Introduction

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In the rat, low doses of amphetamine produce increases in locomotor activity when administered acutely by systemic injection. Considerable evidence now indicates that this effect is mediated by actions of this drug on mesolimbic dopaminergic neurotransmission. Thus, the locomotor activation produced by amphetamine is associated with an increase in extracellular levels of DA in the NAcc, which receives dense axonal projections from mesolimbic DA neurons (Clarke et al., 1988; Kuczenski and Segal, 1989; Meredith et al., 1993). AMPH injections into the NAcc increase locomotion, whereas injections of DA receptor antagonists into the NAcc and lesions of DA nerve terminals in this region block amphetamine-induced locomotion (Roberts et al., 1975; Kelly and Iversen, 1976; Joyce and Koob, 1981; Vezina et al., 1991).

Glutamatergic projections to the NAcc originating from prefrontal cortex, hippocampus and amygdala (Christie et al., 1987; Meredith et al., 1993) also regulate locomotor activity by interacting with dopaminergic neurotransmission. Anatomical studies of the NAcc provide the basis for a possible interaction between DA and glutamate at the level of nerve terminals in this site (Sesack and Pickel, 1990, 1992). Consistent with the results of these studies, it has been shown that agonists of iGluRs, including AMPA and NMDA, increase extracellular levels of DA and locomotor activity when infused into the NAcc and these effects are inhibited by drugs that interfere with dopaminergic neurotransmission (Donzanti and Uretsky, 1983; Boldry and Uretsky, 1988; Imperato et al., 1990; Youngren et al., 1993). Furthermore, the application of glutamate receptor antagonists to the NAcc attenuates the locomotor effects of psychomotor-stimulant drugs. For example, it has been shown that the infusion of AMPA/kainate glutamate receptor antagonists into the NAcc reduces the increases in locomotor activity and in the levels of extracellular DA in this site produced by either AMPH or cocaine (Pulvirenti et al., 1989; Willins et al., 1992; Kaddis et al., 1993; Pap and Bradberry, 1995). NMDA receptor antagonists have also been reported to decrease such psychomotor stimulant-induced effects (Pulvirenti et al., 1991; Kelley and Throne, 1992; Burns et al., 1994; Moghaddam and Bolinao, 1994). These results suggest that increases in glutamatergic neurotransmission as well as in dopaminergic neurotransmission contribute to AMPH-induced locomotion.

The NAcc expresses high amounts of mGluRs (Albin et al., 1992; Shigemoto et al., 1992; Testa et al., 1994; Romano et al., 1995). The mGluRs represent a large family of G protein-coupled receptors that activate multiple second messenger systems leading to various signal transduction processes (Nakanishi, 1994). The recent development of mGluR-selective ligands (Eaton et al., 1993; Birse et al., 1993; Hayashi et al., 1994) has introduced important roles for mGluRs in the modulation of voltage- and ligand-gated ion channels, the induction of long-term changes in synaptic strength and in spatial and olfactory memory formation (Schoepp and Conn, 1993; Pin and Bockaert, 1995; Pin and Duvoisin, 1995; Miller et al., 1995). It has also been shown that the local application of the selective mGluR agonist, (1S,3R)-ACPD, to the NAcc can modulate extracellular levels of DA in this site (Ohno and Watanabe, 1995; Taber and Fibiger, 1995) and increase locomotor activity in a DA-dependent manner (Kim and Vezina, 1997). These results provide evidence for functional interactions between mGluRs and DA terminals in the NAcc. However, whereas the contribution of NAcc iGluRs to AMPH-induced locomotion is well established, there have been no studies of the potential role of NAcc mGluRs in the mediation of such an effect. The present experiment, therefore, used the selective mGluR antagonist, MCPG, to investigate the contribution of this receptor to the locomotor effects produced by acute injections of AMPH into the NAcc.

	Materials and Methods

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Subjects and surgery. Male Sprague-Dawley rats weighing 250 to 275 g on arrival from Harlan Sprague-Dawley (Madison, WI) were used. They were housed individually in a 12-hr light/dark reverse cycle room, with food and water available at all times. Four to seven days after arrival, rats were anesthetized with ketamine (1 mg/kg i.p.) followed by xylazine (0.3 mg/kg i.p.) and placed in a stereotaxic instrument with the incisor bar at 5.0 mm above the interaural line (Pellegrino et al., 1979). They were then implanted with chronic bilateral guide cannulae (22 gauge, Plastics One, Roanoke, VA) aimed at the NAcc (A/P, +3.4; L, ±1.5; D/V, 7.5 from bregma and skull). Cannulae were angled at 10° to the vertical and positioned 1 mm above the final injection site. All cannulae were secured with dental acrylic cement anchored to stainless steel screws fixed to the skull. After surgery, 28 gauge obturators were inserted to a depth 1 mm below the guide cannula tips and rats were returned to their home cages for a 10-day recovery period. All procedures involving animals were conducted according to an approved Institutional Animal Care and Use Committee protocol.

Drugs. D-Amphetamine sulfate (National Institute on Drug Abuse, Rockville, MD) was dissolved in sterile 0.9% saline. (RS)-MCPG (Tocris Cookson, St. Louis, MO) was dissolved in equimolar NaOH (0.1 N; Fisher Scientific, Fair Lawn, NJ) and small aliquots were stored at 80°C. Immediately before use, frozen aliquots of the drugs were diluted in sterile 0.9% saline. They were prepared either separately or as a cocktail. R()-Apomorphine hydrochloride (Research Biochemicals Internationals, Natick, MA) was dissolved either in oxygen-free boiled water or in 50 mM (RS)-MCPG solution.

Intracranial microinjection. Bilateral intracranial microinjections into the NAcc were made in the freely moving rat. Injection cannulae (28 gauge) connected to 1-µl syringes (Hamilton, Reno, NV) via PE-20 tubing were inserted to a depth 1 mm below the guide cannula tips. Injections were made in a volume of 0.5 µl/side during 30 sec. Sixty seconds later, the injection cannulae were withdrawn, the obturators replaced and the rat placed immediately in an activity box.

Locomotor activity. A bank of 12 activity boxes was used to measure locomotor activity. Each box (22 × 43 × 33 cm) was constructed of opaque plastic (rear and two side walls), a Plexiglas front-hinged door and a tubular stainless steel ceiling and floor. Two photocells, positioned 3.5 cm above the floor and spaced evenly along the longitudinal axis of each box, estimated horizontal locomotion. Two additional photocells, positioned on the side walls 16.5 cm above the floor and 5 cm from the front and back walls, estimated rearing. Separate interruptions of photocell beams were detected and recorded via an electrical interface by a computer situated in an adjacent room. The activity boxes were kept in a room lighted dimly with red light.

Design and procedure. In all experiments, rats were injected and tested only during their dark cycle (between 10:00 A.M. and 6:00 P.M.). Brief exposure to light was unavoidable during transport from the housing to the testing room and at the time of injection. Rats were randomly assigned to five different groups. On each of two occasions, rats in each group were administered a bilateral microinjection into the NAcc of one of five doses of (RS)-MCPG (0, 0.025, 0.25, 2.5 and 25 nmol/side). On one occasion, these microinjections were made alone with saline. On the other, they were made in a cocktail with AMPH (6.8 nmol [2.5 µg]/side). The two microinjections were separated by 3 days, and their order (MCPG alone or in cocktail with AMPH) was counterbalanced in all groups. Individual rats were administered only one dose of the mGluR antagonist. Immediately after the microinjections, rats were placed in activity boxes, and their locomotor activity was measured for 2 hr. Additional groups of rats were tested to assess the possible contribution of mGluRs to postsynaptic components of DA neurotransmission. Three different groups of rats received either saline (0.5 µl/side), apomorphine (32.9 nmol [10 µg]/side) or a cocktail of apomorphine (32.9 nmol/side) plus (RS)-MCPG (25 nmol/side). Rats were placed in the activity boxes immediately after injection and their locomotion was measured for 2 hr. The behavior of some rats was simultaneously filmed with a closed-circuit video system to permit the subsequent observation and recording of stereotyped, seizure-like (e.g., motor convulsions, foreleg clonus, wet dog shakes and hyper salivation; see Burns et al., 1994) and other behaviors not detected by photobeam interruptions.

Histology. After completion of the experiments, the rats were anesthetized and perfused via intracardiac infusion of saline and 10% formalin. Brains were removed and postfixed further in 10% formalin for 3 to 5 days. Coronal sections (40 µm) were subsequently stained with cresyl violet for verification of cannulae tip placements.

Data analyses. The data were analyzed either with one-way ANOVA or with between-within ANOVA with dose of (RS)-MCPG as the between factor and injection [(RS)-MCPG alone or co-injected with AMPH] as the within factor. Post hoc Scheffé comparisons were made according to Kirk (1968).

	Results

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(RS)-MCPG modulates the locomotor-activating effects of NAcc AMPH. Consistent with previous reports (Taylor and Robbins, 1984; Vezina and Stewart, 1990; Burns et al., 1994) microinjection of AMPH into the NAcc significantly increased both horizontal and vertical locomotor activity when compared with saline control injections (fig. 1). These effects of AMPH were either unaffected or progressively and significantly enhanced by increasing concentrations of the four lower doses of (RS)-MCPG tested (0, 0.025, 0.25 and 2.5 nmol/side). Co-injection with a moderately high dose of (RS)-MCPG (25 nmol/side) completely blocked the locomotor-activating effects of NAcc AMPH. None of the doses of (RS)-MCPG tested when administered alone into the NAcc produced effects on locomotion that differed significantly from those observed after injections of saline. Time-course analyses showed that the locomotor-activating effects of NAcc AMPH persisted for approximately 1 hr of testing, consistent with previous reports (Vezina and Stewart, 1990). The ability of (RS)-MCPG to block these effects was apparent throughout this time course. The ANOVA conducted on the data obtained in the first 30 min of testing revealed significant effects of groups [between different doses of (RS)-MCPG; F(4,50) = 5.26, P < .002 and F(4,50) = 4.28, P < .005 for horizontal and vertical activity, respectively], injections [saline versus AMPH at all levels of (RS)-MCPG; F(1,50) = 100.99, P < .0001 and F(1,50) = 72.58, P < .0001] and a groups × injections interaction [F(4,50) = 4.57, P < .004 and F(4,50) = 4.45, P < .004]. Post hoc Scheffé comparisons revealed that AMPH produced significantly greater locomotor activity than saline at the four lower doses (P < .001) but not at the highest dose of (RS)-MCPG tested. In addition, 0.25 nmol (RS)-MCPG significantly enhanced (P < .05, horizontal and approached, P < .075, statistical significance for vertical) and 25 nmol (RS)-MCPG significantly decreased (P < .05, horizontal and vertical) the locomotor activity produced by AMPH relative to that observed in animals having received AMPH with 0 nmol (RS)-MCPG. The ANOVA conducted on the data obtained in the second 30 min of testing also revealed significant effects of injections [F(1,50) = 36.73, P < .0001 and F(1,50) = 28.84, P < .0001 for horizontal and vertical locomotion, respectively]. Again, post hoc Scheffé comparisons revealed that AMPH produced significantly greater locomotor activity than saline (P < .05-0.01) but not when it was co-injected with either the 2.5 (vertical) or the 25 nmol (horizontal and vertical) doses of (RS)-MCPG.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Effects of (RS)-MCPG on the locomotor-activating effects of NAcc AMPH. Data are shown as group mean (+S.E.M.) horizontal and vertical locomotor activity counts for each 30-min period of the 2-hr test after microinjection of the indicated dose of (RS)-MCPG (0, 0.025, 0.25, 2.5 and 25 nmol/side) with saline or AMPH (6.8 nmol/side). Numbers of rats in each group are indicated at the base of the different columns. Individual animals were tested with only one concentration of (RS)-MCPG, once co-injected with saline and once co-injected with AMPH. Symbols indicate significant differences as revealed by post hoc Scheffé comparisons after between-within ANOVA. ***P < .001, **P < .01 and *P < .05, AMPH compared with saline at the indicated dose of (RS)-MCPG. P < .05, AMPH at the indicated dose of (RS)-MCPG compared with AMPH at (RS)-MCPG (0 nmol/side).

(RS)-MCPG blocks the locomotor-activating effects produced by NAcc infusions of the direct DA receptor agonist apomorphine. When microinjected into the NAcc, apomorphine (32.9 nmol [10 µg]/side) produced a significant increase in horizontal locomotor activity during the second hour of testing. This finding, together with the lack of effect of these infusions on vertical activity, is consistent with those reported previously by others (Jackson et al., 1975; Cools, 1986). As shown in figure 2, co-injecting (RS)-MCPG (25 nmol/side) with apomorphine into the NAcc completely blocked this effect. The ANOVA conducted on these data revealed a significant effect of groups [F(2,16) = 5.54, P < .02] during the second hour of testing. Apomorphine, at the dose tested, produced significantly greater locomotion than that produced by saline or apomorphine + (RS)-MCPG (P < .05 as revealed by post hoc Scheffé comparisons).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   The mGluR antagonist, (RS)-MCPG, blocks the locomotor-activating effects of NAcc apomorphine (APO). Data are shown as group mean (+S.E.M.) horizontal locomotor activity counts obtained in the second hour. Note that when microinjected into the NAcc, apomorphine (32.9 nmol/side) compared with saline produced a significant increase in locomotor activity during the second hour of testing in a manner consistent with previous reports (Jackson et al., 1975; Cools, 1986). The same dose of (RS)-MCPG (25 nmol/side) found to be effective for blockade of AMPH-induced locomotor activity was used. The number of rats in each group is indicated at the base of each column. Symbols indicate significant differences as revealed by post hoc Scheffé comparisons after one-way ANOVA. *P < .05, apomorphine compared with saline and apomorphine + (RS)-MCPG.

Histology. Figure 3 shows the location of the injection cannula tips in the NAcc for all animals tested and included in the statistical analyses. Only data from rats with injection cannula tips located bilaterally in the NAcc were considered. Eight rats were excluded for failing to meet this criterion. Also shown are representative photomicrographs illustrating the guide and injection cannula tracks (aimed at the rostral pole, A, and more caudally into the core, B, subregions of the NAcc) of two of the animals tested. Little evidence for neurotoxicity beyond the mechanical damage produced by penetration of the cannulae was detected. Most injections were made bilaterally into the rostral pole (fig. 3A) and core (fig. 3B) subregions of the NAcc. Some animals received injections either bilaterally into the shell subregion of this nucleus or into the core on one side and the shell on the other. No evidence was obtained from the small number of such animals tested to indicate that infusions of (RS)-MCPG either alone or in combination with AMPH into the shell subregion produced differential effects on horizontal and vertical locomotion relative to infusions into other subregions of the NAcc.

View larger version (71K): [in this window] [in a new window]   Fig. 3.   Location of the microinjection cannula tips in the NAcc of rats included in the data analyses (C). The line drawings are from Paxinos and Watson (1986). Numbers to the right indicate millimeters rostral to bregma. The different symbols refer to the different microinjections administered: open circles, (RS)-MCPG (0 nmol/side) microinjected either alone (with saline) or in combination with AMPH; filled circles, the remaining doses of (RS)-MCPG microinjected either alone (with saline) or in combination with AMPH; open triangles, apomorphine; filled triangles, apomorphine + (RS)-MCPG. Also shown are representative photomicrographs illustrating the guide and injection cannula tracks of one rat that received microinjections into the rostral pole subregion (A) and another that received microinjections more caudally into the core subregion (B) of the NAcc. Note the lack of neurotoxicity beyond the mechanical damage produced by penetration of the injection cannulae.

	Discussion

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High-dose (RS)-MCPG blocks the locomotor-activating effects of NAcc AMPH. The present findings are consistent with those of previous studies showing that glutamate receptor antagonists microinjected into the NAcc attenuate the locomotor effects of psychomotor-stimulant drugs (Pulvirenti et al., 1989; Willins et al., 1992; Kaddis et al., 1993; Burns et al., 1994). For example, the selective NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid has been shown to dose-dependently reduce the effectiveness of NAcc AMPH to produce locomotion (Kelley and Throne, 1992). The intra-accumbens administration of the AMPA/kainate antagonist, DNQX, has also been shown to antagonize the locomotor-stimulant response to cocaine administered either systemically or directly into the NAcc (Kaddis et al., 1993). The present study extended these findings to mGluRs. In a manner similar to iGluR antagonists, a moderately high dose (25 nmol/side) of (RS)-MCPG completely blocked the locomotor effects of NAcc AMPH. The dose dependence of this effect and the effective dose of (RS)-MCPG (25 nmol/side) found to produce it is reasonably similar to the effects obtained with iGluR antagonists and the effective doses of these antagonists reported. For instance, 4 nmol/side of DNQX and 21 nmol/side of GAMS, both AMPA/kainate receptor antagonists, have been used to inhibit the locomotor stimulation produced by AMPH (0.5 mg/kg s.c.) (Willins et al., 1992), whereas 98 nmol/side of GDEE, a quisqualate receptor antagonist, was used to significantly reduce AMPH (0.75 mg/kg s.c.) locomotor effects (Pulvirenti et al., 1989). For the NMDA receptor, 2.5 to 5 nmol of 2-amino-5-phosphonopentanoic acid was shown to effectively reduce the locomotor effects of AMPH (13.6 nmol [5 µg]) microinjected into the NAcc (Kelley and Throne, 1992). More importantly, when injected alone into the NAcc, the dose of (RS)-MCPG (25 nmol/side) used to effectively block the locomotor effects of NAcc AMPH (as well as all other doses tested) in this study produced no significant effects on various types of behaviors including locomotor activity and stereotypy. As revealed from the time-course analyses, this blockade by (RS)-MCPG of NAcc AMPH-induced locomotion persisted for the duration of the latter drug's effect (approximately 1 hr). A similar persistence in the ability to block NAcc AMPH-induced locomotion was reported previously for the NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (Kelley and Throne, 1992).

(RS)-MCPG blocks the locomotor-activating effect of NAcc apomorphine. Consistent with previous reports (Cools, 1986), infusion into the NAcc of the direct DA receptor agonist, apomorphine, produced a significant increase in horizontal locomotion during the second hour of a 2-hr test. Again, (RS)-MCPG when co-injected with apomorphine into the NAcc blocked this effect, which suggests that (RS)-MCPG can act postsynaptically at mGluRs expressed by intrinsic NAcc neurons. High concentrations of mGluRs are known to be expressed on both pre- and postsynaptic sites in the NAcc (Albin et al., 1992; Ohishi et al., 1993; Testa et al., 1994; Romano et al., 1995). The mGluRs are known to activate multiple second messenger systems via G proteins, including phosphotidylinosite hydrolysis and inhibition or activation of cAMP (Nakanishi, 1994). Thus, it is possible that mGluRs located on the dendritic processes of intrinsic NAcc cells may regulate incoming dopaminergic signals by acting on second messenger systems with which mGluRs are coupled. Recent ultrastructural studies of the NAcc have indicated that some of the terminals of the descending excitatory amino acid projections from cortex and those of ascending DA mesencephalic projections not only come in close apposition to each other but form synaptic contacts with the same intrinsic NAcc neurons as well (Sesack and Pickel, 1990, 1992). This anatomical arrangement provides the basis for a possible interaction between glutamate and DA at the same synaptic sites in the NAcc. However, it does not preclude the possibility that receptor antagonists such as (RS)-MCPG can modulate the release of DA by AMPH by acting at mGluRs located presynaptically on dopaminergic nerve terminals in the NAcc. Indeed, it was recently shown that the local application of (1S,3R)-ACPD (1 mM) to the NAcc via reverse dialysis can regulate the extracellular levels of DA in this site (Ohno and Watanabe, 1995; Taber and Fibiger, 1995). Although such results may reflect the contribution of mGluRs located on nondopaminergic (e.g., serotonergic) afferents to the NAcc or, as recently suggested for iGluRs (Taber et al., 1996), of mGluRs expressed by intrinsic NAcc cells sending reciprocal projections to DA perikarya in the ventral tegmental area, they are also consistent with a contribution by mGluRs located on DA neuron terminals. This latter possibility, together with the ability of AMPH to release DA in an action potential-independent manner, suggests that mGluRs may be linked to postreceptor pathways within DA neuron terminals that are capable of influencing the stimulation of DA release by AMPH. Thus, although the present results support a contribution by mGluRs located postsynaptically to DA neuron terminals to dopaminergic neurotransmission in the NAcc, they cannot exclude an equally important contribution by mGluRs located presynaptically on DA neuron terminals in this site.

Different subtypes of mGluRs exist in the NAcc. Based on their sequence homology, transduction mechanisms and pharmacology, the mGluR family, consisting of at least eight different subtypes of receptors, has been divided into three groups (Pin and Bockaert, 1995; Pin and Duvoisin, 1995; Miller et al., 1995). Whereas receptors belonging to group I (mGluR1 and mGluR5) are coupled to G-proteins which lead to activation of phospholipase C, those in group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7 and mGluR8) are negatively coupled to G-proteins and inhibit adenylyl cyclase. Among these, mGluR5 and mGluR3 mRNAs are expressed most abundantly in the NAcc (Testa et al., 1994). It is known that (RS)-MCPG acts as an antagonist at both mGluR5 (group I) and mGluR3 (group II) (Pin and Bockaert, 1995). Thus, it is possible that either or both of these receptors may be involved in mediating the effect of (RS)-MCPG in blocking the locomotor-activating effects of DA agonists in the NAcc. The mechanisms mediating these effects, however, remain to be determined.

Low-dose (RS)-MCPG potentiates the locomotor-activating effects of NAcc AMPH. In the lower range of doses of (RS)-MCPG tested (0, 0.025, 0.25 nmol/side), increasing the dose of this mGluR antagonist progressively enhanced the locomotor-activating effects of NAcc AMPH. Although the magnitude of this effect was small, it did achieve statistical significance at the 0.25 dose of (RS)-MCPG for horizontal activity (P < .05, AMPH + 0.25 (RS)-MCPG vs. AMPH + 0 (RS)-MCPG) and approached statistical significance at this dose (P < .075) for vertical activity, which suggests that this mGluR antagonist may have biphasic effects on NAcc AMPH-induced locomotion. Such a possibility is not without precedent. For example, it was recently reported that whereas a low concentration of the mGluR agonist (1S,3R)-ACPD decreased levels of extracellular DA in the NAcc, a higher concentration produced the opposite effect (Taber and Fibiger, 1995). The present findings (enhancement of NAcc AMPH-induced locomotion by a low dose of (RS)-MCPG and its blockade by a higher dose) are entirely consistent with these results. The mechanisms underlying these effects are unknown. It is conceivable, however, as suggested by some (Schoepp and Conn, 1993; Taber and Fibiger, 1995; Kim and Vezina, 1997), that opposing actions by different concentrations of mGluR agonists and antagonists may reflect the different affinities for such ligands displayed by mGluR subtypes (Pin and Bockaert, 1995; Pin and Duvoisin, 1995) and their differential activation by the different doses. Given the different transduction mechanisms associated with mGluR3 and mGluR5 described above, it is likely that their selective recruitment would lead to opposing actions (Schoepp and Conn, 1993; Taber and Fibiger, 1995; Kim and Vezina, 1997).

Conclusion. The present results demonstrate that the mGluR antagonist, (RS)-MCPG, can block the locomotor-activating effects of both direct and indirect DA agonists when these are infused into the NAcc. Although these results can be interpreted to suggest that mGluRs located on intrinsic NAcc cells postsynaptic to DA neuron terminals mediate this effect, a contribution by presynaptically expressed mGluRs remains a possibility. The additional finding that low and high doses of (RS)-MCPG produced opposing effects indicates, in a manner consistent with previous reports, that this mGluR antagonist exerts biphasic effects on NAcc AMPH-induced locomotor activity. The contribution of the locally expressed mGluR3 and mGluR5 subtypes to these effects as well as their pre- and postsynaptic locus and site of action in the NAcc remain to be elucidated.