Introduction

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The striatum is the main input nucleus of the basal ganglia, subcortical nuclei involved in behavioral control. The striatum receives excitatory, glutamate input from the cerebral cortex and thalamus (Kemp and Powell, 1971; Kitai et al., 1976), and modulatory, dopamine input from the substantia nigra pars compacta (Arluison et al., 1984; Freund et al., 1984). The activity of striatal efferent neurons, and therefore the rest of the basal ganglia, is determined by glutamate acting through both NMDA and non-NMDA receptors (Herrling, 1985; Jiang and North, 1991). However, dopamine alters the response of spiny efferent neurons to glutamate input (Cepeda et al., 1993). In particular, dopamine acting through D2 receptors seems to inhibit the functioning of striatopallidal neurons (Ferre et al., 1993; Gerfen et al., 1991, 1995). Consequently, D2 dopamine receptor antagonists are thought to increase striatopallidal neuron function (Dragunow et al., 1990; Robertson and Fibiger, 1992).

Numerous studies have provided evidence for interactions between dopamine D2 and NMDA receptor-mediated processes in the regulation of striatal neuron function. For example, electrophysiological studies show that stimulation of D2 dopamine receptors decreases NMDA receptor-mediated currents in striatal neurons (Cepeda et al., 1993). In addition, the induction of immediate early genes by D2 dopamine receptor antagonists is attenuated by administration of NMDA receptor antagonists (Boegman and Vincent, 1996; Dragunow et al., 1990; Ziolkowska and Hollt, 1993). These effects suggest that the ability of dopamine D2 receptor activation to modulate striatopallidal neuron function under a number of conditions is dependent on its ability to modulate the response of those neurons to ongoing NMDA receptor activation.

Although these dopamine-mediated effects in the striatum are dependent on NMDA receptor activation, the extent to which different types of NMDA receptors are involved in these interactions is unknown. The NMDA receptor is a multimeric receptor comprised of an NR1 subunit and one or more NR2 subunits (Hollmann and Heinemann, 1994). The NR2 subunits, termed NR2A-2D in the rat, play an important role in determining the functional and pharmacological properties of the resulting NMDA receptor. For example, incorporation of an NR2A subunit yields an NMDA receptor that has been referred to as "antagonist" preferring, because such receptors show a higher affinity for competitive NMDA receptor antagonists and there is a high degree of correlation between receptors labeled in vivo with competitive NMDA receptor antagonists and the distribution of the NR2A subunit (Buller et al., 1994). However, incorporation of the NR2B subunit yields an "agonist-preferring" receptor. Such receptors have higher affinity for glutamate than do receptors containing the NR2A subunit (Buller et al., 1994; Laurie and Seeburg, 1994). In addition, the distribution of NMDA receptors labeled with glutamate or the use-dependent, noncompetitive NMDA receptor antagonist MK-801 correlates highly with the distribution of the NR2B subunit (Buller et al., 1994).

Although both NR2A and NR2B subunits are expressed in spiny efferent neurons of the striatum, they differ in their regional distribution (Standaert et al., 1994; Watanabe et al., 1993). The NR2A subunit shows a lateral-to-medial gradient, with greater expression in lateral striatum and very limited expression in medial striatum. However, the NR2B subunit is expressed uniformly throughout the striatum. To the extent that these NMDA receptor subunits confer different pharmacologies to the NMDA receptor, these regional differences in expression suggest that different types of NMDA receptors may be differentially involved in regulating striatal neuron function.

Several lines of evidence support such an hypothesis. First, NMDA-induced efflux of dopamine, -amino-butyric acid, acetylcholine and spermidine is differentially affected by various NMDA receptor antagonists (Nankai et al., 1995; Nankai et al., 1996; Nicolas et al., 1994). Second, noncompetitive NMDA receptor antagonists inhibit, whereas competitive NMDA receptor antagonists potentiate, D2 dopamine receptor agonist-induced behaviors in rats (Loschmann et al., 1997; Morelli et al., 1992; Svensson et al., 1992; Wachtel et al., 1992). Third, we recently have shown that D1 dopamine receptor agonist-induced immediate early gene expression in the striatum is inhibited by competitive and channel-blocking NMDA receptor antagonists, but potentiated by the NR2B-selective, polyamine-site antagonist of the NMDA receptor, ifenprodil (Keefe and Ganguly, 1998). Finally, previous work has shown that both a competitive and noncompetitive NMDA receptor antagonist can block haloperidol-induced immediate early gene expression (Boegman and Vincent, 1996); however, blockade of gene expression by the competitive antagonist was obtained only with a very high dose (33 mg/kg, i.p.), supporting the possible involvement of different types of NMDA receptors.

Our study was therefore designed to test the hypothesis that different types of NMDA receptor antagonists will have different effects on dopamine D2 receptor antagonist-induced increases in striatopallidal neuron function. Changes in immediate early gene expression in the striatum were used as markers for altered striatopallidal neuron function, as D2 receptor-induced changes in gene expression are correlated with altered -amino-butyric acid release and 2-deoxyglucose utilization in the globus pallidus (Ferre et al., 1993; Trugman and Wooten, 1987). Three different NMDA receptor antagonists that vary in their mechanism of action and selectivity for NMDA receptors containing different NR2 subunits were used.

	Methods

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Animals. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 225 to 300 g were used in all experiments. Rats were housed in groups of four in hanging wire-mesh cages in a temperature-controlled room. Rats were on a 12:12 light:dark cycle, and had free access to food and water. All animal care and experimental manipulations were approved by the Institutional Animal Care and Use Committee of the University of Utah and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Drugs. (S)-Eticlopride hydrochloride, (+)-MK-801 hydrogen maleate and ifenprodil tartrate were obtained from Research Biochemicals International (Natick, MA). CGS 19755 was kindly donated by Ciba-Geigy Corporation (Summit, NJ). The doses of the NMDA receptor antagonists were calculated as the salt, whereas the dose of eticlopride was calculated as the free base. Ifenprodil was dissolved in deionized water, CGS 19755 in phosphate-buffered saline and eticlopride and MK-801 in normal saline. All drugs were administered i.p. in a volume of 1 ml/kg, with the exception of the highest dose of ifenprodil, which was given in a volume of 8 ml/kg.

Pharmacological manipulations. On the day of the experiment, rats were rehoused in plastic tub cages (four to five per cage) and transferred from their home cages to the laboratory. Rats were weighed and then received injections of either an NMDA receptor antagonist or the appropriate vehicle solution. After injection of the NMDA receptor antagonist or vehicle, each rat received an injection of the D2 dopamine receptor antagonist eticlopride (1 mg/kg, i.p.). The time between the injection of the NMDA receptor antagonist and the injection of eticlopride was 30 min for MK-801 and ifenprodil, and 60 min for CGS 19755. These time delays and the doses of NMDA antagonists used were chosen on the basis of previously published studies showing effective NMDA receptor blockade within these dose-ranges and at these time points (Cain et al., 1997; Carter et al., 1990; De Sarro and De Sarro, 1993; Koerner et al., 1996). The dose of eticlopride used was based on pilot studies conducted in our laboratory that indicated that this dose produced significant, yet nonsaturating, induction of the immediate early genes, affording us the ability to see both increases and decreases in response to the NMDA receptor antagonist treatment. Rats were killed 40 min after the injection of eticlopride, a time at which there is significant induction of immediate early genes in both the medial and lateral striatum (H. Steiner, personal communication).

In situ hybridization histochemistry. Forty min after the second injection, rats were euthanized by exposure to CO₂ and decapitated. The brain was rapidly removed and frozen in isopentane chilled on dry ice. Brains were stored at 20°C until they were cut in 12-µm sections in a cryostat (Cryocut 1800, Cambridge Instruments, Germany). Sections were thaw-mounted onto gelatin-chrome alum-subbed slides and stored at 20°C. Once all brains from an experiment had been sectioned, slides from all animals in that experiment were postfixed in 4% paraformaldehyde/0.9% NaCl, acetylated in fresh 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl, dehydrated in alcohol, delipidated in chloroform and then rehydrated in a descending series of alcohols. Slides were air-dried and stored at 70°C.

Data analysis. Film autoradiograms were analyzed using the image analysis program Image (Wayne Rasband, National Institutes of Health, Bethesda, MD), yielding average density (gray) values over regions of interest. Before the measurement of brain sections, the linearity of the video camera and video capture card to increasing signal intensity was determined by measuring the average gray value of signals of known optical density from a photographic step tablet (Eastman Kodak Co.). The intensity of the illuminating light then was adjusted so that the values measured from film autoradiograms of brain sections fell within the linear portion of the system's response. The images of sections from all experimental and control groups within a given experiment that were processed and hybridized in parallel were captured and measured under constant lighting and camera conditions. Measurements were made over the medial, central and lateral thirds of the right, middle striatum (0.5 mm anterior to bregma) from its dorsal aspect to the level of the anterior commissure ventrally. The average gray value of the white matter overlying the striatum was subtracted from the average gray value of the striatal regions of interest to correct for background labeling.

	Results

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Effects of the competitive NMDA receptor antagonist CGS 19755. Systemic administration of the D2 dopamine receptor antagonist eticlopride increased immediate early gene expression in the striatum, the increase being more apparent in the lateral striatum (figs. 1, 3, and 5). Systemic administration of the competitive NMDA receptor antagonist CGS 19755 one hr before the administration of eticlopride produced a significant dose-dependent suppression of both c-fos (figs. 1 and 2) and zif268 (fig. 1; table 1) in the striatum. This suppression was most apparent in the medial striatum, with some significant attenuation also seen in the central third of the striatum. Although there was a slight trend toward suppression of the gene expression in the lateral striatum (fig. 2; table 1), this suppression was small and never reached statistical significance.

View larger version (114K): [in this window] [in a new window]   Fig. 1.   In situ hybridization film autoradiograms showing the expression of c-fos and zif268 in the striatum of rats receiving two vehicle injections (control), the D2 dopamine receptor antagonist eticlopride (1 mg/kg, i.p.) 1 hr after injection of vehicle, or eticlopride (1 mg/kg, i.p.) 1 hr after injection of the competitive NMDA receptor antagonist CGS 19755 (10 mg/kg, i.p.). Rats were killed 40 min after the second injection.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of systemic administration of CGS 19755 ("CGS"; 1, 5 and 10 mg/kg, i.p.) on c-fos expression induced in the mid-striatum (approximately 0.5 mm anterior to bregma) by administration of eticlopride ("etic"; 1 mg/kg, i.p.). CGS 19755 was administered 1 hr before the injection of eticlopride. Control rats received two vehicle injections 1 hr apart. Rats treated with CGS 19755 alone received the highest dose of CGS 19755 (10 mg/kg, i.p.) followed 1 hr later by a vehicle injection. Rats treated with eticlopride alone received a systemic injection of vehicle followed 1 hr later by eticlopride (1 mg/kg, i.p.). All rats were killed 40 min after the second injection. Values are mean gray values (±S.E.M.; arbitrary units) measured in the medial, central, and lateral thirds of the striatum. Numbers in parentheses indicate the number of animals per group. * Significantly different from control, P < .05. + Significantly different from eticlopride alone, P < .05.

Effects of the noncompetitive, channel blocking antagonist MK-801. As with CGS 19755, systemic administration of the noncompetitive, channel blocking antagonist MK-801 also produced a dose-dependent suppression of eticlopride-induced c-fos expression in the medial third of the striatum (figs. 3 and 4). The central and lateral thirds, although again showing a trend toward being suppressed, were not significantly attenuated at either the rostral or middle striatal levels (fig. 4). In this experiment, neither the eticlopride-induced increase in zif268 expression in the medial striatum, nor the effects of MK-801 on eticlopride-induced zif268 expression were statistically significant (table 2). However, the highest dose of MK-801 did tend to suppress the response in all three regions, consistent with the effects on c-fos expression.

View larger version (113K): [in this window] [in a new window]   Fig. 3.   In situ hybridization film autoradiograms showing the expression of c-fos and zif268 in the striatum of rats receiving two vehicle injections (control), the D2 dopamine receptor antagonist eticlopride (1 mg/kg, i.p.) 30 min after injection of vehicle, or eticlopride (1 mg/kg, i.p.) 30 min after injection of the noncompetitive NMDA receptor antagonist MK-801 (1 mg/kg, i.p.). Rats were killed 40 min after the second injection.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effects of systemic administration of MK-801 (0.01, 0.1 and 1 mg/kg, i.p.) on c-fos expression induced in the mid-striatum (approximately 0.5 mm anterior to bregma) by eticlopride ("etic"; 1 mg/kg, i.p.). MK-801 was administered 30 min before the injection of eticlopride. Control rats received two vehicle injections 30 min apart. Rats treated with MK-801 alone received the highest dose of MK-801 (1 mg/kg, i.p.) followed 30 min later by a vehicle injection. Rats treated with eticlopride alone received a systemic injection of vehicle followed 30 min later by eticlopride (1 mg/kg, i.p.). All rats were killed 40 min after the second injection. Values are mean gray values (±S.E.M.; arbitrary units) measured in the medial, central and lateral thirds of the striatum. Numbers in parentheses indicate the number of animals per group. * Significantly different from control, P < .05. + Significantly different from eticlopride alone, P < .05.

Effects of the noncompetitive, polyamine-site antagonist ifenprodil. The administration of ifenprodil 30 min before eticlopride also produced a dose-dependent suppression of c-fos expression in the striatum (figs. 5 and 6). This inhibition was again most evident in the medial and central thirds of the striatum, although the decrease in the medial striatum did not quite reach statistical significance on the post hoc analysis (difference = 2.0; critical difference = 2.1). As with the other NMDA receptor antagonists, there also was a trend for a decrease in the lateral striatum, but the induction remained significantly elevated relative to controls and was not different from that seen in animals receiving eticlopride alone. Similar results were seen with zif268 induction (fig. 5; table 3) in that the induction of zif268 tended to be decreased in the medial third of the striatum. In addition, the induction in the central third of the striatum was significantly decreased by the highest dose of ifenprodil. There also was a nonsignificant and not dose-dependent decrease of zif268 induction in the lateral striatum. The highest dose of ifenprodil tended to decrease the basal expression of the immediate early genes (fig. 6; table 3).

View larger version (117K): [in this window] [in a new window]   Fig. 5.   In situ hybridization film autoradiograms showing the expression of c-fos and zif268 in the striatum of rats receiving two vehicle injections (control), the D2 dopamine receptor antagonist eticlopride (1 mg/kg, i.p.) 30 min after injection of vehicle, or eticlopride (1 mg/kg, i.p.) 30 min after injection of the NMDA receptor polyamine-site antagonist ifenprodil (10 mg/kg, i.p.). Rats were killed 40 min after the second injection.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effects of systemic administration of ifenprodil ("ifen"; 1 and 10 mg/kg, i.p.) on c-fos expression induced in the mid-striatum (approximately 0.5 mm anterior to bregma) by eticlopride ("etic"; 1 mg/kg, i.p.). Ifenprodil was administered 30 min before the injection of eticlopride. Control rats received two vehicle injections 30 min apart. Rats treated with ifenprodil alone received the highest dose of ifenprodil (10 mg/kg, i.p.) followed 30 min later by a vehicle injection. Rats treated with eticlopride alone received a systemic injection of vehicle followed 30 min later by eticlopride (1 mg/kg, i.p.). All rats were killed 40 min after the second injection. Values are mean gray values (±S.E.M.; arbitrary units) measured in the medial, central, and lateral thirds of the striatum. Numbers in parentheses indicate the number of animals per group. * Significantly different from control, P < .05. + Significantly different from eticlopride alone, P < .05.

	Discussion

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Our experiments show that blockade of D2 receptors with eticlopride increases the expression of the immediate early genes c-fos and zif268 in the striatum, and that this effect can be blocked by administration of an NMDA receptor antagonist. These findings therefore confirm the results of previous studies showing induction of immediate early genes in the striatum by D2 receptor antagonists and the dependence of this effect on NMDA receptors (Boegman and Vincent, 1996; Dragunow et al., 1990; Robertson and Fibiger, 1992; Robertson et al., 1992; Ziolkowska and Hollt, 1993). However, our findings extend those previous observations by showing that NMDA receptor blockade selectively blocks eticlopride-induced gene expression in the medial-central aspects of the striatum, but not in the lateral striatum (see also Wagstaff and Gerfen, 1997). In addition, our findings failed to provide support for the hypothesis that different types of NMDA receptor antagonists exert differential effects on D2 antagonist-induced activation of striatopallidal neurons.

Our overall purpose was to examine the hypothesis that different types of NMDA receptor antagonists would have different effects on dopamine D2 receptor antagonist-induced increases in striatopallidal neuron function, as evidenced by changes in immediate early gene expression. This hypothesis was based on the observation that the NR2 subunits of the NMDA receptor show regional and cellular differences in their expression in the striatum and also confer different pharmacologies to the receptors in which they are expressed. Both MK-801 and CGS 19755 are effective antagonists of NMDA receptors containing NR2A or NR2B subunits, although CGS 19755 has slightly higher affinity for receptors containing the NR2A vs. the NR2B subunit (Laurie and Seeburg, 1994). However, ifenprodil, a putative polyamine-site antagonist, is up to 400 times more potent as an antagonist of NMDA receptors containing the NR2B subunit (Williams, 1993). Despite these differences in NMDA receptor selectivity, all three of these antagonists exerted similar effects on eticlopride-induced immediate early gene expression. The uniformity of their effects thereby suggests that the NMDA receptors involved in eticlopride-induced immediate early gene expression are a uniform population with respect to their pharmacology. Of course, the development of NMDA receptor antagonists with greater subunit selectivity may lead to finer insight into the potential roles that distinct NMDA receptors might play in regulating the effects of dopamine on striatopallidal neuron gene expression.

Although the different types of NMDA receptor antagonists did not exert markedly different effects on eticlopride-induced immediate early gene expression, the blockade of eticlopride's effects in the striatum by the NMDA antagonists was not uniform. Significant decreases in c-fos induction were seen only in the medial (CGS 19755 and MK-801) and central (CGS 19755 and ifenprodil) thirds of the striatum. Similarly, zif268 induction by eticlopride was significantly decreased in the medial (CGS 19755) and central (CGS 19755 and ifenprodil) thirds. For all three antagonists at the highest doses tested, the expression of both immediate early genes in the medial and central striatum was not significantly different from control. However, in the lateral striatum, although there was a consistent trend for all of the drugs to decrease the expression of both genes, these effects were never statistically significant and the expression was still significantly greater than that seen in controls. Previous studies have reported decreases in D2 antagonist-induced gene expression in the lateral striatum after NMDA receptor blockade (Boegman and Vincent, 1996), suggesting that with higher doses of the NMDA receptor antagonists we might have observed a significant attenuation in the lateral striatum. However, we think that this is unlikely for two reasons. First, only in the case of MK-801 was this trend dose dependent. Second, the antagonists used have clearly been shown in other studies to block NMDA-evoked responses when administered systemically and in the dose-range used here (Cain et al., 1997; Carter et al., 1990; De Sarro and De Sarro, 1993; Koerner et al., 1996), suggesting that NMDA receptor blockade was achieved in our studies. Even if higher doses of antagonists were to produce a significant attenuation, our findings would still indicate that this eticlopride-induced gene expression in the striatum is differentially regulated by excitatory input through NMDA receptors in different regions of the striatum.

The mechanisms accounting for the regional differences in the effects of the NMDA receptor antagonists on eticlopride-induced immediate early gene expression are not clear. It seems unlikely that the distribution of NMDA receptors is responsible, as the NR2B subunit is uniformly distributed across the medial-lateral extent of the striatum (Standaert et al., 1994; Watanabe et al., 1993), yet ifenprodil, with significant affinity for NMDA receptors containing an NR2B subunit, still preferentially exerted its effects in the medial and central aspects of the striatum. Conceivably, NMDA receptors containing the NR2A subunit might continue to subserve immediate early gene expression in the lateral striatum in the presence of blockade of receptors containing the NR2B subunit. However, in our study CGS 19755 and MK-801, antagonists with significant affinity for receptors containing both the NR2A and NR2B subunits (Buller et al., 1994; Laurie and Seeburg, 1994), also had no significant effect on the induction in the lateral striatum, suggesting that NR2A-containing NMDA receptors do not mediate the response in the lateral striatum either. Finally, the fact that NMDA receptor antagonists with different mechanisms of action exerted similar effects further argues against differences in NMDA receptor subtypes or NMDA receptor modulation being responsible for the observed regional effects.

It is interesting that the regions most sensitive to NMDA receptor blockade are also the regions in which the induction of immediate early genes by eticlopride or other D2 antagonists is less. Thus, D2 antagonist-induced gene expression predominates in the lateral striatum, a pattern that has been attributed to the greater concentration of D2 receptors in that region (Robertson and Fibiger, 1992). The relative lack of effect of NMDA receptor blockade in the lateral striatum may thus be due to greater D2 receptor blockade leading to greater increases in intracellular cAMP and cAMP-mediated processes in those neurons expressing greater numbers of D2 receptors. Studies in primary cultures of striatal neurons have revealed that immediate early gene induction in striatal neurons by low vs. high doses of forskolin is differentially dependent on glutamate input through NMDA receptors (Konradi, 1998). When high doses of forskolin are used, the induction of immediate early genes is not affected by NMDA receptor antagonists, suggesting that sufficient increases in cAMP levels can directly induce immediate early gene expression in the striatum independent of NMDA receptor activation. The dose of eticlopride used in our study may thus produce such high levels of activation in the adenylate cyclase pathway in the lateral striatum, but not in the medial striatum, leading to differences both in the amount of immediate early gene induction as well as in the dependence on NMDA receptor activation. The blockade of c-fos induction in the lateral striatum by MK-801 in the Boegman and Vincent study (1996) may therefore reflect lesser activation of the adenylate cyclase pathway by a relatively low dose of haloperidol (0.2 mg/kg) and consequent NMDA receptor dependence. Interestingly, Dragunow et al. (1990) have reported that MK-801 blocks induction of Fos immunoreactivity in the dorsal striatum by a low (0.5 mg/kg), but not a high (4 mg/kg), dose of haloperidol. Whether such differences in the magnitude of the response to D2 receptor blockade determine the regional differences in the dependence of D2 antagonistinduced gene expression on NMDA receptors remains to be determined.

In conclusion, our data indicate that different types of NMDA receptor antagonists do not exert differential effects on eticlopride-induced immediate early gene expression in the striatum based on their potential NMDA receptor subunit selectivity. The findings do suggest, however, that the induction of immediate early genes in different regions of the striatum by the dose of eticlopride used in this study is differentially dependent on NMDA receptor activation. These differences may be related to the differential magnitude of the induction in the medial and lateral aspects of the striatum. These findings imply that the mechanisms underlying the induction of immediate early genes throughout the striatum by D2 receptor blockade may not be uniform.