Introduction

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Opioids inhibit adenylyl cyclase via the activation of a pertussis toxin-sensitive G protein, G_o/G_i (reviewed by Childers, 1991). Many G proteins-linked receptors have been found to exhibit a diminished responsiveness (desensitization or tolerance) in the continued presence of agonists (Law et al., 1983; Benovic et al., 1986; Puttfarcken et al., 1988; Lefkowitz et al., 1990; Simmons et al., 1990). Chronic morphine treatment induces adaptations in G-proteins and cAMP system in numerous brain regions known to be involved in the chronic and acute effects of opiates (Duman et al., 1988; Nestler and Tallman, 1988; Nestler et al., 1989; Terwilliger et al., 1991; Harris and Williams, 1991; Matsuoka et al., 1994).

Previous studies have also reported that chronic cocaine treatment and chronic morphine treatment induce related adaptive responses in the mesolimbic system, affecting G proteins, protein kinase activity and neurofilament proteins in a similar manner (Terwillinger et al., 1991; Beitner-Johnson et al., 1992). Furthermore, a selective desensitization of  opioid receptors (defined as a reduced ability of opioid agonist to inhibit adenylyl cyclase activity) in the caudate putamen and the nucleus accumbens, without modification in the ability of a µ opioid agonist to inhibit this enzyme, may be induced by some subchronic cocaine or morphine treatment regimens (Unterwald et al., 1993; Noble and Cox, 1996). Because the caudate putamen and nucleus accumbens receive major dopaminergic projections from the substantia nigra and the VTA, respectively, the involvement of dopamine in this adaptive response appears likely.

In the present study, we have used the selective D₁ dopamine antagonist SCH 23390 (Hyttel, 1983; Barnett et al., 1986) and the selective D₂ dopamine antagonist eticlopride (Hall et al., 1985) to examine the role of the dopaminergic system in mediating selective desensitization of  opioid receptors after chronic morphine treatment in the caudate putamen and the nucleus accumbens. In striatum, adenylyl cyclase activity is subject to regulation by agonist with high selectivity for both µ and  receptors (Izenwasser et al., 1993). Pharmacological analysis suggests that the activity of this enzyme in striatal membranes is inhibited by agonist acting through either the ₁ or the ₂ subtype of opioid receptor (Bùzàs et al., 1994; Noble and Cox, 1995), although the molecular basis for the observed differences in ligand selectivities in functional assays for these  receptor subtypes is not yet established. Furthermore, the various opioid receptors that regulate adenylyl cyclase in caudate putamen show an apparent differential neuronal location that corresponds to the differential location of D₁ and D₂ dopamine receptors on striato-nigral and striato-pallidal efferent neurons (Gerfen et al., 1990; Le Moine et al., 1990; Noble and Cox, 1995). These observations suggest a neuroanatomical explanation of the effects induced by chronic morphine treatment.

	Materials and Methods

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Animals and surgery. The experiments reported herein were conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals (Institute of Animal Resources, National Research Council, NIH publication 85-23).

Membrane preparation. On the seventh day after implantation of the osmotic pump, rats were killed by decapitation. Their brains were rapidly removed. Caudate putamen and nucleus accumbens were obtained by gross dissection. Dissected tissues from isolated brain regions of one rat were homogenized separately and diluted into buffer [20 mM Tris-HCl (pH 7.4), 2 mM EGTA, 1 mM MgCl₂ and 250 mM sucrose] and centrifuged at 27,000 × g for 15 min at 4°C. The pellets were resuspended in fresh buffer and centrifuged again for 15 min. The supernatants were discarded, and the pellets were homogenized in 30% (w/v) ice-cold buffer [2 mM Tris-HCl (pH 7.4) and 2 mM EGTA] for determination of adenylyl cyclase activity.

Determination of adenylyl cyclase activity. Tissue homogenate (15-30 µg of protein in 10 µl) was added on ice to assay tubes (final volume 60 µl) containing 80 mM Tris-HCl (pH 7.4), 10 mM theophylline (or 150 µM papaverine in experiments performed in the presence of CGS 21680), 1 mM MgSO₄, 0.8 mM EGTA, 30 mM NaCl, 0.25 mM ATP, 0.01 mM GTP and either the drug being tested or water (all drugs tested in this assay were soluble in water at the concentrations used). Triplicate samples for each treatment were incubated at 30°C for 5 min. Adenylyl cyclase activity was terminated by placing the tubes into boiling water for 2 min. The amount of cAMP formed was determined by a [³H]cAMP protein binding assay (Brown et al., 1971). [³H]cAMP (final concentration 4 nM) in citrate-phosphate buffer (pH 5.0) and then binding protein prepared from bovine adrenal glands were added to each sample. Additional samples were prepared, without tissue, containing known amounts of cAMP; these served as standards for quantification. The binding reaction was allowed to reach equilibrium by incubation for 90 min at 4°C, and the assay was terminated by the addition of charcoal and centrifugation (1000 × g for 10 min, at 4°C) to separate the free tritiated cAMP from that which was bound to the binding protein. Aliquots from the supernatant containing bound cAMP were placed into scintillation vials to which Beckman Ready Value Scintillation Cocktail was added, and radioactivity was determined by liquid scintillation spectrometry. Radioactivity was converted to picomoles of cAMP by comparison to the curve derived from the standards. Protein values were determined by a modification of the Lowry procedure, using bovine serum albumin as standard (Peterson, 1977). Results are expressed as percentage of the respective basal or stimulated activity (i.e., naive or morphine-dependent rats) measured in the absence of opioid.

Chemicals. The following drugs and chemicals were used in this study: DAMGO, DPDPE, DT-II, Tyr-D.Pen-Gly-Phe-D.Pen, ATP (disodium salt), GTP (lithium salt), cAMP (sodium salt), theophylline, EGTA (Sigma Chemicals Co., St. Louis, MO), SCH 23390, eticlopride, SKF 38393, CGS 21680, papaverine HCl, quinelorane (Research Biochemicals Inc., Natick, MA), morphine sulfate (Merck Chemical Div., (Rahway, NJ) and [³H]cyclic AMP (ammonium salt; specific activity 28.1 Ci/mmol) (Du Pont NEN Research Products, Boston, MA).

Statistical analysis. Dose-response curves from adenylyl cyclase assays were analyzed with a two-way ANOVA. If a significant effect was observed, a one-way ANOVA was used, followed by a Newman-Keuls' test, to determine the significance at each concentration. The level of significance was set at P < .05.

	Results

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Effects of chronic morphine treatment on inhibition of adenylyl cyclase activity induced by opioid agonists and dopamine agonists in the caudate putamen. We have previously reported (Noble and Cox, 1996) the selective impairment of  opioid receptor-mediated inhibition of basal adenylyl cyclase activity in the rat caudate putamen after chronic morphine treatment. We now show that this morphine treatment does not affect the ability of either the D₁-selective dopamine agonist SKF 38393 or the A_2a-selective adenosine agonist CGS 21680 to stimulate adenylyl cyclase activity in the rat caudate putamen (table 1) [F(1,17) = 0.205, P > .05 and F(1,17) = 0.068, P > .05, respectively]. The inhibitory effects of the ₂ receptor agonist DT-II were tested against adenylyl cyclase activity stimulated by SKF 38393 or CGS 21680 (fig. 1). The chronic morphine pretreatment attenuated the ability of DT-II to inhibit CGS 21680 (0.1 µM)-stimulated adenylyl cyclase (when compared with the inhibitory effects of DT-II in caudate putamen from saline-treated rats) [F(1,24) = 22.722, P < .001] but had no effect on the efficacy or potency of DT-II in inhibiting SKF 38393 (10 µM)-stimulated adenylyl cyclase activity [F(1,18) = 1.091, P > .05].

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effects of DT-II on D1 dopamine receptor-stimulated adenylyl cyclase activity or A2a adenosine receptor-stimulated adenylyl cyclase activity in saline-treated rats () or morphine-treated rats () in the caudate putamen. Results are expressed in percentages of SKF 38393 (10 µM)-stimulated adenylyl cyclase activity or in percentages of CGS 21680 (0.1 µM)-stimulated adenylyl cyclase activity, as means ± S.E.M. from three or more independent experiments, each performed in triplicate.  indicates significant differences between treatments (P < .05) at the indicated concentration of agonist (Newman-Keuls' test).TOT indicates activity in the absence of inhibitor.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of quinelorane on A2a adenosine receptor-stimulated adenylyl cyclase activity in saline-treated rats () or morphine-treated rats () in the caudate putamen. Results are expressed in percentages of CGS 21680 (0.1 µM)-stimulated adenylyl cyclase activity, as means ± S.E.M. from three or more independent experiments, each performed in triplicate.  indicates significant differences between treatments (P < .05; Newman-Keuls' test).

Effects of chronic dopamine antagonist treatments on inhibition of adenylyl cyclase activity induced by opioid agonists and dopamine agonists in the caudate putamen. Chronic treatment with the D₁ dopamine antagonist SCH 23390 (0.6 mg/rat/day, for 7 days) induced a desensitization of  opioid receptors in the caudate putamen (fig. 3). The ₁ opioid agonist DPDPE and the ₂ opioid agonist DT-II appeared unable to inhibit basal adenylyl cyclase activity in SCH 23390-treated rats as compared with saline-treated rats [F(1,22) = 22.856, P < .001 and F(1,20) = 72.818, P < .001, respectively]. In contrast, no modification of ability of the µ opioid agonist DAMGO to inhibit basal adenylyl cyclase activity was observed in SCH 23390-treated rats as compared with saline-treated rats [F(1,20) = 2.891, P > .05] (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effects of DPDPE and DT-II on basal adenylyl cyclase activity or of quinelorane on CGS 21680-stimulated adenylyl cyclase activity in saline-treated rats () or SCH 23390-treated rats () in the caudate putamen. Results are expressed in percentages of basal adenylyl cyclase activity or in percentages of CGS 21680 (0.1 µM)-stimulated adenylyl cyclase activity, as means ± S.E.M. from three or more independent experiments, each performed in triplicate.  P < .05 and P < .01 indicate significant differences between treatments (Newman-Keuls' test).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effects of DT-II on D1 dopamine receptor-stimulated adenylyl cyclase activity or A2a adenosine receptor-stimulated adenylyl cyclase activity in saline-treated rats () or SCH 23390-treated rats () in the caudate putamen. Results are expressed in percentages of SKF 38393 (10 µM)-stimulated adenylyl cyclase activity or in percentages of CGS 21680 (0.1 µM)-stimulated adenylyl cyclase activity, as means ± S.E.M. from three or more independent experiments, each performed in triplicate.  P < .05 indicates significant differences between treatments (Newman-Keuls' test).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effects of SKF 38393 on basal adenylyl cyclase activity in saline-treated rats () or eticlopride-treated rats () in the caudate putamen. Results are expressed in percentage of basal adenylyl cyclase activity as means ± S.E.M. from three or more independent experiments, each performed in triplicate.  P < .05 and P < .01 indicate significant differences between treatments (Newman-Keuls' test).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effects of DPDPE and DT-II on basal adenylyl cyclase activity in rats given saline (), morphine () or morphine and eticlopride (), in the caudate putamen. Enzyme activity in the presence of inhibitor is expressed as a percent of basal adenylyl cyclase activity. Values are means ± S.E.M. from three or more independent experiments, each performed in triplicate.  P < .05 and P < .01 as compared with saline group,  P < .05 and P < .01 as compared with morphine/eticlopride group (Newman-Keuls' test). The data points connected by dashed lines have previously been reported (Noble and Cox, 1996).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effects quinelorane on CGS 21680-stimulated adenylyl cyclase activity in the caudate putamen from rats given saline (), morphine () or morphine and eticlopride (). Enzyme activity in the presence of inhibitor is expressed as a percent of CGS 21680 (0.1 µM)-stimulated adenylyl cyclase activity. Values are means ± S.E.M. from three or more independent experiments, each performed in triplicate.  P < .05 as compared with saline group,  P < .05 as compared with morphine/eticlopride group (Newman-Keuls' test).

Effects of chronic D₂ dopamine receptor antagonist treatment on inhibition of adenylyl cyclase activity induced by opioid agonists and dopamine agonists in the nucleus accumbens. As reported previously (Noble and Cox, 1996), chronic morphine treatment induced a selective impairment of  opioid receptors (₁ and ₂) but had no effect on the ability of the µ opioid agonist to inhibit basal adenylyl cyclase activity (fig. 8). Two-way ANOVA analysis revealed a significant difference in the ability of the ₁ opioid agonist DPDPE to inhibit adenylyl cyclase activity in morphine-treated rats as compared with saline-treated rats [F(1,30) = 28.529, P < .001], as well as in the ability of the ₂ opioid agonist DT-II to inhibit the enzyme [F(1,31) = 28.749, P < .001], whereas no difference could be observed in the inhibition induced by the µ opioid agonist DAMGO [F(1,35) = 0.001, P > .05].

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Effects of DT-II, DPDPE or DAMGO on basal adenylyl cyclase activity in the nucleus accumbens of rats treated with saline (), morphine () or morphine and eticlopride (). Enzyme activity in the presence of inhibitor is expressed as a percent of basal adenylyl cyclase activity. Values are means ± S.E.M. from three or more independent experiments, each performed in triplicate.  P < .05 as compared with saline group;  P < .05 as compared with morphine/eticlopride group (Newman-Keuls' test). The data points connected by dashed lines have previously been reported (Noble and Cox, 1996).

	Discussion

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A selective impairment of  opioid receptor function in the caudate putamen and the nucleus accumbens after chronic morphine treatment was confirmed in the present study. Moreover, the results obtained demonstrate that the D₂ dopamine antagonist eticlopride, administered chronically throughout the chronic morphine treatment, abolished this effect in both structures.

Several laboratories have obtained evidence supporting the hypothesis of heterogeneity of  opioid receptors (₁ and ₂) (Jiang et al., 1991; Mattia et al., 1991; Sofuoglu et al., 1991; Bùzàs et al., 1994; Noble and Cox, 1995). Furthermore, in the caudate putamen it has been shown that D₁ dopamine and A_2a adenosine receptors, which both increase adenylyl cyclase activity by stimulating G_s (Stiles, 1992; reviewed by Angulo and McEwen, 1994), are preferentially expressed by striato-nigral neurons and striato-pallidal neurons, respectively (Gerfen and Young, 1988; Gerfen et al., 1990; Le Moine et al., 1990; Schiffmann et al., 1991; reviewed by Gerfen, 1992; Augood and Emson, 1994). We have measured the ability of opioid agonists to inhibit selectively adenylyl cyclase activity measured in the presence of one activator of adenylyl cyclase but not in the presence of the other activator, and we suggest that such selective inhibitory activity is consistent with the hypothesis that the inhibitory receptor is located on the same cell membranes (and therefore on the same neuron population) as the activating receptor. Using this approach, differential neuronal locations in the caudate putamen of µ, ₁ and ₂ opioid receptors have been proposed (Noble and Cox, 1995). Thus µ opioid receptors that regulate adenylyl cyclase in caudate putamen appear to be preferentially expressed by striato-nigral neurons and ₁ opioid receptors by striato-pallidal neurons, whereas ₂ opioid receptors appear to be expressed by both of these striatal efferent neuron populations. It is important to note that this differential distribution of µ and ₁ receptors may apply only to the opioid receptor types that regulate adenylyl cyclase activity. If separate subsets of µ and ₁ receptors mediate opioid regulation of ion channel function, then these receptor subsets may not share the same neuronal distribution. For this reason, immunocytochemical localization of opioid receptor types may not confirm the proposed distribution of the subsets of receptors that regulate adenylyl cyclase.

A reduced ability of the ₁ opioid agonist DPDPE (Mosberg et al., 1983; Jiang et al., 1991; Mattia et al., 1991; Sofuoglu et al., 1991) and of the ₂ opioid agonist DT-II (Erspamer et al., 1989; Jiang et al., 1991; Mattia et al., 1991; Sofuoglu et al., 1991) to inhibit basal adenylyl cyclase activity was observed in the caudate putamen and the nucleus accumbens after morphine treatment as compared with control animals (Noble and Cox, 1996). Because ₂ opioid receptors that regulate adenylyl cyclase appear to be present on both striato-nigral neurons and striato-pallidal neurons (Noble and Cox, 1995), it was interesting to determine whether this desensitization was unique to one of these populations by evaluating the effects of DT-II after selective stimulation of adenylyl cyclase with the D₁ dopamine agonist SKF 38393 (striato-nigral neurons) or the A_2a adenosine receptor agonist CGS 21680 (striato-pallidal neurons). After chronic morphine treatment, DT-II inhibited SKF 38393-stimulated adenylyl cyclase activity in the same intensity and concentration range as in control rats, whereas a reduced ability of the ₂ opioid agonist to inhibit CGS 21680-stimulated adenylyl cyclase activity was observed in tissues from the morphine-treated animals. These results indicate that after chronic morphine treatment, there is a selective impairment of ₂ opioid receptors localized on striato-pallidal neurons without alteration of ₂ opioid receptors expressed by striato-nigral neurons.

In situ hybridization studies have also demonstrated that D₂ dopamine receptors, negatively coupled to adenylyl cyclase via G_i/G_o proteins, are also preferentially expressed by striato-pallidal neurons but not by striato-nigral neurons (Le Moine et al., 1990; Gerfen et al., 1990; reviewed by Gerfen, 1992). In good agreement with this observation, the D₂/D₃ dopamine agonist quinelorane was able to inhibit CGS 21680-stimulated adenylyl cyclase activity in control animals. This inhibitory effect was selectively antagonized by the D₂ dopamine antagonist eticlopride. Quinelorane was significantly less effective in inhibiting adenylyl cyclase activity in morphine-treated animals than in control rats, which suggests that dopamine D₂ receptor function in striato-pallidal neurons is impaired after chronic morphine treatment.

Because morphine is a µ-selective agonist (Matthes et al., 1996), the heterologous desensitization of the adenylyl cyclase-coupled receptors (i.e., of  opioid receptors and D₂ dopamine receptors) in striato-pallidal neurons is probably the result of an indirect mechanism that involves other neurotransmitters. In previous experiments, it has been reported that chronic treatment with cocaine, an indirect dopamine agonist, induced a selective impairment of  opioid receptors in the caudate putamen (Unterwald et al., 1993), a result that suggests involvement of the dopaminergic system in the effects observed in the present study. To examine the possible involvement of the dopaminergic system in these effects, we determined the effects of chronic D₁ dopamine antagonist (SCH 23390) and chronic D₂ dopamine antagonist (eticlopride) treatments on the adaptations in the regulation of adenylyl cyclase induced by chronic morphine treatment.

Chronic treatment with the D₁ antagonist SCH 23390 resulted in a desensitization of D₂ dopamine receptors and of ₁ and ₂ opioid receptors. The desensitization of the ₂ opioid receptors was shown to be uniquely related to adenosine A_2a-activated adenyl cyclase, which indicated that the ₂ receptor desensitization was specific for the ₂ receptors expressed by striato-pallidal neurons. (Previous studies have suggested that ₁ receptors that regulate adenylyl cyclase in caudate putamen are selectively localized on striato-pallidal neurons; Noble and Cox, 1995). Thus it appears that the effects of chronic morphine closely resemble those of chronic D₁ antagonist treatment: a selective impairment of receptors negatively coupled to adenylyl cyclase in striato-pallidal neurons without modification of those localized on striato-nigral neurons (fig. 9). In contrast, chronic treatment with the D₂ antagonist eticlopride did not change D₂ or opioid receptor regulation of adenylyl cyclase in caudate putamen but resulted in a partial desensitization of D₁-stimulated adenylyl cyclase activity.

View larger version (33K): [in this window] [in a new window]   Fig. 9.   Schematic representation of the effects of chronic morphine and SCH 23390 treatment on the regulation of adenylyl cyclase activity (AC) by stimulatory (D1, A2a) and inhibitory (D2, µ, 1, 2) receptors in caudate putamen. It is assumed that dopamine D1 receptors selectively activate AC in striato-nigral neurons, whereas adenosine A2a receptors selectively activate AC in striato-pallidal neurons (Noble and Cox, 1995).

It has previously been shown that activation of µ receptors hyperpolarizes and thus reduces the release of transmitters from GABA neurons in the VTA (Johnson and North, 1992) and other brain regions (Nicoll et al., 1980). In contrast, activation of D₁ dopamine receptors facilitates GABA release in the VTA (Cameron and Williams, 1993) and in primary cultures of embryonic rat striatal neurons (Phillips and Cox, in preparation). We therefore propose that treatment with either the µ agonist morphine or the D₁ antagonist SCH 23390 results in reduced GABA neuron-mediated inhibition of the substantia nigra dopamine neurons and thus induces an increased release of dopamine in caudate putamen. The increased levels of dopamine have little consequence for the striato-nigral neurons, because either their D₁ receptors are blocked by SCH 23390 or the neurons are inhibited by morphine acting on µ receptors. During chronic morphine treatment, it is possible that coincident activation of G_s (through D₁ receptors) counteracts adaptive changes induced by prolonged activation of G_i/o through µ receptors in the striato-nigral neurons. In contrast, in the striato-pallidal neurons, the increased level of released dopamine after chronic morphine or chronic D₁ antagonist treatment may result in increased activation of the D₂ receptors, because these neurons are not directly regulated by either µ agonists or D₁ antagonists (Noble and Cox, 1995). In the striato-pallidal neurons, D₂ and  receptor activation have similar consequences: an inhibition of adenylyl cyclase activity via G_i activation. Treatments that result in enhanced activation of the striato-pallidal neuron D₂ receptors (e.g., chronic morphine or D₁ antagonist) result in a heterologous desensitization in inhibitory receptor regulation of AC in this pathway. Mechanisms that might account for the heterologous desensitization of  and D₂ receptors include a reduction in the activity or levels of G_i in these neurons (Childers, 1991; Izenwasser et al., 1993; Noble and Cox, 1995). However, direct measurement of G_i subunits in striatum after chronic morphine treatment failed to demonstrate any change in their amounts (Dr. T. E. Cote, personal communication). Another possibility is a D₂ receptor-mediated sensitization of AC to activation through G_s (Watts and Neve, 1996), although this mechanism seems less likely because GCS 21680-stimulated AC activity was not changed by morphine or SCH 23390 treatment in the present study. The role of an initial activation of D₂ receptors in the desensitization of  opioid receptors is confirmed by our observation that blockade of D₂ dopamine receptors prevented the  opioid receptor desensitization normally observed after morphine treatment.

Chronic treatment of rats with cocaine or morphine also leads to long-term biochemical and functional changes in the nucleus accumbens, a brain region implicated in mediating the reinforcing effects of drugs of abuse (Goeders et al., 1984; Bozarth, 1986; Terwilliger et al., 1991; Unterwald et al., 1993; Izenwasser et al., 1996). Chronic morphine treatment also induced a selective impairment of  opioid receptors that regulate adenylyl cyclase activity in this brain structure (Noble and Cox, 1996). In nucleus accumbens, as in caudate putamen, chronic eticlopride treatment administered concurrently with the chronic morphine treatment blocked the selective desensitization of ₁ and ₂ opioid receptors induced by morphine treatment. This result is consistent with previous reports that indicated an involvement of the mesolimbic dopamine system in mediating the motivational effects of opioids (Hand and Franklin, 1985; Smith et al., 1985; Shippenberg and Herz, 1988; Cador et al., 1991). Our results indicate a critical role for dopamine in the mediation of morphine-induced opioid receptor desensitization in the nucleus accumbens.

In conclusion, it appears that chronic morphine treatment induced a selective desensitization of  opioid receptors in the caudate putamen and the nucleus accumbens via an indirect mechanism that involves the dopaminergic system. It is possible that under more intense chronic morphine treatments than those used in the present study, a direct desensitization of µ opioid receptors might also be observed in both structures.