Download M100,907, a selective 5-HT antagonist, attenuates dopamine

Brain Research 888 (2001) 51–59
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Research report
M100,907, a selective 5-HT 2A antagonist, attenuates dopamine release
in the rat medial prefrontal cortex
a,
a,b
a
a
a
E.A. Pehek *, H.G. McFarlane , K. Maguschak , B. Price , C.P. Pluto
a
Department of Psychiatry, Case Western Reserve University School of Medicine, Kenyon College, Gambier, OH 43022, USA
b
Department of Psychology, Kenyon College, Gambier, OH 43022, USA
Accepted 19 September 2000
Abstract
Previous research has suggested that serotonin 5-HT 2A receptors modulate the functioning of the mesocortical dopamine (DA) pathway.
However, the specific role of 5-HT 2A receptors localized within the medial prefrontal cortex (mPFC) is not known. The present study
employed in vivo microdialysis to examine the role of this receptor in the modulation of basal and K 1 -stimulated (Ca 21 -dependent) DA
release. The selective 5-HT 2A antagonist M100,907 was infused directly into the mPFC of conscious rats. This resulted in a
concentration-dependent blockade of K 1 -stimulated DA release. Intracortical application of M100,907 also blocked increases in DA
release produced by the systemic administration of the 5-HT 2A / 2C agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). These
findings demonstrate that local 5-HT 2A antagonism has an inhibitory effect on stimulated, Ca 21 -dependent DA release. They suggest that
cortical 5-HT 2A receptors potentiate the phasic release of mesocortical DA.  2001 Elsevier Science B.V. All rights reserved.
Theme: Neurotransmitters, modulators, transporters and receptors
Topic: Interactions between neurotransmitters
Keywords: Antipsychotic; In vivo; Mesocortical DA pathway; Microdialysis; Schizophrenia; Serotonin; DOI
1. Introduction
The mesocortical dopamine (DA) system has been
implicated in a wide range of emotional, motivated, and
cognitive behaviors. Neurons in this pathway originate in
the ventral tegmental area of the midbrain and terminate in
the medial prefrontal cortex (mPFC) [40]. Several in vivo
studies have demonstrated that cortical DA neurochemistry
is modulated by serotonin (5-HT) receptors [5,10,12].
Examination of the interactions between DA and 5-HT is
thus central to the understanding of psychotropic drug
action in the prefrontal cortex.
In vitro and in vivo neurochemical studies in the dorsal
and ventral striatum indicate that 5-HT 2 receptors regulate
DA function [9,26]. There is also evidence that 5-HT 2
*Corresponding author. VA Medical Center GMH(B), 10000 Brecksville Rd., Brecksville, OH 44141, USA. Tel.: 11-440-526-3030 ext.
6610; fax: 11-440-546-2713.
E-mail address: [email protected] (E.A. Pehek).
receptors regulate DA function in the mPFC. For example,
administration of the atypical antipsychotic drug clozapine,
a potent 5-HT 2 antagonist [20], increases extracellular DA
concentrations in vivo when administered either systemically or directly into the mPFC [15,21,28]. In addition,
systemic administration of amperozide, a 5-HT 2A antagonist [33,37], and intracortical administration of ritanserin, a
5-HT 2 antagonist, were both found to elevate dialysate DA
levels in the mPFC [15,24,29,30]. Thus, each of these
potent 5-HT 2A antagonists increased cortical DA efflux
suggesting that 5-HT 2A receptors inhibit DA release from
the mesocortical DA system.
Although there are three known subtypes of the 5-HT 2
receptor, the 2A, the 2B, and the 2C [32], many of the
studies that have attempted to examine the role of these
receptors in mesocortical DA function have used ligands
that are not subtype selective. Those experiments that have
used selective ligands have generally employed the systemic administration of such agents [7,10]. Since systemically administered drugs can have actions at multiple
sites in the brain, the literature remains unclear as to the
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52
E. A. Pehek et al. / Brain Research 888 (2001) 51 – 59
specific role of cortical 5-HT 2A receptors in the modulation
of mesocortical DA function.
In the present study, we examined the role of prefrontocortical 5-HT 2A receptors through the use of the
highly selective antagonist M100,907 (R-(1)-a-(2,3dimethoxyphenyl) - 1 - [2 - (4 - fluorophenylethyl)] - 4 - piper idine-methanol). This ligand has 100 fold selectivity for
the 5-HT 2A (Ki50.36 nM) over the 5-HT 2C (Ki5105 nM)
receptor [25] and has negligible affinity for DA receptors
(Ki.540 nM) [14]. M100,907 was infused directly into
the mPFC through reverse microdialysis. Effects on both
basal and potassium (K 1 )-stimulated DA release were
examined. Additionally, the effects of intracortical
M100,907 on alterations in DA release produced by the
systemic
administration
of
1-(2,5-dimethoxy-4iodophenyl)-2-aminopropane (DOI), a 5-HT 2 agonist,
were investigated. We hypothesized that intracortical infusion of a selective 5-HT 2A antagonist would increase DA
release in the mPFC.
2. Materials and methods
2.1. Animals and surgery
Male Sprague–Dawley rats (Zivic Miller, Hillson, PA,
USA), weighing from 200 to 400 g at the time of surgery,
were used throughout this study. Rats were housed in pairs
in a temperature-controlled room on a 12 / 12 light / dark
cycle. Food and water were available ad libitum. Prior to
surgery, the rats were anesthetized with a mixture of
ketamine (70 mg / kg) and xylazine (6 mg / kg) injected
i.m., and then mounted in a stereotaxic frame. After dura
was removed, 21 gauge stainless steel guide cannulae were
chronically implanted on the brain surface above the
mPFC (AP 3.2, ML 0.8) [27]. The guide cannulae were
secured to the skull with three skull screws covered with
cranio-plastic cement. Animals were then housed individually for the 3–5 day period between surgery and
microdialysis experiments.
Each rat was used once, and after the experiment was
concluded, probe placements were verified histologically.
Only animals whose probe placements were verified to be
in the mPFC (see below) were used in the study. All
animal use procedures were in strict accordance with the
NIH Guide for the Care and Use of Laboratory Animals
and were approved by the local animal care committee.
2.2. Microdialysis
Microdialysis probes of a concentric flow design were
used [43]. Probes were constructed to dialyze the mPFC
from the dorsally located anterior cingulate cortex, through
the prelimbic cortex, and including the ventrally located
infralimbic subregion. The active dialyzing surface of the
membrane (Spectra / Por Hollow, MW cutoff513 000,
diameter5200 mm) was 5.0 mm in length. At 18 to 24 h
before the start of the experiments, microdialysis probes
were lowered carefully through the guide cannulae into
awake rats and secured in place with Krazy Glue GelE.
Immediately following probe implantation, animals were
placed in clear Plexiglas test chambers and tethered to
counterbalance arms that permitted relatively free movement. They remained there with food and water until the
start of the experiments.
A micro-infusion pump (PHD 2000E, Harvard Apparatus) and liquid swivels were used to perfuse the buffer
through the probes at a constant rate. Although each
experiment is self-contained with its own appropriate
controls, perfusion flow rates differed between some
experiments. An initial experiment (Experiment 2) employed a flow rate of 1.5 ml / min. In order to increase the
recovery of DA, the flow rate was decreased to 1.0 ml / min
in all subsequent experiments. Dialysate samples were
collected every 30 min until basal DA concentrations were
stable for at least 3 baseline samples. Drugs or artificial
cerebrospinal fluid (aCSF) buffers with altered ionic
compositions were then administered by manually switching tubing connections. This was performed rapidly and
flow rate and collection volumes were maintained. Sample
collections then continued every 30 min for another 2.5 to
3.5 h depending on the experiment.
For experiments examining the effects of M100,907 on
basal or DOI-induced DA release (Experiments 2 and 4), a
modified, commercially available, aCSF was employed:
Dulbecco’s phosphate buffered saline (137 mM NaCl, 2.7
mM KCl, 0.5 mM MgCl 2 , 1.5 mM KH 2 PO 4 , 8.1 mM
Na 2 HPO 4 , pH: 7.4). CaCl 2 (1.2 mM) and glucose (10
mM) were added to this solution. All previous work in the
present laboratory has employed this buffer which has
excellent pH stability and produces stable DA levels in
control animals. However, the high K 1 studies (Experiments 1 and 3) required an increased KCl concentration
(80 mM) and, to maintain solution osmolarity, a simultaneous decrease in NaCl (to 60 mM). Both adjustments
were not possible using the Dulbecco’s buffer solution.
Thus, a laboratory prepared Krebs–Ringer buffer (137 mM
NaCl, 3 mM KCl, 1.2 mM MgSO 4 , 0.4 mM KH 2 PO 4 , 1.2
mM CaCl 2 and 10 mM glucose, pH: 7.4) was employed.
The ionic concentrations of this normal Ringers was then
modified (KCl 80 mM, NaCl 60 mM) to create a high K 1
Ringers solution.
For high K 1 experiments, drug treated rats were pretreated for 30 min with the appropriate concentration of
M100,907 dissolved in normal Ringer’s. This was followed by 30 min perfusion with the drug dissolved in the
high K 1 Ringers solution. During these experiments,
control rats received infusions of normal Ringers for 30
min followed by high K 1 Ringers for 30 min.
2.3. Drugs
M100,907 (Hoechst Marion Roussel, free base) was
administered intracortically via reverse dialysis. Solutions
E. A. Pehek et al. / Brain Research 888 (2001) 51 – 59
were made by dissolving the drug in 2.5 ml of glacial
acetic and 1 ml deionized water. aCSF was then utilized to
dilute this solution to the appropriate concentrations (100
nM, 1.0 mM, 10 mM, and 100 mM, pH 7.4). (6)-DOIhydrochloride (Research Biochemicals Incorporated, MA,
USA) was administered subcutaneously (s.c). DOI (2.5
mg / kg) or vehicle (deionized water) were injected in 1.0
ml / kg volumes.
2.4. Chromatography
DA content of dialysate samples was measured by
HPLC coupled with electrochemical detection. Twentymicroliter dialysis samples were injected onto a 2 mm
Phenomenex column (UltracarbE, 3 mm particle size,
ODS 20). The mobile phase consisted of 32 mM citric
acid, 54 mM sodium acetate, 0.074 mM EDTA, 0.215 mM
octylsulfonic acid, and 3% methanol (vol / vol), pH 4.2. To
maintain separation of DA from its metabolites and 5hydroxyindoleacetic acid, the pH of the mobile phase and
the concentration of octylsulfonic acid were adjusted as
needed. A BAS LC-4C electrochemical detector with a
glassy carbon electrode, maintained at a potential of 10.60
V relative to an Ag /AgCl reference electrode, was employed. The limit of detection for dopamine was 0.1 pg / 20
ml.
2.5. Experimental design
2.5.1. Experiment 1
2.5.1.1. Effects of M100,907 on K 1 -stimulated cortical
DA release. This experiment tested the ability of a high
K 1 infusion to increase extracellular DA in the mPFC.
Furthermore, it tested the Ca 21 -dependency of this effect
and the ability of M100,907 to modulate the K 1 -evoked
release. Baseline samples were collected from all rats
utilizing normal Ringer’s. One group of rats then received
infusions of a high K 1 buffer with normal Ca 21 (1.2 mM)
while a separate group was perfused with a Ca 21 -free / high
K 1 solution. Two other groups were pretreated with either
10 or 100 mM M100,907 before infusions of high K 1
(both with normal Ca 21 ).
2.5.2. Experiment 2
2.5.2.1. M100,907 effects on basal DA. Microdialysis
samples were collected from three separate groups of rats:
vehicle controls and those treated with either 10 or 100
mM M100,907. After stable baseline samples were collected, the animals were perfused for 60 min with
M100,907, dissolved in aCSF, followed by an additional 3
53
h of perfusion and sample collection without drug. The
vehicle group was perfused with aCSF throughout.
2.5.3. Experiment 3
2.5.3.1. Effects of lower concentrations of M100,907 on
DA efflux. Experiment 3A examined the effects of 1.0 mM
M100,907 on K 1 -stimulated DA efflux. Drug treated rats
were compared to high K 1 controls. Procedures were the
same as in Experiment 1. Experiment 3B determined the
effects of lower concentrations of M100,907 on basal
dialysate DA. Following the collection of stable baselines,
100 nM concentrations were infused for 2 h. This was
followed by perfusion with 1.0 mM concentrations for 2 h
in the same rats.
2.5.4. Experiment 4
2.5.4.1. Effects of DOI and M100,907 on cortical DA
release. This study examined the effects of a systemic
injection of DOI on DA efflux in the mPFC. The receptor
specificity was examined by investigating the ability of
intracortical infusions of M100,907 (10 mM) to attenuate
the effects of DOI. Four groups of rats were utilized in this
study: DOI alone, vehicle, M100,907 alone, M100,9071
DOI. The first group received a s.c. injection of DOI while
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
a systemic injection of DOI. DOI was administered 30 min
after the start of perfusion with M100,907.
2.6. Data analysis
Data were expressed, analyzed, and graphed as the
percentage of the last 3 baseline samples. Statistical
analyses were performed using repeated measures
ANOVAs. For two-way ANOVAs, time was the repeated
measures factor and drug condition was the independent
factor. For one-way ANOVAs, time was the repeated
factor. Post-hoc comparisons employed Dunnett’s test for
comparing treatment means with a control value.
3. Results
3.1. Experiment 1
3.1.1. K 1 effects on DA concentrations
Fig. 1 shows that treatment with high K 1 increased
extracellular mPFC DA levels to 265% of baseline
[F(9,81)54.54, P,0.001, one-way repeated measures
ANOVA]. Post hoc tests indicated that DA efflux was
significantly increased at the 30 min time point after
infusion with high K 1 . Removing Ca 21 from the medium
abolished this effect [F(9,36)51.57, n.s.]. Basal DA
concentrations were 0.6660.08 pg / 20 ml (n510) for the
54
E. A. Pehek et al. / Brain Research 888 (2001) 51 – 59
Fig. 1. The effects of intracortical infusions of high K 1 and Ca 21 -free /
high K 1 Krebs–Ringer solution on 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
1
were infused for 30 min from time 0 to 30. * indicates that high K
significantly increased extracellular cortical DA concentrations (P,0.05).
21
This was blocked by removal of Ca from the perfusion medium (no
significant increase in DA). Group ns were: high K 1 510; Ca 21 -free / high
K 1 55.
high K 1 group and 0.7560.14 pg / 20 ml (n55) for the
group that was subsequently treated with a Ca 21 -free / high
K 1 solution.
3.1.2. Effects of M100,907 on K 1 -induced DA release
Treatment with M100,907 attenuated K 1 -stimulated DA
release in a concentration-dependent manner (see Fig. 2).
Dialysate DA only increased to 126% and 155% of
baseline values following infusions of 100 mM or 10 mM
M100,907, respectively. When the 100 mM concentration
plus high K 1 was compared to the high K 1 alone group,
there was a significant drug3time interaction [F(9, 153)5
2.38, P,0.015, two factor repeated measures ANOVA].
Post-hoc analyses of each condition over time revealed that
the high K 1 infusion did NOT significantly increase DA
efflux when 100 mM M100,907 was co-infused [F(9,
72)51.99, P50.053, one-way, repeated measures
ANOVA]. The trend towards a significant F value was due
to decreases (maximal decrease563% of baseline) in DA
concentrations at the 120 and 150 min timepoints (i.e.
90–120 min after the termination of the M100,907 perfusion). The 10 mM concentration also blocked the effects of
high K 1 , as demonstrated by the lack of a significant time
effect for this group [F(9, 45)51.93, P50.07]. The trend
towards significance was again due to a decrease in DA to
60% at the 120 min time point. Basal DA concentrations
Fig. 2. The effects of intracortical infusions of M100,907 on high
K 1 -induced DA release. Values are expressed as the percentage of 3
pre-drug baselines and are the means6S.E.M. of the subjects. For the
drug group, the bar indicates that M100,907 was infused for 60 min from
time 230 to time 30, and high K 1 was co-infused for 30 min from time 0
to time 30. For the non-drug group, the high K 1 solution was infused for
30 min from time 0 to time 30. * indicates that high K 1 significantly
increased extracellular mPFC DA concentrations (P,0.05). Co-administration of M100,907 at either concentration blocked this increase. Group
ns were: high K 1 510; 10 mM1high K 1 56; 100 mM1high K 1 59.
were 0.9760.17 pg / 20 ml (n59) for the 100 mM MDL
100,907 group and 0.5460.08 pg / 20 ml (n56) for the 10
mM group. The high K 1 group is from Fig. 1.
3.2. Experiment 2
3.2.1. MDL effects on basal DA efflux
Fig. 3 demonstrates the effects of intracortical infusions
of 10 and 100 mM M100,907 on basal DA outflow. There
was a significant main effect for drug [F(2,23)53.41,
P50.051). This was due to decreases in DA efflux after
the drug perfusion was stopped (maximal decreases, 100
mM: 55% of baseline at the 120 time point; 10 mM: 63%
of baseline at the 150 time point). Basal DA concentrations
were 0.8260.21 pg / 20 ml (n56) for the 10 mM group,
0.5560.09 pg / 20 ml (n511) for the 100 mM group, and
0.6860.07 pg / 20 ml (n57) for the vehicle group.
3.3. Experiment 3
3.3.1. Effects of lower concentrations of M100,907 on
DA efflux
Infusions of 1.0 mM M100,907 did not alter K 1 -stimulated DA efflux (Fig. 4A). Likewise, perfusion with 100
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. 4B). Basal dialysate concentrations were 0.4660.09
(n55) for the M100,9071high K 1 group, 0.5560.05 (n5
6) for the high K 1 controls, and 0.5260.11 (n58) for the
100 nM / 1.0 mM group.
3.4. Experiment 4
3.4.1. MDL effects on DOI-stimulated DA release
Statistical analyses performed on the raw data (pg / 20
ml) demonstrated that systemic administration of 2.5 mg /
kg DOI increased dialysate DA concentrations [F(6,24)5
3.69, P50.01]. Post-hoc tests revealed a significant increase 30 min post-injection (Fig. 5). DA concentrations in
vehicle animals did not change over time [F(6,18)50.62,
n.s.]. Pretreatment with an intracortical infusion of
M100,907 (10 mM) attenuated the increase in DA produced by DOI [no change over time: F(6,24)51.10, n.s.].
Infusions of M100,907 (10 mM) alone for 3 h decreased
basal DA efflux [F(6,24)54.63, P50.003]. Post hoc tests
demonstrated that this decrease was significant at all time
points except for the 90 min interval following drug
administration. Basal DA concentrations were 0.5760.10
(n55) for the DOI alone group, 0.5060.17 (n54) for the
vehicle group, 0.5760.09 (n55) for the M100,907 alone
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
subjects. A: Effects of 1.0 mM M100,907 on K 1 -stimulated DA efflux.
For the drug group, the bar indicates that M100,907 was infused for 60
min from time 230 to time 30, and high K 1 was co-infused for 30 min
from time 0 to time 30. For the non-drug group, the high K 1 solution was
infused for 30 min from time 0 to time 30. This concentration of
M100,907 did not alter high K 1 -induced DA release. Group ns were:
high K 1 56; M100,9071high K 1 55. B: Effects of 100 nM and 1.0 mM
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
mM, for 2 h. There were no significant effects on DA efflux. Group n was
8.
group, and 0.4060.03 (n55) for the DOI1M100,907
group.
4. Discussion
The present findings indicate that, contrary to our initial
hypothesis, 5-HT 2A receptor antagonism does not increase
cortical DA release. In fact, the opposite was observed,
namely that intracortical administration of the selective
5-HT 2A antagonist M100,907 blocked DA release induced
by the infusion of a high K 1 solution. Furthermore,
intracortical M100,907 also blocked DA release induced
by the systemic administration of the 5-HT 2 agonist DOI.
56
E. A. Pehek et al. / Brain Research 888 (2001) 51 – 59
Fig. 5. The effects of intracortical M100,907 (10 mM) administration on
DOI-induced DA release. Values are expressed as the percentages of 3
pre-drug baselines and are the means6S.E.M. of the subjects. DOI (2.5
mg / kg s.c.) or vehicle were injected at the time indicated by the arrow.
The timing of M100,907 perfusion is indicated by the bar. Perfusion
began 30 min before the DOI injection and lasted to the end of the
experiment (another 2.5 h). DOI administration significantly increased
cortical DA efflux (P50.01) and this was blocked by intracortical
M100,907 (no significant increase in DA). Infusions of M100,907 alone
decreased basal DA release (P50.003).
Local infusion of M100,907 also produced small but
significant decreases in basal DA concentrations. These
results suggest that, under certain conditions, 5-HT 2A
receptors, localized in the mPFC, may function physiologically to augment mesoprefrontocortical DA release.
Most previous work has employed the systemic administration of drugs, which does not elucidate the neuroanatomical localization of the relevant receptors. Theoretically, 5-HT 2A receptors could regulate mesocortical DA
activity, and subsequent transmitter release, through actions on the DA cell bodies in the ventral tegmental area
and / or on neurons in the mPFC. Gobert and Millan [10]
have recently published microdialysis studies demonstrating that systemic administration of the 5-HT 2 agonist DOI
increased cortical DA release by 50% and this release was
blocked by systemic administration of M100,907. These
authors suggested that 5-HT 2A receptors are involved in
facilitating the phasic release of frontocortical DA. Our
results demonstrating a significant blockade of high K 1
and DOI stimulated DA release by M100,907 agree with
this interpretation. The present data further suggest that
systemically administered 5-HT 2A ligands alter mesocortical DA release by acting, at least in part, on 5-HT 2A
receptors localized in the mPFC, where they are abundant
[4,6,31,41].
Recent microdialysis work in the ventral and dorsal
striatum using the selective 5-HT 2A antagonist SR 46349B
[7,18] has demonstrated findings that also agree with those
observed in the present study. These investigators found
that systemic administration of SR 46349B blocked DA
release in the nucleus accumbens that was induced by the
stimulation of the dorsal raphe nucleus. They also found
that local infusions of this drug blocked DA release in the
striatum that was induced by treatment with haloperidol.
Haloperidol induces impulse-dependent DA release by
blocking somatodendritic DA autoreceptors that regulate
DA cell firing [40]. These results are similar to ours in the
mPFC employing high K 1 as the depolarizing agent. They
indicate that the 5-HT 2A receptor regulates phasic DA
release in both the striatum and the mPFC. This agrees
with the suggestion by Lucas and Spampinato [18] that
5-HT 2A receptors may modulate DA release only when DA
neurons are activated. These investigators, as well as
others [10,45], found no effect of selective 5-HT 2A antagonism on basal DA efflux. In the present work, there
were relatively slight decreases in dialysate DA following
reverse dialysis with M100,907. These decreases may
reflect a neuromodulatory, conditional, role of 5-HT 2A
receptors on DA release that is more apparent when the
mesocortical system is stimulated (e.g. with high K 1 or
haloperidol administration).
Administration of M100,907 has also been shown to
attenuate striatal DA release induced by the administration
of the psychostimulant 3,4-methylenedioxymethamphetamine (MDMA) [36]. In contrast, administration of
DOI potentiated MDMA-induced DA release [11].
Schmidt and colleagues have proposed that these ‘‘results
suggest a permissive role for 5-HT2 receptors in the
activation of the dopamine system which occurs during
states of high serotonergic activity . . . ’’ [35]. The present
results agree with this suggestion since infusions of high
K 1 should increase the release of all transmitters, including DA and 5-HT. In addition, it has recently been
demonstrated that administration of M100,907 attenuates
the increases in prefrontocortical DA observed following
the systemic administration of fluoxetine [45]. Fluoxetine
blocks the 5-HT transporter and thereby increases synaptic
concentrations of 5-HT. Thus, considered together, the past
and present results provide support for the suggestion that,
at least under conditions of activated dopaminergic and
serotonergic systems, 5-HT 2A receptors potentiate DA
release in the mPFC.
Microdialysis studies utilizing the acute, systemic administration of nonselective 5-HT 2 antagonists, have generally observed increases in cortical extracellular DA
levels. For example, amperozide [24,29], ritanserin [30]
and clozapine [21,28] have all been shown to increase
cortical dialysate DA levels. However, ritanserin could
have actions on 5-HT 2C receptors [33]. Amperozide is
selective for the 5-HT 2A over the 5-HT 2C receptor [33] but
also blocks the DA transporter [42]. As is well known,
clozapine has high to moderate affinity for multiple
receptor subtypes [20]. The present work indicates that the
preceding agents do not increase cortical DA release solely
E. A. Pehek et al. / Brain Research 888 (2001) 51 – 59
as a consequence of selective 5-HT 2A receptor blockade in
the cortex.
The cellular localization of cortical 5-HT 2A receptors
regulating DA release remains to be determined. Evidence
indicates that 5-HT 2A receptors are not localized presynaptically on DA terminals [16,17]. Thus, 5-HT 2A
receptors likely modulate DA release indirectly, either
through neuronal circuits located within the mPFC, or
through connections to other brain areas. Recent immunohistochemical work has shown that prefrontocortical 5HT 2A receptors appear to be located principally on the
apical dendrites of pyramidal cells as well as on GABA
interneurons in the rat and monkey [13,39]. 5-HT could
theoretically act on 5-HT 2A receptors localized to pyramidal cells or GABAergic interneurons that, in turn, regulate
mesocortical DA release.
One limitation of the present approach is that the
concentration of M100,907 entering the brain is unknown.
The current study, like most microdialysis studies, employed mM concentrations to produce effects equivalent to
low to moderate doses of systemically administered ligands. In particular, the present work with 10 and 100 mM
M100,907 agrees with the previous finding that systemic
injections of a moderate dose (0.4 mg / kg) of this compound blocked DOI-stimulated cortical DA release [10].
Empirical evidence suggests that the concentrations of
drugs crossing the dialysate membrane are extremely
small. For example, the amounts of various drugs (DA
uptake blockers) that crossed the dialysate membrane in
vitro range from 2.0 to 8.6% [23]. However, in vitro
studies of recovery fail to account for impediments to drug
diffusion that normally occur in vivo in the brain tissue. In
fact, there is evidence that ‘‘the tissue is normally more
important than the membrane in determining the performance of a microdialysis probe’’ [8]. One study that
calculated the true in vivo diffusion of an antiviral
nucleoside demonstrated that the recovery was one-third of
that observed in vitro [38]. Furthermore, even if the drug
concentrations immediately surrounding the membrane are
relatively high, there is significant tissue damage in this
vicinity [44]. Studies with smaller in vivo voltammetric
electrodes have shown that pharmacological effects on DA
efflux are absent in this zone [44]. Other work has shown
that there is a gradient of drug concentration around the
probe and that DA measured by microdialysis is diffusing
from areas close to, but away from, the probe [2]. Thus,
the changes in DA release in the present study are likely
due to actions at sites slightly distal to the probe and not in
the vicinity of mM concentrations.
The concentrations of M100,907 employed in the present research were chosen on the basis of work by others
employing reverse dialysis of serotonergic ligands. For
example, 100–300 mM concentrations of 5-HT 1B ligands
altered DA release in the mPFC [12]. Perfusion with 50
mM 8-OH-DPAT, a 5-HT 1A agonist, stimulated DA release
in the striatum [1]. This latter study also employed 10–
57
1000 mM and 0.25–4 mM concentrations of other 5-HT 1
agonists. In the present study, 10 mM M100,907 significantly attenuated basal, K 1 -stimulated, and DOI-stimulated DA release. This concentration of M100,907 is 1 / 10
of that employed in prior work with local infusions of
clozapine [28]. This ratio of 10:1 roughly corresponds to
the affinities of clozapine (4 nM) versus M100,907 (0.36
nM) for the 5-HT 2A receptor ([34,25], respectively). 100
nM and 1.0 mM concentrations M100,907 did not alter DA
efflux, suggesting that the present work reflects the left
side of the concentration–response curve. This also agrees
with prior work demonstrating that 100 nM M100,907 did
not alter 5-HT-stimulated cortical DA release [12].
In summary, local infusions of the selective 5-HT 2A
antagonist M100,907 into the mPFC attenuated K 1 -induced DA release in a concentration-dependent manner.
Systemic administration of the 5-HT 2 agonist DOI increased cortical DA efflux. This increase was attenuated by
intracortical administration of M100,907. These results
indicate that, under activated conditions, cortical 5-HT 2A
receptors potentiate the phasic release of DA. They raise
questions as to whether 5-HT 2A receptors modulate DA
that is released physiologically (e.g., in response to novelty
or stress) or in a non-impulse dependent manner (e.g.,
amphetamine-induced). This, in turn, raises the issue of
whether 5-HT 2A receptors regulate DA-mediated behaviors. In fact, it has been demonstrated that M100,907
blocks both cocaine [19] and amphetamine-mediated behaviors [22]. The present data suggest that when cortical
DA is released physiologically, 5-HT, via a 5-HT 2A
mechanism, may act to further stimulate DA release and
thus increase the salience of the neurochemical signal. This
corresponds with recent reports demonstrating that 5-HT
may act as a paracrine transmitter [3].
Acknowledgements
We wish to thank Dr. Christine Nocjar for her helpful
insights and discussions in the preparation of this manuscript. This work was supported by grant MH52220 to
E.A.P. The authors wish to thank Hoechst Marion Roussell
for their kind donation of M100,907.
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