In
Vivo Microdialysis and HPLC
1) Microdialysis Collection Protocol
Probes
& Collection Setup: CMA/10 (Carnegie/Medicin) dialysis probes
pictured at left are used in our laboratory to collect dialysates from
discrete brain nuclei. The probes have a relative recovery of approximately
20%. 
The
inlet arm of the probe is connected by PE tubing to a 1.0 ul syringe (Exmire/CMA)
that is driven by a CMA/100 microinfusion pump. The flow rate of the pump
is adjusted to 1.0 ul per minute and the probes are perfused continuously
during microdialysis experiments with artificial cerebrospinal fluid (aCSF)
(189 mM NaCl, 3.9 mM KCl, 3.37 mM CaCl adjusted to pH 6.3). The outlet
arm of the probe is connected by PE tubing to collection vials that contain
0.1 N acetic acid in 13 ul of DHBA (1.5 pg/ul) which serves as an internal
standard during HPLC analysis.
Microdialysis
Test Chamber: The test chamber consists of a clear plastic bowl (CMA/120
System for Freely Moving Animals) that measures 115 cm in diameter and
35 cm in height. The floor of the chamber is covered with identical shaving
chips that are used in the animals home cage. Food and water are available
ad libitum during the microdialysis procedures.
Collection Procedures: The microdialysis experiments are generally
divided into 3 phases consisting of habituation, baseline collection and
experimental treatment. Habituation: On the day of testing the
animals are transported to the lab and left undisturbed for 20 minutes.
Afterwards, the microdialysis probe is inserted through the guide cannula
and perfused continuously with aCSF at a flow rate of 1 ul per minute
by a CMA/100 microinfusion pump. No microdialysate samples are collected
during this one-hour period.Baseline Collection: Baseline collection
of dialysate samples generally begin 1 hour and 20 minutes after the start
of the experiment. One baseline sample is collected every 20 minutes for
a period of 1 hour. Each of these samples contain 20 ul of dialysate and
13 ul of 0.1 N acetic acid and DHBA for a total volume of 33 ul. Experimental
Treatment: Drug or placebo injections are initiated two-hours and
20 minutes into the experiment. The substances to be administered and
the sequence of their delivery during the treatment period vary in each
experiment. Dialysate samples of norepinephrine are collected every 20
minutes. After collection, all samples are sealed with parafilm and stored
on ice until they are assayed with HPLC. The entire experiment lasts approximately
4-6 hours.
2)
Norepinephrine Assay with HPLC
Dialysate
samples (33 ul) of norepinephrine are analyzed using reverse-phase liquid
chromatography with electrochemical detection (Shown on the left). Norepinephrine
is assayed by an HPLC system with a Waters 510 pump, Waters 717 autosampler,
Axxiom Chromatography column (5 micron ODS, 25 cm) and an ESA Coulochem
Model 5100A electrochemical detector. The mobile phase consist of 50 mg
disodium EDTA, 13.8 mg monobasic sodium phosphate and 58 mg octane sulfonate
adjusted to pH 3.2 by adding 85% phosphoric acid. The flow rate is adjusted
to 0.9 ml per minute. At the end of each experiment the microdialysate
samples are loaded into the Waters 717 auto sampler (temperature 7°
Celsius) and automatically injected and analyzed with an average analysis
period of 35 minutes per sample. The limits of detection for catecholamines
with this HPLC system is approximately 2 pg. Norepinephrine concentrations
and peak heights are measured in comparison to peak heights and concentration
of an internal norepinephrine standard (2, 4, 16 and 20 pg/ul). The concentration,
peak height, and retention time for dialysate samples of norepinephrine
are saved and stored into a computer by the Waters Millenium database
program. A sample chromatogram illustrating the peak heights and retention
time of separate norepinephrine standards is shown below.
The findings from a representative in vivo microdialysis study conducted
in our lab (Williams, Men, & Clayton, 2002) is shown in the figure
below:
Figure Legend. Mean percent change in amygdala norepinephrine concentrations
produced by microinfusion of PBS or clenbuterol (CLN: 50 or 100 ng/0.5
ml) into the NTS, escapable footshock (0.6 mA, 1s), or ip injection of
saline or epinephrine (Epi. 0.3 mg/kg). Injection into the NTS of a dose
of CLN which improves cognitive functioning (i.e. 100ng) produced a significant
increase in amygdala norepinephrine concentrations that exceeded that
collected during baseline (** p < .01) or that produced by NTS infusion
of PBS (*p < .05). Administration of Epi. in doses that improve memory
produced a significant elevation in extracellular norepinephrine concentrations
20 and 40 min post injection (** p < .01, * p < .05). The concentration
of norepinephrine sampled following Epi. injection was also significantly
greater than that produced by saline (p < .05, t-tests).
Cedric
Williams
The University of Virginia
Psychology Department