the brain. Most calcium signals arise from voltage-dependent calcium channels which are activated by neural activity. Extensive research has identified how particular patterns of activity drive plasticity. For example, in the case of spike-timing dependent plasticity, the timing between glutamatergic input and postsynaptic APs determines the sign and magnitude of plasticity. Although calcium signals evoked by activity are necessary for the generation of plasticity, the mapping between calcium signals and specific plasticity responses is still unclear. In this proposal, we hypothesize that accounting for distortions in measurements of calcium signals imposed by the use of fluorescent calcium indicators will elucidate how calcium signals activate synaptic plasticity. To address this hypothesis, we have established a method of measuring calcium currents, which are independent of the distortion imposed by fluorescent calcium indicators. In Aim 1, we will develop a complete model of synaptic calcium handling to enable measurements of physiological calcium signals. In Aim 2, we will apply these methods to understand how coincident activation of glutamatergic input and postsynaptic action potentials drives calcium signals and whether it can explain the time course of spike-timing dependent plasticity. Inhibition can spatially restrict and prevent plasticity from occurring. This function is important for associative learning and is disrupted in human neurological diseases. However, methodological challenges have prevented investigations of the mechanisms of inhibitory control of plasticity. In Aim 3, we will combine our method of measuring calcium currents with two new technologies that enable extremely detailed investigations of inhibitory control of synaptic calcium signals. We will measure how inhibition modulates synaptic calcium signals and determine the effect of this modulation on the induction of plasticity. In summary, this proposal provides an important methodological innovation that will significantly progress our understanding of fundamental neural functions. The results of these investigations will provide a framework for understanding how synaptic plasticity is regulated and may guide the development of therapies for neurogenerative disease.