Cyclic Voltammetry for the Detection of Dopamine in vivo

1. Cyclic Voltammetry for the Detection of Dopamine in vivo

2. Dopamine • Neurotransmitter • small molecule chemical messenger • Important for motor and cognitive functions • Deficts in dopamine levels cause Parkinson Disease • Regulates reward • Dopamine increases after drugs of abuse like cocaine Structure of Dopamine. 4-(2-aminoethyl) benzene-1,2-diol

3. Dopaminergic Neurons • Dopamine is synthesized in dopaminergic neurons and packaged into membrane bound vesicles • Electrical action potential initiates the release of dopamine • Dopamine vesicles undergo exocytosis • Spills out into the extracellular space • Can be detected The dompaminergic neuron can excytos dopamine vesicles into the extracellular space where its contents can be detected by target neurons.

4. Detection of Dopamine • Exocytosis of dopamine from vesicle occurs on a milisecond time scale • Sensor must be fast, sensitive, and selective since dopamine concentrations are low • Fast scan cyclic voltammetry is the dominant electrochemical technique used

5. Volametry • Electrochemical technique in which the current (I) is measured as a function of voltage (Eapp). • There are several kinds: • Linear Scan voltammetry (Polarography) • Differential pulse polarography • Square-wave voltammetry • Cyclic Voltametry

6. Three Electrode Set Up 1. Working Electrode: Redox reaction • limited reaction surface area in order to limit current flow 2. Auxillary Electrode: Consists of an inert material (Hg, Pt) • does not participate in redox reaction • completes electrical circuit • Reference Electrode: constant potential reference (SCE) • establishes electrical potential Modern Three Electrode Set Up

7. Cyclic Voltammetry • A potential is applied between the reference electrode and the working electrode. • The electrode potential ramps linearly at the experimental scan rate (V/s) and the current is measured between the working electrode and auxillary electrode • Data is plotted as Current (I) vs. Potential(E). Forward scan produces a current peak for the analytes that have been reduced in scanned potential range.

8. Cyclic Voltammetry • The direction of the potential is reversed at the end of the each scan in cyclic voltammetry • Current increases as the potential reached the reduction potential of the anlayte • Current decreases as the analyte is depleted in the redox reaction

9. Experimental Results • In a recent study by Venton and Wightman (2003), rats were implanted the carbon-fiber microelectrode electrode at thieir nucleus accumbens, a part of the brain associated with reward among other functions. • A biopolar stimulating electrode was implanted in the substantia nigraventra until its electrically evoked release was observable at the carbon-fiber microelectrode. • After collection, background subtraction and digital filtering were performed. • To remove the background and charging currents, the collected set of cyclic voltammograms was subtracted from the average of the first 10. • The peak oxidation potential for dopamine is 0.65 V, which was the condition use.

10. Results Electrical Stimulation • Subtracted voltamogram taken at the end of stimulation (above) resembles one recorded in a solution containing dopamine. • Subtracted voltamogram taken five sconds after stimulation ended (below) is due to changes in the residual current caused by a basic pH change that originates from a change in local blood.

11. Results Cocaine Influsion 52s after infusion 22s after infusion 62s after infusion • Subject rats were infused with saline (i.v., 20 s per infusion), followed by 0.3 mgkg, 1.0 mgkg, and 3.0 mgkg cocaine infusions at 15-min intervals. • Subtracted cyclic voltammograms recorded at various times after the infusion as seen above. • The cyclic voltammograms represented with the dashed lines are those obtained with electrical stimulation.

12. Results • The similarities between subtracted cyclic voltamograms of electrical stimulation and cocaine infusion indicate a similar change in electroactive species after both events. • While this change in electroactive species is a composite of multiple substances, the two major contributors are dopamine and pH, determined through regressional analysis.

13. Advantages • Allows for the product of the electron transfer reaction that occurred in the forward scan to be probed again in the reverse scan • Can be used to determine of formal redox potentials, detect of chemical reactions that occur before or after a electrochemical reaction, and evaluate electron transfer kinetics

14. Carbon Fiber Microelectrodes • Carbon Fiber Microelectrodes (CFME) are electrodes in which a carbon fibers serve as the electroactive area • Advantages: • Carbon fibers are biological compatible to cells • Small size (less than 10um in diameters) allows for implantation in vivo • Commonly used with cyclic voltammetry Carbon Fiber Microelectrode