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Repeated administration of the D1-dopamine agonist SKF-38393 to adult rats having had dopaminergic neurons destroyed early in development results in an increasing enhancement of the behavioral response to SKF-38393 with each dose until a maximum is reached. This increased sensitivity lasts for at least 6 months. In the present study, this long-lasting change in behavioral responsiveness to repeated treatment with SKF-38393, referred to as D1-dopamine receptor priming, was shown to be dose dependent with smaller doses requiring an increased number of administrations to produce a maximal response when compared to higher doses. In addition, priming occurred equally well when treatment intervals ranged from 1 day to 14 days. These latter data reinforced the view that activation of D1-dopamine receptors results in a prolonged change in neural function. In subsequent experiments, D1-dopamine receptor priming was blocked by pretreatment with the NMDA-receptor antagonist MK-801. This antagonism of priming could not be attributed to a blockade of D1-dopamine receptors by MK-801 or to the induction of interfering behaviors. Because an NMDA antagonist interfered with D1-receptor priming as it does with other long-term neural messages, a common requirement for these diverse forms of neuronal plasticity appears to involve activation of the NMDA receptor. This functional link between NMDA receptors and dopaminergic function and its relationship to neuronal plasticity could have relevance to the biochemical mechanisms involved in learning and to symptoms in central disorders during development that worsen over time, particularly those proposed to involve malfunctioning dopaminergic mechanisms.
Recently, our laboratory has reported an increasing functional sensitivity of D1-dopamine receptors upon repeated stimulation with the D1-dopamine receptor agonist SKF-38393 in rats lesioned as neonates with 6-hydroxydopamine (6-OHDA)6,14. The behavioral activation that follows SKF-38393 administration to neonatal 6-OHDA-lesioned rats becomes greater with each dose and reaches a maximum intensity after 3–5 weekly treatments6. This change in sensitivity is still present 6 months after the last exposure to the D1-dopamine agonist and is not dependent on cues associated with drug administration14 or to a change in dopamine receptor number or affinity7. This long-lasting change in the responsiveness to D1-dopamine receptor activation is referred to as priming6,14. There are two aspects of priming that have not been defined. These are the dose and the time parameters that will result in priming of the D1-dopamine receptor response. Therefore, one purpose of the present manuscript was to define these parameters for priming.
D1-dopamine receptor priming shares many properties with the enduring changes in synaptic sensitivity resulting from transient events which are thought to be the biological substrate for learning1–3,23. Recent work has demonstrated that spatial learning20, as well as models of long-term neural plasticity such as long-term potentiation15 observed upon stimulating pathways to the hippocampus12,13,16,18,20 and kindling produced by multiple stimulations of the amygdala4,10,19 can be blocked by N-methyl-d-aspartate (NMDA) receptor antagonists. These findings intimate that activation of the NMDA receptor, a glutamate receptor subtype, may be necessary for some types of neuronal plasticity. The similarity between the parameters controlling D1-dopamine receptor priming and those controlling other forms of neuronal plasticity suggested that these processes may share a common mechanism. Consequently, another set of investigations was undertaken to determine if blockade of NMDA receptors by MK-801 would alter priming of D1-dopamine receptors associated with repeated administration of SKF-38393.
Pregnant Sprague-Dawley rats obtained from Charles River Laboratories Inc. (Raleigh, NC) were individually housed with Wayne Lab Blox laboratory chow and water available ad libitum until delivery of neonatal rats. At day 3 after delivery, each neonate was injected intracisternally under ether anesthesia with 100 µg (free base) of 6-OHDA hydrobromide (Regis Chemical, Chicago, IL) dissolved in 10 µl of saline8,22. Neonates were then returned to their mothers with litter size limited to 10. Some neonates were pretreated with 20 mg/kg desmethylimipramine (Regis Chemical) 1 h prior to 6-OHDA injections to restrict the lesion to dopamine-containing neurons22. Rats were weaned at day 30. Both male and female rats were used to evaluate the dose and time parameters in relationship to priming of D1-dopamine receptors. All other experiments used female rats. Because Criswell et al.14 did not find differences in priming between rats having only brain dopamine or both brain catecholamines reduced, rats from both of these treatment groups were used for the experiments in this investigation.
Locomotor behavior was measured by placing rats in a circular photocell activity monitor17. Six photo sensors were placed about the periphery and counts from each sensor were collected by a microcomputer and recorded at 10 min intervals for 150 min. Rats were habituated to the activity chamber for 1 h before administration of the dopamine agonist.
In one experiment, various behaviors were quantified by observing whether a behavior was seen during a 1-min period at 10 min intervals5. Each 1-min interval was divided into four 15-s units to allow calculation of the percentage of the 15-s intervals a behavior was observed. This score was totalled for each behavior for the twelve 1-min observation periods.
Testing of rats was begun at 40–50 days of age. For the experiments dealing with time of administration, animals were given 3 mg/kg of SKF-38393, i.p., repeatedly at 1, 2, 7 or 14 day intervals for 4 doses. In order to evaluate dose relationships, rats were given a total dose of 9 mg/kg of SKF-38393 divided such that one group received 9 mg/kg of SKF-38393 as a single dose, another group received three 3.0 mg/kg doses and a final group received six 1.5 mg/kg doses of SKF-38393. When more than one dose was necessary, the treatment interval was 4 days. Once these latter groups had been treated with an accumulated dose of 9 mg/kg, 3 additional doses of 3 mg/kg SKF-38393 were administered at 4 day intervals and activity was measured after each drug dose.
In order to evaluate the effect of MK-801 on the priming of the SKF-38393 induced locomotor response, rats were given 0.1, 0.3 and 1 mg/kg of MK-801 30 min prior to the injection of SKF-38393 (3 mg/kg). Another group of rats received saline rather than MK-801 with the SKF-38393. These drug combinations were given for a total of 4 treatments at weekly intervals. Once this regimen was completed, all rats were given 4 additional SKF-38393 (3 mg/kg) treatments to evaluate whether rats showing blockade of priming after the MK-801/SKF-38393 combination could now be primed. In addition, a group of rats was treated with MK-801 (0.3 mg/kg) plus SKF-38393 (3 mg/kg) and behavioral responses were recorded for 2 h after the administration of the D1-dopamine agonist as previously described5,14. This combination was given for 3 cycles at 3 day intervals, at which time SKF-38393 was administered alone and behavior was measured. Two additional doses of SKF-38393 were administered to establish which of the rats met our criteria for inclusion in the final statistical evaluation (see below). The behavioral responses for the MK-801/SKF-38393 treatment group were compared to the behavioral responses obtained after SKF-38393 administration to a group receiving their first dose and a group of neonatal-6-OHDA-lesioned rats already primed with SKF-38393. Another set of rats showing maximal behavioral responses to SKF-38393 were treated with 0.1, 0.3 or 1.0 mg/kg of MK-801 in order to see if the MK-801 blocked or influenced the SKF-38393 activity response. Additionally, the effect of 0.3 mg/kg MK-801 on the response to quinpirole was measured in rats showing a supersensitive response to this D2-dopamine agonist.
SKF-38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben-zepine-7,8-diol (Smith Kline and French Laboratories, Philadelphia, PA); MK-801 ((+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine; Merck, Rahway, NJ); quinpirole (Lilly Research Laboratory, Indianapolis, IN); and Ro 4-4602 (Hoffmann-La Roche, Nutley, NJ); were dissolved in saline and administered intraperitoneally. l-DOPA (Hoffmann-La Roche, Nutley, NJ) was suspended in 0.5% carboxymethylcellulose and injected i.p.
Locomotor activity was analyzed by summing the activity counts for 150 min and performing a repeated measures ANOVA on the totals. Individual comparisons of the experimental groups to controls were made with the Dunnett test24. Since previous data has documented that only rats which reach an asymptotic response to SKF-38393 totaling at least 10,000 counts/150 min have dopamine depletions of at least 90%5,7, only those rats meeting this behavioral criteria were used for analysis. The ratio of rats omitted to those retained was determined and the ratio for the groups that received only SKF-38393 was compared to that for the groups that were dosed with MK-801 + SKF-38393 using the test for the difference between proportions9. Behavioral series for groups were compared using analysis of variance. For each analysis that yielded a significant F ratio, a Tukey HSD was applied to allow mean comparison of each behavior24.
Increasing locomotor activity as a function of repeated administration of the D1-dopamine agonist SKF-38393 at varying time intervals to adult rats lesioned with 6-OHDA as neonates is shown in Fig. 1. There was no difference in the rate of D1-dopamine receptor priming when the 1, 2, 7 and 14 day injection intervals were compared. These results indicated that the neural signal from a single injection can be observed when animals are challenged at 1 day intervals and that the consequence of the single treatment will last for at least 14 days.
Since a significant effect on activity can be observed after 3 doses of 3 mg/kg of SKF-38393, doses of 1.5, 3 or 9 mg/kg of SKF-38393 to accumulate a total dose of 9 mg/kg were administered at 4 day intervals except for 9 mg/kg where only a single dose was injected prior to additional challenges. The effects of increasing doses of SKF-38393 on the D1-dopamine receptor response with each dose are shown in Table I. The response to the first dose of SKF-38393 was dose dependent, but even the 9 mg/kg dose did not produce a response as great as that seen after multiple doses of 1.5 or 3.0 mg/kg (Fig. 2). It can also be seen that one dose of 9 mg/kg produced a greater increment in activity following a second dose of 3 mg/kg of SKF-38393 than did a single 3 mg/kg dose (Fig. 2; Table I). However, following 3 doses of 3 mg/kg or 6 doses of 1.5 mg/kg of SKF-38393 (i.e. for a total cumulative dose of 9 mg/kg) the difference between groups disappeared when given an additional challenge dose of 3 mg/kg (see after 9 mg/kg, Fig. 2). No further significant increase occurred following 3 additional doses of 3 mg/kg (see asymptote, Fig. 2).
Documentation of the long-term consequences of repeated doses of SKF-38393 led us to speculate that this priming phenomenon may share characteristics with other long-term adaptive changes. Fig. 3 shows that 0.3 mg/kg of MK-801 administered 30 min before each of 4 doses of SKF-38393 (3 mg/kg, i.p.) blocked the increasing behavioral activation usually produced by this compound (see Fig. 1 for increasing activity with each dose of SKF-38393 in the absence of MK-801). The fifth dose of SKF-38393 (S5; Fig. 3) administered without MK-801 likewise produced no major increase in activity, indicating that D1-dopamine receptor priming of this behavioral response was blocked. As shown in Fig. 4B, higher and lower doses of MK-801 (1.0 and 0.1 mg/kg) also prevented the expected response to a fifth dose of SKF-38393. The activity response to SKF-38393 immediately following the MK-801/SKF-38393 treatment (i.e. fifth dose) did not differ dramatically from that seen when a first dose of SKF-38393 was administered to naive rats (Fig. 4A). These observations indicated that D1-dopamine receptor priming did not occur in the presence of MK-801.
Following the observation that MK-801 would antagonize the consequence of repeated administration of SKF-38393 to neonatal 6-OHDA-lesioned rats, it was then asked whether the animals that received the MK-801/SKF-38393 treatment would show D1-dopamine receptor priming if they then received an additional series of weekly injections of SKF-38393 alone. Responses to these additional doses of SKF-38393 in the absence of 0.3 mg/kg MK-801 are presented in Fig. 3 (e.g. S5–S9). It is apparent that by the third additional dose of SKF-38393 (3 mg/kg) in the absence of MK-801 (i.e. S7; Fig. 3) SKF-38393 produced a significant increase in activity compared to the first dose after MK-801 treatment (i.e. S5; Fig. 3). This procedure of administering SKF-38393 without MK-801 was performed for the two other groups of rats previously dosed with the MK-801 (0.1 and 1.0 mg/kg)/SKF-38393 combination. Both groups demonstrated a significant increase in activity after 3–5 additional doses of SKF-38393, indicating that D1-dopamine receptor responses could be primed in these rats (see legend Fig. 3).
Since some rats could not be retained for analysis because they did not reach our behavioral criteria for supersensitivity after repeated administration of SKF-38393, the ratio of rats omitted to those retained was evaluated for the saline- and MK-801-pretreated rats. Six of 8 saline-treated rats subsequently primed with repeated SKF-38393 treatment, while the ratio for rats showing priming to those that did not when pretreated with 0.1, 0.3 and 1 mg/kg of MK-801 were 5 of 7, 7 of 8, and 4 of 5, respectively. None of the MK-801/SKF-38393 treated groups differed significantly from saline/SKF-38393 treated control group (P > 0.1).
An additional experiment was performed to determine if behaviors other than locomotion were being primed during repeated administration of the SKF-38393/MK-801 combination. As shown in Table II, the behavioral responses to SKF-38393 after the 3 MK-801/SKF-38393 treatments were similar to those observed after the first dose of SKF-38393 administered in the absence of MK-801 and were less than seen when SKF-38393 was administered alone to primed rats. This finding indicated that the priming of all behaviors induced by SKF-38393 repeat injection was being blocked when coadministered with MK-801.
Because the D1-dopamine receptor antagonist SCH-23390 blocks both priming and the behavioral effects produced by SKF-3839314, studies were undertaken to see if the blockade of priming by MK-801 might be due to an antagonism of SKF-38393-induced behavioral responses. This possibility was examined by using primed rats that had reached maximal supersensitivity to SKF-38393. As shown in Table III, 0.3 and 1.0 mg/kg of MK-801 produced ataxia and retarded SKF-38393-induced activity, whereas 0.1 mg/kg of MK-801 did not antagonize and actually increased the activity response following SKF-38393 administration. The reduced locomotor response to SKF-38393 after 0.3 mg/kg could not be interpreted as a specific action on D1-dopamine receptor because the increased locomotion induced by the D2-dopamine agonist quinpirole was also reduced by this dose of MK-801 (Table III). In addition, it should be noted that the activity response to the first dose of SKF-38393 in the presence of 0.1 mg/kg of MK-801 was significantly increased (Fig. 4A). This latter effect is due to a stimulant action of this dose of MK-801 (Criswell and Breese, unpublished data). In spite of the elevated activity when SKF-38393 and 0.1 mg/kg of MK-801 were co-administered, the response to SKF-38393 alone following the repeated administration of the combination was significantly lower than that for the group treated with saline rather than MK-801 (Fig. 4B).
Previous work from this laboratory has demonstrated that repeated administration of the D1-dopamine agonist SKF-38393 at weekly intervals results in an increasing response with each dose — a phenomenon referred to as priming6,14. In the present investigation, additional information about this phenomenon was sought. It was demonstrated that priming could be exhibited if doses of the agonist were administered at one to 14 day intervals. The fact that priming could be observed when doses of SKF-38393 were administered at 14 day intervals clearly documents the long term contribution of each dose to the priming process. Further, the effectiveness of the accumulated action of SKF-38393 indicates that decay of the signal must be minimal. Previous data have demonstrated that once the maximal response is reached, the primed response persists for at least 6 months14. The present investigation also documented that priming of the D1-dopamine receptor response occurred when the SKF-38393 was administered at one-day intervals. This observation provides additional evidence that this enhanced sensitivity with repeated dosing of SKF-38393 differs from such a change in sensitivity observed after repeated administration of d-amphetamine. In contrast to SKF-38393, weekly inter-injection intervals of d-amphetamine produce greater behavioral sensitization than does daily administration21.
The D1-dopamine antagonist SCH-23390 blocks priming of the D1-dopamine receptor response, whereas the D2-dopamine receptor antagonist haloperidol blocks neither the behavioral effects of SKF-38393 nor the subsequent priming of D1-dopamine receptor supersensitivity14. This observation points out that not all dopamine antagonists block priming and emphasizes the importance of D1-dopamine receptor activation to this phenomenon. In the present study, MK-801 was found to antagonize priming. However, there are several observations suggesting that the action of MK-801 is not due to a direct action on dopamine receptors. For example, MK-801 does not displace [3H]spiperone or [3H]apomorphine from striatal tissue11. The lowest dose of MK-801 employed in the present study blocked D1-dopamine receptor priming without diminishing the behavioral activating effect of SKF-38393, indicating that a blockade of the behavioral activating effect of SKF-38393 is not necessary for the prevention of priming. The fact that a higher dose of MK-801 antagonized the response of both SKF-38393 and quinpirole and caused ataxia suggests that the inhibition of SKF-38393-induced locomotion is the result of the motor incoordination produced by this dose of MK-801 and not a specific action on D1-dopamine receptor function. Another potential explanation for the data demonstrating an MK-801 antagonism of priming of SKF-38393-induced locomotion was that the treatment with MK-801 enhanced SKF-38393-induced priming of other behaviors that would be incompatible with the occurrence of activity (i.e. behavioral competition). An experiment to test this view indicated that specific behaviors induced by SKF-38393 were not being enhanced or preferentially primed when SKF-38393 was given in combination with MK-801. Thus, neither a blockade of D1-dopamine receptor function nor the production (i.e. priming) of interfering behaviors with repeated MK-801/SKF-38393 treatment can explain the reduced activity found for the first dose of SKF-38393 given after the MK-801/SKF-38393 combination.
As described in the introductory remarks, several examples of long-term change in neuronal sensitivity have been postulated to depend upon activation of the NMDA receptor. The ability of the NMDA-receptor antagonist MK-80125,26 to block development of supersensitivity to repeated D1-dopamine receptor agonist administration suggests that activation of the NMDA receptor is necessary for the occurrence of D1-dopamine receptor priming. Since previous work14 has shown that the D1-dopamine antagonist SCH-23390 blocks priming, the concomitant activation of both D1-dopamine receptors and NMDA receptors appear necessary for priming to occur. The indication that NMDA receptor activation is also a necessary prerequisite for other responses that convey a long-term neural message, such as learning20 and long-term potentiation12,13,16,18, suggests that these phenomena and D1-dopamine receptor priming may share a common biochemical mechanism.
Because the increasing behavioral sensitivity to SKF-38393 induced locomotor activation (i.e. priming) in neonatal 6-OHDA-lesioned rats is associated with the activation of a specific neural receptor6,14 as well as a defined anatomical locus14, priming can be easily controlled and investigated. These characteristics and the finding that priming shares a relationship with other types of long-lasting neural plasticity through an involvement of NMDA receptors may facilitate efforts to delineate the nature of the cellular biochemical change(s) that underlie such long-term neural events. Obviously, the most important of these processes is learning. It should be understood that this conclusion is based upon the assumption that the biochemical mechanism by which neurons induce a long lasting neural plasticity is common for differing types of neurons. Regardless, the data presented here concerning the enhanced behavioral sensitivity observed with repeated stimulation of D1-dopamine receptors provide further functional evidence for the view that NMDA receptors may be involved in critical biochemical processes responsible for long-term plasticity changes in the CNS.
The authors acknowledge the excellent assistance of Ms. Doris Lee in the typing of this manuscript. Work in this manuscript is supported by USPHS Grants NS-21345, HD-23042 and HD-03110.