Real-Time Measurements of Phasic Changes in Extracellular Dopamine Concentration in Freely Moving Rats by Fast-Scan Cyclic Voltammetry - Drugs of Abuse: Neurological Reviews and Protocols - page 440

is then lowered by small increments until electrically evoked dopamine release is
optimized. The electrode can be secured in position by the locking device on the
micromanipulator. At this time the rat is allowed to habituate to the environment
(~30 min) before commencing the experiment ( see Note 8 ).

3.5.3. Synchronization of Electrochemical Data with Behavior

The electrochemical and behavioral data must be synchronized for precise
correlation of neurochemistry and behavior. A video character generator
superimposes the fi le and scan number from the data acquisition computer onto
the behavioral video recording in real time ( see Note 9 ).

3.6. Electrode Calibration
Carbon fi ber microelectrodes are calibrated in vitro with known concentra-
tions of dopamine ( see Note 10 ). Electrodes are lowered via the micromanipula-
tor into the fl owstream of a fl ow injection apparatus, perfused with Tris-HCl
buffer. The fl ow injection apparatus is equipped with an injection loop, in which
the analyte of interest (dopamine) is loaded. The carbon fi ber microelectrode
and a reference electrode (submerged in the buffer) are connected to the
headstage and the voltammetric waveform is applied. The timing of the injec-
tion of dopamine into the fl owstream is controlled by the data acquisition
software. Because of the linearity of the response, one or two concentrations of
dopamine (~1-2 M ) are suffi cient to calibrate the electrode. Calibrations are

done in triplicate and the average value for the current at the peak oxidation
potential is used to normalize in vivo signals to dopamine concentration.

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3.7. Data Analysis
Data analysis is carried out using software locally written in LabVIEW. The
chemical integrity of each signal is checked from the cyclic voltammogram.
Once a signal is confi rmed to be dopamine, the concentration is determined
from the current at the peak oxidation potential ( see Notes 11-13 ).

3.8. Verifi cation of Electrode Placement
To obtain unequivocal evidence of placement, histological methods must
be used to verify the sampling site. Because the fi ber is so small, a noticeable
tract will not be seen using standard histological methods. However, a lesion
can be made at the tip of the electrode that can be visualized using histological
techniques ( see Note 14 ).

1. The rat is anesthetized with 160 mg/kg of intramuscular ketamine and 24 mg/kg
of xylazine, and an electrolytic lesion is made with a stimulator (100 A, 5 s;

anode connected to the carbon fi ber microelectrode, cathode connected to a
rectal probe).

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