Introduction

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The nucleus accumbens (NA) is located within the ventral striatum and is an important neural substrate in motivation, reward, and behavioral activation (Mogenson et al., 1980; Kalivas et al., 1993). Within the NA, emphasis has been placed on the mesoaccumbens dopaminergic afferents as a primary regulator of the motor activation frequently associated with motivation and reward. The ventral tegmental area (VTA) and medial substantia nigra are the sources of dopamine projections to the accumbens (Fallon and Moore, 1978). Supporting a role by these projections in motor activation, the administration of dopamine receptor agonists into the NA elicits locomotor activity (Swanson et al., 1997), and indirect dopamine agonists such as cocaine and amphetamine have been shown to produce their behavioral activating effects by enhancing extracellular dopamine levels in accumbens (Kuczenski and Segal, 1989).

In addition to dopaminergic innervation, the NA receives glutamatergic input from prefrontal cortex, amygdala, and hippocampus (Meredith et al., 1993), and a growing body of evidence suggests that these two neurotransmitter systems may converge to regulate motor activity. For instance, microinjection of glutamate agonists into the NA increases locomotor activity, and antagonists of glutamate or dopamine receptors can inhibit this response (Donzanti and Uretsky, 1983; Pulvirenti et al., 1994). Moreover, glutamate agonists modulate extracellular dopamine levels in the NA, and locomotor activation produced by psychostimulants can be inhibited by glutamate receptor antagonists (Imperato et al., 1990; Burns et al., 1994). Study of the synaptic ultrastructure in the NA has disclosed that excitatory afferents form synaptic contacts on the same dendritic spines as dopaminergic nerve terminals (Sesack and Pickel, 1992). This juxtaposition supports the likelihood that the dopamine and glutamate neurotransmitter systems not only share common postsynaptic targets but also may interact via a paracrine-like heterosynaptic modulation. The prefrontal cortex sends glutamatergic projections not only to NA but also to the VTA (Sesack et al., 1989). This anatomical organization provides an additional mechanism by which glutamatergic innervation of the mesoaccumbens projection can regulate dopamine transmission in the NA and influence the expression of motor behaviors. For example, stimulation of the projection from prefrontal cortex to the VTA or the administration of glutamate agonists directly into VTA increases extracellular dopamine levels in the NA and increases locomotor activity (Suaud-Chagny et al., 1992).

Glutamatergic neurotransmission is mediated by both ionotropic and metabotropic receptors (mGluRs). Most information accrued regarding glutamatergic involvement in behavioral activation involves ionotropic glutamate receptors (Burns et al., 1994; Pulvirenti et al., 1994; Pap and Bradbury, 1995). As such, relatively few studies have evaluated the role of mGluRs in the mesoaccumbens projection to modulate motor activity. The few studies that have been conducted reveal that mGluR agonists stimulate motor activity when microinjected into the NA; however, these studies have used the nonselective mGluR agonist (Attarian and Amalric, 1997; Vezina and Kim, 1998, for review), which has been shown to display nearly equal affinity for both group I and group II mGluRs (Conn and Pin, 1997). Eight mGluR genes have been identified, and the protein products are divided into three subgroups based on sequence homology, pharmacology, and coupling to intracellular transduction systems (Conn and Pin, 1997). Group I receptors consist of mGluR1 and mGluR5, group II consists of mGluR2 and mGluR3, and group III includes mGluR4 and mGluR6 through mGluR8. Over the past 5 years, drugs have been developed that are relatively selective for the mGluR subgroups, making it possible to discern which subgroup or subgroups of receptor may be mediating the motor stimulation produced by the less selective mGluR agonists. In the present report, we investigate a role for each mGluR subgroup in motor behavior and offer an overview of their action in two important nuclei within the motive circuit. It is important to note that although the compounds used in this study have shown in vitro selectivity, there are no existing data on the nature of their actions in vivo.

	Materials and Methods

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Animals and Surgery. Male Sprague-Dawley rats weighing between 250 and 300 g (Simonsen Laboratories, Gilroy, CA) were individually housed with food and water available ad libitum. A 12-h light/dark cycle (7 AM to 7 PM lights on) was used to regulate the animal photocycle. All experimentation was carried out during the light cycle.

Drugs. All mGluR agonists and antagonists used in this study were purchased from Tocris Cookson (Ballwin, MO). (S)-3,5-Dihydroxyphenylglycine (DHPG) and (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) were dissolved in 0.9% sterile saline. 7-(Hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) was dissolved in 50% dimethyl sulfoxide and sterile water. The control injection for the CPCCOEt experiment consisted of 50% dimethyl sulfoxide in water. L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4) and (RS)--methyl-4-phosphonophenylglycine (MPPG) were dissolved in 1 Eq NaOH, neutralized with 0.1 N HCl, and diluted with sterile water (Sigma Chemical Co., St. Louis, MO). All drugs were made up in bulk volume and stored at 80°C. For all drugs, nanomole doses represent the total amount administered per bilateral injection (i.e., 5 nmol = 2.5 nmol/0.5 µl/side).

Microinjection and Experimental Design. Immediately before testing, the obturators were removed and the injection cannulas (33 gauge, 15 mm) fitted to a 1-µl Hamilton syringe via 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 cannulas 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). Animals were allowed to recover 2 days after each test day to ensure clearance of drug and recovery from the microinjection procedure. For all experiments, we used a counterbalanced design across days over the complete test period, resulting in each animal receiving a maximum of five microinjections.

Histology and Data Analysis. The rats were administered an overdose of pentobarbital (>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 repeated measures ANOVAs within this study. On the discovery of statistical significance, post hoc analyses were performed on dose-response data with Dunnett's test to compare each dose to saline controls. For antagonist studies, pair-wise post hoc comparisons were made with Fisher's Protected Least Significant Difference. All evaluations of horizontal time course data included a two-way ANOVA with repeated measures over time followed by inspection of individual time points with use of Fisher's Protected Least Significant Difference.

	Results

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Effect of Group I mGluR Agonist on Motor Activity. Figure 1 illustrates the behavioral activation produced by the microinjection of the group I agonist DHPG into the NA and VTA. A significant effect of dose by DHPG in the NA was measured for horizontal (F_4,21 = 4.085; P = .013) counts and total distance traveled (F_4,21 = 3.568; P = .023). Analysis of vertical counts revealed only a trend toward a significant effect (F_4,21 = 2.33; P = .089). Post hoc comparison with Dunnett's test showed a significant difference from controls at the 5-nmol dose for horizontal (t₂₁ = 3.763) and vertical counts (t₂₁ = 2.889), as well as for total distance traveled (t₂₁ = 3.505). No interaction was measured in any of the three parameters tested. Figure 1B illustrates the time course for horizontal counts with DHPG. As can be seen, the motor stimulant response after the 5 nmol DHPG infusion was sustained for the duration of the experiment. Visual inspection might suggest that 3 and 10 nmol of DHPG also produced significant behavioral activation; however, due to the typically large variance observed in the DHPG response and the relatively low number of determinations used in this study, these doses did not prove to be significant when analyzed with the use of Dunnett's test.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Stimulation of group I mGluRs with DHPG in the VTA or NA elicits dose-dependent motor activation. A and C, total amount of activity generated during the 2 h after microinjection. The number of vertical counts was multiplied by 1000 for illustrative purposes. B and D, time course of the horizontal data shown on the left. The number of determinations at each dose is shown in parentheses, and the data are shown as mean ± S.E. *P < .05, comparing 5 nmol with saline. P < .05, comparing 10 nmol with saline. #P < .05, comparing 3 nmol with saline.

Effect of Group II mGluR Agonist on Motor Activity. The motor effects of local infusion of the specific group II agonist DCG-IV into the NA and VTA are shown in Fig. 2. Figure 2A shows that the microinjection of DCG-IV into the NA produced a dose-dependent elevation in all three parameters of motor behavior (horizontal: F_3,23 = 4.837, P = .009; distance: F_3,23 = 4.186, P = .017; vertical: F_3,23 = 3.751, P = .025). Post hoc analysis with Dunnett's test revealed a significant difference from control for all three parameters at the 0.1-nmol dose. The time course shown in Fig. 2B reveals a significant interaction (F_7,,21 = 3.391; P = .0001). Microinjection of 0.1 nmol produced an increase in horizontal counts that endured for roughly the first 45 min after microinjection.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Dose-dependent elevation of motor activity in the VTA and NA by the group II agonist DCG-IV. A and C, total number of horizontal photocell counts generated during the 2 h after microinjection. The number of vertical counts was multiplied by 1000 for illustrative purposes. B and D, time course of the horizontal data shown on the left. The number of determinations at each dose is shown in parentheses, and the data are shown as mean ± S.E. *P < .05, comparing all doses of DCG-IV with saline. #P < .05, comparing 0.01 nmol of DCG-IV with saline. P < .05, comparing 1 nmol of DCG-IV with saline.

Effect of Group III mGluR Agonist on Motor Activity. Figure 3 illustrates that the effect of the group III agonist L-AP4 on motor activation was less robust than that of either the group I or group II agonists. Statistical significance was found only in the NA for horizontal activity and only at the highest dose tested (F_3,20 = 3.992; P = .022; Fig. 3). There were no significant effects seen in distance traveled or vertical counts. The time course data in Fig. 3B revealed a near-significant interaction (F_7,21 = 1.593; P = .0589); motor activity was elevated by the highest dose of L-AP4 (10 nmol) at 15 and 30 min after microinjection. L-AP4 microinjection into the VTA produced no significant alteration in horizontal, distance traveled, or vertical activity.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Effect on motor activity of the group III agonist L-AP4 microinjection into the VTA or NA. Left, total number of horizontal photocell counts generated during the 2 h after microinjection. The number of vertical counts was multiplied by 1000 for illustrative purposes. Right, time course of the horizontal data shown on the left. The number of determinations at each dose is shown in parentheses, and the data are shown as mean ± S.E. *P < .05, comparing all doses of L-AP4 with saline. #P < .05, comparing 0.1 nmol of L-AP4 with saline.

Blockade of Motor Activation with Subgroup-Specific Antagonists. Verification that the effect of DHPG was mediated by mGluR1 is illustrated in Fig. 4, A and B, which shows that the specific mGluR 1 antagonist CPCCOEt (10 nmol; Litschig et al., 1999) abolished the motor activation elicited by DHPG (5 nmol) in the NA (treatment: F_3,24 = 4.161, P = .0161; interaction: F_7,21 = 1.864, P = .0163). DHPG microinfusion elicited an expected increase in motor activity that lasted the duration of the 2-h test period, whereas the coadministration of CPCCOEt completely abolished the effect.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Evaluation of the ability of distinct antagonists to block motor-stimulating effects of DHPG and DCG-IV in the NA. A and B, ability of group I antagonist CPCCOEt to block the motor response elicited by DHPG in the NA. The data are shown as mean ± S.E. of total horizontal photocell counts and horizontal time course and reveal that blockade of group I receptors by CPCCOEt (10 nmol) blocked the increase in photocell counts by DHPG (5 nmol). C and D, effects of concurrent administration of the NMDA antagonist CPP on the behavioral-activating ability of DCG-IV in the NA. CPP was unable to block motor activity produced by DCG-IV microinfusion in this region. A and B, *P < .05 when comparing 5 nmol CPCCOEt with vehicle, C and D, *P < .05 when comparing DCG-IV with saline; P < .05 when comparing DCG-IV + CPP with saline; #P < .05 when comparing CPP with saline.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Determination of the ability of MPPG to block the motor-stimulating effects of DCG-IV in the NA and VTA. A and B, ability of group II/III antagonist MPPG to block the motor stimulant response to DCG-IV microinjection into the NA. The data are shown as mean ± S.E. of total horizontal photocell counts and horizontal time course and reveal that blockade of group II/III receptors by MPPG (10 nmol) partially blocked the increase in photocell counts by DCG-IV (0.1 nmol). The number of determinations are shown in parentheses. C and D, ability of group II/III-specific antagonist MPPG to block the increased motor activity elicited by DCG-IV in the VTA. The data are shown as mean ± S.E. of total horizontal photocell counts and horizontal time course. The number of determinations is shown in parentheses. *P < .05 when comparing DCG-IV with saline; P < .05 when comparing DCG-IV + MPPG with saline.

Histology. Figure 6 illustrates the location of cannulas tips placed into the VTA and NA. Guide cannula placements were located throughout the rostrocaudal and mediolateral extent of the VTA. In the NA, the cannulas tended to terminate near the medial border between the shell and core. No overt differences in behavior were detected with respect to rostrocaudal placement of injection cannulas.

View larger version (45K): [in this window] [in a new window]   Fig. 6.   Location of cannula tips in the NA and VTA. Coordinates are representative of stereotaxic location with respect to interaural zero according to the atlas of Paxinos and Watson (1986), The majority of NA microinjections fell on the medial border of the core and shell. VTA placements tended to be located throughout the rostrocaudal VTA.

	Discussion

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Previous research reveals that the stimulation of mGluR receptors in the NA elicits a dose-dependent elevation in motor activity (Attarian and Amalric, 1997; Vezina and Kim, 1999). These studies were conducted with the nonselective mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid. The present research shows that stimulation of either group I or group II mGluR subgroups produced a motor stimulant response in either the VTA or NA, whereas the stimulation of group III receptors was relatively ineffective.

Motor Activity Elicited by mGluR Receptor Stimulation in NA. The group I agonist DHPG elicited a robust and enduring motor stimulant response when administered into the NA. Both of the group I mGluR subtypes are found in the NA, with mGLuR5 being in particularly high abundance (Testa et al., 1994). In general, both group I receptor subtypes are located perisynaptically, where mGuR1 is located at both presynaptic and postsynaptic sites and mGLuR5 appears to be more postsynaptic (Fotuhi et al., 1993; Lujan et al., 1997). In this study, increased motor activity was abolished by an mGLuR1-selective receptor antagonist, suggesting that the motor response was mediated primarily by these receptors. Interestingly, there is evidence in other brain regions that the stimulation of group I mGluRs increases glutamate release via a presynaptic mechanism (Herrero et al., 1992; Moroni et al., 1998). It is then possible that DHPG microinfusion in the NA acts primarily on presynaptic mGluR 1 receptors and results in glutamate release. The potential release of glutamate within the NA could contribute to the observed motor stimulation. Alternatively, some studies indicate that spiny cells in the NA projecting to either the VTA or ventral pallidum express a high density of group I mRNA (Testa et al., 1995; Romano et al., 1995). However, because mGluR5 seems to be the dominant subtype on spiny cells and DHPG-induced motor activation was abolished by a selective mGluR1 antagonist, the present data argue against this postsynaptic mechanism.

Motor Activity Elicited by mGluR Receptor Stimulation in VTA. Similar to the NA, both group I and group II, but not group III, mGluR agonists stimulated motor activity when microinjected into the VTA. Interestingly, the opposite profile exists for the expression of mRNA encoding the mGluR subtypes. Thus, mGluR7 mRNA and protein are relatively abundant in the VTA (Ohishi et al., 1995; Kinoshita et al., 1998), whereas mRNA encoding the group I and group II mRNAs is absent or minimal (Ohishi et al., 1993a,b). However, mGluR1 and mGluR2/3 protein content is moderate in the ventral mesencephalon, indicating a presynaptic localization (Romano et al., 1995; Petralia et al., 1996). This implies that the behavioral effects of the group I and group II agonists are mediated by presynaptic modulation of transmitter release. Because group I agonists presynaptically augment glutamate release (Herrero et al., 1992; Macek et al., 1998; Moroni et al., 1998), a reasonable mechanism for DHPG-induced motor activity in the VTA is enhanced glutamate release, resulting in the stimulation of ionotropic glutamate receptors on dopamine (Suaud-Chagny et al., 1992). Moreover, the ventral portion of the prefrontal cortex is a primary source of glutamatergic innervation of the VTA (Sesack et al., 1989), and this cortical region is relatively enriched in mRNA encoding mGluR1 (Testa et al., 1995). Alternatively, it was recently reported that mGluR receptor stimulation of dopamine cells produces a decrease followed by an increase in cell membrane potential or firing frequency (Meltzer et al., 1997; Fiorillo and Williams, 1998). This appears to be a postsynaptic effect mediated by group I mGluRs (Fiorillo and Williams, 1998). Thus, despite the low abundance of group I mRNA in the VTA, the latter studies support a direct postsynaptic action to stimulate dopamine cells and elicit motor activation.

Summary. The present report demonstrates that group I and group II mGluR stimulation in the VTA and NA elicits motor activation. To a lesser extent, group III receptor stimulation in the NA was also behaviorally active. Based on the present findings and the literature reviewed, it is proposed that the primary action of DHPG is on mGluR1 located presynaptically on glutamatergic afferents, where the stimulation of these receptors results in the release of glutamate. In contrast, the present data and current literature are most consistent with DCG-IV action on the spiny cells of the NA, whereas a preferential presynaptic-versus-postsynaptic action of DCG-IV in the VTA is not implied by extant data.