NTs - University of New Mexico

Journal of Neurochemistry, 2004, 91, 852–859
doi:10.1111/j.1471-4159.2004.02763.x
Serotonin–GABA interactions modulate MDMA-induced
mesolimbic dopamine release
Michael G. Bankson and Bryan K. Yamamoto
Laboratory of Neurochemistry, Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine,
Boston, Massachusetts, USA
Abstract
3,4,-Methylenedioxymethamphetamine (MDMA; ‘ecstasy’)
acts at monoamine nerve terminals to alter the release and
re-uptake of dopamine and 5-HT. The present study used
microdialysis in awake rats to measure MDMA-induced
changes in extracellular GABA in the ventral tegmental area
(VTA), simultaneous with measures of extracellular dopamine
(DA) in the nucleus accumbens (NAC) shell. (+)-MDMA
(0, 2.5, 5 and 10 mg/kg, i.p.) increased GABA efflux in the
VTA with a bell-shaped dose–response. This increase was
blocked by application of TTX through the VTA probe. MDMA
(5 mg/kg) increased 5-HT efflux in VTA by 1037% (p < 0.05).
The local perfusion of the 5-HT2B/2C antagonist SB 206553
into the VTA reduced VTA GABA efflux after MDMA from a
maximum of 229% to a maximum of 126% of basal values
(p < 0.05), while having no effect on basal extracellular GABA
concentrations. DA concentrations measured simultaneously
in the NAC shell were increased from a maximum of 486% to
1320% (p < 0.05). The selective DA releaser d-amphetamine
(AMPH) (4 mg/kg) also increased VTA GABA efflux (180%),
did not alter 5-HT and increased NAC DA (875%) (p < 0.05),
but the perfusion of SB 206553 into the VTA failed to alter
these effects. These results suggest that MDMA-mediated
increases in DA within the NAC shell are dampened by
increases in VTA GABA subsequent to activation of 5-HT2B/2C
receptors in the VTA.
Keywords: dopamine, gamma amino butyric acid, 3,
4-methylenedioxymethamphetamine, nucleus accumbens,
serotonin, ventral tegmental area.
J. Neurochem. (2004) 91, 852–859.
3,4,-Methylenedioxymethamphetamine (MDMA; ‘e’; ‘x’;
‘ecstasy’) is an amphetamine derivative that is being
increasingly abused across the US and worldwide. Some of
the unique properties of MDMA that make it subjectively
different from the parent compound, d-amphetamine
(AMPH), and probably account for its continued popularity,
include cognitive enhancement, feelings of warmth toward
others, abatement of anxiety and enhanced perceptual ability
(Vollenweider et al. 1998).
The stimulant and rewarding properties of MDMA, as well
as the other amphetamines, are thought to arise in part from
the ability of these drugs to activate mesolimbic dopamine
(DA) neurons in the ventral tegmental area (VTA) that
project to the nucleus accumbens (NAC) (Hoebel 1985; Gold
et al. 1989a; Kelley and Delfs 1991). Both MDMA and
AMPH act primarily by releasing DA from nerve terminals
via reversal of the dopamine transporter (Fischer and Cho
1976; Holmes and Rutledge 1976). The enhancement of DA
release in NAC is thought to mediate their locomotor
activating and rewarding properties (Gold et al. 1989b).
Compared with AMPH, MDMA binds with higher affinity to
the serotonin re-uptake transporter to release 5-HT (Johnson
et al. 1991; Rudnick and Wall 1992). Thus, the unique
properties of MDMA that differentiate it from the effects of
other amphetamines in both human (Greer and Tolbert 1986)
and animal (Paulus and Geyer 1992) studies may arise from
the simultaneous release of 5-HT and DA.
Another difference between MDMA and amphetamine
that may account for the unique pharmacological profile of
MDMA is that MDMA, unlike AMPH, causes both transporter-mediated and impulse-mediated DA release. The
impulse-mediated release of DA produced by MDMA
852
Received June 11, 2004; revised manuscript received July 15, 2004;
accepted July 16, 2004.
Address correspondence and reprint requests to Bryan K. Yamamoto
PhD, Department of Pharmacology and Experimental Therapeutics,
L-613, Boston University School or Medicine, Boston, MA 02118,
USA. E-mail: [email protected]
Abbreviations used: AMPH, d-amphetamine; DA, dopamine; GABA,
gamma amino butyric acid; 5-HT, 5-hydroxytryptamine, serotonin;
MDMA, 3,4-methylenedioxymethamphetamine; VTA, ventral tegmental
area.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 852–859
MDMA and VTA GABA 853
appears to be dependent on 5-HT transmission (Koch and
Galloway 1997) and may be explained by the stimulatory
effects of 5-HT1B/1D (Hallbus et al. 1997), 5-HT2A (Schmidt
et al. 1992a), 5-HT3 (De Deurwaerdere et al. 1998), and
5-HT6 (Minabe et al. 2004) receptors on DA release. The
impulse dependency of MDMA-induced DA release is
consistent with the findings that MDMA-induced increases
in extracellular DA in the striatum are attenuated by the
sodium channel blocker TTX (Yamamoto et al. 1995) and
that pharmacological inhibition of MDMA-induced 5-HT
release attenuates MDMA-induced striatal DA release
(Gudelsky and Nash 1996; Koch and Galloway 1997).
The premise that MDMA-induced DA release is secondary, in part, to MDMA-induced 5-HT release is also
consistent with locomotor studies showing that MDMAinduced activity is blocked by 5-HT1B (Callaway et al. 1992;
McCreary et al. 1999) or 5-HT2A (Kehne et al. 1996)
antagonists. Alternatively, blockade of 5-HT2B/2C receptors
has been shown to greatly enhance MDMA-induced locomotion (Bankson and Cunningham 2002). Thus, there are
opposing effects of 5-HT on the behavioral effects of
MDMA, a 5-HT1B/2A stimulatory effect and a 5-HT2C
inhibitory effect. One explanation for these behavioral
findings is that activation of 5-HT2C receptors may act in
opposition to the stimulatory effects of 5-HT1B and 5-HT2A
receptors by limiting MDMA-induced DA release in NAC
(Gold et al. 1989). While the stimulatory role of the 5-HT2A
receptor is consistent with these receptors being located on
and directly stimulating mesolimbic DA neurons (Doherty
and Pickel 2000), the role of 5-HT1B receptors in facilitating,
and the role of 5-HT2C receptors in dampening MDMAinduced motor activation, may be mediated through changes
in mesolimbic GABA.
GABA is known to decrease DA cell firing in the VTA
(Kiyatkin and Rebec 1998). 5-HT2C receptors located on
non-dopaminergic (presumably GABA) neurons in the VTA
increase GABA transmission (Stanford and Lacey 1996).
Therefore, 5-HT2C receptors may decrease DA cell firing
through increases in GABA release in the VTA. Consistent
with these findings, selective stimulation of 5-HT2C receptors
decreases DA release in the NAC shell and decreases the
firing rate of mesolimbic DA neurons (Di Giovanni et al.
2000). Conversely, antagonism of this receptor increases the
firing rate of VTA DA neurons and increases DA release in
the NAC (Di Matteo et al. 1999). While these studies are
suggestive of a modulatory role of 5-HT2C receptors on VTA
neurons and DA release in the NAC shell, the systemic
administration of 5-HT agonists and antagonists was used
and thus do not address the specific locus of action or
whether 5-HT2C receptors dampen DA transmission directly
or indirectly through the enhancement of GABAergic
transmission within the VTA.
Previous work from this laboratory has shown that the
non-selective 5-HT2A/2C receptor antagonist ritanserin
attenuates MDMA-induced increases in DA release in the
nigrostriatal pathway (Yamamoto et al. 1995) and suggests
that 5-HT2A receptor activation is necessary for MDMAinduced DA release (Schmidt et al. 1992b); however, this
study did not address the specific contribution of 5-HT2C
receptors. Furthermore, this prior work did not examine how
MDMA-induced DA release is regulated in the mesolimbic
pathway. Di Giovanni et al. (2000) have shown that
mesolimbic 5-HT2C receptors exert greater inhibitory control
over DA release than do nigrostriatal 5-HT2C receptors, but
no studies to date have used localised blockade of 5-HT2C
receptors in the VTA to evaluate their involvement in
controlling mesolimbic DA release after MDMA administration. Based on the aforementioned behavioral, neurochemical and anatomical studies, the 5-HT2C receptor in the
VTA may oppose and modulate the stimulatory effects of
5-HT1B and 5-HT2A receptors to limit the impulse-mediated
release of DA in the NAC shell, and thereby contribute to the
unique neurochemical and behavioral profile of MDMA
relative to the actions of AMPH. The hypothesis of the
current study is that MDMA-induced DA release in the NAC
shell differs from that of AMPH-induced DA release in that
DA release produced by MDMA is dampened by the
enhancement of GABA transmission in the VTA via the
activation of the 5-HT2C receptor. To test this hypothesis,
extracellular DA within the NAC shell produced by MDMA
or AMPH was measured simultaneously with changes in the
extracellular concentrations of GABA within the VTA in the
presence or absence of the local perfusion of the VTA with a
5-HT2B/2C antagonist.
Materials and methods
Subjects
Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis,
IN, USA) weighing 175–250 g at the beginning of experimental
procedures were housed in groups of three in a temperature (21–
23C) -controlled environment for at least 2 days prior to
experiments. Food and water were available ad libitum. Lighting
was maintained under a 12-h light-dark cycle (lights on 07.00–
19.00 h). All experimental procedures were performed between
07.00 and 19.00 h and were carried out in accordance with the
National Institutes of Health Guide for the Care and Use of
Laboratory Animals.
Drugs
SB 206553 [N-3-pyridinyl-3,5-dihydro-5-methylbenzo(1,2-b:4,5b¢)dipyrrole-1(2H)carboxamide] and TTX (tetrodotoxin) were
obtained from Research Biochemicals Inc. (Natick, MA, USA).
(+)-MDMA (3,4-methlenedioxymethamphetamine) was obtained
from the National Institutes on Drug Abuse (NIDA, Research
Triangle Park, NC, USA). Doses refer to the weight of the salt.
MDMA was administered intraperitoneally in a volume of 1 mL per
kg of body weight. SB 206553 (5 lM) and TTX (1 lM) were
2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 852–859
854 M. G. Bankson and B. K. Yamamoto
administered by reverse dialysis in modified Dulbecco’s buffered
saline (137 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 8.1 mM
Na2HPO4, 1.5 mM KH2PO4, 1.2 mM CaCl2 and 5 mM d-glucose,
pH 7.4).
Surgical procedures
After acclimation to the colony room, all rats were anesthetized with
a combination of xylazine (12 mg/kg) and ketamine (80 mg/kg) and
placed into a Kopf stereotaxic frame. The skull was exposed and a
microdialysis probe placed in the ventral tegmental area (VTA)
(4.8 mm posterior, ±0.8 mm medial, 9 mm ventral to bregma). For
dual probe studies, probes were placed in both the VTA and nucleus
accumbens shell (NAC) 1.7 mm anterior, ±1.1 mm medial and
9.1 mm ventral to bregma at an angle of 15 degrees. The probes
were constructed as previously described (Pehek et al. 1990) and
have an active membrane length of 1.75 mm. The probes and a
metal male connector were secured to the skull with three stainless
steel screws and cranioplastic cement.
Microdialysis procedures
The day after probe insertion, the modified Dulbecco’s phosphatebuffered saline medium was pumped through the microdialysis
probes with a Harvard Model 22 syringe infusion pump (Hollison,
MA, USA) at a rate of 2 lL/min. A 1.5-h perfusion period was
allowed prior to sample collection. Thirty-minute samples were then
collected. Four baseline samples were collected after which
SB 206553 or TTX was added to the perfusion medium of the
probe positioned in the VTA. MDMA or saline was injected 2 h
after initiation of the perfusion of TTX (1 lM) or SB 206553 (5 lM)
into the VTA. In the single probe experiments that measured GABA
in VTA, MDMA was injected 30 min after SB 206553 infusion into
VTA. Samples from the VTA and NAC shell were collected for 3 h.
For the experiments that involved a single probe implantation in the
VTA, MDMA, AMPH or saline control was injected immediately
after four baseline samples and samples taken every 30 min for 3 h.
HPLC analysis of monoamines and GABA
Microdialysis samples were assayed for dopamine, 5-HT, and
GABA by high-performance liquid chromatography with electrochemical detection. Three-point standard curves were evaluated
periodically to determine peak identity and linearity of the
concentration–response. Single point calibration was used on a
daily basis to quantify peak height. Separation was achieved with a
C18 column (100 · 2.0 mm, 3 lm particle size; Phenomenex,
Torrance, CA, USA). The mobile phase for detection of DA, 5-HT
and metabolites (pH 4.2) consisted of 32 mM citric acid, 54.3 mM
sodium acetate, 0.074 mM EDTA, 0.215 mM octyl sodium sulfate
and 3% methanol. GABA was derivatized with o-phthaldialdehyde
and sodium sulfite (Smith and Sharp 1994). Briefly, 2 lL of the
stock derivatization reagent containing 22 mg of OPA, 0.5 mL of
100% ethanol, 0.5 mL of 1 M sodium sulfite and 9 mL of sodium
borate buffer (0.4 M boric acid, pH 10.4) was added to 20 lL of
dialysate or standard, vortexed and allowed to react for 5 min before
injecting onto a C18 column (100 · 2.0 mm, 3 lm particle size;
Phenomenex). GABA was eluted using a mobile phase consisting of
0.1 M Na2HPO4 and 0.1 mM EDTA in 10% methanol at pH ¼ 4.4.
Compounds were detected with an LC-4C amperometric detector
(Bioanalytical Systems, West Lafayette, IN, USA) or DecadeR.
detector (Antec-Leyden, the Netherlands) with a 6-mm glassy
carbon working electrode maintained at a potential of +0.65 V (DA,
5-HT) or 0.7 V (GABA) relative to a Ag/AgCl reference electrode.
Statistical analyses
One-way way repeated-measures analyses of variance (ANOVA)
(AUC), or two-way ANOVAs (time-course) were computed to
compare rats treated with drugs across all sample collection times.
Post-hoc Tukey’s HSD tests were used to analyze any significant
treatments at specific time points. Average baseline values for all
experiments was calculated from the four samples prior to control,
TTX or SB 206553 pre-treatment.
Results
MDMA effects on VTA GABA: interaction with
SB 206553 and TTX
Across groups, basal GABA in the VTA was 22.8 ± 2.3 nM;
basal DA in the NAC shell was 0.48 ± 0.05 nM; and basal
5-HT in the VTA was 0.14 ± 0.03 nM. MDMA significantly
increased extracellular GABA concentrations in the VTA
(F3,24 ¼ 6.31, p < 0.05) compared with vehicle controls
(Fig. 1). MDMA (5 mg/kg) had the greatest effect on VTA
GABA release and was therefore used in all subsequent
experiments. Pre-treatment with SB 206553 or TTX into the
VTA before systemic administration of MDMA (5 mg/kg)
significantly attenuated the increases in GABA produced by
MDMA (F2,17 ¼ 7.41, p < 0.05). No differences were
observed in basal VTA GABA efflux during SB 206553 or
TTX infusion prior to MDMA administration.
Fig. 1 Extracellular GABA concentrations in VTA: area under the
curve (AUC). MDMA was injected at the indicated dose after a 2-h
baseline. n ¼ 6–8 rats/group. SB 206553 (SB) (5 lm) was added to
dialysis medium 30 min before systemic MDMA. TTX was added to
the dialysis medium 2 h before systemic MDMA. Bars represent area
under the curve (± SEM) for 3-h post MDMA injection (six samples).
*p < 0.05 compared with saline treatment; #p < 0.05 compared with
MDMA (5 mg/kg, i.p.) treatment by one way ANOVA. Dose of MDMA in
mg/kg i.p. (2.5, 5, or 10); SB-MDMA 5: SB 206553 with MDMA at
5 mg/kg i.p.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 852–859
MDMA and VTA GABA 855
Time course of SB 206553 on MDMA-induced GABA in
VTA
MDMA produced an overall increase in extracellular GABA
concentrations in the VTA compared with saline controls
(F1,151 ¼ 6.86, p < 0.05) (Fig. 2) MDMA-induced GABA
concentrations were significantly different from saline controls at all time points after MDMA administration
(p < 0.05). Pre-treatment with SB 206553 into the VTA
significantly attenuated the MDMA-induced increases in
GABA over the time course of the experiment (F13,151 ¼
1.87, p < 0.05). SB + MDMA produced increases in VTA
GABA which were significantly less than MDMA alone at
the 5-, 5.5-, 6- and 7-h time points (Tukey post hoc,
p < 0.05).
Time course of SB 206553 effects on MDMA-induced DA
in NAC shell
MDMA produced an overall increase in extracellular DA
concentrations in the NAC shell compared with saline
controls (F1,134 ¼ 12.73.86, p < 0.05) and over the time
course of the dialysis experiment (F13,134 ¼ 7.72,
p < 0.05) (Fig. 3). MDMA produced an increase in DA
which was significantly different compared with saline
controls at all time points after MDMA administration,
except for 7 h, p < 0.05. Pre-treatment with SB 206553
into the VTA significantly augmented MDMA-induced
increases in DA concentrations in NAC shell (F1,137 ¼
6.59, p < 0.05), and was significantly different from
MDMA alone at the 4.5-, 5- and 5.5-h time points,
p < 0.05.
Fig. 2 MDMA effects on GABA concentrations in VTA. Dialysis
samples were collected from the VTA of rats treated with SB 206553
(SB) (5.0 lm) or vehicle was reverse dialyzed into the VTA 2 h prior to
systemic administration of MDMA (5 mg/kg i.p.). n ¼ 6–8/group.
#p < 0.05 compared with vehicle + MDMA; *p < 0.05 compared with
vehicle + saline (SAL) rats; two-way repeated measures ANOVA. Hatched horizontal bar indicates the duration of SB into the VTA. Arrow
indicates time of systemic MDMA administration. Error bars represent ± SEM.
Fig. 3 MDMA effects on DA concentrations in NAC. Dialysis samples
were collected from the NAC of rats treated with SB 206553 (SB)
(5.0 lm) or vehicle that was reverse dialyzed into the VTA for 2 h prior
to systemic administration of MDMA (5 mg/kg i.p.) n ¼ 6–8/group.
#p < 0.05 compared with vehicle l + MDMA; *p < 0.05 compared with
vehicle + saline (SAL) rats; two-way repeated measures ANOVA. Hatched horizontal bar indicates duration of SB into the VTA. Arrow
indicates time of systemic MDMA administration. Error bars represent ± SEM.
Comparison of MDMA and AMPH-induced 5-HT
concentrations in VTA
MDMA produced an overall increase in extracellular 5-HT
concentrations in the VTA compared with AMPH (F1,105 ¼
41.89, p < 0.05) and over the time course of the dialysis
experiment (F13,105 ¼ 7.57, p < 0.05) (Fig. 4). MDMAinduced 5-HT levels were significantly different from AMPH
control for all time points after MDMA administration except
at 7 h, p < 0.05.
Fig. 4 MDMA and AMPH effects on 5-HT concentrations in the VTA.
Dialysis samples were collected from the VTA of rats treated with
systemic administration of MDMA (5 mg/kg i.p.) or d-amphetamine
(AMPH) (4 mg/kg i.p.). n ¼ 5/group. *p < 0.05 by one-way ANOVA.
Arrow indicates time of systemic MDMA or AMPH administration. Error
bars represent ± SEM.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 852–859
856 M. G. Bankson and B. K. Yamamoto
Fig. 5 AMPH effects on GABA concentrations in VTA. Dialysis samples were collected from the VTA of rats treated with SB 206553 (SB)
(5.0 lm) or vehicle that was reverse dialyzed into the VTA 2 h prior to
systemic administration of d-amphetamine (AMPH) (4 mg/kg i.p.).
n ¼ 6–8/group. #p < 0.05 compared with vehicle + AMPH; *p < 0.05
compared with vehicle + saline (SAL) rats by two-way repeated
measures ANOVA. Hatched horizontal bar indicates duration of SB into
the VTA. Arrow indicates time of systemic AMPH administration. Error
bars represent ± SEM.
Time course of SB 206553 on AMPH-induced GABA in
VTA
AMPH produced an overall increase in extracellular GABA
concentrations in the VTA compared with saline controls
(F1,151 ¼ 6.86, p < 0.05) and over the time course of the
experiment (F13,151 ¼ 3.29, p < 0.05) (Fig. 5). GABA levels were significantly different from saline control at the 4.5-,
5.5- and 7-h time points, p < 0.05. Pre-treatment with
SB 206553 alone into the VTA had no effect on basal
concentrations of GABA compared with vehicle-infused
controls during the time period (2–4 h time points) prior to
the injection of AMPH. Pre-treatment with SB 206553 into
the VTA had no effect over time or compared with AMPH
alone.
Time course of SB 206553 effects on AMPH-induced DA
in NAC
AMPH produced an increase in extracellular DA concentrations in the NAC compared with saline controls (F1,146 ¼
8.84, p < 0.05) and over the time course of the dialysis study
(F13,146 ¼ 13.54, p < 0.05) (Fig. 6). AMPH produced DA
levels that were significantly different from saline controls
for all time points after drug injection except for 6.5 and 7 h,
p < 0.05. Pre-treatment with SB 206553 into the VTA had
no effect over time or compared with AMPH alone.
Discussion
These studies are the first to demonstrate that MDMA elicits
a dose- and impulse-dependent increase in extracellular
Fig. 6 AMPH effects on DA concentrations in NAC. Dialysis samples
were collected from the NAC of rats treated with SB 206553 (SB)
(5 lM) or vehicle that was reverse dialyzed into the VTA for 2 h prior to
systemic administration of d-amphetamine (AMPH) (4 mg/kg i.p.)
n ¼ 6–8/group. #p < 0.05 compared with vehicle + saline (SAL) by
two-way repeated measures ANOVA. Hatched horizontal bar indicates
duration of SB into the VTA. Arrow indicates time of systemic AMPH
administration. Error bars represent ± SEM.
GABA concentrations in the VTA (Fig. 1). The results also
show that unlike d-amphetamine (Figs 5 and 6), the increase
in extracellular concentrations of 5-HT in the VTA produced
by MDMA (Fig. 4) acts at 5-HT2C receptors to control both
VTA GABA as well as NAC shell DA release (Figs 2 and 3).
Although the neuronal origin of GABA has been
questioned (Timmerman and Westerink 1997), the current
findings suggest that the increase in GABA produced by
MDMA and 5-HT2C activation is dependent upon impulse
flow from GABA afferents or depolarization of GABAergic
soma. The addition of TTX to the dialysis medium blocked
the MDMA-induced increase in VTA GABA but did not
alter the basal concentration of GABA. This complete
blockade of MDMA-induced GABA efflux suggests that
MDMA elicits GABA release through impulse-mediated,
neuronal mechanisms, while the basal concentrations of
GABA are derived from impulse-independent or non-neuronal sources (Matuszewich and Yamamoto 1999).
The effect of MDMA on extracellular GABA concentrations in the VTA produced a bell-shaped dose–response
curve (Fig. 1). Although increasing doses in the lower range
resulted in progressive increases in extracellular GABA, the
higher dose (10 mg/kg) was less effective, possibly due to
rapid desensitization of 5-HT2C receptors (Berg et al. 2001)
and the predicted greater 5-HT release and stimulatory effect
on 5-HT2C receptors. In contrast to the bell-shaped dose–
response for MDMA-induced changes in VTA GABA,
several studies have shown a positive correlation between
striatal and accumbal DA release and dose of MDMA across
the range of doses tested (Nash 1990; Kalivas et al. 1998;
Kankaanpaa et al. 1998), indicating that the 5 mg/kg dose of
2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 852–859
MDMA and VTA GABA 857
MDMA used in the present study is submaximal for eliciting
the release of DA. Although the present study only measured
DA release after 5 mg/kg of MDMA, this dose produced a
maximal effect on VTA GABA, suggesting that at lower
doses of MDMA, DA release occurs despite elevated VTA
GABA. The 10 mg/kg dose of MDMA produced smaller
increases in VTA GABA, suggesting that the impact of of
5-HT2C receptors and GABA release in the VTA may have
greater importance at lower doses of MDMA.
Previous work from this laboratory has shown an MDMAinduced decrease in GABA concentrations in the substantia
nigra (Yamamoto et al. 1995). This result is different from
the present study showing increases in GABA in the VTA.
Thus, MDMA may be affecting extracellular GABA concentrations by different mechanisms in the substantia nigra as
compared with the VTA. This premise is consistent with
reports that the 5-HT2C receptor exerts a greater inhibitory
influence over mesolimbic DA than it does over the
nigrostriatal DA system (Di Giovanni et al. 2000). Furthermore, ritanserin, a non-selective 5-HT2A/2C antagonist, was
used in the previous study that focused on the nigrostriatal
pathway and suggested a stimulatory effect of 5-HT2A
receptors on DA release in contrast to the present study
showing an inhibitory effect of 5-HT2B/2C receptors in the
VTA mediated through the increase in GABA. The contrasting effects on mesolimbic versus nigrostriatal dopaminergic
systems are particularly interesting given the findings that
MDMA is less readily self-administered compared with other
psychostimulant drugs of abuse (Schenk et al. 2003; Fantegrossi et al. 2004).
SB 206553 blocks both 5-HT2B and 5-HT2C receptors
(Kennett et al. 1996) and blockade of either may account for
the attenuation of MDMA-induced GABA efflux in the VTA
and attenuation in DA release in the NAC shell. However,
modest densities of 5-HT2B receptors are found in brain
compared with the more highly expressed 5-HT2C receptor
(Duxon et al. 1997; Barnes and Sharp 1999). Furthermore,
another selective 5-HT2C receptor antagonist, SB 242084,
which has little affinity for 5-HT2B receptors (Kennett et al.
1997), also increases basal mesolimbic DA efflux (Di Matteo
et al. 1999) and further supports the conclusion that 5-HT2C
blockade modulates DA release in the NAC shell.
The application of the 5-HT2B/2C antagonist SB 206553
into the VTA did not alter basal concentrations of GABA but
attenuated the MDMA-induced increase in extracellular
GABA while dramatically and simultaneously increasing
MDMA-induced DA efflux in the NAC shell (Figs 2 and 3).
The lack of an effect of SB 206553 on basal extracellular
concentrations of GABA suggests that 5-HT via 5-HT2B/2C
receptors do not have a tonic stimulatory effect on extracellular GABA in the VTA. While these studies do not provide
direct evidence of a link between VTA GABA release and
MDMA-induced DA release, several reports indicate that
GABA exerts an inhibitory influence on VTA DA neurons.
Activation of 5-HT2C receptors in VTA is known to inhibit
stress-induced, but not basal DA release and blockade of
these receptors in the VTA increases DA release (Pozzi et al.
2002). The GABAB agonist baclofen inhibits DA release in
the prefrontal cortex (Westerink et al. 1998) and NAC (Xi
and Stein 1998, 1999) when infused into the VTA. These
findings in combination with the current results suggest that
5-HT2C mediated elevations of GABA concentrations in the
VTA may act to limit the magnitude of MDMA-induced DA
release.
At first glance, these results showing an inhibition of
VTA-NAC DA transmission via 5-HT2C receptors may
appear to be inconsistent with previous studies showing a
5-HT mediated, impulse dependent enhancement of MDMAinduced DA release (Gudelsky and Nash 1996; Koch and
Galloway 1997). However, the inhibitory effect of 5-HT2C
activation on VTA-NAC DA release is countered by an
opposing action of 5-HT1B receptors. In fact, 5-HT1B
activation in the VTA has been shown to inhibit local
GABA efflux (Yan and Yan 2001) and elicit DA release in
the NAC (Guan and McBride 1989). This suggests that
MDMA-induced increases in extracellular 5-HT concentrations can also stimulate 5-HT1B receptors to decrease VTA
GABA concentrations and disinhibit VTA DA neurons
projecting to the NAC. This is consistent with locomotor
studies indicating that MDMA-induced hyperactivity
requires activation of 5-HT1B receptors (McCreary et al.
1999) and can be augmented or unmasked by 5-HT2B/2C
antagonism (Bankson and Cunningham 2002). Thus, the
pharmacological action of MDMA on DA release is a
balance of the stimulation and inhibition of 5-HT1B and
5-HT2C receptors on GABA efflux, respectively.
The lack of a complete reversal of MDMA-induced
GABA by SB 206553 suggests that D1-mediated activation
of mesolimbic GABA neurons also contributes to MDMAinduced GABA release (Cameron and Williams 1993, 1995).
The resultant expression of MDMA-induced DA release may
be dependent upon the relative balance of 5-HT2C, 5-HT1B,
and D1 receptor activation. Thus, the impulse-dependency of
MDMA-induced DA release in forebrain regions (Yamamoto
et al. 1995) is mediated by a 5-HT1B receptor stimulatory
and a 5-HT2C/D1 receptor inhibitory effect via a decrease and
an increase, respectively, in GABA efflux in midbrain DA
nuclei.
In contrast to the partial modulation of DA release by
impulse flow caused by MDMA, AMPH releases DA via an
impulse-independent reversal of the DAT (Fischer and Cho
1976; Hurd and Ungerstedt 1989; Westerink et al. 1989).
This transporter-mediated release of DA does not appear to
be significantly influenced or counteracted by a presumed
simultaneous inhibition of impulse-mediated NAC shell DA
release produced by a D1-mediated increase in GABA
transmission in the VTA. This lack of an influence of
impulse flow on AMPH-induced DA release is consistent
2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 852–859
858 M. G. Bankson and B. K. Yamamoto
with the finding that AMPH causes the release of DA in the
absence of DA cell firing (Bunney et al. 1973). The present
finding that blockade of 5-HT2C receptors in the VTA does
not effect AMPH-induced GABA or DA release (Figs 5 and
6) highlights the significant difference between this drug
and MDMA-induced DA release in the NAC shell (Figs 3
and 6).
In conclusion, MDMA-induced DA release in the NAC
shell is dampened by a 5-HT2C receptor-mediated increase in
GABA within the VTA and is in marked contrast to the
primarily impulse-independent and 5-HT-independent effects
of AMPH on DA release. These findings may have important
implications for the possible influence of prior exposure to
MDMA-induced neurotoxicity to 5-HT neurons. The longterm decrease in 5-HT transmission produced by neurotoxic
doses of MDMA could lead to a decreased activation of
5-HT2C receptors, a consequent loss of inhibitory GABAergic
tone on mesolimbic DA transmission and, subsequently, an
increased vulnerability to the DA-mediated rewarding effects
of this and other psychostimulant drugs of abuse.
Acknowledgements
This work was supported by DA07427, DA16501 and a gift from
Hitachi America Inc.
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