Microdialysis Coupled with Electrochemical Detection - Drugs of Abuse: Neurological Reviews and Protocols - page 412

neurotransmitter measurement also can be accomplished by implanting a probe
and perfusing it each day for several days. We assayed DA in the nucleus
accumbens from 9 A . M . to 7 P . M . for 8 d. We noticed that at d 1 and 2 basal levels
of DA were stable, but from d 3 they kept decreasing to become undetectable at
d 8, probably due to reactive gliosis clogging of the fi ber (1 , 7) .

3.1.5. Two-Probe Experiment
Two probes can be implanted in an animal when the difference of the drug
effect in two different regions of brain has to be ascertained. Recently this type
of study has established a different response to drugs of abuse between the shell
and the core region of the nucleus accumbens. No particular diffi culties are
encountered in implanting two probes but a two-channel swivel is mandatory
to avoid twisting of perfusion tubing (16) .

3.2. General Considerations
3.2.1. Working Day
A typical microdialysis working day begins with preparation of perfusion
Ringer. It can be quickly prepared, and diluted single salt stock solutions (100X)
kept in the refrigerator. Stock solutions prepared and fi ltered under vacuum
(0.22

m) can last up to 3 mo. Filling syringes and connecting tubing may
require about 1 h if four to six animals are used. Because samples recovered
from the probe in the fi rst hour of perfusion do not refl ect a steady state of the
neurotransmitter assayed, the samples collected during the fi rst hour can be
discarded. This hour can be used to prepare and inject some standards into the
HPLC. The DA solution can be prepared daily and kept shielded from light
under ice. To extend the life of standards, 0.1 M perchloric acid and 0.5 m M
sodium metabisulfi te can be added to 10 m M DA stock solution, which can to
be kept as individual samples in a deep freezer (-80°C). At least four or
fi ve concentrations, in the range of that found in the sample recovered from the
area investigated, must be injected. Thereafter samples can be assayed. Usu-
ally after a sharp decrease at beginning of perfusion, the concentration of
neurotransmitter tends to remain constant, although it depends on the brain
area under investigation. When the last three samples differ from each other
<10% the animal can be tested and the mean of the last three samples can
be considered as a reference basal. Stable levels of neurotransmitters can be
obtained in about 2 or 3 h after start of dialysis ( see Notes 5-7) .

µ

3.2.2. Collecting Samples
In the last decade we have collected samples using PE tubing (0.58 i.d.,
0.96 o.d.; Portex, Hythe, UK) used to connect probes to the perfusion syringe.
A 12-cm long coil is positioned in the probe outlet fi tting tightly the 23-gauge