Repeated cocaine administration results in supersensitivity of striatal D-2 dopamine autoreceptors to pergolide

Life Sciences, Vol. 42, pp. 255-262 Printed in the U.S.A.

Pergamon Journals

REPEATED COCAINE ADMINISTRATION

RESULTS IN SUPERSENSITIVITY OF STRIATAL D-2 DOPAMINE AUTORECEPTORS TO PERGOLIDE

Linda P. Dwoskin, Joanna Peris, Robert P. Yasuda, Katie Philpott and Nancy R. Zahniser

Department of Pharmacology University of Colorado Health Sciences Center Denver, Colorado 80262 (Received in f i n a l ~ r m N o v e m b e r

16, 1987)

Summary

Groups of rats administered cocaine-HC1 (10 mg/kg, i.p.) or saline either acutely or once daily for 8 or 14 days were killed 24 hrs after the last dose. In striatal slices prelabelled with [3H]DA, modulation of [3H]-overflow by pergollde was used to measure D-2 autoreceptor activity. Compared to the contemporaneous control group pergolide produced a greater inhibition only in striatal slices from rats treated repeatedly with cocaine. In radlollgand binding studies using striatal membranes from control rats, pergolide had a 500-fold greater affinity for the D-2, as opposed to the D-I, dopamine (DA) receptor subtype. These results indicate that repeated treatment with cocaine produces supersensitive strlatal D-2 release-modulating autoreceptors consistent with a compensatory change to diminish the effect of elevated synaptic concentrations of DA produced by cocaine. In contrast, supersensitivity of D-2 receptors was not detected in [3H]splperone binding assays.

The increasing recreational use of cocaine as a euphorlant and drug of abuse has aroused interest in understanding the neurochemleal mechanisms and functional consequences of chronic cocaine administration. Acutely, cocaine blocks neuronal DA uptake, prolongs the presence of DA in the synapse and thus acts indirectly as a DA agonist (I). After chronic administration of cocaine, tolerance develops to the cocaine-lnduced blockade of DA uptake in strlatum (2). Increased synaptic concentrations of DA produced by cocaine may interact with several subtypes of DA receptors. In the strlatum, binding assays measure mainly postsynaptic D-2 receptors (3). Following chronic administration of cocaine, the number of D-2 receptoPs measured with binding assays has been reported to increase (4,5) and more recently to decrease (6) in striatum. Presynaptic D-2 receptors (7), termed autoreceptors (8), have been suggested to be impulse-regulating, synthesls-modulatlng or release-modulating (9). D-2 release-modulating autoreceptors appear to be very sensitive to the concentration of synaptic DA (10,11). However, regulation of these receptors has not been examined following repeated administration of cocaine. In response to repeated cocaine, D-2 autoreceptors may become subsensitive leading to higher synaptic concentrations of DA and an enhanced activation of postsynaptlc DA receptors. Alternatively, these

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0024-3205 88 $3.00 + .00 (c) 1988 Pergamon Journals Ltd.

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receptors may become supersensitive and more efficiently inhibit release, thereby, reducing elevated synaptic concentrations of DA. These hypotheses were tested in the present study by determining the ability of the D-2 receptor agonist pergolide to inhibit DA release in striatal slices from rats repeatedly administered cocaine. Although several laboratories ( 1 1 - 1 3 ) u s e pergolide as a selective D-2 agonist, its selectivity has been questioned (14-16). In order to reconcile this dispute, the selectivity was determined by comparing the ability of pergolide to compete with selective D-2 ([3H]spiperone, ref. 17) and D-I ([3H]SCH 23390, ref. 18) receptor antagonists for their respective binding sites on rat striatal membranes. Additionally, the binding characteristics of striatal D-2 receptors were examined following repeated cocaine administration.

Methods

Animals and treatment schedule: Male Sprague-Dawley rats (150-250 g) were obtained from Sasco (Omaha, NE) and housed in a controlled environment under a 12-hr light cycle. Water and food were freely available. Rats were administered cocaine-HCl (10 mg/kg, i.p.) or saline either acutely or once daily for 8 or 14 consecutive days. Twenty-four hours after the last dose, rats were sacrificed. Strlata were dissected and used in release and binding assays.

[~H]DA release assay: This assay has been described recently (11). Briefly, seven coronal striatal slices (400 um thick) were incubated for 30 min in Krebs' buffer (118 mM NaC1, 4.7 mM KCI, 11.1 mM glucose, 25 mM NaHC03, 1.2 mM MgCI 2, I mM NaH2PO 4, 1.3 mM CaCI2, 0.11 mM L-ascorbic acid, 4 UM disodium EDTA, saturated with 95% 02/5% C02; pH 7.4) in a metabolic shaker at 34°C. Subsequently, slices were incubated for 25 min in 3 ml of fresh buffer containing 0.1 uM [3H]DA. Each slice was placed in a glass chamber containing two platinum electrodes and was superfused at a rate of I ml/min with Krebs' buffer. Sample collection (5-min/sample) began after 50 min of superfusion. The first electrical field stimulation (S I) was applied 70 min after the beginning of the superfusion and the second (S 2) was applied 60 min after S I. A Grass stimulatior (Model SD9) delivered trains of unipolar, rectangular pulses (I Hz, 20 mA, 2 msec duration for I min; 60 pulses). Pergolide was included in the buffer 20 min prior to S 2 and until the end of the experiment. Duplicate control chambers were exposed to buffer only. Radioactivity in Superfusate samples and solubilized tissue was determined by liquid scintillation counting. Radioactivity in superfusate represents a combination of [3H]DA and [3H]-metabolites (19). Fractional release of radioactivity was determined for each sample (i.e., the amount of tritium in each sample was divided by the total tritium in the tissue at the time of collection). Basal outflow was the average of the two samples just before S I and S 2. Stimulation-evoked increase in radioactivity above basal is termed "overflow". The effect of pergolide was assessed by comparing the overflow during S I in the absence of pergolide to the overflow during S2 in its presence. The overflow ratio ($2/S I) was calculated for control and pergolide-treated slices in each experiment.

[_~H]Spiperone and [~H]SCH 23390 binding assays: The method used has been described (17). Briefly, striatal membranes were isolated by homogenization with a Brinkmann Polytron in 500 volumes of 20 mM HEPES containing 154 mM NaC1 (pH 7.5; tissue buffer) and by centrifugation at 20,000 x ~ for 10 min at 4°C. For the pergolide competition curves with either [SH]spiperone or

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[3H]SCH 23390, the tissue buffer was 50 mM Tris HC1, 10 mM MgSO4, 2 mM EDTA and 154 mM NaCI (pH 7.5). Assays were initiated by adding 1850 ul of resuspended membranes (0.05 mg protein) in tissue buffer to 150 ul of I mM HCI containing 40 nM ketanserin ([3H]spiperone binding assays only, to preclude binding to S 2 serotonin receptors, ref. 20), pergolide (15 concentrations ranging from 0.1 nM - 31.5 uM, competition curves only) and radiollgand (nine concentrations from 10-1000 pM for saturation curves and 200 pM for competition curves). Assays were incubated at 37°C for 60 min, terminated by adding 10 ml of cold 10 mM Tris buffer containing 154 mM NaCI (pH 7.5), filtered through glass-fiber filters to separate bound from free radioligand, and finally washed with 10 ml of buffer. Radioactivity was determined by liquid scintillation counting. The nonllnear, least-squares curve-fitting program LIGAND (21) defined nonspecific binding (22) and determined equilibrium dissociation constants (Kd or Ki values) and maximum number of binding sites (Bmax). In separate experiments, Kd and Bmax values for specific [3H]splperone binding to striatal membranes were determined to be the same whether nonspecific binding was measured experimentally in the presence of S-sulpirlde (10 uM) or was fit mathematically by LIGAND (data not shown).

Statistics: Two-way ANOVAs (cocaine x treatment duration) determined if cocaine treatment altered outflow or overflow. Three-way ANOVAs (cocaine x treatment duration x pergolide concentration) with repeated measures on one factor (pergolide concentration) determined if cocaine treatment altered the effect of pergolide. Cocaine-lnduced differences in D-2 receptor binding parameters were analyzed using two-way ANOVAs (cocaine x treatment duration). Duncan's New Multiple Range test determined significant differences between pairs of mean values. Data were analyzed using two-tailed comparisons.

Drugs: [3H]DA (dihydroxyphenyethylamine-3,4-[7-3H(N)]; 29-31 Ci/mmol) and [3H]spiperone (spiperone, [benzene ring-3H-]~ 24 Ci/mmol) were obtained from New England Nuclear (Boston, MA). [SH]SCH 23390 was a gift from Dr. Richard Mailman, University of North Carolina. Pergollde and ketanserin were kindly provided by Lilly Research Laboratories (Indianapolis, IN) and Janssen Pharmaceutlca (Beerse, Belgium), respectively. Cocaine HCI was purchased from Mallinckrodt, Inc. (St. Louis, MO).

Results

Competition curves using [3H]spiperone and [3H]SCH 23390 were used to evaluate the ability of pergollde to interact with D-2 and D-I DA receptor subtypes, respectively. Pergolide was observed to have a 500-fold greater affinity for the D-2 DA receptor (Figure I). Therefore, low nanomolar concentrations of pergolide interact selectively with D-2 receptors and were used to evaluate alterations in DA autoreceptor activity produced by repeated cocaine administration.

In vivo administration of cocaine either acutely or repeatedly did not alter basal outflow (1.8 + .10) from the slices. Cocaine treatment also did not alter overflow (Table-- I). Overflow was greater after 8 injections of either saline or cocaine, than after either I or 14 injections, as indicated by the main effect of treatment duration (F(2,45)= 8.45, P<.O05). The interaction between treatment and treatment duration was not significant. Therefore, the increase in overflow at the 8-day time point is not related to

258

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Cocaine Regulates D-2 DA Autoreceptors

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Pergolide Exhibits a 500-Fold Greater Selectivity for D-2, Rather than D-I, DA Receptors in Strlatal Membranes from Control Rats. The selectivity of pergolide for the D-2 and D-I receptors was determined b[ comparing its ability to compete with binding of [3H]spiperone or [3H]SCH 23390, respectively. Ki values were determined using LIGAND. Values shown are means + S.E.M. for N=3 independent determinations.

TABLE I Stlmulatlon-Evoked [3H]Overflow and Striatal D-2 Receptor Binding Parameters Are Not Changed After Either Acute, 8 or 14 Days of Cocaine Treatment In Vivo.

Acute Saline SI

8 Day Treatment Saline Cocaine

Cocaine

1.76 (7) + .192

1.77 (6) + .135

2.50 (7) + .255

Kd value (pM)

18 (4) + 1.5

20 (8) + 5.3

28 (8) + 2.6

26 (8) + 3.0

30 (4) + 1.3

Bmax (fmol/mg protein)

540 (4) + 48

480 (8) + 49

450 (8) + 25

420 (8) + 18

520 (4) + 42

Data are expressed as mean + S.E.M.

2.30 (10) + .126

14 Day Treatment Saline Cocaine

Numbers

1.97 (11) + .125

1.87 (10) + .133

in parenthesis are N.

29 (4) + 4.5 490 (4) + 22

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Cocaine Regulates D-2 DA Autoreceptors

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FIG. 2 Pergolide-Induced Inhibition of Stimulation-Evoked Overflow from Rat Striatal Slices Is Enhanced Following Repeated Cocaine Administration. Pergolide-induced inhibition of overflow ($2/S I) is presented as a percentage of the overflow evoked from control slices within the same experiment exposed to buffer only. Values shown are means + S.E.M. for N= 6-10 animals for each point. ~ SALINE; ~ COCAINE; * P<0.05.

cocaine treatment and is not directly related to the number of injections. Variablility in overflow between experiments dictated that comparisons between cocaine and saline treatment be restricted to within-experlment comparisons.

Pergolide-induced inhibition of overflow from striatal slices was greater in rats repeatedly treated with cocaine compared to the contemporaneous control group (Figure 2). Main effects of treatment, treatment duration, and dose of pergolide (F(1,45)= 12.9, P<.005; F(2,45)= 6.05, P<.O05; F(1,45)= 755, P<.001; respectively) were significant. Interactions between factors were not significant. After the acute injection of cocaine, the effect of pergolide was not significantly different from that after acute saline. In contrast, after 8 cocaine injections inhibition of overflow produced by pergolide (0.5 and 10 nM) was greater than after 8 saline injections. Similarly, the effect of pergolide (0.5 nM) was greater after 14 cocaine injections than after the same number of saline injections. Although there was a trend for pergolide (10 nM) to be more effective in slices from rats treated with 14 cocaine injections, the difference was not significant. This may be due to the nearly maximal inhibition produced by this concentration of pergolide in the control slices (i.e. ceiling effect). It should be noted that pergolide was slightly less effective after 8 saline injections than after either I or 14 injections of saline. The latter finding may be related to the greater absolute amount

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of overflow observed after 8 injections of saline (Table I), and therefore, to a greater competition between pergolide and DA for the autoreceptor.

In contrast to the functional studies of D-2 autoreceptor activity, neither acute nor repeated treatment with cocaine altered the Kd or Bmax values for [3H]spiperone binding to D-2 receptors on striatal membranes (Table I). The main effects of treatment and of duration, as well as the interaction of treatment x duration, for Kd and Bmax values were not significant. Additionally, as tested only in the 14-day treatment group, the ability of pergolide to inhibit [3H]spiperone binding was not different in control and treated rats (Ki values: saline 6.3 + 1.5 nM, N=4; Cocaine 6.1 + .32 riM, N=4).

Discussion

Although pergolide has been used as a selective D-2 DA receptor agonist, its selectivity has been questioned by functional studies (14-16). Pergolide has been reported to stimulate adenylate cyclase activity, via activation of D-I DA receptors (14). However, micromolar concentrations of pergolide are necessary for this action. Pergolide has been suggested to act as a nonselective D-I/D-2 receptor agonist in behavioral (15) and electrophysiological (16) studies. In the these studies pergolide was admlnisterd in vivo and could have been present in relatively high concentrations at the receptors. An alternative interpretation is that pergolide interacts with D-2 receptors selectively and that in vivo activation of D-2 receptors subsequently influence D-I receptors to affect the functions measured. We attempted to settle this dispute concerning the selectivity of pergolide by performing competition binding assays using selective D-2 and D-I receptor antagonists, [3H]spiperone and [3H]SCH 23390, respectively. The results demonstrated that pergolide was 50D-fold selective for the D-2 receptor subtype. Therefore, low nanomolar concentrations of pergolide, which do not interact with D-I receptors, were used to evaluate alterations in D-2 autoreceptor activity produced by repeated cocaine administration. Pergolide was found to inhibit overflow to a greater extent in striatal slices from rats repeatedly administered cocaine compared to their contemporaneous control groups injected with saline. Acute cocaine treatment did not augment pergolide-induced inhibition of overflow. These results suggest that the nigrostriatal dopaminergic system may compensate for repeated stimulation induced by cocaine by increasing the sensitivity of inhibitory D-2 autoreceptors, thereby decreasing the elevated synaptic concentrations of DA produced by cocaine. Whether this change is a reflection of an increased number of receptors, an increased receptor affinity or an increased coupling of the autoreceptor with its transduction mechanism remains to be determined.

These results cannot be explained by the presence of cocaine in the tissue (ti/2= 20 min, ref. 23) because experiments were performed 24 hr after the laSt cocaine injection. Furthermore, if cocaine had been present in the tissue, then overflow would be expected to be greater in the cocaine-treated than in the control rats (11). This was not the case (Table I). Moreover, in vitro pergolide is less effective at inhibiting overflow, when the synaptic concentration of DA is high, as is the case in the presence of an uptake blocker such as cocaine (10,11).

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26]

After repeated cocaine treatment, overflow was not lower than in the contemporaneous controls (Table I), as would be predicted if the autoreceptors were supersensitive to endogenously released DA. If after repeated cocaine treatment overflow had been reduced, then the increased effectiveness of pergolide could have been due to a lower concentration of released DA in the synapse and not to supersensitive DA autoreceptors. The fact that in the present study there is an increased effectiveness of pergolide without changes in overflow after repeated cocaine treatment indicates that D-2 autoreceptors are actually supersensitive to pergolide.

Whereas convincing evidence exists regarding the development of supersensitive pre and postsynaptic DA receptors as a result of chronic administration of the DA receptor antagonist haloperidol (24), chronic treatment with DA agonists appears to be more complex (for review, 25, 26). Results from several experimental approaches indicate that DA autoreceptors become subsensitive in response to direct and indirect agonists. Binding assays have been used (27); however, in striatum these assays do not measure autoreceptors but rather reflect postsynaptic receptors (3). Indirect measures of autoreceptor function have also been used (28); but the relationship between the indirect measures employed and the autoreceptor(s) involved is not clear. Impulse-regulating DA autoreceptors on neurons in the zona compacta of the substantia nigra (29) and on neurons in the ventral tegmental area projecting to the nucleus accumbens (30) become subsensitive following repeated administration of amphetamine and cocaine, respectively. Therefore, it appears that impulse-regulating and release-modulating DA autoreceptors may be regulated differently in response to chronic administration of indirect DA agonists. However, generalizations obtained from experiments using amphetamine and cocaine must be made with caution. Moreover, the effect of repeated cocaine treatment on impulse-regulating DA autoreceptors in the substantia nigra has not as yet been investigated.

In the present study, direct binding of [3H]spiperone to striatal D-2 DA receptors and its inhibition by pergolide were not changed by the repeated cocaine administration. These data support the idea that [3H]splperone binding to D-2 receptors in striatum does not reflect D-2 autoreceptors modulating DA release. Our results from these binding studies are in contrast to a recent report (6) of decreased D-2 receptor number in striatum after repeated cocaine. The discrepancy may be explained in that in the latter study the rats were sacrificed 20 min after the last dose of cocaine, when cocaine would be present and synaptic concentrations of DA would be maximal. In the present study, the cocaine-induced increase in synaptic DA may not have been great enough or for a long enough period of time to result in regulation of these postsynaptic receptors. Furthermore, our results are in accord with our observation that striatal DA levels were not depleted in the rats repeatedly administered cocaine (data not shown). Cocaine-induced DA depletion would be expected to result in an upregulation of postsynaptic DA receptors (31). In conclusion, repeated treatment with cocaine produces supersensitive striatal D-2 release-modulating autoreceptors to pergolide consistent with a compensatory change to diminish elevated synaptic concentrations of DA produced by cocaine.

Acknowledgement This work was supported by U.S. Public Health Service Grants NS 09199 (NRZ), AM 07391 (LPD) and MH 09387 (JP).

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