Introduction

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The NAcc receives a dense dopaminergic innervation from the ventral mesencephalon (Meredith et al., 1993). This dopaminergic system, consisting of perikarya in the ventral tegmental area and their axonal projections to the NAcc, plays a critical role in the mediation of the incentive and locomotor stimulating properties of psychomotor-stimulant drugs such as amphetamine and cocaine (Robbins et al., 1989; Koob, 1992). These drugs produce an increase in locomotor activity when given either systemically or intracranially into the NAcc by increasing extracellular levels of DA in this site (Hurd et al., 1989; Kuczenski and Segal, 1989). The NAcc also receives extensive glutamatergic projections directly from the prefrontal cortex and limbic structures such as the hippocampal formation and amygdala (Christie et al., 1987; Meredith et al., 1993). These projections have also been implicated in the regulation of locomotor activity and evidence suggests that they may produce their effects by interacting with dopaminergic neurotransmission. For example, agonists of glutamatergic ionotropic receptors, such as AMPA, kainic acid, and NMDA, stimulate locomotor activity when injected into the NAcc, and drugs that interfere with dopaminergic neurotransmission inhibit these effects (Donzanti and Uretsky, 1983; Hamilton et al., 1986; Boldry and Uretsky, 1988; Burns et al., 1994). 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 DA and glutamate at the level of nerve terminals in the NAcc. Several studies have also shown that excitatory amino acid agonists increase extracellular levels of DA when infused into the NAcc in vivo or when applied to NAcc slices in vitro (Jones et al., 1987; Imperato et al., 1990; Youngren et al., 1993). Glutamatergic involvement in the regulation of locomotor activity is also inferred from observations that infusions of glutamate receptor antagonists into the NAcc attenuate both the locomotor and DA-activating effects of psychomotor-stimulant drugs (Pulvirenti et al., 1989, 1991; Willins et al., 1992; Kaddis et al., 1993; Pap and Bradberry, 1995).

The NAcc contains not only iGluRs but also relatively high concentrations of mGluRs (Albin et al., 1992; Ohishi et al., 1993; Testa et al., 1994; Romano et al., 1995). The mGluRs mediate relatively slow glutamate responses by coupling to intracellular signal transduction pathways via GTP-binding proteins and have been shown to modulate a wide variety of ionic conductances in diverse neuronal cell types (Nakanishi, 1994; Gerber and Gahwiler, 1994). The development of mGluR-selective ligands (Palmer et al., 1989; Eaton et al., 1993) has permitted the characterization of important roles for mGluRs in the tuning of fast synaptic transmission, in 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). Recently, it was shown that the selective mGluR agonist, (1S,3R)-ACPD, injected acutely into the striatum in moderate to high doses produces DA-dependent contralateral rotation (Sacaan et al., 1991, 1992; Smith and Beninger, 1996). It was also shown recently that the local application of (1S,3R)-ACPD to the NAcc via reverse dialysis can regulate the extracellular levels of DA in this site (Ohno and Watanabe, 1995; Taber and Fibiger, 1995). These results provide evidence for functional interactions between mGluRs and DA terminals in the NAcc. However, there have been no studies of the behavioral consequences of the selective activation of mGluRs in the NAcc in vivo. The results of such activation of mGluRs via bilateral microinjection of (1S,3R)-ACPD into the NAcc were characterized in the present experiment.

	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 positioned 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 placed in the guide cannulae and rats were returned to their home cages for a 10-day recovery period.

Drugs. (1S,3R)-ACPD and (RS)-MCPG (Tocris Cookson, St. Louis, MO) were 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. Fluphenazine dihydrochloride (Research Biochemicals Internationals, Natick, MA) was dissolved either in 0.9% saline or in a 1 mM (1S,3R)-ACPD solution. The doses of fluphenazine refer to the weight of the salt.

Intracranial microinjections. 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 of 1 mm below the guide cannula tips. Injections were made in a volume of 0.5 µl/side over 30 sec. Sixty seconds later, the injection cannulae were withdrawn, the obturators replaced and rats placed immediately in the activity boxes.

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. Different groups of rats were administered either saline or one of four doses of (1S,3R)-ACPD (0.005, 0.05, 0.5 or 2.5 nmol/side) bilaterally into the NAcc. Immediately after the injection, rats were placed in activity boxes and their locomotor activity was measured for 2 hr. The behavior of some rats was filmed simultaneously with a closed-circuit video system to permit the subsequent observation and recording of stereotyped (e.g., repetitive movements in one location; see Creese and Iversen, 1973), seizure-like (e.g., motor convulsions, foreleg clonus, wet dog shakes and hypersalivation; see Burns et al., 1994) and other behaviors not detected by photobeam interruptions. Animals were observed for 15 sec every 2 min throughout the 2-hr period. When a specific behavior occurred within an observation period, it was given a score of 1, making 60 the maximum possible frequency score for each behavior during the 2 hr of testing.

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 in 10% formalin for 1 week. Coronal sections (40 µm) were subsequently stained with cresyl violet for verification of cannulae tip placements.

Data analyses. The data were analyzed with one-way between analyses of variance (ANOVA) and pairwise comparisons were subsequently made by Bonferroni t tests with SigmaStat statistical software.

	Results

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Microinjection of (1S,3R)-ACPD into the NAcc dose-dependently increases locomotor activity. Figure 1 shows the locomotor activity counts produced by the microinjection of (1S,3R)-ACPD into the NAcc. It can be seen that, when injected into this site, (1S,3R)-ACPD dose-dependently increased both horizontal and vertical locomotor activity. Time-course anaylses revealed that, in a manner similar to the locomotor hyperactivity observed after infusions of amphetamine into the NAcc, this effect was most pronounced in the initial 30 min after injection. The ANOVA conducted on these data revealed a significant effect of groups [F(4,39) = 6.16 and 3.48, P < .0007 and P < .02] for horizontal and vertical locomotor activity, respectively. This effect, present throughout the initial 30 min of testing, subsequently diminished and was no longer present after 1 hr of testing. Subsequent comparisons made with Bonferroni t tests revealed that, although the lowest dose of (1S,3R)-ACPD tested increased horizontal and vertical movements somewhat, these increases did not achieve statistical significance. However, the remaining doses of this agonist together produced significantly greater horizontal and vertical (P < .001) movements when compared with those produced by the saline vehicle. Interestingly, 50-fold (0.05-2.5 nmol/side) increases in the threshold dose of (1S,3R)-ACPD required to produce significant increases relative to saline did not lead to further increases in locomotor activity.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   The effect of microinjections into the NAcc of (1S,3R)-ACPD on horizontal and vertical components of locomotor activity. Data are shown as group means (+S.E.M.) of horizontal and vertical locomotor activity counts for the first three 30-min periods of the 2-hr test after microinjection of the indicated dose of (1S,3R)-ACPD (0, 0.005, 0.05, 0.5 and 2.5 nmol/side). Data for the remaining 30 min are not shown because locomotor effects were no longer present after 90 min of testing. Numbers of rats in each group are indicated at the base of the different columns. Symbols indicate significant differences as revealed by Bonferroni t test comparisons after one-way ANOVA. ***P < .001 indicated doses of (1S,3R)-ACPD compared to saline control.

mGluR and DA receptor antagonists block NAcc (1S,3R)-ACPD-induced locomotor activity. To confirm the receptor specificity of the locomotor-activating effects of NAcc (1S,3R)-ACPD as well as the contribution of local changes in DA neurotransmission, additional animals were tested after bilateral microinjection into the NAcc of 0.9% saline (0.5 µl/side), (1S,3R)-ACPD (0.5 nmol/side), (RS)-MCPG (2.5 nmol/side), the DA receptor antagonist fluphenazine (2.0 or 9.8 nmol/side), a cocktail of (1S,3R)-ACPD (0.5 nmol/side) and (RS)-MCPG (2.5 nmol/side) or a cocktail of (1S,3R)-ACPD (0.5 nmol/side) and one of two doses of fluphenazine (2.0 or 9.8 nmol/side). As shown in figure 2, co-injecting (RS)-MCPG blocked both the increased horizontal and vertical locomotion produced by (1S,3R)-ACPD. In addition, it can be seen that the effect of (1S,3R)-ACPD on horizontal and vertical locomotion was also blocked by the co-injection of either dose of fluphenazine. The ANOVA conducted on the MCPG data (fig. 2A) revealed a significant effect of groups [F(3,24) = 3.03 and 8.85, P < .05 and 0.0005] for horizontal and vertical locomotion, respectively. The ANOVA conducted on the fluphenazine data (fig. 2B) also revealed a significant effect of groups [F(5,34) = 5.64 and 9.66, P < .0008 and 0.0001] for horizontal and vertical locomotion, respectively. As expected, rats that received (1S,3R)-ACPD alone showed significantly higher levels of locomotion than those observed in rats having received saline (P < .025 and P < .001, for horizontal and vertical locomotion, respectively). Animals having received (1S,3R)-ACPD with one of the receptor antagonists exhibited levels of locomotion that did not differ significantly from those observed after saline. (RS)-MCPG and fluphenazine, at the doses used, did not produce significant locomotor effects, compared with saline, when microinjected alone [or with (1S,3R)-ACPD] into the NAcc. The higher dose of fluphenazine used (9.8 nmol/side) did appear to decrease horizontal locomotion relative to saline, although this effect only approached statistical significance (P < .08).

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Co-injecting either the mGluR antagonist, (RS)-MCPG (A), or the DA receptor antagonist, fluphenazine (B), blocks the locomotor-activating effects of NAcc (1S,3R)-ACPD. Data are shown as group means (+S.E.M.) of 1 hr total horizontal and vertical locomotor activity counts. Doses microinjected for the different compounds were 0.5 nmol/side for (1S,3R)-ACPD, 2.5 nmol/side for (RS)-MCPG and 2.0 or 9.8 nmol/side for fluphenazine. The number of rats in each group is indicated at the base of each column. Symbols indicate significant differences as revealed by Bonferroni t test comparisons after one-way ANOVA. *P < .05 and **P < .01 indicated group compared with saline control.

Histology. Only data from rats with injection cannula tips located bilaterally in the NAcc were considered. These are illustrated in figure 3C with representative photomicrographs showing the guide and injection cannulae tracks of two of the animals tested (fig. 3, A and B). Aside from the mechanical damage produced by cannula implantation, little evidence for neurotoxicity was detected at the injection cannula tips. 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 (1S,3R)-ACPD into the shell subregion produced differential effects on horizontal and vertical locomotion relative to infusions into the core subregion of the NAcc.

View larger version (67K): [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). Also shown are representative photomicrographs exemplifying the guide and injection cannula tracks of one rat that received microinjections into the rostral pole subregion of the NAcc (A) and another that received microinjections more caudally into the core subregion (B). Note the lack of neurotoxicity beyond the mechanical damage produced by penetration of the injection cannulae. The line drawings in C are from Paxinos and Watson (1986). Numbers to the right indicate millimeters from the bregma. Symbols represent the different microinjections administered: , saline; , (1S,3R)-ACPD; , (1S,3R)-ACPD + (RS)-MCPG; , (1S,3R)-ACPD + fluphenazine.

	Discussion

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The present results demonstrate that activation of mGluRs in the rat NAcc with (1S,3R)-ACPD increases both horizontal and vertical locomotor activity in a dose-dependent and receptor-specific manner. No evidence of stereotyped or seizure-like behaviors was detected in the dose range tested. Co-injection of the DA receptor antagonist, fluphenazine, completely blocked these locomotor stimulating effects of (1S,3R)-ACPD, which suggests that they are in some way dependent on dopaminergic neurotransmission. These findings indicate that mGluRs in the NAcc figure importantly in the production of locomotor behaviors involving DA-excitatory amino acid interactions in this site.

The effects of (1S,3R)-ACPD on locomotor activity observed in the present study are comparable, in some ways, to those of AMPH. For example, in a manner similar to the locomotor effects produced by injection of AMPH into the NAcc (Vezina and Stewart, 1990; Kim and Vezina, in press), (1S,3R)-ACPD increased locomotor activity predominantly in the initial 30 min of testing with no significant differences from saline apparent 1 hr after injection. Similar effects were reported recently by Attarian and Amalric (1997). In addition, a similar time course was observed for the contralateral turning produced by the unilateral infusion of (1S,3R)-ACPD (0.25 nmol/0.5 µl) into the dorsal striatum (Smith and Beninger, 1996). A longer time course (1-6 hr) was reported by others, but after the infusion of doses 400 to 20,000 times higher than those used in the present and above-mentioned studies (Sacaan et al., 1991, 1992), which introduces the possibility that, in these cases at least, the behaviors produced may not have resulted from normally functioning physiological processes (see Smith and Beninger, 1996, for discussion). In the present study, the threshold dose of (1S,3R)-ACPD required to produce significant increases in locomotion relative to saline when injected into the NAcc (0.05 nmol/side) was orders of magnitude lower (>60-fold) than those reported for AMPH in this site (2.7-4.0 nmol/side; e.g., Vezina et al., 1991). In addition, and unlike AMPH, 50-fold increases in this threshold dose of (1S,3R)-ACPD did not lead to further increases in locomotor activity (cf., Attarian and Amalric, 1997), reflecting possible opposing effects of stimulation at different subtypes of the mGluR in the NAcc (see below). The maximal increases in locomotor activity produced by (1S,3R)-ACPD were 47% and 73% greater than those produced by saline, for horizontal and vertical movements, respectively, compared with increases of 113% and 167% greater than saline produced by the submaximal dose of 6.8 nmol (2.5 µg)/side of AMPH. When co-injected into the NAcc, however, the locomotor effects of these two drugs were additive (Kim J.-H. and Vezina, P., unpublished observations). In a manner similar to AMPH, it was found recently that the local application of (1S,3R)-ACPD (1 mM) to the NAcc via reverse dialysis produces an increase in extracellular DA in this site (Ohno and Watanabe, 1995; Taber and Fibiger, 1995). In addition, the locomotion (present study; see also Attarian and Amalric, 1997) and contralateral turning (Sacaan et al., 1992; Smith and Beninger, 1996) produced by (1S,3R)-ACPD is blocked by the co-administration of DA receptor antagonists. Taken together, these results provide evidence for functional interactions between mGluRs and DA terminals in the NAcc and can be interpreted to suggest that (1S,3R)-ACPD produces increased locomotor activity by acting at mGluRs located on DA neuron terminals in the NAcc to stimulate DA release in this site. The possibility that (1S,3R)-ACPD might produce such effects by acting at mGluRs expressed on cells postsynaptic to DA neuron terminals in the NAcc must also be considered, however. 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 form synaptic contacts with the same intrinsic NAcc neurons (Sesack and Pickel, 1990, 1992), and DA-excitatory amino acid receptor interactions in such neurons have been proposed to contribute to the production of behaviors by respective agonists for these receptors (Kelley and Delfs, 1994). Although it is true that electrophysiological studies conducted in slices and anesthetized rat preparations showed that (1S,3R)-ACPD reduced the excitatory amino acid-induced activation of neurons in the NAcc and striatum (Hu and White, 1996; Lovinger, 1991; Lovinger et al., 1993), observations in this laboratory (Kim and Vezina, in press) have also shown that the blockade of NAcc mGluRs in vivo interferes with the locomotion produced by infusions of the direct DA receptor agonist apomorphine into this site.

Recent studies of the mGluR have revealed a family of at least eight receptor subtypes (mGluR1-8; Pin and Bockaert, 1995; Pin and Duvoisin, 1995; Miller et al., 1995) which can be divided into three groups based on their sequence homology, transduction mechanisms and pharmacology. Whereas receptors in group I (mGluR1 and 5) activate phospholipase C, those in group II (mGluR2-3) and group III (mGluR4 and mGluR6-8) are negatively coupled to adenylyl cyclase. In the NAcc, mRNA of both mGluR5 and 3 are expressed most abundantly (Testa et al., 1994), and it is known that (1S,3R)-ACPD acts as an agonist and (RS)-MCPG acts as an antagonist at both of these receptor subtypes. The present finding, therefore, that co-injection into the NAcc of (RS)-MCPG blocked the locomotor-activating effects of (1S,3R)-ACPD at an antagonist-agonist concentration ratio used successfully by others (Ohno and Watanabe, 1995) strongly suggests a role for NAcc mGluRs in the mediation of these behaviors. Notably, infusions of (1R,3S)-ACPD, the inactive isomer of (1S,3R)-ACPD, have been found to have no effect when injected into other sites (Ohno and Watanabe, 1995; Sacaan et al., 1991; Pin and Duvoisin, 1995). In addition to the above-mentioned differences between the three groups of mGluR, subtypes of this receptor are known to display different affinities for (1S,3R)-ACPD (Pin and Duvoisin, 1995) and their selective activation has been suggested to produce opposing actions on NAcc DA release (Taber and Fibiger, 1995) and NMDA receptor-mediated excitotoxicity (Schoepp and Conn, 1993). It is conceivable, therefore, that the concurrent stimulation of NAcc mGluR5 and 3 by the higher doses of (1S,3R)-ACPD tested in the present study also led to opposing actions on the production of locomotor activation. As a result and consistent with the present results, as well as those of others (Smith and Beninger, 1996), substantial increases in the threshold effective dose of this agonist would not necessarily be expected to lead to further increases in locomotion.

There have been some reports that high doses of (1S,3R)-ACPD produce limbic seizures and neuronal damage when injected into either the hippocampus or the striatum. In the adult rat, for example, a (1S,3R)-ACPD dose of 1 µmol/2 µl/side produced such effects when injected into the hippocampus (Sacaan and Schoepp, 1992) but not into the striatum (Sacaan et al., 1991). However, in the neonatal rat striatum, significant neuronal injury has been observed after 0.5 to 2.0 µmol doses of (1S,3R)-ACPD (McDonald et al., 1993). By comparison, the doses of (1S,3R)-ACPD injected into the NAcc in the present study were 100 to 50,000 times smaller than the dose found to produce injury in the hippocampus and 200 to 10⁶ times smaller than those found to produce injury in the neonatal rat striatum. In addition, behavioral observations of animals revealed no evidence of stereotyped or seizure-like behaviors at any of the doses tested. Finally, cresyl-violet-stained brain tissue sections revealed little evidence of neuronal damage other than that produced by implantation of the cannulae.

In conclusion, the present findings indicate that mGluRs play an important role in the production of locomotor behaviors involving DA-excitatory amino acid interactions in the NAcc. Such a role for mGluRs is consistent with the well-documented contributions of the kainate, AMPA and NMDA subtypes of the iGluR to locomotor activation (Donzanti and Uretsky, 1983; Hamilton et al., 1986; Boldry and Uretsky, 1988; Burns et al., 1994) and NAcc DA release (Jones et al., 1987; Imperato et al., 1990; Youngren et al., 1993). Although detailed neuronal mechanisms remain to be determined, these results strongly suggest that glutamate plays an important role in the regulation of locomotor activity by interacting with dopaminergic neurotransmission. The present findings confirm and extend this important role to the mGluR. Because previous reports have indicated that mGluRs can modulate voltage- and ligand-gated ion channels (Schoepp and Conn, 1993; Nakanishi, 1994; Pin and Duvoisin, 1995) as well as glutamate neurotransmission through iGluRs in the NAcc (Hu and White, 1996), it will be interesting to determine whether these two types of glutamate receptor interact functionally to influence the production of locomotor behaviors and the release of DA from the NAcc.