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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
JPET 298:226–233, 2001
Vol. 298, No. 1
3583/911044
Printed in U.S.A.
Dopamine-Independent Locomotion Following Blockade of
N-Methyl-D-aspartate Receptors in the Ventral Tegmental Area
JENNIFER L. CORNISH,1 MISTU NAKAMURA, and PETER W. KALIVAS
Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina
Received November 21, 2000; accepted March 13, 2001
This paper is available online at http://jpet.aspetjournals.org
A role for the mesocorticolimbic dopamine projection has
been proposed for a number of psychiatric disorders, including schizophrenia and drug addiction (Robinson and Berridge, 1993; Yang et al., 1999). The mesocorticolimbic dopamine system arises from dopaminergic neurons in the ventral
tegmental area (VTA), which send axonal projections to a
number of forebrain nuclei such as the nucleus accumbens
and prefrontal cortex (Fallon and Moore, 1978). As a result of
the therapeutic interest, characterizing afferent regulation of
dopamine cells in the VTA has been a research focus for over
two decades (for review, see Kalivas, 1993). Arising from
these investigations is the demonstration that glutamatergic
afferents to the VTA provide potent excitation of dopamine
neurons (Johnson et al., 1992; Smith et al., 1996). Excitation
This work was supported in part by U.S. Public Health Service Grants
DA-03906 and MH-40817.
1
Current address: Department of Psychobiology, National Institute on
Drug Abuse/National Institutes of Health Building C, Room 321, 5500 Nathan
Shock Dr., Baltimore, MD 21224. E-mail: [email protected]
2) Stimulating orphanin receptors in the ventral tegmental area
selectively inhibits dopamine cells, and this did not alter NMDA
antagonist-induced motor activity. Whereas, stimulating ␥-aminobutyric acid (GABA)B receptors hyperpolarizes both dopamine and GABA cells in the ventral tegmental area, and this
abolished NMDA antagonist-induced motor activity. 3) The microinjection of an NMDA antagonist into the ventral tegmental
area did not increase dopamine metabolism in dopamine terminal fields, including the accumbens, striatum, or prefrontal
cortex. Also consistent with a lack of dopamine involvement,
repeated administration of NMDA antagonist into the ventral
tegmental area did not produce behavioral sensitization. These
data identify a mechanism to elicit a motor stimulant response
from the ventral tegmental area that does not involve activating
dopamine transmission.
of dopamine cells in the VTA is associated with behavioral
activation (Kalivas, 1993; Robinson and Berridge, 1993). Accordingly, pharmacological stimulation of ionotropic or
metabotropic glutamate receptors in the VTA elicits an increase in exploratory motor behavior and promotes the release of dopamine in mesocorticolimbic axon terminal fields
in the ventral striatum and prefrontal cortex (Suaud-Chagny
et al., 1992; Swanson and Kalivas, 2000). However, some
laboratories (Kalivas and Alesdatter, 1993; Narayanan et al.,
1996; Vezina and Queen, 2000), but not others (Kretschmer,
1999) have shown that blockade of the NMDA glutamate
receptor subtype in the VTA also stimulates motor activity.
The present study was designed to further investigate the
capacity of NMDA and non-NMDA glutamate receptor antagonists to produce a motor stimulant response when microinjected into the VTA.
Two populations of neurons can be distinguished in the
VTA, dopaminergic and GABAergic neurons (Johnson et al.,
1992). The dopaminergic and a portion of the GABAergic
ABBREVIATIONS: VTA, ventral tegmental area; NMDA, N-methyl-D-aspartate; GABA, ␥-aminobutyric acid; AMPA, ␣-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; AP-5, 2-amino-5-phosphonopentanoic acid; MCPG, (S)-␣-methyl-4carboxyphenylglycine; CPP, (3-(R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid; DAMGO, D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin; OFQ,
orphanin FQ-nociceptin; DOPAC, 3,4-dihydroxyphenyl acetic acid; HVA, homovanillic acid; ANOVA, analysis of variance; mGluR, metabotropic
glutamate receptor.
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ABSTRACT
Compounds acting in the ventral tegmental area to increase
motor activity are thought to do so by activating mesolimbic
dopamine transmission. The present report demonstrates that
the microinjection of N-methyl-D-aspartate (NMDA) antagonists
into the ventral tegmental area produces a dose-dependent
increase in motor activity. This effect was not mimicked by
antagonizing either ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate or metabotropic glutamate receptors in
the ventral tegmental area. Three experiments were conducted that indicated that the capacity of NMDA receptor
antagonists to elevate motor activity did not involve increased
dopamine transmission. 1) The systemic administration of a D1
dopamine receptor antagonist did not inhibit the motor stimulant response to NMDA antagonist injection into the ventral
tegmental area except at doses that also inhibited motor activity after an injection of saline into the ventral tegmental area.
NMDA Antagonists in VTA
Materials and Methods
Animal Housing and Surgery. All experiments were conducted
according to specifications of the National Institutes of Health Guide
for the Care and Use of Laboratory Animals. Male Sprague-Dawley
rats (Harlan Laboratories, Indianapolis, IN) weighing between 250
and 300 g were individually housed with food and water available ad
libitum. A 12-h light/dark cycle (7:00 AM–7:00 PM light) was used to
regulate the animal photocycle. All experimentation was carried out
during the light cycle.
Surgeries were performed 5 to 7 days after the arrival of the
subjects to the ALAC-approved housing facility and all experimentation began 1 week following the operative procedure. Animals were
anesthetized using ketamine HCl (100 mg/kg, Ketaset; Fort Dodge
Animal Health, Fort Dodge, IA) and xylazine (12 mg/kg, Rompun;
Bayer, Shawnee Mission, KS) and chronic indwelling guide cannulae
(26-gauge, 14 mm; Small Parts, Roanoke, VA) were aimed 1 mm
above injection site in the VTA (A-P, ⫹2.5 mm; L-M, ⫾0.6 mm; D-V,
⫺1.5 mm, from interaural zero angled 6o from the midline in accordance with Pellegrino et al., 1979). The guide cannulae were fixed to
the skull with three stainless steel screws (Small Parts) and dental
acrylic, and were fit with obturators (33-gauge, 14 mm; Small Parts)
between testing periods to prevent blockage by debris.
Drugs. All glutamate receptor antagonists used in this study were
purchased from Tocris Cookson (St. Louis, MO), including 6-cyano-7nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonopentanoic
acid (AP-5), (S)-␣-methyl-4-carboxyphenylglycine (MCPG), and (3-(R)2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). The D1 dopamine receptor antagonist (R)-SCH-23390 HCl and the ␮-opioid receptor
agonist {[D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO)} were purchased from RBI/Sigma (Natick, MA), and orphanin FQ-nociceptin
(OFQ) was a gift from Dr. David Grandy (Vollum Institute, Portland,
OR). All drugs were dissolved in sterile isotonic saline except CNQX,
which was dissolved in dimethyl sulfoxide and diluted in sterile water
to a 10% dimethyl sulfoxide solution. All drugs except SCH-23390 were
made up in bulk volume and stored at ⫺80°C. For all intracranial
injections, nanomolar concentrations represent the total amount administered per bilateral injection.
Microinjection and Experimental Design. Immediately prior
to testing, the obturators were removed and the injection cannulae
(33-gauge, 15 mm) fitted to a 1-␮l Hamilton syringe by PE-20 tubing
were inserted to a depth 1 mm below the tip of the guide cannula.
Bilateral infusions were made over 60 s in a total volume of 0.5
␮l/side. The infusion pump was turned off and the injection cannulae
were left in place for an additional 60 s to prevent backflow of drug
at which time animals were placed into the photocell cages (Omnitech, Columbus, OH). Twenty-four hours prior to the first microinjection, animals were preadapted to the photocell apparatus for 1 h,
given a sham microinjection (needle inserted but no injection made),
and returned to the photocell cage for another hour. Each test period
consisted of a 1-h habituation where animals were placed in photocell cages prior to testing. Following microinfusion, motor activity
was monitored in 15-min intervals for 2 h. Animals were returned to
their home cages at the close of each session and left undisturbed for
2 days following each test day to ensure clearance of drug and
recovery from the microinjection procedure. All experiments used a
counterbalanced design across days over the complete test period,
resulting in each animal receiving a maximum of six microinjections.
For the sensitization study, rats were microinjected into the VTA
with saline (N ⫽ 13) or CPP (0.03 nmol, N ⫽ 11, or 0.1 nmol, N ⫽ 17)
once a day for 3 days. All injections were made in the photocell
apparatus. One week later all animals were microinjected with CPP
(0.1 nmol) and 1 to 2 weeks later all rats were injected with cocaine
(15 mg/kg i.p.).
Motor activity was monitored in clear Plexiglas boxes measuring
22 ⫻ 43 ⫻ 33 cm. A series of 16 photobeams (eight on each horizontal
axis) tabulated horizontal movements, whereas a series of eight
beams located 8 cm above the floor spanned each box to vertical
activity (rearing). Photobeam breaks were recorded by computer
interface and the data was stored after each test day. Total horizontal activity, distance traveled (an estimate of locomotion where only
consecutive breaking of adjacent photocell beams is quantified), and
vertical activity (an estimate of rearing behavior) were monitored
during each test period.
Measurement of Dopamine and Metabolites. Subjects were
microinjected with AP-5 (5.0 nmol), DAMGO (0.1 nmol), or saline
into the VTA, placed in a photocell box to monitor motor activity, and
decapitated 30 min after injection. The nucleus accumbens, striatum, and prefrontal cortex were dissected (Latimer et al., 1987) and
the levels of dopamine and its metabolites 3,4-dihydroxyphenylacetic
acid (DOPAC) and homovanillic acid (HVA) were measured using
high-performance liquid chromatography with electrochemical detection as described elsewhere (Latimer et al., 1987). Peak heights
were measured and normalized to the internal standard isoproterenol for data analysis.
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neurons project out of the VTA (Fallon and Moore, 1978;
Thierry et al., 1980; Van Bockstaele and Pickel, 1995; Steffensen et al., 1998). The remainder of the GABAergic cells
are interneurons that provide inhibitory tone onto dopamine
cells (Johnson et al., 1992). Pharmacologically stimulating a
variety of neurotransmitter receptors in the VTA elicits a
motor stimulant response, including ␮-opioid, neurotensin,
Substance P, ionotropic glutamate (NMDA, AMPA, and kainate subtypes), and GABAA receptors (for review, see Kalivas, 1993). In all instances, the motor stimulant response has
been shown to be blocked by dopamine receptor antagonists
and/or associated with enhanced dopamine transmission in
the nucleus accumbens (Kelley et al., 1979; Cador et al.,
1989; Yoshida et al., 1997). Thus, to date the initiation of
motor activity by modulating neurotransmission in the VTA
is dopamine-dependent, arising from either a direct activation of dopamine neurons or disinhibition of dopamine cells
by inhibiting GABAergic interneurons (Kalivas, 1993). The
present study investigated whether the activation of motor
activity produced by administering NMDA antagonists into
the VTA is dopamine-dependent. This was accomplished by
evaluating the effect of dopamine antagonists on NMDA antagonist-induced motor stimulation, and by examining the
capacity of NMDA antagonist to alter dopamine transmission
in mesocorticolimbic dopamine axon terminal fields. If dopamine were found to mediate the NMDA antagonist-induced
behavioral activation it could be hypothesized that removing
excitatory tone from GABAergic interneurons was disinhibiting dopamnergic cells. Alternatively, if dopamine was determined not to mediate the effect of NMDA antagonists, this
would implicate disinhibition of GABAergic projections cells.
In addition to producing motor activity, the repeated administration of compounds directly into the VTA results in
behavioral sensitization (Elliott and Nemeroff, 1986; Kalivas
and Duffy, 1990). Thus, daily microinjection of ␮-opioid or
neurotensin agonists into the VTA elicits a progressive increase in motor activity that shows cross-sensitization with
systemically administered amphetamine-like psychostimulants and is associated with sensitized release of dopamine in
the nucleus accumbens. The present study also examined
whether the repeated administration of NMDA antagonist
into the VTA elicits behavioral sensitization to a subsequent
microinjection of NMDA antagonist or to the systemic administration of cocaine.
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Histology and Data Analysis. The rats not used for biochemical
analysis (see above) were administered an overdose of pentobarbitol
(⬎100 mg/kg i.p.) and transcardially perfused with 0.9% saline followed by a 10% formalin solution. The brain was removed and placed
in 10% formalin for at least 1 week to ensure proper fixation. Brains
were then blocked and coronal sections (100 ␮m) were made through
the site of cannula implantation with a vibratome. The brains were
subsequently stained with cresyl violet and anatomical placement
was verified by an individual unaware of the animal’s behavioral
response. The StatView statistics package was used to conduct oneway or two-way repeated measures analyses of variance (ANOVA) on
all behavioral data. The biochemical measures were statistically
evaluated using a one-way ANOVA. The levels of dopamine and its
metabolites were evaluated as the dopamine metabolite ratio calculated as follows: (DOPAC ⫹ HVA)/(DOPAC ⫹ HVA ⫹ dopamine).
Upon discovery of statistical significance, pairwise factorial analysis
was performed using a least-significant difference test (Milliken and
Johnson, 1984).
Fig. 2. Lack of behavioral effect of blocking AMPA/kainate and metabotropic glutamate receptors in the VTA. The data are shown as the total
photocell counts (mean ⫾ S.E.M.) obtained over the 2 h after microinjection. In each drug treatment group all animals received every dose in a
counterbalanced design and the data were evaluated using a one-way
repeated measures ANOVA. CNQX: N ⫽ 6, horizontal F(5,15) ⫽ 0.35, p ⫽
0.792; distance, F(5,15) ⫽ 0.31, p ⫽ 0.819; vertical, F(5,15) ⫽ 3.03, p ⫽ 0.034.
MCPG: N ⫽ 5, horizontal F(4,12) ⫽ 1.88, p ⫽ 0.187; distance, F(4,12) ⫽ 2.18,
p ⫽ 0.143; vertical, F(1,12) ⫽ 1.08, p ⫽ 0.395. *p ⬍ 0.05, compared with
saline using a Dunnett’s test for post hoc comparisons.
Results
Motor Stimulation by Blocking NMDA Receptors in
the VTA. Figure 1 shows the dose-dependent motor stimulant response elicited by the microinjection of AP-5 into the
VTA. The motor response occurred with a threshold dose
between 0.5 and 1.5 nmol and at the highest dose (5.0 nmol)
Fig. 3. Blockade of D1 dopamine receptors antagonizes AP-5-induced
motor activity only at doses that also inhibit spontaneous motor activity.
Data are shown as the mean ⫾ S.E.M. horizontal photocell counts over
the 120 min after the injection of intra-VTA injection of AP-5 (5.0 nmol)
or saline. Rats were pretreated 15 min prior to intracranial injection with
saline (1.0 ml/kg i.p.) or various doses of SCH-23390. A, upper lines refer
to the mean ⫾ S.E.M. photocell counts after saline (i.p.) and AP-5 in the
VTA, and the filled squares correspond to AP-5 plus increasing doses of
SCH-23390. The lower lines refer to treatment saline (i.p.) and saline
(intra-VTA) and the open squares refer to increasing doses of SCH-23390
plus saline (intra-VTA). B, data in A normalized to percentage of change
from saline control. This was done to more readily compare the effect of
D1 blockade between the control and AP-5-induced motor responses.
Data were evaluated using an overall two-way ANOVA (treatment,
F(1,4) ⫽ 0.89, p ⫽ 0.348; dose, F(4,57) ⫽ 11.98, p ⬍ 0.001; interaction,
F(4,57) ⫽ 0.30, p ⫽ 0.874) followed by individual comparisons of different
doses of SCH-23390 to saline within each treatment (i.e., saline or AP-5)
using a paired Student’s t test with probability values adjusted according
to the Bonferonni method (Milliken and Johnson, 1984). *p ⬍ 0.05,
comparing each dose of SCH-23390 with saline.
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Fig. 1. Blockade of NMDA receptors in the VTA with AP-5 produces a dose-dependent elevation in motor activity. Rats were adapted to the photocell
box for 60 min, injected with various doses of AP-5 into the VTA at time zero, and motor activity was monitored for 2 h. Left, time course (mean value).
Right, total photocell counts (mean ⫾ S.E.M.) accumulated over 2 h after injection. All subjects (N ⫽ 7) received each dose in counterbalanced order.
The time course data were evaluated using a two-way ANOVA with repeated measures over time [dose F(3,24) ⫽ 3.98, p ⫽ 0.020; time F(11,264) ⫽ 113.84,
p ⬍ 0.001; interaction F(33,264) ⫽ 4.72, p ⬍ 0.001], and the total counts were evaluated using a one-way repeated measures ANOVA [horizontal F(3,27) ⫽
12.67, p ⬍ 0.001; distance F(3,27) ⫽ 16.13, p ⬍ 0.001; vertical F(3,27) ⫽ 5.57, p ⫽ 0.007]. *p ⬍ 0.05 comparing AP-5 to saline using a Dunnett’s test for
post hoc comparison.
NMDA Antagonists in VTA
coadministration of the ␮-opioid DAMGO into the VTA (Fig.
4C). DAMGO microinjection into the VTA did not elevate
rearing behavior.
Figure 5 shows that while the microinjection of either AP-5
or DAMGO into the VTA elicited a motor stimulant response,
only DAMGO microinjection was associated with an increase
in dopamine metabolism in the nucleus accumbens and striatum. Neither AP-5 nor DAMGO elevated dopamine metabolism in the prefrontal cortex.
Lack of Behavioral Sensitization Produced by Repeated NMDA Antagonist Microinjection into the VTA.
Similar to DAMGO, other compounds such as neurotensin or
Substance P elicit a dopamine-dependent activation of motor
activity when microinjected into the VTA, and the repeated
administration of all of these compounds results in long-term
behavioral sensitization of the motor stimulant effect (for
review, see Kalivas, 1993). Figure 6 shows that the daily
administration of the competitive NMDA antagonist CPP
into the VTA did not produced behavioral sensitization to a
challenge microinjection of CPP made 7 days later. Figure 6A
shows that, similar to AP-5, the acute administration of CPP
produced a significant dose-dependent elevation in motor
activity. Figure 6B shows that 1 week after the last daily
injection of CPP a subsequent microinjection of CPP (0.1
nmol) produced a similar increase in motor activity in all
three daily treatment groups, indicating that neither tolerance nor sensitization of the motor response was elicited by
daily CPP injections. One to 2 weeks after the CPP challenge
microinjection (i.e., at 2–3 weeks after the last daily microinjection of saline or CPP) rats were injected with cocaine (15
mg/kg i.p.). Figure 6C shows that the increase in horizontal
photocell counts and distance traveled elicited by cocaine was
significantly reduced in rats retreated with the highest daily
dose of CPP. The time course data in Fig. 6D reveal that the
blunted behavioral response to cocaine occurred during the
first hour after cocaine administration. Three days following
the final injection of cocaine, some rats were decapitated and
the level of dopamine quantified in the nucleus accumbens to
determine whether repeated administration of CPP into the
VTA had damaged the dopamine projection. It was found
Fig. 4. Behavioral activation by AP-5 is blocked by coadministration of baclofen, but not OFQ. Subjects were injected with a mixture of OFQ or baclofen
plus AP-5 or baclofen into the VTA. A, mean ⫾ S.E.M. photocell counts over 2 h following baclofen plus AP-5. Each animal received all four treatments
in each group (N ⫽ 6) and the data were evaluated using a one-way repeated measures ANOVA (horizontal, F(3,23) ⫽ 13.60, p ⬍ 0.001; distance, F(3,23) ⫽
16.13, p ⬍ 0.001; vertical, F(3,23) ⫽ 5.57, p ⫽ 0.007). B, data for subjects treated with OFQ plus AP-5 (N ⫽ 7; horizontal, F(3,27) ⫽ 10.81, p ⬍ 0.001;
distance, F(3,27) ⫽ 10.87, p ⬍ 0.001; vertical, F(3,27) ⫽ 3.84, p ⫽ 0.028). C, data for subjects treated with OFQ plus DAMGO (N ⫽ 6; horizontal, F(3,23) ⫽
13.35, p ⬍ 0.001; distance, F(3,23) ⫽ 12.63, p ⬍ 0.001; vertical, F(3,23) ⫽ 0.46, p ⫽ 0.986). *p ⬍ 0.05, comparing all treatments to saline/saline using a
least-significant difference test (Milliken and Johnson, 1984). ⫹ p ⬍ 0.05, comparing saline plus AP-5 or DAMGO with OFQ or baclofen plus AP-5 or
DAMGO.
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significant behavioral activation endured for 45 min after
AP-5 administration. A similar dose-dependent psychomotor
stimulant effect by AP-5 was measured in estimates of both
locomotion (distance) and rearing.
Figure 2 reveals that intra-VTA administration of the nonNMDA ionotropic glutamate receptor antagonist CNQX did
not alter locomotor activity over a dose range from 0.03 to
10.0 nmol. However, a biphasic effect was measured on rearing behavior with a significant increase being induced by 1.0
nmol of CNQX. The nonselective mGluR competitive antagonist MCPG was without effect on locomotor or rearing behavior over a dose range of 1.0 to 30.0 nmol.
Lack of Dopamine Involvement in Motor Activity
Induced by Blocking NMDA Receptors. The systemic
administration of the dopamine D1 receptor antagonist SCH23390 has been shown previously to block the motor response
elicited by dopamine-dependent psychomotor stimulants
such as amphetamine and cocaine (White et al., 1998). Figure
3 shows that only at doses that suppress spontaneous motor
activity was the systemic administration of SCH-23390 effective at inhibiting the motor response elicited by intra-VTA
administration of AP-5 (5.0 nmol). Evaluation of the data
transformed to percentage of change from systemic saline
administration (Fig. 3B) reveals that at a dose as low as 0.03
mg/kg SCH-23390 significantly reduced both spontaneous and AP-5-induced motor activity. However, a dose of0.3 mg/kg was required to completely block AP-5-induced
locomotion.
Previous studies have shown that stimulating GABAB receptors inhibits the firing frequency of both dopaminergic
and GABAergic cells in the VTA (Johnson and North, 1992).
In contrast, stimulating OFQ receptors in the VTA selectively inhibits dopamine neurons (Murphy and Maidment,
1999). Figure 4 shows that while the coadministration of
baclofen with AP-5 into the VTA inhibited AP-5-induced locomotor activity, OFQ was without effect on AP-5-induced
locomotion. In contrast to locomotion, OFQ partly antagonized AP-5-induced rearing behavior. The biological effectiveness of the dose of OFQ used was verified by showing that
OFQ inhibited the motor stimulant response elicited by the
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that the repeated injection of CPP did not alter the levels of
dopamine (saline, N ⫽ 4, 301 ⫾ 36 pmol/mg of protein; 0.1
nmol CPP, N ⫽ 5, 376 ⫾ 73 pmol/mg).
Histology. Figure 7A reveals the location of cannulae tips
in the VTA of rats used in this study. The cannulae tips were
in the nucleus parabrachialis pigmentosus and nucleus paranigralis, ranging mediolaterally from the lateral aspects of
the interpeduncular nucleus to the medial edge of the substantia nigra, pars compacta. The micrograph in Fig. 7C
shows an injection site from an animal receiving daily CPP
(0.1 nmol) in the sensitization study (see above), and the
high-power micrograph in Fig. 7D reveals no neurotoxicity
produced by this treatment beyond the mechanical damage
caused by penetration of the injection needle. The micrograph in Fig. 7B is an example of an animal having cannulae
placement dorsal to the VTA that did not demonstrate a
motor stimulant response after microinjection of AP-5.
Discussion
The present study reveals that inhibition of NMDA receptors in the VTA elicits a dose-dependent stimulation of motor
activity. Consistent with previous studies (Mathe et al., 1998;
Svensson et al., 1998), except for a biphasic effect on rearing
the microinjection of the AMPA/kainate antagonist CNQX
did not alter motor activity over the dose range examined
(see below). Likewise, blockade of mGluRs in the VTA with
the subtype nonselective antagonist MCPG was without effect on motor behavior.
Lack of Involvement by Dopamine Neurons in the
Motor Stimulant Response to Intra-VTA AP-5 Administration. The VTA contains dopamine cells that project to
forebrain nuclei (Fallon and Moore, 1978), and the microinjection of a variety of neurotransmitter agonists and antagonists into the VTA stimulates motor activity by increasing
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Fig. 5. Motor response elicited by the NMDA antagonist AP-5 is not
associated with increased dopamine metabolism in the nucleus accumbens, striatum, or prefrontal cortex. A, neurochemical response 30 min
following microinjection of saline (N ⫽ 8), AP-5 (5.0 nmol; N ⫽ 5), or
DAMGO (0.1 nmol; N ⫽ 7). The data are shown as the mean ⫾ S.E.M.
dopamine metabolite ratio (DOPAC ⫹ HVA)/(DOPAC ⫹ HVA ⫹ dopamine). The raw data (pmol/mg of protein) following saline treatment for
each brain region are as follows: accumbens, dopamine ⫽ 390.7 ⫾ 36.0,
DOPAC ⫽ 71.9 ⫾ 7.6, HVA ⫽ 24.0 ⫾ 1.3; striatum, dopamine ⫽ 731.3 ⫾
53.6, DOPAC ⫽ 53.9 ⫾ 2.7, HVA ⫽ 25.7 ⫾ 2.1; and prefrontal cortex,
dopamine ⫽ 5.9 ⫾ 0.7, DOPAC ⫽ 1.8 ⫾ 0.1, HVA ⫽ 2.0 ⫾ 0.2. B, motor
stimulant response (mean ⫾ S.E.M. horizontal photocell counts) over 30
min following microinjection before subjects were decapitated for neurochemical analysis. All data were evaluated using a one-way ANOVA and
drug treatments compared with saline using a Dunnett’s test. *p ⬍ 0.05,
comparing AP-5 or DAMGO with saline.
mesocorticolimbic dopamine transmission (Kalivas, 1993;
Karreman et al., 1996; Westerink et al., 1996). Surprisingly,
three experiments in the present study revealed that the
motor activity elicited by NMDA antagonists in the VTA is
likely independent of effects on dopamine transmission. 1)
Systemic or intra-accumbens administration of the dopamine
D1 receptor antagonist SCH-23390 inhibits the motor stimulant effect of amphetamine-like psychostimulants (Baker et
al., 1998; White et al., 1998), and the motor response to
intra-VTA administration of AP-5 was antagonized only in
doses sufficient to inhibit spontaneous motor activity. 2) The
administration of OFQ into the VTA is known to selectively
inhibit dopamine cells (Murphy and Maidment, 1999) and
OFQ did not inhibit AP-5-induced locomotor activity, but did
antagonize the motor stimulant response elicited by the
␮-opioid DAMGO, which is known to increase motor activity
via activation of dopamine cells (Cador et al., 1989; Kalivas
and Duffy, 1990). 3) Also, in contrast with stimulating ␮-opioid receptors, inhibiting NMDA receptors in the VTA elicited
a motor stimulant response that did not increase measures of
dopamine metabolism in dopamine axon terminal fields.
A Role for GABAergic Neurons in the VTA in the
Motor Stimulant Response to Intra-VTA AP-5. The apparent lack of involvement by increased activity of dopamine
neurons is consistent with the findings that competitive
NMDA antagonists administered into the ventral mesencephalon decrease burst firing of dopamine neurons (Overton
and Clark, 1992; Chergui et al., 1993; Christoffersen and
Meltzer, 1995), and a decrease in burst firing would be expected to decrease axon terminal field dopamine release
(Suaud-Chagny et al., 1992). Also, in vivo microdialysis studies find that the administration of AP-5 into the VTA does not
alter the extracellular levels of dopamine in the VTA, nucleus
accumbens, or prefrontal cortex (Enrico et al., 1998; Svensson et al., 1998; Kretschmer, 1999; Fu et al., 2000). The
substantive data that the administration of competitive
NMDA antagonists into the VTA does not alter dopamine
firing frequency or mesocorticolimbic dopamine transmission
point to possible involvement of GABAergic neurons in the
VTA. While the synaptic organization and function of
GABAergic interneurons in the VTA is partly understood,
there is a significant population of GABAergic projection
neurons that have been less well characterized. GABAergic
neurons in the ventral mesencephlon project to a number of
nuclei in parallel with dopamine neurons, including the prefrontal cortex and nucleus accumbens (Thierry et al., 1980;
Van Bockstaele and Pickel, 1995; Steffensen et al., 1998). The
pharmacological administration of glutamate agonists or
stimulating cortical glutamatergic afferents depolarizes and
stimulates firing in both nondopaminergic and dopaminergic
neurons in the VTA (Johnson et al., 1992). Moreover, NMDA
antagonists reduce the spontaneous firing activity of nondopamine cells in the VTA (Steffensen et al., 1998; Bonci and
Malenka, 1999). If these nondopaminergic cells are GABAergic interneurons this action would be expected to increase
dopamine cell activity via disinhibition; however, the apparent lack of dopamine involvement in AP-5-induced motor
activity argues that this is not a primary action. Rather, it
can be postulated that the NMDA antagonist is decreasing
the activity of GABAergic projection neurons, thereby removing inhibitory tone in forebrain nuclei where GABAergic tone
NMDA Antagonists in VTA
231
is known to inhibit spontaneous and pharmacologically induced behavior (Swerdlow et al., 1990; Xi and Stein, 2000).
If a nondopaminergic mechanism is supported by subsequent studies, it offers an alternative route whereby neurotransmission in the VTA can regulate behavioral activation.
Possible glutamatergic afferents to the VTA that could contribute to such a mechanism include the prefrontal cortex
and medial aspects of the subthalamic nucleus and pedunculopontine region (Smith et al., 1996; Carr and Sesack, 2000a).
The present study cannot distinguish which of the afferents
may provide preferential regulation of GABAergic versus
dopaminergic neurons in the VTA. However, recent studies
from Sesack and coworkers reveal the likelihood that glutamatergic afferents from the prefrontal cortex synapse on
GABAergic projection neurons in the VTA (Carr and Sesack,
2000a,b).
Repeated NMDA Antagonist Administration and Cocaine-Induced Motor Activity. The motor stimulant response elicited by the microinjection of drugs into the VTA
that increase dopamine transmission undergoes sensitization following repeated intra-VTA administration (for review, see Kalivas, 1993). Moreover, behavioral sensitization
produced by repeated intra-VTA injections of neurotensin,
␮-opioid, or D1 agonist results in behavioral cross-sensitization to systemically administered psychostimulants such as
amphetamine and cocaine (Kalivas, 1993; Pierce et al., 1996).
Behavioral sensitization elicited by intra-VTA and systemically administered drugs arises in part from stimulating
NMDA receptors in the VTA (Kalivas and Alesdatter, 1993;
Vezina and Queen, 2000). Given these data it is perhaps not
surprising that repeated administration of NMDA antagonist into the VTA did not elicit behavioral sensitization to a
subsequent challenge with either intra-VTA CPP or systemic
cocaine administration. However, it is important to note that
although the daily dosing regimen is similar to that required
for other drugs to elicit behavioral sensitization when microinjected into the VTA (see Elliott and Nemeroff, 1986, for
discussion of daily versus less frequent repeated intra-VTA
adminstration), and the dose range used has been shown
previously to be behaviorally active (Kalivas and Alesdatter,
1993), a different dosage regimen may have elicited behavioral sensitization. Not only did repeated NMDA antagonist
not elicit behavioral sensitization but also the response to
systemic cocaine was reduced in subjects pretreated with
daily intra-VTA administration of NMDA antagonist. The
mechanism mediating the inhibition of cocaine-induced motor activity was not determined in the present report. However, it does not appear to be related to neurotoxicity produced by repeated intra-VTA injections of CPP for two
reasons. 1) No toxicity was apparent from Nissl-stained tissue containing the injection site, and 2) the tissue levels of
dopamine in the nucleus accumbens were not different between animals injected with daily saline compared with daily
CPP-injected subjects. This argues that long-lasting neuroadaptations are produced by the repeated removal of NMDA
tone to neurons in the VTA. Since the motor stimulant response to an acute injection of cocaine arises to a great extent
by increasing dopamine transmission in the nucleus accumbens (Kelly and Iversen, 1976), it is possible that repeated
removal of NMDA tone on dopamine neurons has reduced the
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Fig. 6. Repeated microinjection of the NMDA antagonist CPP into the VTA does not produce behavioral sensitization, but does produce an enduring
decrease in cocaine-induced motor activity. Rats were injected into the VTA daily for 3 days with saline (N ⫽ 13) or CPP (0.03 nmol, N ⫽ 11, or 0.1
nmol, N ⫽ 17), and all subjects were subsequently challenged with CPP (0.1 nmol) or cocaine (15 mg/kg i.p.). A, mean ⫾ S.E.M. photocell counts over
120 min after the first daily injection. B, data from the CPP challenge. C, data from the cocaine challenge (arrow indicates time of injection). Total
photocell counts were evaluated using a two-way ANOVA with repeated measures over days (horizontal: treatment, F(3,38) ⫽ 1.70, p ⫽ 0.197; day,
F(2,76) ⫽ 74.67, p ⬍ 0.001; interaction, F(4,76) ⫽ 4.06, p ⫽ 0.005; distance: treatment, F(3,38) ⫽ 1.32, p ⫽ 0.279; day, F(2,76) ⫽ 70.38, p ⬍ 0.001; interaction,
F(4,76) ⫽ 3.50, p ⫽ 0.011; vertical: treatment, F(3,38) ⫽ 0.60, p ⫽ 0.554; day, F(2,76) ⫽ 10.30, p ⬍ 0.001; interaction, F(4,76) ⫽ 1.41, p ⫽ 0.240). D, time
course data corresponding to the cocaine challenge data (treatment, F(2,38) ⫽ 3.50, p ⫽ 0.040; time, F(11,418) ⫽ 67.84, p ⬍ 0.001; interaction, F(20,418) ⫽
2.85, p ⬍ 0.001). *p ⬍ 0.05, comparing the CPP with the saline treatment groups using a least-significant difference test (Milliken and Johnson, 1984).
232
Cornish et al.
mimicked by blockade of either the AMPA/kainate or mGluR
subtypes of glutamate receptor. The motor stimulant response is not mediated by enhancing dopamine transmission
and presumably involves the removal of NMDA-mediated
excitatory stimulation of GABAergic projection neurons in
the VTA. The finding that repeated NMDA antagonist administration into the VTA reduced the motor stimulant effect
of a subsequent injection of cocaine may indicate a novel
mechanism for reducing the effects of cocaine by inducing
long-term neuroadaptations that counter the pharmacological action of cocaine. Indeed, many reports have demonstrated that psychostimulant-induced behavioral sensitization is blocked by pretreatment with NMDA antagonists (for
review, see Wolf, 1998).
Acknowledgments
excitability of dopamine cells (Johnson and North, 1992).
Alternatively, repeated reduction in NMDA-mediated excitatory tone to GABAergic neurons in the VTA may alter the
inhibitory tone provided by GABAergic interneurons to dopamine cells.
Rearing versus Locomotion. In two instances distinctions between drug effects on rearing and locomotion were
observed: 1) CNQX administration into the VTA elicited a
dose-related biphasic effect on rearing without altering locomotion, and 2) OFQ antagonized AP5-induced rearing without altering the locomotor response to AP-5. This distinction
between rearing and locomotion reveals that different substrates in the VTA mediate each behavior. Moreover, the
capacity of OFQ to antagonize rearing behavior indicates
likely involvement of dopamine transmission. This could
arise from either 1) direct or indirect activation of dopamine
cells, which seems unlikely given the lack of effect on measures of dopamine transmission (although an effect on dopamine transmission in an axon terminal field not evaluated in
this study is possible); or 2) dopamine transmission is permissive to rearing behavior. While NMDA antagonists do not
stimulate dopamine neurons to increase terminal field dopamine transmission, dopaminergic tone may be necessary to
manifest rearing in response to NMDA receptor blockade in
the VTA.
Conclusions
The inhibition of NMDA glutamate receptors in the VTA
produces an increase in motor activity. This effect is not
We thank Carol Heissenbuttle for assistance in preparing the
manuscript, Dr. David Grandy for the generous gift of orphanin, and
National Institute on Drug Abuse for providing cocaine HCl.
References
Baker DA, Fuchs RA, Specio SE, Khroyan TV and Neisewander JL (1998) Effects of
intraaccumbens administration of SCH-23390 on cocaine induced locomotion and
conditioned place preference. Synapse 30:181–193.
Bonci A and Malenka R (1999) Properties and plasticity of excitatory synapses on
dopaminergic and GABAergic cells in the ventral tegmental area. J Neurosci
19:3723–3730.
Cador M, Rivet J-M, Kelley J-M, LeMoal M and Stinus L (1989) Substance P,
neurotensin and enkephalin injections into the ventral tegmental area: a comparative study on dopamine turnover in several forebrain structures. Brain Res
486:357–363.
Carr DB and Sesack SR (2000a) Projections from the rat prefrontal cortex to the
ventral tegmental area: target specificity in the synaptic associations with mesoaccumbens and mesocortical neurons. J Neurosci 20:3864 –3873.
Carr DB and Sesack SR (2000b) GABA-containing neurons in the rat ventral tegmental area project to the prefrontal cortex. Synapse 38:114 –123.
Chergui K, Charlety PJ, Akaoka H, Saunier CF, Brunet J-L, Buda M, Svensson TH
and Chouvet G (1993) Tonic activation of NMDA receptors causes spontaneous
burst discharge of rat midbrain dopamine neurons in vivo. Eur J Neurosci 5:137–
144.
Christoffersen CL and Meltzer LT (1995) Evidence for N-methyl-D-aspartate and
AMPA subtypes of the glutamate receptors on substantia nigra dopamine neurons:
possible preferential role for N-methyl-D-aspartate receptors. Neuroscience 67:
373–381.
Elliott PJ and Nemeroff CB (1986) Repeated neurotensin administration in the
ventral tegmental area: effects on baseline and d-amphetamine-induced locomotor
activity. Neurosci Lett 68:239 –244.
Enrico P, Bouma M, de Vries JB and Westerink BHC (1998) The role of afferents to
the ventral tegmental area in the handling stress-induced increase in the release
of dopamine in the medial prefrontal cortex: a dual-probe microdialysis study in
the rat brain. Brain Res 779:205–213.
Fallon JH and Moore RY (1978) Catecholamine innervation of basal forebrain. IV.
Topography of the dopamine projection to the basal forebrain and striatum.
J Comp Neurol 180:545–580.
Fu Y, Matta S, Gao W, Brower V and Sharp B (2000) Systemic nicotine stimulates
dopamine release in nucleus accumbens: re-evaluation of the role of N-methyl-Daspartate receptors in the ventral tegmental area. J Pharmacol Exp Ther 294:
458 – 465.
Johnson SW, Seutin V and North RA (1992) Burst firing in dopamine neurons
induced by N-methyl-D-aspartate: role of electrogenic sodium pump. Science (Wash
DC) 258:665– 667.
Kalivas PW (1993) Neurotransmitter regulation of dopamine neurons in the ventral
tegmental area. Brain Res Rev 18:75–113.
Kalivas PW and Alesdatter JE (1993) Involvement of NMDA receptor stimulation in
the VTA and amygdala in behavioral sensitization to cocaine. J Pharmacol Exp
Ther 267:486 – 495.
Kalivas PW and Duffy P (1990) Effect of acute and daily neurotensin and enkephalin
treatments on extracellular dopamine in the nucleus accumbens. J Neurosci 10:
2940 –2949.
Karreman M, Westerink BHC and Moghaddam B (1996) Excitatory amino acid
receptors in the ventral tegmental area regulate dopamine release in the ventral
striatum. J Neurochem 67:601– 607.
Kelley AE, Stinus L and Iversen SD (1979) Behavioral activation induced in the rat
by substance P infusion into the ventral tegmental area: implication of dopaminergic A10 neurons. Neurosci Lett 11:335–339.
Kelly PH and Iversen SD (1976) Selective 6-OHDA-induced destruction of mesolimbic dopamine neurons: abolition of psychostimulant-induced locomotor activity in
rats. Eur J Pharmacol 40:45–56.
Kretschmer BD (1999) Modulation of the mesolimbic dopamine system by glutamate:
role of NMDA receptors. J Neurochem 73:839 – 848.
Latimer LG, Duffy P and Kalivas PW (1987) Mu opioid receptor involvement in
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 5, 2017
Fig. 7. Histological verification of cannulae placement in the VTA and
lack of neurotoxicity by repeated intra-VTA microinjection of CPP. A,
location of cannulae tips in the VTA of animals used in the dose-response
studies and experiments involving baclofen and OFQ. The numbers on
the left of the drawings indicate the distance (mm) of the coronal section
rostral to interaural zero (Paxinos and Watson, 1986). B, micrograph
showing an example of cannulae tips that were located outside of the VTA
where the microinjection of AP-5 did not elicit a motor stimulant response. C, micrograph illustrating cannulae placement in the VTA. D,
high-power micrograph corresponding to the area in the box in C and
demonstrates a lack of cell death at a site of repeated CPP administration
into the VTA. While damage was produced by mechanical penetration of
the injection needle, the adjacent neuropil appears healthy. The solid
arrows indicate healthy cells, while the striped arrows indicate the location of damage by injection needle. IP, interpeduncular nucleus; SNp,
substantia nigra, pars compacta; SNr, substantia nigra, reticulat; VTA,
ventral tegmental area. Scale bar, 1 mm (B and C), 100 ␮m (D).
NMDA Antagonists in VTA
acids in the ventral tegmental area for central actions of non-competitive NMDAreceptor antagonists and nicotine. Amino Acids 14:51–56.
Swanson CJ and Kalivas PW (2000) Regulation of locomotor activity by metabotropic
glutamate receptors in the nucleus accumbens and ventral tegmental area.
J Pharmacol Exp Ther 292:406 – 414.
Swerdlow NR, Braff DL and Geyer MA (1990) GABAergic projection from nucleus
accumbens to ventral pallidum mediates dopamine-induced sensorimotor gating
deficits of acoustic startle in rats. Brain Res 532:146 –154.
Thierry A, Deniau J, Herve D and Chevalier G (1980) Electrophysiological evidence
for non-dopaminergic mesocortical and mesolimbic neurons in the rat. Brain Res
201:210 –214.
Van Bockstaele EJ and Pickel VM (1995) GABA-containing neurons in the ventral
tegmental area project to the nucleus accumbens in the rat brain. Brain Res
682:215–221.
Vezina P and Queen A (2000) Pharmacological reversal of behavioral and cellular
indices of cocaine sensitization in the rat. Psychopharmacology 151:184 –191.
Westerink BHC, Kwint H-F and deVries JB (1996) The pharmacology of mesolimbic
dopamine neurons: a dual-probe microdialysis study in the ventral tegmental area
and nucleus accumbens of the rat brain. J Neurosci 16:2605–2611.
White FJ, Joshi A, Koeltzow TE and Hu X-T (1998) Dopamine receptor antagonists
fail to prevent induction of cocaine sensitization. Neuropsychopharmacology 18:
26 – 40.
Wolf ME (1998) The role of excitatory amino acids in behavioral sensitization to
psychomotor stimulants. Prog Neurobiol 54:679 –720.
Xi Z and Stein E (2000) Increased mesolimbic GABA concentration blocks heroin self
administration in the rat. J Pharmacol Exp Ther 294:613– 619.
Yang C, Seamans J and Gorelova N (1999) Developing a neuronal model for the
pathophysiology of schizophrenia based on the nature of electrophysiological actions of dopamine in the prefrontal cortex. Neuropsychopharmacology 21:161–194.
Yoshida M, Yokoo H, Nakahara K, Tomita M, Hamada N, Ishikawa M, Hatakeyama
J, Tanaka M and Nagatsu I (1997) Local muscimol disinhibits mesolimbic dopaminergic activity as examined by brain microdialysis and Fos immunohistochemistry. Brain Res 767:356 –360.
Address correspondence to: Peter Kalivas, Ph.D., Department of Physiology and Neuroscience, P.O. Box 250677, Medical University of South Carolina,
Charleston, SC 29425. E-mail: [email protected] [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 5, 2017
enkephalin activation of dopamine neurons in the ventral tegmental area. J Pharmacol Exp Ther 241:328 –337.
Mathe J, Nomikos G, Schilstrom B and Svensson T (1998) Non-NMDA excitatory
amino acid receptors in the ventral tegmental area mediate systemin dizocilpine
(MK-801) induced hyperlocomotion and dopamine release in the nucleus accumbens. J Neurosci Res 51:583–592.
Milliken GA and Johnson DE (1984) Analysis of Messy Data. Volume I: Designed
Experiments. Lifetime Learning Publications, Belmont, CA.
Murphy N and Maidment N (1999) Orphanin FQ/nociceptin modulation of mesolimbic dopamine transmission determined by microdialysis. J Neurochem 73:179 –
186.
Narayanan S, Willins D, Dalia A, Wallace L and Uretsky N (1996) Role of dopaminergic mechanisms in the stimulatory effects of MK-801 injected into the ventral
tegmental area and the nucleus accumbens. Pharmacol Biochem Behav 54:567–
573.
Overton P and Clark D (1992) Iontophretically administered drugs acting at the
N-methyl-D-aspartate receptor modulate burst firing in A9 dopamine neurons in
the rat. Synapse 10:131–140.
Paxinos G and Watson C (1986) The Rat Brain in Stereotaxic Coordinates. Academic
Press, New York.
Pellegrino LK, Pellegrino AS and Cushman AJ (1979) A Stereotaxic Atlas of the Rat
Brain. Plenum Press, New York.
Pierce RC, Born B, Adams M and Kalivas PW (1996) Repeated intral-ventral tegmental area administration of SKF-38393 induces behavioral and neurochemical
sensitization to a subsequent cocaine challenge. J Pharmacol Exp Ther 278:384 –
392.
Robinson TE and Berridge KC (1993) The neural basis of drug craving: an incentivesensitization theory of addiction. Brain Res Rev 18:247–291.
Smith Y, Charara A and Parent A (1996) Synaptic innervation of midbrain dopaminergic neurons by glutamate-enriched terminal in the squirrel monkey. J Comp
Neurol 364:231–253.
Steffensen SC, Svingos AL, Pickel VM and Henriksen SJ (1998) Electrophysiological
characterization of GABAergic neurons in the ventral tegmental area. Neuroscience 18:8003– 8015.
Suaud-Chagny MF, Chergui K, Chouvet G and Gonon F (1992) Relationship between
dopamine release in the rat nucleus accumbens and the discharge activity of
dopaminergic neurons during local in vivo application of amino acids in the ventral
tegmental area. Neuroscience 49:63–72.
Svensson T, Mathe J, Nomikos G and Schilstrom B (1998) Role of excitatory amino
233