Lesions of the Nucleus Accumbens in Rats Reduce ... - APA PsycNET

Copyright 1989 by the American Psychological Association, IDC. 0735-7044/89/J00.75

Behavioral Neurosaence 1989, Vol. 103, No. 6. 1327-1334

Lesions of the Nucleus Accumbens in Rats Reduce Opiate Reward but Do Not Alter Context-Specific Opiate Tolerance

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John E. Kelsey, William A. Carlezon, Jr., and William A. Falls Bates College Bilateral electrolytic lesions of the nucleus accumbens in rats eliminated the capacity of 10 mg/kg morphine to produce a conditioned place preference (Experiment 1). However, these lesions did not alter the capacity to establish context-specific tolerance to the analgesic effects of 5 mg/kg of morphine (Experiment 2). This latter finding indicates that rats with nucleus accumbens lesions are not impaired in associating the effects of morphine with a particular location. Thus, the failure of morphine to produce a conditioned place preference in these lesioned rats probably cannot be attributed to an inability to associate the effects of morphine with a particular chamber, i.e., the initially nonpreferred chamber. Rather, morphine may fail to establish a conditioned place preference in these rats because nucleus accumbens lesions disrupt a pathway that is critical in mediating the rewarding effects of opiates.

Wise and Bozarth (1984) have argued that opiates, such as morphine, are rewarding, partly because they stimulate the dopamine-releasing neurons in the ventral tegmental area (VTA) that project rostrally to the nucleus accumbens of the forebrain (Lindvall & Bjorklund, 1974). Implicating the VTA, Bozarth and Wise (19 81) found that rats learn to press a lever for injections of morphine directly into the VTA but not for injections into other areas that also contain high densities of opiate receptors, such as the periventricular gray and caudate nucleus (Bozarth & Wise, 1982). Similarly, Phillips and LePiane (1980) demonstrated that injections of morphine directly into the VTA were sufficient to establish a conditioned place preference, whereas injections dorsal to the VTA were not. Evidence implicating the dopamine projection from the VTA to the nucleus accumbens, however, is not as compelling. For example, damage to the cell bodies of the nucleus accumbens by intra-accumbens injections of kainic acid has been reported to reduce intravenous morphine and heroin self-administration (Dworkin, Guerin, Goeders, & Smith, 1988; Zito, Vickers, & Roberts, 1985). However, Pettit, Ettenberg, Bloom, and Koob (1984) reported that more specific damage to the dopamine neurons that terminate in the nucleus accumbens by intra-accumbens injections of 6-hydroxydopamine (6-OHDA) did not markedly alter intrave-

Experiment 1 was based on research submitted to Bates College by William A. Carlezon, Jr., in partial fulfillment of the requirements of a BS degree in biopsychology. The data from both experiments were presented at the Society for Neuroscience Convention in New Orleans, Louisiana, 1987. William A. Carlezon, Jr., is now at the Department of Biological Research, Hoechst-Roussel Pharmaceuticals, Somerville, New Jersey. William A. Falls is now at Yale University, New Haven, Connecticut. Correspondence concerning this article should be addressed to John E. Kelsey, Department of Psychology, Bates College, Lewiston, Maine 04240.

nous heroin self-administration. On the other hand, Spyraki. Fibiger, and Phillips (1983) reported that intra-accumbens injections of 6-OHDA reduced the ability of systemic heroin to produce a conditioned place preference. However, this reduction was modest, and in the absence of a lesioned group not injected with heroin, it is difficult to interpret. To further examine the hypothesis that the nucleus accumbens is involved in mediating some of the rewarding effects of opiates, in Experiment 1 we compared the ability of both systemic morphine and saline to produce a conditioned place preference in rats with lesions of the nucleus accumbens. In Experiment 2, we examined the alternative hypothesis that, rather than specifically reducing opiate reward, nucleus accumbens lesions reduce the ability to associate the consequences of morphine with a particular environment

Experiment 1: Conditioned Place Preference In this experiment, we examined morphine's capacity to produce a conditioned place preference as an assay of the drug's rewarding effects so that we could avoid some of the practical and theoretical problems associated with other methods, such as self-administration (e.g., Mucha, van der Kooy, O'Shaughnessy, & Bucenieks, 1982). To develop a conditioned place preference, rats are typically allowed to establish a preference for one of two or three interconnecting chambers. The rats are then injected with a presumably rewarding drug, such as morphine, while they are confined to their nonpreferred chamber and then are retested for preference after the effect of the drug has worn off. The assumption is that the association of the rewarding properties of the drug with the nonpreferred chamber will cause the rats to increase their preference for that chamber. Thus, if the nucleus accumbens is involved in mediating some of the rewarding effects of morphine, then lesions of the nucleus accumbens should reduce the capacity of morphine to act as a reward and, thus, to produce a conditioned place preference. 1327

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J. KELSEY, W. CARLEZON, JR., AND W. FALLS

To be certain that changes in place preference were due to the morphine, some rats were injected with saline, instead of morphine, while being confined to the nonpreferred cham-

ber.


Method Subjects. The 48 naive male Sprague-Dawley rats weighed 320440 g at the time of surgery and were housed individually in clear Plexiglas cages with wood shavings for bedding in a colony that was lighted from 7 a.m. to 7 p.m. each day. Food and water were available ad lib except during testing. Surgery. The rats were anesthetized with an injection of sodium pentobarbital (6 5 mg/kg i.p.) supplemented with ethyl ether. Bilateral lesions of the nucleus accumbens were performed in 24 rats by passing 1.0 mA of anodal current for 15 s through stereotaxically positioned electrodes (AP = 3.2 mm anterior to bregma, H = 7.2 mm below the dura mater, and L = ±1.7; Peflegrino & Cushman, 1967). The electrodes were no. 1 insect pins insulated with Epoxylite except for the flattened tip. The remaining 24 rats received one of three types of sham operations. Ten rats underwent surgery as described for the lesions, except that the electrodes were lowered only 6.2 mm below the dura mater and no current was passed through the electrodes. Nine rats received identical operations, except that the electrodes were not lowered into the brain, and 5 rats were simply anesthetized. Apparatus. The 20 x 49 x 31 cm shuttle box was made of galvanized steel except for the clear Plexiglas top. The box was divided into two 20 x 24.3 cm interconnecting chambers by a partition that had a 7 x g cm opening in the center flush with the grid floor. Except for the Plexiglas top and grid floor, one chamber was painted black, and the other chamber was left the natural silver-gray tone of the metal. The silver-gray side was illuminated by a 6-W bulb housed on the center of the Plexiglas top, and a steel mesh screen with 1.0 x 1.0 cm openings was placed over the grid floor on that side. The grid floor was made of 0.6-cm-diameter stainless steel rods separated by 2.0 cm center to center and running across the width of the two chambers. This floor pivoted around a bearing located under the partition and was supported on both ends by adjustable weights and switches. The weights were adjusted for each rat so that when the center of the rat was between the fifth and sixth grids from the center of the box, the floor pivoted and tripped the switch on the opposite end, signalling a crossing response. Speakers located at both ends of the box supplied white noise of 75 dB (SPL). The room containing the shuttle box was illuminated by a 25-W red bulb. Procedure. One to 4 weeks after surgery (M ± SE = 15.8 ± 1.0 days), each rat was weighed and transferred to the experimental room at approximately the same time every day (1-4 p.m.). After a S-min adaptation period during which the shuttle box was cleaned with 0.003 M acetic acid, each rat was placed into the silver-gray, lighted chamber facing toward the passage into the black chamber. A microprocessor automatically recorded the number of crossing responses and the cumulative time (to the nearest 0.1 s) spent in each chamber during each 15-min (900-s) session. Preference testing was continued daily in this fashion until each rat established a stable preference for one chamber for 2 consecutive days. Rats were assumed to have a preference when they spent at least 67% of the time (more than 600 s) in one chamber (black or silver gray). Stable preference was denned as less than 200 s of variation between the 2 days. On the following day, approximately half of the rats in each group were injected with 10 mg/kg i.p. morphine sulfate (0.67 cc/kg), and the other half were injected with an equal volume of isotonic saline. After 15 min, each rat was transferred to the experimental room for

a 5-min adaptation period. Each rat was then confined to its nonpreferred chamber for 30 min. On the next day, all rats were injected with 0.67 cc/kg i.p. isotonic saline, moved to the experimental room after 15 min, and confined to the previously preferred chamber for 30 min after a 5-min adaptation period. Changes in preference were assessed on the next (and final) day by following the procedure used during initial assessment of place preference. Each rat was taken from the colony, and after a 5-min adaptation period in the experimental room, each was placed in the middle of the silver-gray, lighted chamber facing the black chamber and allowed to cross freely between the two chambers for 15 min. Histology. After testing, the lesioned rats were killed with ethyl ether and perfused intracardially with isotonic saline followed by a 10% formol-saline solution. Every fourth 64-pm-thick frozen section in the area of the lesion was saved, stained with cresyl violet, and examined under a microscope. Data analysis. Because there were no differences hi the behavior of the three sham-operated groups, their data were combined for subsequent analyses. The stability of the initial place preference as measured by the time spent in the nonpreferred chamber during the last 2 criterion days was analyzed by a Lesion (control and nucleus accumbens lesion) x Drug (saline and morphine) x Days (2) analysis of variance (ANOVA) with repeated measures on days. Subsequent analyses used the average for these 2 criterion days for each group. Changes in place preference as measured by the time spent in the nonpreferred chamber were analyzed by a Lesion (2) x Drug (2) x Days (initial and final preference) ANOVA with repeated measures on days.

Results Histology.

The lesions produced moderate damage to the

anterior portion of the nucleus accumbens surrounding the anterior commissure (see Figure 1), and thus were likely to have damaged many of the dopamine terminals arising from the VTA (Fallon & Moore, 1978; Lindvall & BjSrklund, 1974). The lesions were centered at AP = 9.4, H = -1.0, and L = ±2.0 (Pellegrino & Cushman, 1967). Damage to the overlying striatum and the underlying preoptic area was minimal. The data from 6 rats were eliminated from the analyses because the lesions of these rats extended well outside the nucleus accumbens. Behavior.

The rats established a stable preference (mean

difference of 74.7 + 8.8 s between the 2 criterion days) for a chamber of the shuttle box in 3.2 ± 0.2 days (M ±

SE).

There were no significant differences among the groups in the number of days required to establish a preference or in the stability of the preference. Also, there were no significant differences among the four groups in their initial preference for the black chamber (see Figure 2). In fact, none of the four groups had an average initial preference that differed from chance (450 s; ps > .20), indicating that approximately half of the rats in each group preferred the black chamber, whereas the other half preferred the silver-gray chamber (see Figure 2). However, when the preference for each rat was expressed in terms of the time spent in the nonpreferred chamber, whichever chamber that was, the rats in all four groups had strong and statistically similar initial preferences, spending approximately 22% of the time in their nonpreferred chamber (see Figure 3). Further analysis of these place preference data (see Figure

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NUCLEUS ACCUMBENS LESIONS REDUCE OPIATE REWARD

80

Figure I. Reconstructions of the smallest (solid), typical (dotted), and largest (hatched) lesions of the nucleus accumbens. (FromA Stereotaxic Alias of the Rat Brain(pp. 1, 9, 11, 13, 15, 17, 19,and21) by L. J. Pellegrino and A. J. Cushman, 1967, New York: Appleton-Century-Crofts. Copyright 1967 by Appleton-Century-Crofts. Adapted by permission.)

3), revealed a marginally significant Lesion x Drug x Days interaction, F(l, 38) = 3.27, p < .08. A subsequent simple Drug x Days interaction, F(l, 38) = 11.51,p *MD

45

60

TIME AFTER MORPHINE Figure 4. Tolerance to the analgesic effects of 5 mg/kg morphine in all four groups as measured by pain reactivity. (The error bars reflect ± 1 SE.)

either 0.33 cc/kg s.c. saline (paired animals) or 5 mg/kg s.c. morphine in an equal volume (unpaired animals) and returned to their home cages. This procedure was followed for 4 consecutive days, with the exception that pain reactivity was measured only on the 1st day. On the 5th day, all of the rats were placed in the restraining tubes and taken to the test environment as on the previous days. All rats were then injected with 5 mg/kg s.c. morphine sulfate (0.33 cc/kg) and tested for pain reactivity 15, 30, 45, and 60 min after the injection. At each interval, each rat received three trials spaced 1 min apart, and the latencies for the three trials were averaged. Data analysis. Because there were no differences in behavior between the rats run in Experiment 1 and the rats run only in this experiment, we combined the data to form four groups. Tail-flick latencies after morphine treatment on Day 5 were analyzed by a Lesion (control and nucleus accumbens lesion) x Pairing (paired and unpaired) x Time After Morphine (15, 30, 45, and 60 min) ANOVA with repeated measures on time.

The lack of a significant main effect of the lesion or a significant Lesion x Pairing interaction CFs < 1.0) indicated that this context-specific tolerance to morphine was not altered by lesions of the nucleus accumbens. The paired rats with nucleus accumbens lesions were as tolerant to the analgesic properties of morphine as were the paired sham-operated control animals, and the rats of both paired groups were equally more tolerant than were the rats of both unpaired groups (see Figure 4). A significant main effect of pairing, F(l, 28) = 7.24, p < .05, indicated that, by chance, the paired rats weighed less than the unpaired rats (see Table 3). A significant Lesion x Days interaction, F(l, 28) = 10.78, p < .01, indicated that the control rats lost more weight than did the rats with nucleus accumbens lesions during the 5 days of this experiment (see Table 3).

Results

Discussion

Histology. The lesions were similar to those described in Experiment 1 (see Figure 1). The data from 3 rats were eliminated from the analyses because the lesions of these rats extended below the nucleus accumbens. Two of the rats already had been eliminated from Experiment 1. Behavior. There were no differences among the four groups in baseline reactivity to pain before the drug injections (see Figure 4). However, after 4 days of morphine injections, the rats that had received morphine in the testing environment (paired animals) were more tolerant to morphine in that environment than were the rats that had received morphine in the colony (unpaired animals). In particular, a significant main effect of pairing, F(l, 28) = 11.62, p < .01, indicated that 5 mg/kg morphine produced less analgesia in the paired rats than it did in the unpaired rats (see Figure 4).

Clearly, the procedure used in the experiment was sufficient to produce context-specific tolerance to the analgesic effects of morphine. The rats tested for pain reactivity in the environment in which they had received morphine (paired animals) were more tolerant to the analgesic effects of morphine than were the rats that had received the same injections of morphine in a different environment (unpaired animals). To examine the possibility that this context-specific tolerance was due to the coincidental differences in body weight between the paired and unpaired rats, we eliminated the lightest rat from each of the two paired groups and the heaviest rat from each of the two unpaired groups. This selection eliminated the differences in weight between the paired and unpaired rats but did not alter the significant main effect of pairing on tail-flick latencies after morphine treatment, F(l,

NUCLEUS ACCUMBENS LESIONS REDUCE OPIATE REWARD Table 3 Body Weight on the First and Last Days of Testing for Context-Specific Tolerance Group

First day

Last day

Control Paired Unpaired

431.6 ± 12.4 466.0 ± 9.8

406.6 ± 10.2 428.5 ± 14.7

Lesion Paired Unpaired

402.2 ± 15.9 443.0 ± 18.6

395.4 ± 11.3 430.0 ± 13.7


Note. Values are in grams (M ± SE); n = 8 for each group.

24) = 12.18, p < .01. Thus, the occurrence of context-specific tolerance clearly did not depend on initial differences in body weight. More important is the finding that nucleus accumbens lesions had no effect on the ability to acquire context-specific tolerance. Insofar as the ability to acquire context-specific tolerance requires the ability to associate the effects of morphine with a particular context (Baker & Tiffany, 1985; Paletta & Wagner, 1986; Siegel, 1979, 1983), these data indicate that rats with nucleus accumbens lesions are as capable as control rats of learning to associate the consequences of morphine with a particular environment. The additional finding that the rats with nucleus accumbens lesions lost less weight than did the controls during tolerance development suggests that morphine has less of a weight- or appetite-suppressing effect on rats with nucleus accumbens lesions. Thus, these data are consistent with the data of Experiment 1, which indicated that rats with nucleus accumbens lesions were less affected by the weight-reducing effects of morphine.

General Discussion Experiment 1 demonstrated that moderately sized bilateral lesions of the nucleus accumbens eliminated the capacity of morphine to produce a conditioned place preference. Experiment 2 indicated that these lesions did not alter the capacity to produce context-specific tolerance to the analgesic effects of morphine. Insofar as the acquisition of context-specific tolerance depends on the ability to associate the effects of morphine with a particular environment, the latter finding indicates that nucleus accumbens lesions do not interfere with the formation of context-morphine associations. Therefore, the failure of morphine to establish a conditioned place preference in the rats with nucleus accumbens lesions in Experiment 1 probably was not due to a more general deficit in the ability to associate the effects of morphine with a particular environment, that is, the initially nonpreferred chamber. However, note that the conditioning procedures used to produce context-specific tolerance in the second experiment differed in several ways from those used to produce a conditioned place preference in the first experiment. Thus, for example, it is possible that nucleus accumbens lesions interfered with context-morphine associations after one conditioning trial (Experiment 1) and this associative deficit was

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overcome after three additional trials (Experiment 2). However, the finding that 6-OHDA lesions of the nucleus accumbens reduced the capacity of 2.0 mg/kg heroin to induce a place preference even after four conditioning trials (Spyraki et al., 1983) suggests that this possibility is unlikely. Consequently, by indicating that lesions of the nucleus accumbens apparently do not disrupt the formation of contextmorphine associations, this study indirectly strengthens the hypothesis that nucleus accumbens lesions reduce the ability of morphine to establish a conditioned place preference because they destroy neurons that are essential for the rewarding effects of opiates. If this interpretation is correct, our study adds to the growing literature that one of the roles of the nucleus accumbens is to mediate the rewarding effects of opiates (Spyraki et al., 1983; Wise & Bozarth, 1984). Further research is needed to determine whether these effects of nucleus accumbens lesions are due to damage to the dopaminergic fibers that project from the VTA (Spyraki et al., 1983; Wise & Bozarth, 1984), to damage to opiate-sensitive cell bodies in the nucleus accumbens (e.g., Olds, 1982; Vaccarino, Bloom, & Koob, 1985), or to damage to other systems (Spyraki, Nomikos, Galanopoulou, & Daifotis, 1988). Further research is also needed to determine whether the nucleus accumbens is involved in mediating other effects of opiates, such as analgesia and withdrawal. The failure of nucleus accumbens lesions to alter baseline pain reactivity or the analgesic effects of morphine in this experiment suggests that the nucleus accumbens is not involved in mediating the analgesic effects of opiates. The additional finding that nucleus accumbens lesions did not alter the development of context-specific or context-independent tolerance suggests that this area is not involved in mediating opiate tolerance and therefore may not be involved in mediating opiate withdrawal. These data suggest that the nucleus accumbens may be rather specifically involved in mediating the rewarding (and weight-reducing) effects of opiates.

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Received September 29, 1988 Revision received May 9, 1989 Accepted June 14, 1989