Cyclic Voltammetry for the Detection of Dopamine in vivo

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

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.

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

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

Three Electrode Set Up

1. Working Electrode: Redox reaction• limited reaction surface area

in order to limit current flow2. Auxillary Electrode: Consists of

an inert material (Hg, Pt)• does not participate in redox

reaction• completes electrical circuit

3. Reference Electrode: constant potential reference (SCE)• establishes electrical

potential

Modern Three Electrode Set Up

•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.

Cyclic Voltammetry

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

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.

Results

•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.

Electrical Stimulation

•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.

22s after infusion 52s after infusion 62s after infusion

ResultsCocaine Influsion

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.

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

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