Voltage Gated Sodium Channels (VGSCs) are the fundamental molecular basis of neuronal excitability. VGSCs render neurons as exquisite sensors of incoming synaptic signals that can be amplified to generate what are classically thought of as ?all-or-none? output signals - action potentials (APs). VGSC functions include initiation and propagation of APs, promotion of repetitive or burst spikes, and boosting of synaptic currents,- actions that depend on localization to distinct subcompartments of the neuron. However, manipulating VGSC function and therefore membrane excitability of spatially confined subcompartments of cells is challenging given currently available tools. In response to this need, we have developed a class of synthetic photocaged saxitoxin compounds (STX-PC) that enable fine spatial, temporal, and reversible control of membrane excitability via VGSC block. We propose 1) to develop first and second generation STX-PC variants for single- and multi-photon uncaging and to characterize their performance for in vitro and in vivo applications, 2) to provide proof of concept evidence that these tools are validated to assess defects in macro- and micro-scale VGSC-mediated axonal AP propagation in wild-type versus genetic epilepsy models with altered VGSC (Scn8a) function. At the macro scale we will test the ability of uncaged STX-PC to block AP propagation in the major axon fiber tract of the corpus callosum in vitro and in vivo. At the micro scale will validate the use of STX-PC to study the roles of VGSCs in defined subcompartments of the cell (e.g., axon initial segment) in promoting spike initiation and back propagation. These tools also have therapeutic potential, which will be piloted in Scn8a mice. This work we view as the critical step in creating and validating tools that can be subsequently used by researchers asking mechanistic questions about VGSC function in normal physiological activity and how this is disrupted in neuropsychiatric diseases. A prime example would be epilepsy in which mutant VGSCs with both gain- and loss-of-function are associated with a variety of seizure disorders.
In the brain, electrical communication between neurons depends on a particular protein called sodium channels that are found along the output fibers of neurons called the axons. In the proposed research, we will develop a new class of light activated chemical that can be used to rigorously study axon function through highly localized blockade of sodium channels in axons. These chemical tools will be useful in studies of how sodium channel disruption can lead to neurologic disease and may someday become useful in treatments that could correct problems with axon function in disease.