Introduction

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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 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 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 µ-opioid, neurotensin, Substance P, ionotropic glutamate (NMDA, AMPA, and kainate subtypes), and GABA_A 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 µ-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.

	Materials and Methods

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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.

Drugs. All glutamate receptor antagonists used in this study were purchased from Tocris Cookson (St. Louis, MO), including 6-cyano-7-nitroquinoxaline-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 µ-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-µl 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 µl/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.

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.

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 µm) 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 one-way 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).

	Results

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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) 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.

View larger version (20K): [in this window] [in a new window]   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.

View larger version (28K): [in this window] [in a new window]   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.

Lack of Dopamine Involvement in Motor Activity Induced by Blocking NMDA Receptors. The systemic administration of the dopamine D1 receptor antagonist SCH-23390 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.

View larger version (18K): [in this window] [in a new window]   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.

View larger version (20K): [in this window] [in a new window]   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.

View larger version (27K): [in this window] [in a new window]   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.

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 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).

View larger version (32K): [in this window] [in a new window]   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).

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.

View larger version (126K): [in this window] [in a new window]   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 µm (D).

	Discussion

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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 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 µ-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 µ-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 is known to inhibit spontaneous and pharmacologically induced behavior (Swerdlow et al., 1990; Xi and Stein, 2000).

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, µ-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 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

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The inhibition of NMDA glutamate receptors in the VTA produces an increase in motor activity. This effect is not 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).