Abstract
Bilateral infusions of d-amphetamine into the rat ventral-lateral striatum (VLS) were previously shown to cause a robust behavioral activation that was correlated temporally with a net increase in firing of substantia nigra pars reticulata (SNpr) neurons, a response opposite predictions of the basal ganglia model. The current studies assessed the individual and cooperative contributions of striatal D1 and D2 dopamine receptors to these responses. Bilateral infusions into VLS of the D1/D2 agonist apomorphine (10 μg/μl/side) caused intense oral movements and sniffing, and an overall increase in SNpr cell firing to 133% of basal rates, similar to effects ofd-amphetamine. However, when striatal D2receptors were stimulated selectively by infusions of quinpirole (30 μg/μl/side) + the D1 antagonistR-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390; 10 μg/μl/side), no behavioral response and only modest and variable changes in SNpr cell firing were observed. Selective stimulation of striatal D1 receptors by (±) 6-chloro-APB hydrobromide (SKF 82958; 10 μg/μl/side) + the D2 antagonistcis-N-(1-benzyl-2-methyl-pyrrolidin-3-yl)-5-chloro-2-methoxy-4-methyl-aminobenzamide (YM 09151-2; 2 μg/μl/side) caused a weak but sustained increase in oral movements and modestly increased SNpr cell firing, but neither response was of the magnitude observed with apomorphine. When the two agonists were infused concurrently, however, robust oral movements and sniffing again occurred over the same time period that a majority of SNpr cells exhibited marked, sometimes extreme and fluctuating, changes in firing (net increase, 117% of basal rates). These data confirm that concurrent striatal D1/D2 receptor stimulation elicits a strong motor activation that is correlated temporally with a net excitation rather than inhibition of SNpr firing, and reveal that D1and D2 receptors interact synergistically within the striatum to stimulate both forms of output.
The basal ganglia functional model predicts that dopamine, by stimulating the striatonigral pathway via D1 receptors and inhibiting the striatopallidal pathway via D2receptors, should inhibit output from the substantia nigra pars reticulata and internal pallidal segment (SNpr/GPi). Inhibition of output from the SNr/GPi should in turn disinhibit the thalamus to facilitate movement (for review, see Alexander and Crutcher, 1990). Many of the predictions of the model have been supported by electrophysiological studies in humans with Parkinson's disease and animal models of this disorder (Filion and Tremblay, 1991; Bergman et al., 1994; Papa et al., 1999; Levy et al., 2001). However, the question of whether these predictions are valid and supported in normal animals without dopaminergic lesions has been less rigorously addressed.
A definitive test of this hypothesis has been made difficult by the complexity of the circuitry, in particular by the fact that D1 and D2 receptors are expressed not only within the striatum but also in other nuclei of the basal ganglia that directly or indirectly influence the SNpr/GPi (Smith and Kieval, 2000). Dopaminergic drugs, when administered systemically, may therefore act at sites besides the striatum to modulate the basal ganglia output nuclei, thereby preventing a clear assessment of the specific role of striatal dopamine receptors in the responses. Another problem arises when attempting to distinguish the individual roles of D1 and D2 receptors because a synergistic interaction between them is normally required for generating a “full” behavioral or electrophysiological response to dopamine (Arnt et al., 1987; Walters et al., 1987; White et al., 1988;Waddington and Daly, 1993; White and Hu, 1993). D1/D2 synergism complicates defining the roles of each receptor even when using D1 and D2receptor-selective agonists because the unintended receptor can still be stimulated by endogenous dopamine to yield a mixed D1/D2 response. For these reasons, in studies where dopamine agonists have been given systemically to animals with an intact dopamine system, it has not been possible to attribute a particular behavioral or electrophysiological response to activation of a specific receptor type at a specific location in the circuitry. Moreover, few attempts have been made to correlate the behavioral consequences of striatal D1 and/or D2 receptor stimulation with the electrophysiological consequences of that same manipulation.
The goal of our studies has been to fill these gaps by assessing how discrete stimulation of striatal D1 and D2 dopamine receptors, individually and concurrently, influences behavioral and electrophysiological output from the basal ganglia in normal rats via the SNpr/GPi. To circumvent the problems posed by systemic routes of administration, we and others have adopted the use of microinjections of dopaminergic drugs directly into the corpus striatum. In agreement with previous reports (Kelley et al., 1988; Wang and Rebec, 1993; Dickson et al., 1994), we found that bilateral (but not unilateral) infusions into the rat ventral-lateral striatum (VLS) of the dopamine releaser d-amphetamine (20 μg/μl/side) cause a robust behavioral activation consisting of primarily oral movements (biting, licking, tongue protrusions, and jaw movements) and sniffing. However, contrary to a key prediction of the basal ganglia model, we showed in parallel electrophysiological studies that these same infusions do not cause an inhibition of firing of SNpr neurons. In fact, during the period of peak behavioral activation, we noted extremely variable changes in the firing of SNpr neurons, but the average response was a significant increase in SNpr activity to approximately 120% of basal firing rates (Martin et al., 1997;Waszczak et al., 2001). The variable nature of the responses and the overall excitation of SNpr cell firing were not the result of anesthesia because similar results were obtained in both anesthetized rats and animals that were awake, locally anesthetized, and paralyzed.
To determine the relative contributions of D1 and D2 receptors to these responses, we have now used bilateral infusions into VLS of drug combinations intended to selectively stimulate D1, D2, or both receptors concurrently. Several strategies were considered to prevent endogenous dopamine from stimulating the unintended receptor. 6-Hydroxydopamine lesions of the nigrostriatal dopamine neurons, or pretreatment of the animals with reserpine or α-methyl-p-tyrosine (AMPT), have been used in previous studies to destroy or deplete endogenous dopamine stores. However, chronic dopamine depletion by either of these treatments seriously disrupts the normal interactions between neurons within the circuitry (Huang and Walters, 1994), altering their expression of dopamine receptors and leading to an uncoupling D1/D2 synergism (Hu et al., 1990; LaHoste et al., 1993; Marshall et al., 1993). Such conditions would be inappropriate for assessing the functional roles of striatal D1 or D2 receptors in the normosensitive system. Acute (2–4 h) depletion of dopamine by AMPT, which avoids the disruptions of long-term depletion, has been valuable in demonstrating the phenomenon of D1/D2 synergism (Walters et al., 1987; White et al., 1988; Hu et al., 1990) and was a reasonable approach for our studies. However, AMPT causes a generalized central and peripheral (autonomic) catecholamine depletion that might interfere in the behavioral responses to striatal dopamine agonist infusions. Consequently, we chose a strategy where selective stimulation of each receptor was accomplished by coadministration of an agonist at one receptor together with an antagonist at the unintended receptor. The antagonist would block the effects of both endogenous dopamine and any nonselective effects of each agonist at the opposing dopamine receptor, thereby allowing stimulation of only a single receptor in the area of the infusion.
To selectively activate D1 receptors, we infused a solution containing the full-efficacy D1 agonist SKF 82958 [(±) 6-chloro-APB hydrobromide] (O'Boyle et al., 1989) together with the potent and selective D2 antagonist YM 09151-2 (Terai et al., 1983; Niznik et al., 1985). In this case, although SKF 82958 displays only about 10-fold selectivity for D1versus D2 receptors (Murray and Waddington, 1989;Gnanalingham et al., 1995), any D2 agonist activity should have been prevented by coinfusion of the D2 blocker. To selectively stimulate D2 receptors, we infused the D2 agonist quinpirole (Tsuruta et al., 1981) plus the selective D1 antagonist SCH 23390 (Hyttel, 1983; Iorio et al., 1983). For concurrent stimulation of both D1 and D2 receptors, we infused either the mixed D1/D2 agonist apomorphine, or SKF 82958 and quinpirole together. As in our earlier studies with amphetamine, we monitored the effects of individual and concurrent stimulation of the two receptors on behavior and electrophysiological output from the SNpr in separate but parallel studies following identical drug infusion paradigms.
Materials and Methods
Striatal Drug Infusions.
Male rats (250–275 g) were implanted bilaterally with 23-gauge stainless steel guide cannulae into the VLS (coordinates: A, 9.8 mm to λ; L, 3.8 mm; V, −3.3 mm) 1 week before behavioral testing or just before electrophysiological experiments. For the placement of chronic guide cannulae (behavioral studies only), three screws were set in the skull near the cannula, and the assembly was secured to the skull by using dental acrylic. For striatal drug or saline infusions, a 30-gauge injection cannula was inserted through the guide, and extended 3 mm beyond it, so that its tip reached the target site in the VLS. Infusions were made from a pair of 10-μl Hamilton syringes by using a Harvard Apparatus dual infusion pump. For behavioral studies, the pump was remotely activated and controlled by a computer to avoid behavioral effects due to handling. After completion of experiments, rats were sacrificed and cannulae placements in the VLS were confirmed histologically.
Behavioral Monitoring Techniques.
Behavior was monitored in a test chamber with clear plastic walls, a glass floor (50 × 40 cm), transverse infrared photobeams 10 cm from the end walls, and a video camera mounted below the cage floor. The chamber was housed in a small, darkened room separated from the main laboratory. The rat was handled briefly at the beginning of each test session to remove the dummy cannulae and insert the bilateral injection cannulae through the guides. The injection cannulae were attached via thin polypropylene tubing to a liquid swivel held by a spring arm over the chamber, and the swivel was in turn connected to syringes controlled by the infusion pump. The entire infusion assembly was out of reach of the rat, but allowed the animal complete freedom of movement within the chamber. After a 20-min acclimation period, the infusion pump was remotely activated by the experimenter from outside the testing room, and a drug combination or saline (1 μl/side) was infused bilaterally over 2 min into the VLS. Behavior was monitored for 45 min by videotaping through the cage floor, and by computer acquisition of consecutive (A/B) photobeam breaks. All rats were observed during two baseline sessions (no infusions) and were then randomly given two infusions of each drug combination and saline with at least a 2-day washout between trials. In one group of rats, drug combinations were SKF 82958 (10 μg/μl/side) + YM 09151-2 (2 μg/μl/side) and SKF 82958 (10 μg/μl/side) + quinpirole (30 μg/μl/side). In a second group of rats, drug combinations were quinpirole (30 μg/μl/side) + SCH 23390 (10 μg/μl/side) and quinpirole (30 μg/μl/side) + SKF 82958 (10 μg/μl/side).
Observers blind to the treatments that the animals received viewed videotapes to assess the behavioral effects of the striatal drug infusions. Inter-rater reliability between the observers, and criteria for rating the various behaviors, were established in mock sessions before data collection. Specific behaviors (oral movements, sniffing, grooming, and rearing) were rated for their frequency of occurrence during a 1-min interval every 5 min (0, none; 1, few; 2, often; 3, extreme), and averaged for the two trials at each treatment. Locomotor activity scores (expressed as A/B beam breaks) were summed over 2- or 4-min intervals and then averaged for the two trials at each treatment. The statistical significance of differences in behavior after striatal infusions was determined by two-way ANOVA (drug treatment × time) (GraphPad Prism, version 3.0; GraphPad Software, San Diego, CA) for the mean responses of six rats per treatment group.
Extracellular Single Unit Recording Techniques.
Activities of SNr neurons were recorded in rats anesthetized with chloral hydrate (400 mg/kg i.p.). All surgical procedures were carried out in strict compliance with the National Research Council's Guide for the Care and Use of Laboratory Animals, 1996. We have previously shown that chloral hydrate anesthesia does not blunt or otherwise alter the nature of responses of SNpr neurons to striatal infusions ofd-amphetamine, a dopamine releaser (Martin et al., 1997;Waszczak et al., 2001). SNr neurons were located within the pars reticulata region of the substantia nigra, within the following stereotaxic coordinates: L, 2.0 to 2.4 mm; A, 2.8 to 3.2 mm; V, >7.0 mm. These neurons are distinguished electrophysiologically by their sharp, biphasic extracellular waveforms, duration (<1 ms), firing rates (10–40 spikes/s), and location just ventral to the pars compacta dopamine neurons, according to criteria described previously (Waszczak et al., 1980).
Electrodes were glass micropipettes filled with 1% Pontamine Sky Blue dye in 2 M NaCl. Standard methods were used for amplifying, displaying, and discriminating the single unit activities of SNpr neurons (Waszczak et al., 1980). At the end of recording periods, a small amount of the blue dye was iontophoretically deposited in the brain at the recording site. The animal was sacrificed and the brain was removed, sectioned, and mounted on slides to verify location of the blue spot within the pars reticulata. All neurons included in these studies were located in the SNpr within the following stereotaxic boundaries, according to the atlas of Paxinos and Watson (1986): L, 2.0 to 3.0 mm; A, −4.80 to −6.04 mm from bregma; V, 7.5 to 8.5 mm.
The effects of striatal drug infusions on SNpr firing were evaluated by first obtaining a stable 3- to 5-min period of baseline firing then infusing bilaterally into the VLS over 2 min one of the above-mentioned drug combinations or saline (1 μl/side). SNpr neuronal firing was monitored for 30 min after infusions, although in some cases, units were lost before completion of the 30-min sampling interval. Firing rates were averaged over 5-s intervals and plotted as a percentage of the preinfusion baseline firing rate of the cell. For each drug treatment, the average percentage of change in firing of all SNpr neurons after the infusions was compared with their preinfusion (baseline) rates by using Student's t test.
Drugs.
All drugs used in these studies were obtained from Sigma Chemical (St. Louis, MO) and were prepared fresh before use.
Results
Effects of Striatal Infusion of D1/D2Receptor Agonist Apomorphine on Behavior and SNpr Cell Firing.
Bilateral infusions of the mixed D1/D2 receptor agonist apomorphine (5 and 10 μg/μl/side) into VLS caused a robust, dose-dependent behavioral activation with an onset ranging from immediate to 20-min postinfusion and persisting for at least 40 min after the infusions (Fig. 1). At the 10-μg/μl/side dose, animals tended to be stationary initially, and engaged primarily in intense oral movements and sniffing. By 20-min postinfusion, these behaviors began to decrease somewhat and locomotor activity began to increase. Both the 5- and 10-μg/μl/side doses of apomorphine caused significant increases in oral movements (two-way ANOVA: p < 0.0001, F = 19.84, df = 2,135 at 5 μg/μl/side and p < 0.0001,F = 15.36, df = 2,135 at 10 μg/μl/side), sniffing (two-way ANOVA: p < 0.0001, F= 36.99, df = 2,135 at 5 μg/μl/side and p < 0.0001, F = 19.44, df = 2,135 at 10 μg/μl/side), and locomotor activity (data not shown; two-way ANOVA:p < 0.0001, F = 27.75, df = 2,20 at 5 μg/μl/side and p < 0.0001, F= 57.42, df = 2,20 at 10 μg/μl/side) relative to bilateral infusions of saline, or baseline behavior with no infusion. Grooming and rearing were not significantly altered at either dose (data not shown), and there was no significant effect of postinfusion time on any of the behaviors.
In parallel electrophysiological studies, all SNpr neurons exhibited >20% changes in firing rates for various periods during the 30 min after apomorphine infusions (10 μg/μl/side). However, contrary to predictions of the basal ganglia model, the responses were not a uniform inhibition of activity. Instead, SNpr cells exhibited changes in firing that were highly variable in both direction and magnitude (Fig. 2). Of 15 cells tested, nine showed only increases in firing, some to >400% of baseline. In cases where unit activity was dramatically increased, the neuron frequently entered a state of apparent depolarization blockade and recording could not be continued to the end of the 30-min observation period (visible in graphs showing changes in firing of individual neurons after the infusion). These extreme changes in firing were probably due to an effect of the drug, rather than to fluid or mechanical damage from the infusion, because similar changes were never observed after identical infusions of saline into the VLS (Martin et al., 1997). An additional four cells showed biphasic increases and decreases in activity after infusions, and only two cells showed a modest, intermittent inhibition of firing. Overall, the population response to bilateral apomorphine infusions was a significant increase in firing to 133 ± 11% of baseline firing rates (p < 0.05), and these changes in neuronal activity peaked during the time of maximal behavioral activation.
Effects of Selective Activation of Striatal D2Dopamine Receptors on Behavior and SNpr Cell Firing.
To clarify the role of D2 receptors in mediating the effects of the mixed D1/D2 agonist apomorphine, we used bilateral infusions into VLS of the D2 agonist quinpirole (30 μg/μl/side) + the D1 antagonist SCH 23390 (10 μg/μl/side). In contrast to the effects of apomorphine (see above) and amphetamine in previous studies, selective stimulation of D2receptors failed to elicit a behavioral response (Fig.3). In fact, oral movements, sniffing, and locomotor activity (data not shown) tended to besuppressed at some time points after quinpirole + SCH 23390 infusions, relative to responses observed after bilateral infusions of saline, or baseline behavior with no infusion. Overall, there was no significant change in any of the behavioral measures after selective stimulation of striatal D2 dopamine receptors.
In parallel electrophysiological studies with identical infusion conditions, quinpirole + SCH 23390 also failed to consistently or significantly alter the firing of SNpr neurons (Fig.4). A few cells (4 of 13) showed predominant increases in firing, offset by a few cells (2 of 13) that showed a predominant inhibition of firing. Of the remaining SNpr neurons tested, five exhibited only minor (<20% of baseline) changes in activity, and two showed biphasic changes in firing during the 20 to 30 min after infusions. Overall, selective, bilateral stimulation of D2 receptors in VLS by quinpirole + SCH 23390 caused no net change in SNpr cell firing (108 ± 7% of baseline rates).
Effects of Selective Activation of Striatal D1 Dopamine Receptors on Behavior and SNpr Cell Firing.
To assess the effects of selective stimulation of striatal D1 receptors on motor and electrophysiological output from the SNpr, we coinfused the D1 agonist SKF 82958 (10 μg/μl/side) and the D2 antagonist YM 09151-2 (2 μg/μl/side) into VLS. Bilateral infusions of this drug combination caused a specific behavioral response, i.e., a modest but highly significant increase in oral movements that was sustained during the 30 min after the infusion (two-way ANOVA: p < 0.0001,F = 18.87, df = 2,195 versus baseline or saline infusion; Fig. 5). The increase in oral movements, although significant, was only about one-third the magnitude of that observed after coinfusions of both the D1and D2 agonists (see below). The behavioral activation was also discrete in that sniffing and locomotor activity were not increased relative to saline or baseline controls.
In parallel electrophysiological studies in which SKF 82958 + YM 09151-2 were coinfused into VLS, 8 of 10 cells tested exhibited changes in firing exceeding ±20% of baseline firing rates (Fig.6). However, of these, five cells exhibited exclusively increases in firing, some to >300% of baseline. Another two cells were exclusively inhibited, and one cell showed biphasic increases and decreases in firing. Two neurons of the 10 evaluated showed no changes (±20% of baseline) in firing. Overall, the average population response of SNpr neurons to selective D1 receptor stimulation by SKF 82958 + YM 09151-2 infusions was a nonsignificant increase in firing to 117 ± 14% of basal firing rates.
Effects of Concurrent Activation of D1 + D2Receptors on Behavior and SNpr Cell Firing.
In both groups of animals that were evaluated for behavioral responses to selective D1 or D2 receptor stimulation, we also examined the effects of concurrent stimulation of both receptors by coinfusions into VLS of quinpirole (30 μg/μl/side) + SKF 82958 (10 μg/μl/side). Bilateral infusions of both the D1 + D2 agonist produced a profound behavioral activation similar to that seen previously with apomorphine, and well in excess of responses to either D1 or D2 receptor activation alone (Figs. 3 and 5). Oral movements, sniffing, and locomotor activity (data not shown) were all significantly increased in both groups of animals given the combined agonist infusions [two-way ANOVA: for oral movements, p < 0.001,F = 174.25, df = 2,195 and p < 0.0001, F = 29.33, df = 2,135; for sniffing,p < 0.0001, F = 117.44, df = 2,195 and p < 0.001, F = 109.23, df = 2,135; and for locomotor activity, p < 0.0001, F = 12.30, df = 2,64 and p< 0.0436, F = 3.68, df = 2,20].
In electrophysiological studies, the changes in firing of SNpr neurons after combined D1 + D2 agonist infusions were extremely variable in both the direction and magnitude. As illustrated in Fig.7, the majority of SNr neurons exhibited marked changes in firing during the 30 min after infusions of SKF 82958 + quinpirole. As was true with apomorphine infusions, most cells (6 of 10) exhibited only increases in firing. Another two cells showed dramatic biphasic increases and decreases in firing exceeding 20% of baseline, and two cells showed no changes in firing (<±20% of baseline). There were no cells that showed an exclusively inhibitory response. Overall, the average population response after bilateral SKF 82958 + quinpirole infusions into VLS was a net increase in SNr firing to 117 ± 12% of baseline rates.
Discussion
The basal ganglia functional model put forth more than a decade ago by Albin et al. (1989) and Gerfen et al. (1990) has provided a valuable framework for understanding how disruptions of striatal dopamine transmission give rise to the motor symptoms of basal ganglia disorders, most notably Parkinson's disease. It is now well accepted that striatal release of dopamine regulates motor function via its influence over striatal efferent pathways that ultimately control output from the SNpr/GPi. Specifically, the model postulates that D1 and D2 receptors are predominantly segregated to striatonigral and striatopallidal neurons, respectively, so that each receptor independently regulates the activity of one striatal efferent pathway. These basic elements of the model give rise to three testable hypotheses that we have attempted to address in these studies. First, selective stimulation of striatal D1 receptors is expected to activate the striatonigral pathway to directly inhibit the firing of SNpr neurons. This response presumably involves D1receptor-mediated potentiation of cortical glutamatergic inputs to striatal neurons viaN-methyl-d-aspartate receptors (Hernández-Lopez et al., 1997; Cepeda et al., 1998). Second, selective stimulation of striatal D2 receptors on striatopallidal neurons should have no direct effect on SNpr neurons, but is expected to indirectly reduce their firing by withdrawal of excitation from the subthalamic nucleus inputs to the SNpr. Finally, if D1 and D2 receptors are segregated on the two striatal efferents, as proposed, then the phenomenon of D1/D2synergism must be mediated by interactions between neurons possessing the different receptors, rather than by interactions between the two receptors on the same striatal neurons.
To test these basic predictions of the model, it is necessary to restrict stimulation to a single dopamine receptor subtype within the striatum, and only the striatum, thus ruling out the use of systemically administered dopamine agonists. We chose an acute means of preventing dopamine's activation of the unintended receptor within the field of the agonist infusion to avoid any disruptions to the circuitry. Finally, to determine whether SNpr output is suppressed when movement is facilitated, we monitored the effects of striatal dopamine receptor stimulation on SNpr cell firing under conditions that were known to elicit motor activation in behaving animals. We chose the VLS striatum as the target for our infusions because bilateral amphetamine infusions at this site are known to cause intense oral movements (Kelly et al., 1988; Delfs and Kelley, 1990; Dickson et al., 1994;Waszczak et al., 2001), a behavior postulated to specifically involve output from the SNpr (Childs and Gale, 1983; DeLong et al., 1983;Redgrave et al., 1984).
The results of our studies corroborate and extend those of our earlier work with bilateral infusions of d-amphetamine into the VLS (Martin et al., 1997; Waszczak et al., 2001). We now conclude that concurrent bilateral stimulation of both D1 and D2 dopamine receptors by the dopamine releaserd-amphetamine, the mixed D1/D2 agonist apomorphine, or coinfusion of a D1 + D2agonist produces an intense motor activation consisting of repetitive mouth movements, tongue protrusions, and vacuous chewing movements. Sniffing and locomotor activity were also increased by each treatment. These behavioral changes were correlated temporally with changes in the activity of neurons in the SNpr. However, the changes in firing were extremely variable in direction and magnitude, and included both dramatic increases and decreases in SNpr unit activity. These fluctuations in firing were particularly pronounced after coinfusions of the D1 agonist SKF 82958 and the D2 agonist quinpirole (Fig. 7), and occurred during the time when behavior was maximally stimulated (Fig. 3 and 5). When the responses of all neurons were averaged, the net response to concurrent D1 and D2receptor stimulation by each drug regimen was an increase in SNpr cell firing (to 117–133% of baseline firing rates). It is possible that the electrophysiological responses we observed might not mimic changes that occur in awake, behaving animals because cortical activation of striatal neurons may be dampened in anesthetized rats. It seems unlikely, however, that consistent inhibitory effects would have occurred even in conscious rats because similarly variable changes in firing and a net increase in SNpr cell activity were also seen in awake, locally anesthetized rats given bilateral infusions of amphetamine into the VLS (Waszczak et al., 2001).
Our electrophysiological results are in conflict with a fundamental prediction of the basal ganglia functional model that stimulation of both striatal D1 and D2 receptors should lead, independently and concurrently, to an inhibition of SNpr output. We are not the first to have observed this discrepancy. Other investigators who have examined changes in SNpr activity during spontaneous movement (Gulley et al., 1999), or in response to unilateral striatal infusions of apomorphine (Murer et al., 1997a,b), amphetamine (Olds, 1988; Timmerman et al., 1998; Gulley et al., 1999), or a combination of SKF 38393 and quinpirole (Murer et al., 1997a) have found a mixed pattern of responses. Although drug doses and striatal infusion sites differed, and each of these studies used unilateral rather than bilateral infusions, the results consistently showed that concurrent stimulation of striatal D1 and D2receptors leads to variable changes in SNpr cell firing. Collectively, these findings and our own reveal that the output signal from the SNpr coincident with movement is normally a complex, highly variable one consisting of increases, decreases, and biphasic changes in activity.
To determine whether the complexity of the electrophysiological response was caused by opposing and competing influences of D1 and D2 receptor mechanisms on striatal output, behavioral and electrophysiological responses were evaluated after coinfusions of selective agonist-antagonist pairs, thereby restricting stimulation to a single dopamine receptor subtype. With the same doses of the D1 and D2 agonist that caused profound behavioral effects when given together, neither agonist alone caused a behavioral response of the magnitude observed with concurrent D1/D2 receptor stimulation. Indeed, selective D2 receptor stimulation actually suppressed all forms of behavior, whereas selective D1 receptor stimulation caused a modest increase in oral movements to roughly one-third of that observed after combined D1/D2 receptor stimulation. Oral movements may therefore be unique among the array of dopaminergic behaviors in that they can be elicited to a modest extent by D1 receptor stimulation alone. These findings are in good agreement with the early literature concerning the behavioral effects of systemically administered D1 and D2receptor-selective agonists. For instance, i.p. administration of the partial D1 agonist SKF 38393 has been shown to cause a mild increase in oral movements (Johansson et al., 1987) similar to that which we observed after striatal coinfusion of SKF 82958 + YM 09151-2. Conversely, i.p. administration of low doses of quinpirole were found to inhibit all movement, including oral movements (Johansson et al., 1987; Delfs and Kelley, 1990; Canales and Iversen, 1998), similar to the suppression of behavior that we observed after coinfusions of quinpirole plus SCH 23390. Numerous studies have corroborated the modest behavioral effects of systemically administered D1- and D2-selective agonists when given alone (for reviews, see Clark and White, 1987;Waddington and Daly, 1993). The additional information contributed by our studies is that these behaviors, or the lack thereof, can be entirely attributable to striatal actions of the drugs, rather than mediated in part by their actions at extrastriatal D1 and D2 receptors.
Consistent with the lack of a behavioral response with selective D2 receptor stimulation, we observed only minor fluctuations in SNpr cell firing and no net change from baseline rates. Conversely, the mild oral behavior seen after selective D1 receptor stimulation correlated with moderate and variable changes in SNpr cell firing, and a slight increase in activity similar to that observed with concurrent D1/D2 receptor stimulation. It is conceivable that the modest effects of SKF 82958 we observed were not entirely due to D1 receptor stimulation because this agonist exhibits only 10-fold D1:D2 receptor selectivity (Murray and Waddington, 1989; Gnanalingham et al., 1995). In fact, SKF 82958 has been shown to inhibit substantia nigra dopamine cell firing, a response attributed to D2 autoreceptor stimulation and reversed by D2 antagonists (Ruskin et al., 1998). It has also been found to inhibit slowly inactivating K+ currents in rat striatal neurons by a non-D1 receptor mechanism (Gabel and Nisenbaum, 1998; Nisenbaum et al., 1998). In our studies, however, it is unlikely that D2 receptor stimulation contributed to the responses observed because such effects should have been prevented by coinfusion of the D2 antagonist YM 09151-2. We cannot rule out the possibility, however, that the agonist and antagonist might have differed in their spread in tissue, thereby exposing some striatal neurons to the agonist without concurrent blockade of the opposing receptor. Similarly, we cannot rule out the possibility that in some animals a small amount of the drug solution might have spread caudally into the adjacent pallidum, a nucleus of the “indirect” pathway.
Previous investigators who have examined the effects of unilateral infusions of selective D1 and D2 receptor agonists on SNpr cell firing have made observations similar to ours. Timmerman et al. (1998) reported that striatal infusions of neither the D1 agonist CY 208243 nor the D2 agonist quinpirole (LY171555) significantly altered the firing rates of SNpr neurons.Akkal et al. (1996) and Murer et al. (1997a) both found that a subset of SNpr neurons did exhibit changes in firing after intrastriatal infusions of SKF 38393 or quinpirole, but the responses consisted of both excitations and inhibitions and appeared to be generally quite modest. The low incidence of responding neurons and the minor changes in firing in these reports may have been due in part to much lower doses of the agonists than we used, and/or to the use of unilateral rather than bilateral infusions. However, as in our studies, infusions of the D1 and D2 agonists concurrently at the same doses given alone invariably caused greater responses than occurred with either drug individually. Moreover, the different magnitude of responses seen with unilateral and bilateral infusions suggests that the contralateral side can play a role in regulating output from the basal ganglia via the SNpr, a point made in our earlier report (Waszczak et al., 2001).
Several interpretations can be drawn from these data. First, stimulation of neither striatal D1 nor D2 receptors leads to a consistent inhibition of SNpr cell firing, as is predicted by the basal ganglia model. Indeed, when behavior is activated, SNpr neurons are more frequently excited than inhibited. Second, the complex and variable effects of concurrent D1/D2 receptor stimulation are not due to separate and competing effects on striatal output mediated by the individual receptors, because their individual effects on SNpr output are also variable, modest, and mildly excitatory. These findings draw into question fundamental predictions of the basal ganglia model, at least in its most simplistic form. Nevertheless, it is important to note that aspects of the model may still be valid under conditions different from those of our study. For instance, it is possible that the ventral lateral striatum (where our infusions were targeted) may differ from the dorsal striatum in the manner by which D1 and D2 receptors regulate striatal output, and in turn SNpr output. It is also conceivable that neurons of the SNpr and GPi, the two output nuclei of the basal ganglia, are not equivalent in their striatal inputs and therefore differ in their responses to striatal dopamine receptor stimulation. Because we did not evaluate GPi neurons, it is unclear whether these neurons were inhibited by our striatal drug infusions.
A third interpretation of our results is that electrophysiological and behavioral output from the basal ganglia requires coactivation of both striatal dopamine receptors for full expression, in accordance with the findings of numerous previous investigators (Arnt et al., 1987; Walters et al., 1987; White et al., 1988; Waddington and Daly, 1993; White and Hu, 1993). The synergism between the receptors draws into question another tenet of the basal ganglia model, i.e., that D1 and D2 receptors are segregated in their expression and independently control the activities of the two striatal efferent populations. For instance, if D1 receptors predominate in regulating the activity of the “direct” striatonigral γ-aminobutyric acidergic pathway, as predicted by the model then our data suggest that D1 receptor activation inhibits striatonigral efferents to a greater extent than it activates them, a conclusion contrary to predictions of the model. Conversely, if D2 receptors exert predominant (inhibitory) control over the activity of striatopallidal neurons, as predicted by the model, then our data imply that striatal D2receptors and the indirect pathway must have little influence over the tonic activity of the SNpr, also contrary to predictions of the model. Indeed, neither the proposed functional roles of striatal D1 and D2 receptors nor the segregated pattern of their expression offers a tenable explanation for our findings, and both appear to be overly simplistic.
A more parsimonious explanation, supported by a growing body of evidence, is that D1 and D2receptors coexist on a substantial proportion of striatonigral and striatopallidal neurons, and interact synergistically on these neurons to generate a complex, highly variable output to SNpr/GPi. Indeed, a significant colocalization of the two receptors on striatal efferents now seems clear. Surmeier et al. (1992, 1993, 1996) showed that both efferent groups respond electrophysiologically to both D1 and D2 receptor agonists, and many contain mRNA for both classes of receptor.Meador-Woodruff et al. (1991) and Lester et al. (1993) found that D1 and D2 mRNAs were coexpressed by 27 to 33% of striatal efferents, whereas Ariano et al. (1992) observed D2 receptor immunoreactivity on a minimum of 60% of striatonigral neurons. More strikingly, Aizman et al. (2000) showed complete colocalization of the two receptors on neurons from embryonic rat striatum. The means of interaction between the coexpressed receptors giving rise to the dramatic and fluctuating changes in striatal output transmitted to the SNpr is less clear. Possible mechanisms include modulation of neuronal excitability by D1/D2 synergistic regulation of Na+ fluxes through tetrodotoxin-sensitive Na+ channels and the Na+/K+ ATPase (Bertorello et al., 1990; Aizman et al., 2000). By whatever the mechanism, our results suggest that stimulation of striatal D1and D2 receptors gives rise to a dynamic and complex signal that is transmitted, in turn, to the thalamus and other premotor nuclei to facilitate movement. It is increasingly apparent that movement is signaled by discrete changes in the firing of subpopulations of SNpr/GPi neurons, rather than by a uniform inhibitory response of the entire population. In view of these findings, the basal ganglia functional model needs to be revised to accommodate the fact that, at least in normal animals with an intact circuitry, dopamine does not produce simple, unidirectional changes in the activities of striatal efferents or their targets in the SNpr/GPi.
Footnotes
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These studies were supported by National Institutes of Neurological Disorders and Stroke Grant NS 23541 to B.L.W. The behavioral studies reported in this article served as the Senior Honors Thesis of Heather Finlay in the Behavioral Neuroscience program at Northeastern University. A preliminary report of this work was published in 1997 in Abstr Soc Neurosci23:190.
- Abbreviations:
- SNpr
- substantia nigra pars reticulata
- VLS
- ventral-lateral striatum
- AMPT
- α-methyl-p-tyrosine
- ANOVA
- analysis of variance
- SKF 82958 ((±)6-chloro-APB hydrobromide)
- (±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzapenine hydrobromide
- YM 09151-2
- cis-N-(1-benzyl-2-methyl-pyrrolidin-3-yl)-5-chloro-2-methoxy-4-methyl-aminobenzamide
- SCH 23390
- R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine
- CY 208243
- (−)-4,6,6a,7,8,12b-hexahydro-7-methyl-indolo[4,3-ab]-phenanthridine
- Received September 27, 2001.
- Accepted December 5, 2001.
- The American Society for Pharmacology and Experimental Therapeutics