Presenter: Anna Everett
Authors: Anna Everett, Ben Graul, Daniel Watts, Kayden Robinson, Jordan Yorgason
Faculty Advisor: Jordan Yorgason
Institution: Brigham Young University
Fast-scan cyclic voltammetry (FSCV) is a highly effective electrochemistry technique for measuring rapid dopamine (DA) release and clearance, particularly in the nucleus accumbens and dorsal striatum. The DA terminals in these areas are abundant, and DA concentrations are heavily regulated by the dopamine transporter (DAT). Historically, the Michaelis-Menten model has been a standard in measuring DAT enzymatic kinetics. However, this approach has historically used experimenter-fitted modeling and is therefore extremely subject to experimenter bias. This study presents a novel, unbiased analysis technique using the first (velocity) and second (acceleration) derivatives of evoked DA signals. Maximal upwards derivatives predict changes in DA release due to D2 and GABAB agonist-mediated DA release inhibition. Maximal downwards derivatives are indicative of DA reuptake that is relatable to the Michaelis-Menten Vmax, an indirect measure of DAT activity based on the number of expressed DATs. When evoked DA release in DATKO mice was subtracted from DA release in WT mice, DAT kinetics were accurately modeled. Increasingly negative velocities correlated with higher DAT activity. Critically, the Michaelis-Menten model is dependent upon an enzyme-specific constant, precluding its usage in DATKO (or similar enzymatic knockout) mice. In addition to DATKO data, the effect of two competitive, high-affinity DAT inhibitors (PTT and WF23) and the lower affinity inhibitor cocaine were analyzed to study relative effects on velocity and acceleration measures. All three inhibitors had clear effects on increasing upward velocity and decreasing downward velocity, with greatest effects on downward velocity at highest concentrations. These tools provide opportunities to study DA kinetics in situations previously inaccessible using current methodologies.