SYNAPSE 11:47-57 (1992)

Dopamine Depletion Produces Augmented Behavioral Responses to a Mu-, But Not a Delta-Opioid-ReceptorAgonist in the Nucleus Accumbens: Lack of a Role for Receptor Upregulation LYNN CHURCHILL AND PETER W. KALIVAS Alcohol and Drug Abuse Program; Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, Washington 99164-6520

KEY WORDS

Receptor autoradiography, Mesolimbic dopamine system, Opioid-induced locomotion

ABSTRACT Microinjection of either p- or d-opioid agonists into the nucleus accumbens produces a n increased locomotor activity, and when the dopaminergic innervation of the nucleus accumbens is bilaterally lesioned, the locomotor response to the microinjection of mixed p- and a-opioid agonists is augmented. To determine whether the lesioninduced augmentation to opioids is specific to )*- or a-opioid receptor activation, dopamine innervation of the nucleus accumbens was lesioned with 6-hydroxydopamine (6-OHDA), and the motor stimulant response to intra-accumbens microinjection of the selective p-opioid agonist, Tyr-D-Ala-Gly-mePhe-Gly-OH(DAMGO), was compared to that of the (DPDPE). The lesions caused a 95% d-opioid agonist, [~-penicillamine~~~l-enkephalin depletion of tissue dopamine levels in the nucleus accumbens of the DAMGO-injected rats compared to sham-lesioned rats. Horizontal and vertical photocell counts were significantly increased in response to DAMGO in 6-OHDA-lesioned compared t o the sham-lesioned rats. This behavioral augmentation was dose dependent and blocked by naloxone. In rats with similar accumbal dopamine depletions (94%),the locomotor response to DPDPE was not enhanced. The augmentation in the behavioral response to DAMGO was not associated with a change in the B,,, or & of [1251]DAMG0binding in nucleus accumbens homogenates from lesioned rats. Likewise, using quantitative receptor autoradiography, no difference between 6-OHDA- and sham-lesioned rats was observed in [lz5I1DAMGOor [lz5I1DPDPE binding. Therefore, the augmented behavioral response to opioids in the nucleus accumbens following dopamine depletion relies predominately on p-opioid receptor stimulation. However, this augmentation is not mediated by a n alteration in the number or affinity of these receptors. o 1992 Wiley-Liss, Inc. INTRODUCTION

in motor activity (Dauge et al., 1988; Kalivas e t al., The nucleus accumbens receives dopaminergic inner- 1983; Pert and Sivit, 1977; Vezina et al., 1987). Furvation from the ventral tegmental area in the ventro- thermore, the intra-accumbens administration of the medial mesencephalon (Fallon and Moore, 1978; Ger- quaternary opioid antagonist, methylnaloxonium, infen et al., 1987; Swanson, 1982). The mesoaccumbens hibits the locomotor stimulant effect of peripherallydopamine projection has been characterized as a sys- administered heroin (Amalric and Koob, 1985). Dopamine- and opioid-induced locomotion appear to tem important in the modulation of spontaneous and result from parallel, independent inputs to accumbal pharmacologically stimulated locomotion (Clarke et al., neurons. Thus, dopamine receptor antagonists do not 1988; Delfs et al., 1990; Fink and Smith, 1980; Kelly alter the motor stimulant effect elicited by the microinand Iversen, 1975; Koob et al., 1981; Pijnenburg et al., jection of opioids into the nucleus accumbens (Kalivas 1976). Similar to the dopaminergic input, enkephalinergic innervation of the nucleus accumbens modulates locomotor activity. The microinjection of p- and a-opioid agonists into the nucleus accumbens elicits a n increase Received May 16,1991;accepted in revised form September 23,1991 0 1992 WILEY-LISS, INC.

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L. CHURCHILL AND P.W. KALIVAS

et al., 1983; Pert and Sivit, 1977). Likewise, the microinjection of naloxone in and adjacent to the nucleus accumbens did not alter the motor effect of peripheral amphetamine administration while preventing peripheral morphine-induced motor activity (Stevens et al., 1986).Also, Swerdlow et al. (1985) found that a fivefold greater dose of systemic naloxone was required to inhibit the motor stimulant effect of a low peripheral dose of amphetamine (0.25 mgkg, s.c.) than was necessary to prevent heroin-induced locomotion. Although these data argue against an important interaction between these two transmitters in eliciting locomotion in the nucleus accumbens, chronic disruption of accumbal dopamine transmission results in a dramatic alteration in opioid transmission. Lesions of dopamine fibers in the nucleus accumbens, or chronic administration of dopamine receptor antagonists increases the tissue concentration of both enkephalin and preproenkephalin mRNA (Hong et al., 1978, 1979; Morris et al., 1988; Sabol et al., 1983). These treatments also markedly augment the locomotor stimulant effect following the microinjection of enkephalin analogues into the nucleus accumbens (Kalivas and Bronson, 1985; Stinus et al., 1985). More recently, the rate of acquisition of opioid self-administration in rats was shown to be increased in animals sustaining dopamine lesions in the nucleus accumbens (Stinus et al., 1989). The present study was designed t o further characterize the augmentation of enkephalin-induced locomotion following 6-OHDAlesions of dopaminergic inputs to the nucleus accumbens. The effect of specific opioid receptor agonists was examined on the rat's behavioral responses at least 10 days after 6-OHDA lesions of the nucleus accumbens. Tyr-D-Ala-Gly-NmePhe-Gly-OH (DAMGO)was utilized as a p-selective agonist having a 10,000-foldgreater affinity for y-opioid receptors than 6-opioid receptors (Handa et al., 1981; Lutz et al., 1985), and the &selective enkephalin analogue, [D-penicillamine2%enkephalin (DPDPE) was selected because it has about 3000-fold greater affinity for the a-opioid receptor than for the p-opioid receptors (Mosberg et al., 1983).We also evaluated the effects of dopamine depletion on opioid receptor binding in the nucleus accumbens to determine if the neurochemical basis for the behavioral augmentation was due t o receptor upregulation. METHODS Animal housing, surgery, and microinjections Male Sprague-Dawley rats (250-350 g; Laboratory Animal Research Center, Pullman, WA) were injected with desmethylimipramine (25 mg/ml, i.p.1 a t least 20 min prior to anesthetizing with Equithesin (2 ml/kg). Bilateral 6-OHDA lesions of the nucleus accumbens were performed by injecting 3 pl of 6-OHDA (4 pg/pl free base in 0.25 mg/ml ascorbic acid in sterile saline) through 30 gauge cannulae inserted in the nucleus ac-

cumbens (A/P 8.9; M/L 1.7; D N -0.4 mm, relative to intraaural line; Pellegrino et al., 1979) at the rate of 0.2 pU10 sec with a 40-sec pulse between injections. Following a 3-min delay for absorption, the 30-gauge cannulae were removed and 26-gauge guide cannulae implanted 1mm above the lesion site. The cannulae were secured to the skull with dental acrylate and stainless steel screws, and the rats allowed to recover for a minimum of 10 days after surgery. Simultaneous bilateral microinjections were performed in the unrestrained rat by inserting a 33-gauge needle into each guide cannulae. The injection needle was connected by PE-20 tubing to a 1-p1 syringe mounted on a Sage infusion pump and the needles left in place for 20 sec after discontinuing infusion. For the dose-response curves, 6-OHDA- (n = 15) and sham-lesioned (n = 13) rats were analyzed for their behavioral responses after microinjections of saline or the p-opioid agonist, DAMGO (0.03-1.0 nmob'0.5 pl sterile saline/ side). The behavior for another group of 6-OHDA(n = 9) or sham-lesioned (n = 6) rats was analyzed after microinjections of saline or the a-opioid agonist, DPDPE (0.3-3.0 nmoU0.5 pUside). In another experiment, the behavioral response of 6-OHDA-(n = 4) or sham-lesioned (n = 6) rats was analyzed after naloxone (3.0 mgkg, s.c.) was injected 4-5 min prior to microinjection of saline or DAMGO (1.0 nmoU0.5 pb'side). Each rat received a maximum of 6 microinjections, including one saline injection in random order. Behavioral measurements Spontaneous motor activity was measured using a photocell apparatus (Omnitech Electronics, Columbus, OH) operated via an Apple IIe computer. Two rows of photocells located 5 and 8 cm off the cage floor were distributed around the cage. The plexiglass cages were isolated in separate wooden boxes equipped with light and air supply and a 2-way mirror for behavioral observation. The rats were preadapted to the cages and injection procedure 24 hr prior to starting the experiment by placing them into the cages for 30 min, making sham injections by inserting a needle into the injection site, and then returning them to their cages for another hour. When injections were to be made, the rats were adapted to the cages for 1 hr prior to injection, and behavioral measurements were conducted for 2 hr after injection. After the behavioral analyses, the rats were returned to their home cages for a minimum of 3 days. Measurement of biogenic amines Dopamine and serotonin were measured using highpressure liquid chromatography (HPLC) with electrochemical detection. Injection cannulae were inserted into the guide cannulae just prior to decapitation in order to verify the anatomical position of the guide cannulae. The nucleus accumbens in all rats and the dorsolateral and ventromedial striatum and prefrontal cor-

6-OHDA LESION ENHANCES MU-OPIOID RESPONSE

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buffer and recentrifuged at 40,OOOg for 30 min. This pellet was suspended in 5 mM Tris-HC1, pH 7.7 and rocked gently for 30 rnin in a cold room prior to centrifugation at 40,OOOg for 30 min. The pellet was then washed and resuspended in 50 mM Tris-HC1, pH 7.4 prior to freezing at -80°C. Binding assays were performed in silanized glass tubes. Each tube contained 70 pg homogenate protein, 0.1-3 nM [12511DAMG0in 50 mM Tris-HC1, pH 7.4, 10 pM bestatin, 0.2% BSA with or without 1 p,M naloxone in a volume of 240 pl. Assays were completed by filtration over GF/B filters presoaked in 1%polyethylimine and squirted with 2% BSA just prior to filtration. Washes of the filters consisted of 10 mM Tris-HC1, pH 7.4, 0.2% polyethylimine. The filters were counted using a Beckman Model 5000 Receptor autoradiography and filtration assays gamma counter with an efficiency of 6045%. The re[1251]DAMG0binding to the p-opioid receptors was sults were analyzed using LIGAND on an IBM-PC analyzed autoradiographicallyin coronal sections (10 pm) (Munson and Rodbard, 1980). throughout the nucleus accumbens of both 6-OHDAStatistics (n = 6) and sham-lesioned (n = 6) rats. [1251]DPDPE binding to the a-opioid receptors was analyzed in 5 of All animals with one or both of the cannula tips outthese same 6-OHDA- and sham-lesioned rat brains. side the nucleus accumbens or with less than 70% Brain sections were simultaneously incubated 1 hr at dopamine depletion were excluded from the data analyroom temperature in 0.4 nM ['2511DAMG0, 50 mM ses. Only 2 out of 46 rats were excluded for cannulae Tris-HC1, pH 7.4, 1 pM bestatin, 0.2% bovine serum placement, and 2 out of 46 rats were excluded for albumin (BSA),or in 1.0 nM [lZ5I]DPDPE,50 mM Tris- dopamine depletion less that 70%. The dose-response HCl, pH 7.4, 10 pM bestatin, 10 pM carboxymethyl- effects were statistically evaluated with a two-way phenylalanine-leucine, 0.2% BSA. Preincubation analysis of variance (ANOVA)using a Newman-Keuls washes in 100 mM NaC1, 50 pM GTP for 15 min, and multiple comparison test, while the time course was the incubation buffer minus radioactive ligand or dis- evaluated separately for the sham- or 6-OHDA-lesioned placer for 5 min were conducted to remove endogenous rats with a two-way repeated measure ANOVA folpeptides. The displacers were 1 pM naloxone or 1 pM lowed by a multiple comparison test using the method DPDPE, respectively. The sections were washed briefly described by Milliken and Johnson (1984). The effects in hypotonic rinses and then for 10 min in 50 mM Tris- of the antagonist on the behavioral response was evaluHC1, pH 7.4, 0.2% BSA twice prior to dipping in dis- ated separately for the sham- or 6-OHDA-lesionedrats tilled water and drying under cool air. Sections were using a one-way repeated measure ANOVA with a apposed to SB5 X-ray film (Kodak) for 5 days. Film was Fisher least significant difference test. A one-way developed in GBX developer and fixer (Kodak).Autora- ANOVA was used to compare optical density in quantidiograms were quantified in the medial shell and lat- fying the autoradiograms. An unpaired, two-tailed Stueral accumbens core using the template previously pub- dent's t-test was used to evaluate any differences in the lished (Churchill et al., 1990) and a Nikon photometer dopamine or serotonin concentration with the lesion. with an 0.25 mm aperture. Three spots were measured RESULTS within each of 3 sections. Computer-generated linear of the p-opioid agonist, DAMGO Effect regression analysis of log optical density vs. log radioacInjections of DAMGO into the nucleus accumbens tivity for a series of [12511standards was used to compute fmol/mg tissue. The linear regression analysis produced a dose-dependent increase in horizontal phoshowed a linear relationship over a range of 4-5 stan- tocell counts in the rats bilaterally lesioned with dards within the optical density range of 0.08-0.8. The 6-OHDA in the nucleus accumbens (Fig. 1A). The increase in photocell counts produced by DAMGO was correlation coefficients were 0.99. Bilateral nucleus accumbens was dissected from a augmented at all doses examined in the 6-OHDA-lefreshly-removed brain from 6-OHDA- (n = 4)or sham- sioned compared to sham-lesioned animals. Although lesioned (n = 4) rats and homogenized in 0.8 ml Tris- DAMGO increased the horizontal photocell counts HC1, pH 7.4, 10% sucrose in a glass-glass homogenizer within the sham-lesioned treatment group, no signifias previously described (Dilts and Kalivas, 1990). The cant differences compared to saline were observed. homogenate was centrifuged a t 1,OOOg for 10 min, the Figure 2A,B shows that the increase in horizontal phosupernatant removed and recentrifuged at 40,OOOgfor tocell counts produced by DAMGO in control animals 30 min. The pellet was washed in the homogenizing was most marked at 45-90 min after injection. The

tex in the DAMGO-naloxone injected rats were dissected on an ice-cooled glass plate. After dissection, bilateral tissue punches were placed in 0.5 ml of mobile phase (0.1M trichloroacetic acid, 0.1 M sodium acetate, 0.1 mM EDTA, 18%methanol (pH 4)and 2 x lop7 M isoproterenol as an internal standard), sonicated and centrifuged (13,OOOg)for 2 min. The supernatant was removed for analysis on the HPLC, while the pellet was assayed for protein using the Lowry method. The biogenic amines were separated on an ODS C-18 reversedphase column and oxidized a t 0.7 V. The concentration of biogenic amines were calculated from a standard curve to l o p l o moles). The detection limit was moles. about 3 x

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DOSE (MOL/SIDE) Fig. 1. Dose-response effects of DAMGO on motor activity. Values represent the mean tSEM of cumulative photocell counts for 120 min following microinjection of DAMGO or saline into the nucleus accumbens of either 6-OHDA- or sham-lesioned rats. The number of rats receiving each dose are in parentheses. A Total horizontal photocell counts (CTS). The F scores were treatment F(1,69) = 41.498, P < 0,0001, dose F(4,69) = 9.166, P < 0.0001 and interaction F(4,69) = 4.395, P = 0.0032. B: Total vertical photocell counts. F values were treatment F(1,69) = 61.461, P < 0,0001, dose F(4,69) = 9.891, P < 0,0001, interaction F(4,69) = 5.39, P = 0.0008. *P < 0.05 comparing all groups with saline or sham-lesioned rats using a two-way ANOVA and a Newman-Keuls multiple comparison test.

augmentation of the locomotor response produced by DAMGO in the 6-OHDA-lesioned rats occurred through most of the 120 min time course. Figure 1B demonstrates that the number of vertical movements also increased significantly in a dose-related fashion after DAMGO injections into the nucleus accumbens of rats previously lesioned by 6-OHDA. As with horizontal photocell counts, the trend toward a n increase in vertical movements produced by DAMGO was not statistically significant in sham-lesioned rats. The vertical movements in the lesioned rats were significantly elevated over those in the control animals a t all doses of DAMGO greater than 0.03 nmovside. The nonspecific p- and d-opioid antagonist, naloxone (3.0 mgkg, i.p.), significantly reduced the horizontal and vertical photocell counts elicited by DAMGO injec-

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TIME (rnin) Fig. 2. Time course of the motor activity elicited by the microinjection of DAMGO or the saline into the nucleus accumbens. Values represent the mean of cumulative photocell counts for 15-min intervals. A Sham-lesioned rats. The F scores were treatment F(4,35) = 0.75, P = 0.565, time F(7,28) = 59.454,P < 0.0001, interaction F(28,245) = 1.028, P = 0.432. B: 6-OHDA lesioned rats. The F scores were treatment F(4,39) = 5.505, P = 0.0013, time F(7,28) = 101.811, P < 0.0001, interaction F(28,273) = 1.873, P = 0.0061. S i g nificant differences ( P < 0.05) from saline injections were observed for DAMGO -0.3 and 1.0 nmol up to 90 min, DAMGO-0.1 from 30-75 min, and DAMGO-0.03 from 30-45 rnin using a two-way repeated measure ANOVA followed by a multiple comparison test by Milliken and Johnson (1984).

tion into either 6-OHDA or sham-lesioned rats (Fig. 3). Naloxone alone did not significantly alter basal activity . Effect of the d-opioid agonist, DPDPE Figure 4 shows that injections of 1.0 and 3.0 nmol of DPDPE into the nucleus accumbens increased horizontal photocell counts in rats bilaterally lesioned with 6-OHDA in the nucleus accumbens relative to the saline injection. However, at a dose of DPDPE that was 30-fold greater than the minimum effective dose of DAMGO, DPDPE did not elicit a significant augmentation in horizontal photocell counts compared to shamlesioned rats. The significant increase in photocell counts induced by DPDPE occurred mainly in the first 45 min after injection in both 6-OHDA- and sham-lesioned rats (Fig. 5A,B). Figure 4B shows that compared to a microinjection of saline, DPDPE produced a significant elevation in vertical photocell counts in 6-OHDA-

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MICROINJECTIONS Fig. 3. Effects of the opioid antagonist, naloxone on motor activity elicited by microinjections of DAMGO or saline into the nucleus accumbens. A. Sham-lesioned rats (n = 6). Values were the mean ?SEM of the total horizontal photocell counts. F values were treatment F(3,23) = 5.647, P = 0.009.B: 6-OHDA-lesioned rats (n = 4). F values were treatment F(3,15) = 5.939, P = 0.016. *P < 0.05 comparing DAMGO injections with the other treatments using a one-way repeated measure ANOVA with a Fisher least significant difference test.

lesioned rats a t the highest dose examined. No significant effect on vertical activity was measured at any dose of DPDPE injected into the nucleus accumbens of sham-lesioned rats.

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DOSE (MOLISIDE) Fig. 4. Doseresponse effects of DPDPE microinjection into the nucleus accumbens on motor activity. Values represent the mean 2SEM of cumulative photocell counts for 120 min following injection of DPDPE or saline into the nucleus accumbens of 6-OHDA or shamlesioned rats. The number of rats receiving each surgical treatment are in parenthesis. A Total horizontal photocell counts. The F scores were treatment F(1,52) = 1.776, P = 0.189; dose F(3,52) = 7.556, P = 0.0003, interaction F(3,52) = 1.335, P = 0.273. B Total number of vertical movements. F values were treatment F(1,52) = 0.216, P = 0.644, dose F(3,52) = 7.258, P = 0.0004, interaction F(3,52) = 0.394, P = 0.758. ‘ ( P < 0.05) comparing all groups with saline using a twoway ANOVA and a Newman-Keuls multiple comparison test.

6-OHDA lesioned rats in either the nucleus accumbens or striatum (Table I). Effect of 6-OHDA lesion on opioid receptors The lack of effect by 6-OHDA lesions on [lZ5I]Using light microscopic measurements of [1251]- DAMGO binding sites observed in the in situ binding DAMGO and [lZ5I]DPDPEbinding in the nucleus ac- assay was also revealed in [12511DAMG0 binding to cumbens and ventromedial striatum, no significant al- membrane preparations from the nucleus accumbens. terations in binding density were found between The best fit curve derived from the four replications in 6-OHDA and sham-lesioned rats. Autoradiograms of duplicate of six concentrations of [12511DAMG0in ho[1251]DAMG0(Fig. 6A,B) and [lZ5I]DPDPE binding mogenates from sham-lesioned rats revealed a single (Fig. 6A,B) in the rostra1 nucleus accumbens showed no binding site having a I(d = 0.70 0.04 nM and a difference between rats lesioned with 6-OHDA (Figs. B,, = 42 * 16 fmoWmg protein (LIGAND; Munson 6B,D) ten days earlier and sham-lesioned rats and Rodbard, 1980).Parallel experiments using accum(Fig. 6A,C). Table I shows that dopamine depletion in bal tissue from 6-OHDA-lesioned rats also revealed a the nucleus accumbens produced no change in single binding site for [1251]DAMG0 with a & = [1251]DAMG0binding density in the patch or the ma- 0.65 0.23 nM and a B, = 45 & 10 fmoWmg protein. trix of the core of the nucleus accumbens, nor in the Dopamine depletion and histology accumbal shell region. [1251]DAMG0binding was also The nucleus accumbens from each rat was examined unaltered in the patch of the dorsolateral striatum. Likewise, [lZ5I]DPDPE binding was unaltered in for tissue content of dopamine. Rats were determined to

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TIME (min) Fig. 5. Time course of the motor activity after microinjection of DPDPE or saline into the nucleus accumbens. Values represent the mean of cumulative photocell counts for 15 min. A Sham-lesioned rats. F values were treatment F(4,20) = 2.12, P = 0.116, time F(7,28) = 88.404, P < 0.0001, interaction F(28,140) = 2.094, P = 0.0027. B: 6-OHDA-lesioned rats. F values were treatment F(4,36) = 6.12, P = 0.0007, time F(7,28) = 48.164, P < 0.0001, interaction F(28,252) = 2.592, P < 0,0001.Significant differences (P< 0.05) comparing all groups with saline were observed for DPDPE-1.0-3.0 nmol up to 45 min and for DPDPE-0.3 nmol at 15 min using a two-way ANOVA with a multiple comparison test as described by Milliken and Johnson (1984).

have correct cannulae placement if the cannula track could be seen to penetrate the dorsal, but not ventral edge of the tissue punch. Visualization of the track was facilitated by making a sham injection immediately prior to decapitation. Using this technique of cannula track verification, 32 out of 34 rats implanted with bilateral accumbal injection cannula were found to have correct cannula placement. Dopamine levels in the nucleus accumbens were depleted by greater than 95% in 6-OHDA pretreated rats that received DAMGO and 94% in 6-OHDA-lesioned rats that received DPDPE; however, the rats that received DAMGO and naloxone only showed an 85% depletion in DA (Table 11). By contrast, the dopamine levels in the prefrontal cortex was not reduced significantly; whereas the dopamine levels in the dorsolateral and ventromedial striatum were reduced by 31% and 71%, respectively, compared to control levels. No significant changes in serotonin were observed in the nucleus accumbens, prefrontal cortex, or dorsolateral or ventromedial striatum.

Cresyl-violet staining of the nucleus accumbens of 6-OHDA-lesionedrats (Fig. 7C) demonstrated that medium-sized neurons were present outside of the glial scar from the cannulae track (arrow) in a concentration similar to that observed in the sham-lesioned rats (Fig. 7A). The higher magnification of the medial nucleus accumbens core (square insert) demonstrates the clusters of medium-sized neurons still evident after the 6-OHDA- or sham-lesion.

DISCUSSION This study extends previous investigations that demonstrated that a chronic decrease in dopamine transmission in the nucleus accumbens elicits an augmentation of the motor stimulant effect produced by an acute intra-accumbens microinjection of opioids (Kalivas and Bronson, 1985; Stinus et al., 1985; Maldonado et al., 1991). The present data provide evidence that the augmented location seen in 6-OHDA-lesioned rats following opioid agonist administration into the nucleus accumbens results predominantly from stimulation of the p-opioid receptor subtype. Thus, augmented locomotion was observed after the intra-accumbens microinjection of the p-specific agonist, DAMGO, but not after the &specific agonist, DPDPE. The augmented motor response in dopamine-lesioned rats was not associated of [1251]DAMG0 with an alteration in the & or B, binding to nucleus accumbens homogenates. This lack of effect on p,-opioid receptors by accumbal dopamine depletion was verified in an anatomically discrete analysis of [12511DAMG0binding in the nucleus accumbens and dorsolateral striatum using light microscopic quantitative autoradiography. The lack of an upregulation of p-opioid receptors is consistent with other studies employing unilateral 6-OHDA dopamine depletions which have demonstrated either no change (Dilts and Kalivas, 1989) or a reduction in receptor density (Pollard et al., 1977; Unterwald et al., 1989). Interestingly, Pan et al. (1985) and Untenvald et al. (1989) have presented evidence that the down-regulation of popioid binding sites in the nucleus accumbens results from a transsynaptic degeneration subsequent to the loss of accumbal dopamine innervation. Consistent with this possibility, the density of radiolabeled DAMGO binding sites in the nucleus accumbens is markedly reduced by quinolinic or ibotenic acid lesions of accumbal neurons (Churchill et al., 1990; Unterwald et al., 1989). The lack of effect by 6-OHDA on a-opioid binding sites in the nucleus accumbens is consistent with a report by Unterwald et al. (1989) but differs from other studies showing an increase (Dilts and Kalivas, 1990; Esposito et al., 1987) in binding density. Dilts and Kalivas (1990) observed an increase in [lZ5I]DPDPEbinding contralateral to a unilateral 6-OHDA lesion made in the ventral tegmental area. Thus, the apparent change in a-opioid receptors may result from a hemispheric imbalance in

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Fig. 6. Autoradiograms of opioid receptor binding in the nucleus accumbens. l'2511DAMG0 (A,B) or [lZ5I]DPDPE(C,D) binding to the rostra1 nucleus accumbens and caudate-putamen in rats that received bilaterally either a sham (A,C) or 6-OHDA lesion (B,D) 10 days earlier. No significant differences were observed between sham- or 6-OHDA-lesioned rats. These autoradiograms are representative examples from sham- (n = 6) and 6-OHDA-lesioned (n = 6) rats.

TABLE I. Quantitative densitometric analyses of [lZ5I]DAMGOor [1251]DPDPEbinding to the nucleus accumbens of rats injected with either ascorbic acid (sham) or 6-OHDA (lesion) 10 days prior to sacrifice 1-4 [1251]DAMG0(.4 nM) (fmol/mg tissue) Sham Lesion (n = 6) Lat NA-mat Lat NA-patch Med NA DL Caud-mat DL Caud-patch

0.171 f 0.120 0.543 f 0.023 0.386 C 0.013 0.525 0.034 0.928 f 0.039

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'The number of rats is in parentheses. Mean SEM. ANo significant differences were observed in receptorconcentration between sham and 6-OHDA-lesionedrats, using two-tailed Student's t-test. 'NA, nucleus accumbens; Lat, lateral; Med, medial; Caud, caudate-putamen, DL, dorsolateral; mat, matrix.

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dopamine innervation that would not appear in the present study where bilateral lesions were used. Esposito et al. (1987) observed a 30% increase in L3H1(DMa2, D-Leu5)enkephalinbinding sites seven days after

6-OHDA administration into the nucleus accumbens. The fact that no change in a-opioid binding density was measured when animals were examined at least 10 days to 2 weeks after 6-OHDA administration (Unter-

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activity without showing any overall change in p-opioid receptor binding. A functional relationship between the dopaminergic Conc and enkephalinergic system has been suggested previ(umol/ma motein) ously by the increase in Met-enkephalin immunoreacExoeriment Treatment N DoDamine Serotonin tivity and preproenkpehalin mRNA after chronic treatDAMGO-doses Sham 9 560.4 f 42.9 40.3 k 3.5 ment with dopamine receptor antagonists or 6-OHDA Nucleus accumbens 6-OHDA 9 28.7 f 5.9* 44.2 t 4.3 DPDPE-doses Sham 6 506.3 33.1 44.5 t 1.8 lesions of the nigrostriatal neurons (Angulo et al., 1990; Nucleus accumbens 6-OHDA 9 31.0 k 5.1* 44.6 rt 2.0 Hong et al., 1979; Tang et al., 1983; Thal et al., 1983; DAMGO-naloxone Sabol et al., 1983; Vernier et al., 1988; Voorn et al., Brain Regions: 1987). A relationship is also suggested by the augmenNucleus accumbens Sham 6 516.0 f 58.5 44.8 rt 4.2 6-OHDA 4 80.0 5 23.8* 55.7 j,4.1 tation in locomotor activity in response to an opioid Ventromedial caud Sham 6 749.3 f 98.4 32.5 rt 3.4 agonist in the nucleus accumbens after chronic dopa6-OHDA 4 218.0 f 80.7* 43.2 rt 7.1 mine antagonist administration or 6-OHDA lesions in 1307.0 f 82.1 29.2 k 3.4 6 Dorsolateral caud Sham 6-OHDA 4 888.0 It 51.4* 19.5 rt 2.7 the nucleus accumbens or ventral tegmental area (KaliPrefrontal cortex Sham 6 21.9 1.6 42.9 * 2.3 vas and Bronson, 1985; Stinus et al., 1985). More re6-OHDA 4 26.6 f 4.5 40.1 t 4.2 cently, Stinus et al. (1989) have demonstrated that the IThe number of rats = N. acquisition of heroin self-administration is facilitated YCaud,caudate-putamen. *P< 0.05, comparing 6-OHDA- to shamdesioned using a two-tailed Student's t-test. in rats pretreated with chronic dopamine receptor antagonists. Since an upregulation in opioid binding sites cannot explain the reciprocal association between dopawald et al., 1989; the present study) argues that the mine transmission and neurochemical and functional increase may be transient, or that a delayed transsyn- measures of enkephalin transmission in the nucleus aptic change may occur which masks an initial increase accumbens, the induction of postsynaptic changes beassociated with the destruction of dopaminergic termi- yond the opioid receptor is a reasonable postulate. The nals. Although some contradiction is present in the lit- stimulation of opioid receptors in the striatum inhibits erature regarding alterations in a-opioid binding sites the stimulation of adenylyl cyclase by forskolin (Duman in the nucleus accumbens following dopamine deple- et al., 1988). However, no evidence was obtained for an tion, an upregulation of a-opioid binding sites is not augmentation of this effect in accumbal tissue after associated with an augmented behavioral response to 6-OHDA lesions of the nucleus accumbens (Churchill the microinjection of a a-opioid agonist into the nucleus and Kalivas, 1990). The predominant effect of iontophoretic ejection of opioids onto accumbal neurons is a accumbens. The interpretation of the results depends on the spec- decrease in the firing frequency of spontaneous or exciificity of 6-hydroxydopamine to destroy dopaminergic tatory amino acid-driven neurons (Hakan and Henrikterminals without nonspecifically destroying intrinsic sen, 1989; McCarthy et al., 1977). In many brain areas, neurons in the nucleus accumbens. Although the dose the inhibitory effect of p-opioids results from a G proof 6-hydroxydopaminewas high, nonspecific damage to tein-dependent increase in potassium conductance the nucleus accumbens was not observed in the tissue (Lacey et al., 1989; North et al., 1987; Van Dongen except at the position of the injection cannulae. Since et al., 1988). If such a mechanism accounts for the in[1251]DAMG0binding to p-opioid receptors decreased hibitory effect of opioids on accumbal neurons, an alterdramatically after quinolinic acid lesions that de- ation in the coupling of the G protein to potassium stroyed intrinsic neurons, nonspecific damage to the channels could be produced by a loss of dopamine innernucleus accumbens after 6-OHDA should result in a vation. The lack of enhanced motor response to the a-opioid decrease in binding in the lesioned area. The fact that no significant decreases in cresyl-violet staining of me- agonist in 6-OHDA-treated rats was surprising in view dium-sized neurons were observed in the area outside of the findings of Dauge et al. (1988) that specific a-opiof the glial scar caused by the cannulae in the 6-OHDA- oid agonists produce a motor stimulant effect when milesioned rats relative to the sham-lesioned rats sug- croinjected into the nucleus accumbens of normal rats. gests that nonspecific damage was not a cause for the These data may indicate that the cellular mechanism(s) differences in the behavioral responses to opioids. How- by which a-opioid agonists elicit motor activity in the ever, the possibility remains that a small population of accumbens diverges from that of p-opioid and dopa6-OHDA-sensitiveneurons containing opioid receptors mine receptor-stimulated locomotion. Similar to the disappeared, while another opioid-receptive group of stimulation of p-opioid receptors, a-opioid agonists inneurons upregulated their opioid receptors in a manner hibit forskolin-activated adenylyl cyclase (Chneiweiss that counterbalanced the loss. Nonetheless, regardless et al., 1988; Duman et al., 1988). Some studies have of the specificity of the lesion, the 6-OHDA-lesioned shown that a-opioid agonists stimulate the in vivo rerats show opioid-induced augmentation in locomotor lease of dopamine in the nucleus accumbens (Pentney TABLE II. Dopamine and serotonin concentratwns in the nucleus accumbens and surrounding brain areas following sham or 6-OHDA injections into the nucleus accumbens 1,2

*

*

6-OHDA LESION ENHANCES MU-OPIOID RESPONSE

Fig. 7. Cresyi-violetstaining ofthe nucleus accumbens in 6-OHDA- (C,D)and sham- (A,B)lesioned rats adjacent to the glial scar resulting from the injection cannulae (arrow). A higher-power magnification within the inset (A or C) illustrates the morphological integrity of medium-sized neurons (B or D). These sections are representative examples from adjacent sections collected for histological analyses from the sham- and 6-OHDA-lesioned rats in Figure 6.

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Delfs, J.M., Schreiber, L., and Kelley, A.E. (1990) Microinjection of cocaine into the nucleus accumbens elicits locomotor activation in the rat. J. Neurosci., 10:303-310. Dilts, R.P., and Kalivas, P.W. (1989) Autoradiographic localization of p-opioid and neurotensin receptors within the mesolimbic dopamine system. Brain Res., 488:311-327. Dilts, R.P., and Kalivas, P.W. (1990) Autoradiographic localization of delta opioid receptors within the mesocorticolimbic dopamine system using radioiodinated [2-~-penicillarnine,5-D-penicillaminelenkephalin ("'I-DPDPE). Synapse, 6:121-132. Duman, R.S., Tallman, J.F., and Nestler, E.J. (1988) Acute and chronic opiate-regulation of adenylate cyclase in brain: Specific effects in locus coeruleus. J . Pharmacol. Exp. Ther., 246:1033-1039. Esposito, E., Cervo, L., Petrillo, P., Sbacchi, M., Tavani, A,, and Samanin, R. (1987) Dopamine denervation of the nucleus accumbens induces a selective increase in the number of a opioid binding sites. Brain Res., 43625-29. Fallon, J.H., and Moore, R.Y.(1978) Catecholamine innervation of the basal forebrain. IV. Topography of the dopamine projection to the basal forebrain and neostriatum. J. Comp. Neurol., 180545-580. Fink, J.S., and Smith, G.P. (1980) Mesolimbocortical dopamine tefminal fields are necessary for normal locomotor and investigatory exploration in rats. Brain Res., 199:359-384. Gerfen, C.R., Herkenham, M., and Thibault, J . (1987) The neostriatal mosaic. 11. Patch- and matrix-directed mesostriatal dopaminergic and non-dopaminergic systems. J . Neurosci., 7:3915-3934. Hakan, R.L., and Henriksen, S.J. (1989) Opiate influences on nucleus accumbens neuronal electrophysiology: Dopamine and non-dopamine mechanisms. J. Neurosci., 9:3538-3546. Handa, B.K., Lane, A.C., Lord, J.A.H., Morgam, B.A., Rance, M.J., and possessing selective Smith, C.F.C. (1981) Analogues of B-LPH,,,, agonist activity at p-opiate receptors. Eur. J . Pharmacol., 70:531540. Heijna, M.H., Padt, M., Hogenboom, F., Portoghese, P.S., Mulder, A.H., and Schoffelmeer, A.N.M. (1990) Opioid receptor-mediated inhibition of dopamine and acetylcholine release from slices of rat nucleus accumbens, olfactory tubercle and frontal cortex. Eur. J. Pharmacol., 18:267-278. Hong, J.S., Yang, M.-Y., Fratta, W., and Costa, E. (1978) Rat striatal met-enkephalin content after chronic treatment with cataleptogenic and non-cataleptogenic antischizophrenia drugs. J. Pharmacol. Exp. Ther., 205141-147. Hong, J.S., Yang, H.-Y.T., Gillin, J.C., Di Giulio, A.M., Fratta, W., and ACKNOWLEDGMENTS Costa, E. (1979) Chronic treatment with haloperidol accelerates the biosynthesis of enkephalins in rat striatum. Brain Res., 160:192We wish to thank Jenny Baylon for assistance in 195. preparing this manuscript. This work was supported in Kalivas, P.W., and Bronson, M. (1985) Mesolimbic dopamine lesions produce a n augmented behavioral response to enkephalin. Neuropart by U S . Public Health Service grants DA-06612 pharmacology 24:931-936. (L.C.), DA-03906 (P.W.K.),and MH-40817 (P.W.K.). Kalivas, P.W., Widerlov, E., Stanley, D., Breese, G., and Prange, A.J., J r . (1983) Enkephalin action on the mesolimbic system: A dopamine-dependent and a dopamine-independent increase in locomotor REFERENCES activity. J . Pharmacol. Exp. Ther., 227:229-237. Amalric, M., and Koob, G.F. (1985) Low doses of methylnaloxonium in Kelly, P.H., and Iversen, S.D. (19751 Selective 6-OHDA-induced destruction of mesolimbic dopamine neurons: Abolition of psychostimthe nucleus accumbens antagonize hyperactivity induced by heroin ulant-induced locomotor activity in rats. Eur. J . Pharmacol., 40:45in the rat. Pharmacol. Biochem. Behav., 23:411-415. 56. Angulo, J.A., Cadet, J.L., Woolley, C.S., Suber, F., and McEwen, B.S. (1990) Effect of chronic typical and atypical neuroleptic treatment Koob, G.F., Stinus, L., and Le Moal, M. 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