Targeted mutagenesis to elucidate the function of understudied ion channels in the central nervous system

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Project Summary/Abstract Many types of neurons utilize complex and highly specific patterns of action potential firing to regulate neurotransmitter release and communicate with downstream cells. Action potential firing is primarily regulated through neurotransmitter input as well as through the expression of ion channel genes that determine the baseline firing properties of an individual neuron. While some of these ion channels are well studied, many remain unexplored or underexplored. Dopamine neurons in the ventral tegmental area (VTA) exhibit tightly controlled activity patterns, including bursts and pauses in activity, which encode information about environmental cues and rewards and influence learning and motivation. It is believed that disruptions in these firing patterns contribute to a variety of behavioral perturbations associated with mental illness. Thus, VTA dopamine neurons provide an excellent model system for investigating the function of understudied ion channels within their native neuronal environment. A recent study identified the entire complement of ion channels expressed in dopamine neurons in mice; I have selected for study three of these channels that also appear on the Illuminating the Druggable Genome (IDG) list of understudied proteins: Cacna2d3, encoding the 2-3 calcium channel auxiliary subunit, Kcna6, encoding the Shaker potassium channel KV1.6, and Kcnab2, encoding the KV2 potassium channel auxiliary subunit. I will utilize a novel, single vector adeno associated viral system that takes advantage of CRISPR/Cas9 gene editing technology to rapidly induce gene mutation in a cell-type specific (Cre-dependent) manner in neurons of adult mice. I will then use slice electrophysiology and fast-scan cyclic voltammetry in combination with optogenetics to characterize the effects of individual ion channel gene knockout and determine how these understudied ion channels regulate dopamine neuron physiology and dopamine release dynamics. Completion of this research will establish a simple, single virus technique for rapidly and specifically inducing gene knockout, which will be widely applicable to investigations of understudied proteins. Additionally, by identifying novel regulators of dopamine firing patterns we will both increase our understanding of the underlying physiology driving these critical neurons and identify new potential targets for precision therapeutics.