E .A. Pehek et al. Brain Research 888 2001 51 –59
53
were made by dissolving the drug in 2.5 ml of glacial h of perfusion and sample collection without drug. The
acetic and 1 ml deionized water. aCSF was then utilized to vehicle group was perfused with aCSF throughout.
dilute this solution to the appropriate concentrations 100 nM, 1.0 mM, 10 mM, and 100 mM, pH 7.4. 6-DOI-
2.5.3. Experiment 3 hydrochloride Research Biochemicals Incorporated, MA,
USA was administered subcutaneously s.c. DOI 2.5 2.5.3.1. Effects of lower concentrations of M100,907 on
mg kg or vehicle deionized water were injected in 1.0 DA efflux. Experiment 3A examined the effects of 1.0 mM
1
ml kg volumes. M100,907 on K -stimulated DA efflux. Drug treated rats
1
were compared to high K controls. Procedures were the
same as in Experiment 1. Experiment 3B determined the 2.4. Chromatography
effects of lower concentrations of M100,907 on basal dialysate DA. Following the collection of stable baselines,
DA content of dialysate samples was measured by 100 nM concentrations were infused for 2 h. This was
HPLC coupled with electrochemical detection. Twenty- followed by perfusion with 1.0 mM concentrations for 2 h
microliter dialysis samples were injected onto a 2 mm in the same rats.
Phenomenex column Ultracarb E, 3 mm particle size,
ODS 20. The mobile phase consisted of 32 mM citric 2.5.4. Experiment 4
acid, 54 mM sodium acetate, 0.074 mM EDTA, 0.215 mM octylsulfonic acid, and 3 methanol vol vol, pH 4.2. To
2.5.4.1. Effects of DOI and M100,907 on cortical DA maintain separation of DA from its metabolites and 5-
release. This study examined the effects of a systemic hydroxyindoleacetic acid, the pH of the mobile phase and
injection of DOI on DA efflux in the mPFC. The receptor the concentration of octylsulfonic acid were adjusted as
specificity was examined by investigating the ability of needed. A BAS LC-4C electrochemical detector with a
intracortical infusions of M100,907 10 mM to attenuate glassy carbon electrode, maintained at a potential of 10.60
the effects of DOI. Four groups of rats were utilized in this V relative to an Ag AgCl reference electrode, was em-
study: DOI alone, vehicle, M100,907 alone, M100,9071 ployed. The limit of detection for dopamine was 0.1 pg 20
DOI. The first group received a s.c. injection of DOI while m
l. the second group received vehicle injections. A third group
was perfused intracortically with 10.0 mM M100,907 for 3 h. The last group received similar M100,907 infusions plus
2.5. Experimental design a systemic injection of DOI. DOI was administered 30 min
after the start of perfusion with M100,907. 2.5.1. Experiment 1
2.6. Data analysis Data were expressed, analyzed, and graphed as the
1
2.5.1.1. Effects of M100,907 on K -stimulated cortical percentage of the last 3 baseline samples. Statistical
DA release. This experiment tested the ability of a high analyses
were performed
using repeated
measures
1
K infusion to increase extracellular DA in the mPFC.
ANOVAs. For two-way ANOVAs, time was the repeated
21
Furthermore, it tested the Ca -dependency of this effect
measures factor and drug condition was the independent
1
and the ability of M100,907 to modulate the K -evoked factor. For one-way ANOVAs, time was the repeated
release. Baseline samples were collected from all rats factor. Post-hoc comparisons employed Dunnett’s test for
utilizing normal Ringer’s. One group of rats then received comparing treatment means with a control value.
1 21
infusions of a high K buffer with normal Ca
1.2 mM
21
while a separate group was perfused with a Ca -free high
1
K solution. Two other groups were pretreated with either
3. Results
1
10 or 100 mM M100,907 before infusions of high K
21
both with normal Ca .
3.1. Experiment 1
1
2.5.2. Experiment 2 3.1.1. K effects on DA concentrations
1
Fig. 1 shows that treatment with high K increased
extracellular mPFC DA levels to 265 of baseline 2.5.2.1. M100,907 effects on basal DA. Microdialysis
[F9,8154.54, P,0.001, one-way repeated measures samples were collected from three separate groups of rats:
ANOVA]. Post hoc tests indicated that DA efflux was vehicle controls and those treated with either 10 or 100
significantly increased at the 30 min time point after
1 21
m M M100,907. After stable baseline samples were col-
infusion with high K . Removing Ca from the medium
lected, the animals were perfused for 60 min with abolished this effect [F9,3651.57, n.s.]. Basal DA
M100,907, dissolved in aCSF, followed by an additional 3 concentrations were 0.6660.08 pg 20 ml n510 for the
54 E
1 21
Fig. 2. The effects of intracortical infusions of M100,907 on high Fig. 1. The effects of intracortical infusions of high K
and Ca -free
1 1
K -induced DA release. Values are expressed as the percentage of 3 high K
Krebs–Ringer solution on extracellular DA concentrations. pre-drug baselines and are the means6S.E.M. of the subjects. For the
Values are expressed as the percentages of 3 pre-drug baselines and are drug group, the bar indicates that M100,907 was infused for 60 min from
the means6S.E.M. of the subjects. The bar indicates that the solutions
1 1
time 230 to time 30, and high K was co-infused for 30 min from time 0 were infused for 30 min from time 0 to 30. indicates that high K
1
to time 30. For the non-drug group, the high K solution was infused for
significantly increased extracellular cortical DA concentrations P,0.05.
1 21
30 min from time 0 to time 30. indicates that high K significantly
This was blocked by removal of Ca from the perfusion medium no
1 21
increased extracellular mPFC DA concentrations P,0.05. Co-adminis- significant increase in DA. Group ns were: high K 510; Ca
-free high
1
tration of M100,907 at either concentration blocked this increase. Group K 55.
1 1
1
ns were: high K 510; 10 mM1high K 56; 100 mM1high K 59.
1
high K group and 0.7560.14 pg 20 ml n55 for the
21
group that was subsequently treated with a Ca -free high
were 0.9760.17 pg 20 ml n59 for the 100 mM MDL
1
K solution.
100,907 group and 0.5460.08 pg 20 ml n56 for the 10
1
m M group. The high K
group is from Fig. 1.
1
3.1.2. Effects of M100,907 on K -induced DA release
1
Treatment with M100,907 attenuated K -stimulated DA 3.2. Experiment 2
release in a concentration-dependent manner see Fig. 2. Dialysate DA only increased to 126 and 155 of
3.2.1. MDL effects on basal DA efflux baseline values following infusions of 100 mM or 10 mM
Fig. 3 demonstrates the effects of intracortical infusions M100,907, respectively. When the 100 mM concentration
of 10 and 100 mM M100,907 on basal DA outflow. There
1 1
plus high K was compared to the high K
alone group, was a significant main effect for drug [F2,2353.41,
there was a significant drug3time interaction [F9, 1535 P50.051. This was due to decreases in DA efflux after
2.38, P,0.015, two factor repeated measures ANOVA]. the drug perfusion was stopped maximal decreases, 100
Post-hoc analyses of each condition over time revealed that m
M: 55 of baseline at the 120 time point; 10 mM: 63
1
the high K infusion did NOT significantly increase DA
of baseline at the 150 time point. Basal DA concentrations efflux when 100 mM M100,907 was co-infused [F9,
were 0.8260.21 pg 20 ml n56 for the 10 mM group, 7251.99,
P50.053, one-way,
repeated measures
0.5560.09 pg 20 ml n511 for the 100 mM group, and ANOVA]. The trend towards a significant F value was due
0.6860.07 pg 20 ml n57 for the vehicle group. to decreases maximal decrease563 of baseline in DA
concentrations at the 120 and 150 min timepoints i.e. 3.3. Experiment 3
90–120 min after the termination of the M100,907 perfu- sion. The 10 mM concentration also blocked the effects of
3.3.1. Effects of lower concentrations of M100,907 on
1
high K , as demonstrated by the lack of a significant time DA efflux
1
effect for this group [F9, 4551.93, P50.07]. The trend Infusions of 1.0 mM M100,907 did not alter K -stimu-
towards significance was again due to a decrease in DA to lated DA efflux Fig. 4A. Likewise, perfusion with 100
60 at the 120 min time point. Basal DA concentrations nM or 1.0 mM M100,907 did not alter basal DA efflux
E .A. Pehek et al. Brain Research 888 2001 51 –59
55
Fig. 3. The effects of intracortical infusions of M100,907 at 10 mM and 100 mM concentrations on basal extracellular DA concentrations. Values
are expressed as the percentages of 3 pre-drug baselines and are the means6S.E.M. of the subjects. The bar indicates that the solutions were
infused for 1 h from time 0 to time 60. DA concentrations were significantly decreased P50.05. Group ns were: vehicle57; 10 mM58;
100 mM511.
Fig. 4. The effects of intracortical administration of low concentrations of M100,907 on extracellular DA concentrations. Values are expressed as the
percentages of 3 pre-drug baselines and are the means6S.E.M. of the
1
Fig. 4B. Basal dialysate concentrations were 0.4660.09
subjects. A: Effects of 1.0 mM M100,907 on K -stimulated DA efflux.
1
n55 for the M100,9071high K group, 0.5560.05 n5
For the drug group, the bar indicates that M100,907 was infused for 60
1
1
min from time 230 to time 30, and high K was co-infused for 30 min
6 for the high K controls, and 0.5260.11 n58 for the
1
from time 0 to time 30. For the non-drug group, the high K solution was
100 nM 1.0 mM group.
infused for 30 min from time 0 to time 30. This concentration of
1
M100,907 did not alter high K -induced DA release. Group ns were:
1 1
high K 56; M100,9071high K 55. B: Effects of 100 nM and 1.0 mM
3.4. Experiment 4
concentrations on basal DA efflux. The arrows indicate the beginning of each drug infusion. 100 nM was infused first, for 2 h, followed by 1.0
m M, for 2 h. There were no significant effects on DA efflux. Group n was
8.
3.4.1. MDL effects on DOI-stimulated DA release Statistical analyses performed on the raw data pg 20
m l demonstrated that systemic administration of 2.5 mg
kg DOI increased dialysate DA concentrations [F6,245 group, and 0.4060.03 n55 for the DOI1M100,907
3.69, P50.01]. Post-hoc tests revealed a significant in- group.
crease 30 min post-injection Fig. 5. DA concentrations in vehicle animals did not change over time [F6,1850.62,
n.s.]. Pretreatment with an intracortical infusion of
4. Discussion
