Differential effects of morphine andd -amphetamine on self-stimulation from closely adjacent regions in rat midbrain

Brain Research, 136 (1977) 103-117

103

~ Elsevier/North-Holland Biomedical Press

D I F F E R E N T I A L E F F E C T S OF M O R P H I N E A N D D - A M P H E T A M I N E ON S E L F - S T I M U L A T I O N F R O M CLOSELY A D J A C E N T R E G I O N S I N R A T MIDBRAIN

JEFFREY LIEBMAN* and DAVID S. SEGAL Department of Psychiatry, School of Medicine, University of Cali]brnia at San Diego, La Jolla, Calif. 92093 (U.S.A.)

(Accepted February 23rd, 1977)

SUMMARY The effects of morphine were investigated on self-stimulation from numerous electrode placements in the area of the substantia nigra or in the ventral half of mesencephalic central gray matter. Before pharmacological testing, current intensity was reduced to yield stable, submaximal rates of self-stimulation. Rats were then injected daily with morphine for l0 days, and were tested three hours after injection. Between days 5 and 10 of treatment, many rats self-stimulated at more than 1 5 0 ~ of baseline, but some others reduced self-stimulation to as little as 3 ~o of baseline. Histological evaluation revealed that morphine facilitated self-stimulation when the electrode tip was located more than 0.3 m m from substantia nigra or more than 0.2 m m from the midline of central gray. In rats with electrode tips closer to substantia nigra or to the midline of central gray, morphine often reduced or failed to alter self-stimulation rates. The effects of a low dose of D-amphetamine (0.1 mg/kg) were investigated on electrode placements in the substantia nigra area. Placements close to the dorsal border of substantia nigra yielded less facilitation of self-stimulation by D-amphetamine than did placements located more dorsally or medially. Possible catecholaminergic substrates of these differential effects are discussed.

INTRODUCTION The initial demonstration that morphine facilitates intracranial self-stimulation (SS) from the lateral hypothalamus 2~ indicated that SS may be a valuable approach * Now at CIBA-GEIGY Pharmaceutical Co., Division of CIBA-GEIGY Corporation, Department of Pharmacology, 556 Morris Avenue, Summit, N.J. 07901, U.S.A. All correspondence should be addressed to Jeffrey Liebman.

104 to the investigation of morphine's presumed involvement with brain reward mechanisms. Acute, low doses of morphine facilitate SS shortly after injection 11, but moderate to high doses of this narcotic (5-20 mg/kg) enhance SS only upon repeated administration 26. This pronounced facilitatory effect succeeds an initial, depressant phase which lasts for one to two hours after injection and which dissipates with repeated administration 22,26. Morphine- or heroin-induced increases in the rate of SS appear to be unrelated to the concurrent effects of these narcotics on locomotor activity17, z6 particularly since morphine also lowers the current intensity threshold for SS (see ref. 27). In a previous investigation 23, several animals unexpectedly failed to increase SS in substantia nigra (SN) after repeated morphine administration. Responding by these rats declined appreciably during treatment, but returned to pre-morphine levels after cessation of chronic morphine. We have confirmed and extended these observations, and we now report that morphine and D-amphetamine differentially influence SS in closely adjacent brain regions within the SN area, and that morphine similarly differentiates the medial and lateral parts of the mesencephalic central gray matter (CG). EXPERIMENT 1 Morphine was administered daily to rats who self-stimulated from electrodes which had been aimed at SN. The facilitatory effects of morphine on SS were evaluated as a function of electrode placement. Methods

The experimental subjects were 47 male Sprague-Dawley rats (Carworth Farms), 100-120 days old at the time of surgery. Electrodes were implanted bilaterally in SN according to previously described methods 22. Brain stimulation was delivered from an AC power supply through 2 M~] resistance in series with the rat's electrode. A cable and commutator system allowed the animal free movement at all times. The experimental apparatus was similar to that used previously 22. At least one week after surgery, rats were screened for SS performance. During screening, optimal current intensities (typically 40-60 # A r m s ) were used, and the maximal SS rate was determined. Rats which attained a criterion SS rate of at least 1100 bar-presses per 30 min through at least one electrode were used in pharmacological experiments. In some cases, SS through either electrode satisfied the above criterion, and for these rats the electrode which was used for pharmacological testing was arbitrarily selected. None of the experimental animals required priming to initiate SS, and they were never primed during test sessions. All test sessions took place once daily except for weekends, and lasted 30 min. After initial screening for SS, the current intensity was reduced in succeeding sessions so that the SS rate was considerably below the maximum rate elicited by high current intensities. Drug testing began after the animal responded at a rate of between 400 and 1000 bar-presses per 30 min in at least three consecutive test sessions. The current

105 intensities thus derived generally did not exceed 40 #A and typically ranged from 10 to 25 #A. Morphine sulfate (Mallinckrodt) was dissolved in bacteriostatic saline and injected subcutaneously three hours before testing on the basis of a ml/kg volume. Thirtyfive of the subjects received 15 mg/kg morphine daily for 9 days and were tested on days 1, 3, 5, 7 and 9 of treatment. Because two of these animals died of apparent respiratory difficulty before tolerance developed, the dose regimen was modified in 12 additional rats in an attempt to minimize this occurrence. These rats received 10 mg/kg, on day 1, 12.5 mg/kg on day 2 and 15 mg/kg on days 3-10. In these rats, the last two tests took place on days 8 and 10. After cessation of morphine treatment, behavioral testing was continued for at least one week to verify the reversibility of the drug effects. As previously reported23,25, 26, the facilitatory effects of morphine reached a maximal level on the fifth day of treatment, and continued daily administration of morphine did not further augment SS. Whenever morphine reduced SS, this effect persisted through days 5-10 of treatment. Therefore, for purposes of analysis, the SS rates recorded during the last three test sessions were combined and averaged. This average rate was expressed as a percentage of the response rate during the last baseline session prior to morphine treatment. After completion of testing, rats were sacrificed and electrode placements were verified as previously described2L Brain sections were examined without prior knowledge of the behavioral results from the electrode placements in question. Results For various reasons, it was necessary to exclude the results of some animals. One rat failed to resume SS after cessation of morphine treatment. Histological results were not available for another animal for reasons unrelated to the experimental treatment. Three additional animals died of respiratory difficulty during initial morphine treatment. No deaths or overt toxic effects occurred after the third day of treatment. Examination of the histological results showed that the electrode tips for three animals lay within or anterior to the AP ÷ 3430 plane of KSnig and KlippeP 6, which is more than 0.5 mm anterior to SN. These rats were also excluded from further analysis, leaving 39 subjects which yielded usable results. Their electrode placements are shown in Fig. 1. Numerous electrode placements which were close to the dorsal boider of SN failed to yield substantial increases in SS during morphine treatment, although some exceptions were apparent (Fig. 1). In fact, several animals reduced SS strongly in the absence of overt toxic effects during testing. Among placements farther from SN, morphine almost invariably increased SS to 160~ or more of baseline of SN. To evaluate this trend quantitatively, the distance between the center of each electrode tip and the nearest point on the dorsal surface of SN was measured for all electrodes which were located in or between the ~ 1610 and -b 2790 planes of KSnig and KlippeP 6. The dorsal surface of SN was defined as the dorsal border of the zona reticulata, since this margin is readily visualized in all sections. No placements in the present

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Fig. 1. Electrode placements of morphine-treated animals with electrodes in the SN area. At each placement, the self-stimulation rate after morphine treatment is shown as a percentage of the corresponding baseline rate. Frontal sections are from K6nig and KlippelTM. experiments fell within the zona reticulata o f SN. The + 1 6 1 0 and 72790 frontal planes enclose S N in its r o s t r o - c a u d a l extent. In Fig. 2, the distances from S N (z.r.) are plotted against per cent change in SS d u r i n g m o r p h i n e treatment. A p o s t hoc analysis o f the trend was p e r f o r m e d by dividing the placements into two g r o u p s : those located no m o r e t h a n 0.3 m m from S N (designated as the N e a r group) and those located at least 0.4 m m from this stucture (the D i s t a n t group). Because o f heterogeneity o f variance, the Behrens t-test a6 was used. F a c i l i t a t i o n o f SS by m o r p h i n e was significantly less p r o n o u n c e d in the N e a r g r o u p t h a n in the Distant g r o u p (X• = 123.5, S.E.M. 17.5; XD = 241.8, S.E.M. ---- 20.6, t - - 3.57, P 0.01, two-tailed comparison). The effects o f m o r p h i n e were n o t related to the distance o f the electrode from midline o r from medial lemniscus. Similarly, the frontal plane in which the electrode

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from the dorsal border of SN (zona reticulata). was located was unrelated to the effects of morphine. The effects of morphine on SS also did not vary as a function of baseline SS rate, maximal SS rate attained during initial SS training, or the current intensity used for pharmacological testing. Discussion

The effects of morphine on SS three hours after injection were clearly related to the distance of the SS electrode from the dorsal surface of SN. Morphine typically increased SS to more than 160~o of baseline if the electrode was more than 0.4 m m from this structure. In contrast, morphine often failed to increase, and sometimes actually reduced, SS among rats with electrodes closer to SN. It is important to emphasize that, after cessation of morphine treatment, SS from all placements in Fig. 1 returned to baseline levels. EXPERIMENT 2 In an attempt to extend these findings to other brain regions, an additional experiment was performed. The entire ventral half of caudal mesencephalic central gray yields high rates of SS 21,3~. This region contains the medially situated dorsal raphe nucleus, within which serotonergic cell bodies are packed. It is, however, neuropharmacologically heterogenous, as catecholaminergic fibers are distributed throughout this area 24,a9. SS electrodes were aimed at the medial and lateral portions of this structure and the effects of morphine on this behavior were investigated. Methods

The 25 experimental rats used in this experiment were of the same stock as in Experiment l, except that it was necessary to obtain 8 of the animals from Hilltop as the previous supplier ceased production of this strain. The stereotaxic coordinates

108 for surgery were: AP, --0.2 to --0.8 from zero; L, q-0.1 and 0.9 bilaterally; D, 3.5 m m dorsal to zero by reference to Pellegrino and Cushman 29. Surgical procedures were otherwise identical with Experiment 1. The experimental apparatus and procedures were similar to those of Experiment 1. All but 4 of the experimental subjects bar-pressed at a maximal rate which exceeded 1000/30 min. For these 4 rats, the current intensity was reduced so as to yield a barpressing rate between 25 % and 75 % of the maximum rate observed during screening (which exceeded 700/30 min in all cases). The remaining animals were tested at a baseline rate of between 400 and 1000 bar-presses per 30 min, as in Experiment 1. Morphine sulfate was prepared and injected as in Experiment 1. All rats received 10 mg/kg on day 1, 12.5 mg/kg on day 2 and 15 mg/kg daily on days 3-10. They were tested three hours after injection on days 1, 3, 5, 8 and 10 of treatment. It seemed possible that the tendency of some animals to reduce SS three hours after morphine injection might not have been present at other post-injection intervals. Therefore, two rats which had strongly reduced C G SS three hours after treatment were also tested 1.5 and 5 h post-injection. Each continued to receive 15 mg/kg morphine daily between the 10th and 15th days of treatment. On the l lth and 15th days, each was tested at either 1.5 or 5 h after injection, in counterbalanced order. For comparison, the animals were tested at three hours after injection during the intervening days. Five additional rats which increased SS during morphine treatment also continued to receive morphine daily through the 15th day, and they were tested 1.5 h after injection during one session between days 10 and 15. Data analysis and histological evaluation were performed as in Experiment 1. Results One rat failed to resume normal baseline responding after cessation of morphine treatment, and was therefore excluded from the experimental results. The results were comparable for animals from either commercial supplier, and were therefore combined. Electrode placements are shown in Fig. 3. Two placements were either rostral or caudal to the dorsal raphe level of the C G (frontal planes -~0.6 and --1.2, Fig. 3). The distances from midline of the remaining placements were measured as a rough estimate of the distance from the dorsal raphe. These distances are plotted in Fig. 4 against the effects of morphine on SS. Morphine increased SS by more than 160 % in all but one of the electrode placements which were located 0.3 m m or farther lateral to the midline of CG. In contrast, morphine increased SS in only two of the 11 placements which were within 0.25 mm of midline CG. Other placements near the midline manifested strong decrements in SS without any apparent, concomitant toxicity between days 5 and 10. In a post hoc analysis of these results, the placements located within 0.25 mm of midline were grouped and compared with those farther lateral. The two groups differed significantly with respect to the effects of morphine on SS ( X m i d l i n e : - 86.2, S.E.M. = 24.5; X l n t e r a l - - 213.4, S.E.M. -- 39.2; t = 2.62, P < 0.025, two-tailed Student's t-test). Theeffects of morphine on SS did not vary systematically with the anterior-posterior location of the electrode, the baseline SS rate, or the current intensity at the

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110 Discussion

When the CG SS electrode was located at least 0.3 mm from the midline, SS was strongly facilitated by chronic morphine treatment. In contrast, more medially situated electrodes generally failed to yield increases in SS during morphine treatment. In some of these rats, morphine actually induced strong reductions in SS which dissipated several days after cessation of chronic drug treatment. The question arises of whether the placements could be reliably differentiated according to whether or not they were within the dorsal raphe. A number of placements (Fig. 3) were clearly outside the lateral-most extension of the dorsal raphe by up to 0.8 mm, and all but one of these placements yielded an increase in SS after treatment with morphine. Most of the placements which were within or on the border of the dorsal raphe yielded no increase or an actual reduction of SS when morphine was administered. However, seveial exceptions to this trend among the dorsal raphe placements are apparent (Fig. 3). The actual distance of the electrode from the midline of the central gray, rather than whether or not it was located in dorsal raphe, appeared to be most closely related to the lack of effect of morphine, it should be noted that electrodes which are directly situated on the midline would have been expected to stimulate a relatively greater proportion of the dorsal raphe than would the electrodes situated on its lateral border. Thus, the results are consistent with the possibility that morphine reduces SS from the dorsal raphe. The results may also indicate, however, functional anatomical differentiation within ventral central gray, which may not be closely related to dorsal raphe per se. It may be of interest that the one laterally placed electrode which failed to yield an increase in SS was farther lateral than any'other electrode. Therefore, it is possible that the lateral CG zone within which morphine increases SS may be circumscribed laterally as well as medially. These results recall the recent report that morphine has differential effects on e555.5

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111 lateral hypothalamus and medial frontal cortex SS 25. It should be noted, however, that under the same conditions as in the present experiment (15 mg/kg, testing three hours after injection, 5 days of chronic treatment), Lorens 25 found that SS increased significantly in both the medial frontal cortex and lateral hypothalamus. Therefore, the effects of morphine on SS from medial frontal cortex may not be similar to its effects on SS in SN proper or in medial CG. However, the methodology of Lorens 25 differs from that of the present study in several respects, and any comparisons between these results and the present experiments must necessarily be regarded as tentative. Interestingly, D- and L-amphetamine have been reported to exert differential effects on medial and lateral portions of the dorsal tegmentum s. According to these authors, D-amphetamine facilitates SS more strongly from the dorsal tegmentum lateral and ventral to CG than from the medial portion of CG. Moreover, medial frontal cortex and lateral hypothalamus SS also differ with respect to the effects of D-amphetamine 2. It, therefore, appeared pertinent to determine whether D-amphetamine exerts differential effects on SS from electrode placements adjoining SN proper, as opposed to the surrounding area in ventral tegmentum. To evaluate this possibility, the anatomical results from previous investigations of D-amphetamine on SN SS (see refs. 20 and 22) were re-examined, and an additional experiment was conducted. EXPERIMENT 3 Examination of the SN SS electrode placements which had undergone testing with low doses of D-amphetamine in two previous investigations 2°,22 revealed a hitherto undetected trend. Generally, little or no facilitation of SS resulted from treatment with D-amphetamine at a low dose if the electrode tip was very close to SN, but considerable facilitation was observed from electrodes which were farther from SN. To confirm and extend these results, an additional group of rats was tested with a low dose of D-amphetamine. The treatment conditions were comparable to those of Liebman and Sega122 except that treatment with this drug was acute rather than chronic and was preceded and followed by saline controls. Because the peak effects of D- and L-amphetamine on motor activity and SS occur at different post-injection intervals, according to Segal (ref. 34 and unpublished observations), these two isomers were not compared in the present investigation. Methods The rats were prepared for SS testing by the same procedure as in Experiment 1. Of the 12 rats used in this investigation, 5 had previously served in Experiment 1. Pharmacological testing began after three days of stable baseline responding (400-1000 bar-presses per 30 rain). All injections were given subcutaneously 30 min before testing in a ml/kg volume, Bacteriostatic saline was injected prior to the daily test sessions which preceded and followed D-amphetamine treatment. D-Amphetamine (0.1 mg/kg, expressed as the base) was given in bacteriostatic saline. The SS rates during the

112 saline sessions which preceded and followed treatment did not differ, and these sessions were therefore averaged. The effect of D-amphetamine on SS was expressed as a percentage of the average saline SS rate. Histological evaluation was performed as in Experiment 1.

Results A careful comparison of the results from the present rats with those from the 8 animals who received 0.1 mg/kg D-amphetamine in a previous investigation of SN SS (see ref. 22) revealed a similar relationship between electrode placement and drug effect. Both experiments had been performed in the same behavioral apparatus under almost identical conditions. Therefore, these results were combined and are shown in Fig. 5. To facilitate comparison with the present results, the effects of 0.1 mg/kg D-amphetamine 22 were computed as acute rather than chronic effects. For this pur-

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pose, the response rate on the first day of treatment was expressed as a percentage of the rate during the last preceding baseline, and the results for the remaining chronic treatment period were disregarded. The electrode placements which closely adjoined SN appared to be less sensitive to facilitation of SS by D-amphetamine than were more dorsally or medially situated electrodes (Fig. 5). The distance of the tip from the dorsal border of SN was measured and plotted as in Experiment 1 (Fig. 6). The results from the electrode placements located within 0.2 m m of SN were grouped on a post hoc basis and tested against placements distant from SN (0.3 m m or farther). This comparison yielded a significant difference (XNear = 112.3, S.E.M. ---- 7.5; X D i s t a n t = 187.7, S.E.M. -- 20.0; t ---- 2.81, P < 0.05, two-tailed Student's t-test). It was also noted that medial electrode placements (which were largely distant from SN) tended to yield greater enhancement of SS following D-amphetamine, as had been previously reported 2°. This trend was tested by comparing the 6 most medial placements with the others. This comparison also yielded a significant difference in the effects of D-amphetamine on SS (XLat ---- 131.7, S.E.M. = 9.1; XMed ----209.1, S.E.M. -- 31.0; t = 2.94, P < 0.025). N o other relationship was apparent between electrode placement and drug effect, nor were baseline or maximal response rates related to the effects of D-amphetamine. Discussion

A low dose of D-amphetamine (0.1 mg/kg) preferentially facilitated SS from electrodes which were more than 0.2 m m distant from the dorsal border of SN. Electrodes which were closer to the dorsal border of SN failed to yield enhanced SS upon administration of this dose of D-amphetamine. Herberg et al. za investigated the comparative effects of D- and L-amphetamine on SS from the SN area. According to their description of the results, D-amphetamine had a relatively weaker facilitatory effect on SS from two placements that appeared to be within SN, as compared with 4 placements which were located dorsal to SN

114 proper. Thus, their findings appear to be in accord with the present results although they did not discuss this apparent trend. It is possible that some previous inconsistencies with respect to amphetamine effects on SN SS (see refs. 30 and 32) may have been due to small differences in SN electrode placements along a dorsal-ventral gradient. These results appear to parallel the effects of chronic morphine treatment on SN SS (Experiment 1). It should be noted, however, that in no case did o-amphetamine clearly depress responding, whereas chronic morphine treatment severely (but reversibly) reduced SS in several animals with electrodes close to SN. GENERAL DISCUSSION These experiments have demonstrated that morphine increases SS from some brain regions, yet fails to facilitate and may actually depress SS from other, closely adjacent regions. Several considerations suggest that these differential effects reflect heterogeneity in the neural systems which mediate SS, as opposed to other possible interpretations. It might be argued that facilitation of SS by morphine is merely secondary to its antinociceptive action. Morphine reduces escape from aversive brain stimulation, according to Pert 3° and unpublished observations. Such an interpretation, however, fails to explain why tolerance does not develop to the facilitatory effects of morphine on SS for periods of chronic treatment up to 25 days, either in SN (see ref. 23) or CG (unpublished observations). Although tolerance to morphine's effects on SS was noted by Glick and Rapaport 11, the doses employed were much lower than in the present experiments, and may have been subanalgesic. The effects of morphine on SS also do not appear attributable to its effects on locomotor activity, since if this were the case no dependence on electrode placement would have been expected. An alternative interpretation which has been proposed to explain differential effects of morphine on SS is that these effects depend on the baseline rate of SS (see ref. 17). In the present investigation, however, the baseline rates were equated prior to testing and were unrelated to the subsequent effects of morphine. Similarly, the observed maximal rates of SN SS were comparable for animals which increased or reduced this behavior upon treatment with morphine. It remains possible that the observed differential effects of morphine may simply reflect regional variations in its time course, as previously suggested2L Although the present results do not allow definite conclusions regarding possible differential time courses, the results of Experiment 2 suggest that reduction of CG SS by morphine three hours after injection was associated with a similar effect at 5 h and an even stronger reduction of SS at 1.5 h. Thus, the depressant effects of morphine on medial CG SS at three hours appeared to be representative of its effects throughout a 5-h time course. Correspondingly, facilitation of CG SS by 15 mg/kg morphine (n ~ 5) attained a maximum at three hours rather than at 1.5 h after injection. Although these experiments were conducted in a limited number of animals, they do indicate that the three-hour post-injection interval yielded effects which were representative of the direction of morphine's action on SS.

115 The proximity to dorsal raphe of CG placements which failed to increase SS after morphine treatment would suggest that the serotone~gic cell bodies of dorsal raphe might be involved in the effect. In fact, SS from the dorsal raphe appears to be inhibited rather than mediated by the serotonergic cells in this nucleus, because parachloro-phenylalanine has been reported to increase SS from dorsal raphe 35. Perhaps, therefore, morphine enhances the release of both catecholamines and serotonin, resulting in inhibition of SS in dorsal raphe. Lateral CG, which predominantly contains noradrenergic24 as opposed to serotonergic fibers, would yield increases in SS after morphine treatment. This hypothesis, however, does not readily explain the results obtained from SS placements in the SN area, nor does it explain the differential effects of D-amphetamine. Alternatively, reward may result from stimulation of noradrenergic rather than dopaminergic fibers within the regions from which morphine or D-amphetamine facilitates SS. In fact, noradrenergic neurons from the central tegmental tract appear to course between SN and the medial lemniscus, and dorsally to the medial lemniscus, but not within SN proper 24. Other investigators have reported that D-amphetamine more strongly facilitates SS in brain regions which are believed to contain predominantly noradrenergic rather than dopaminergic fibers2,31,32,87. It must be noted, however, that the relative potency of D-amphetamine on various aspects of noradrenergic or dopaminergic neurotransmission remains unclear 12,14,3s. With respect to morphine, biochemical and neuropharmacological investigations have shown that this narcotic influences neurotransmission in noradrenergic, dopaminergic and serotonergic systems4,5,9,to,18,3z,40. However, some indirect evidence suggests that morphine's effects on SS may be mediated by noradrenergic as opposed to dopaminergic neurotransmission systems. The characteristic increases in activity and repetitive forepaw grooming which are induced by morphine appear to be more directly related to noradrenergic than to dopaminergic neurotransmission~,6. Furthermore, the time course of these effects, and their progressive augmentation in tolerant animals, resemble the temporal characteristics of morphine's effects on SS3,23,26,~8. As already noted, the effects of morphine on SS are not secondary to its effects on locomotor activity, but the two effects may be independently mediated by a common, possibly noradrenergic mechanism. Furthermore, opioid-seeking behavior may depend on noradrenergic substratesT, 19. Finally, naloxone has been reported to antagonize the facilitatory effects of D-amphetamine on SS (see ref. 15) as well as those of heroin 17. This hypothesis would require that medial CG contain a non-noradrenergic substrate of SS, which would then fail to be facilitated by morphine or D-amphetamine. In fact, such a possibility has been suggested a, in that Lindvall and Bjorklund24 have noted the existence of catecholaminergic cell bodies in dorsal raphe which apparently belong to the A10 dopaminergic cell group. At present, however, the possibility of noradrenergic involvement in the differential effects of morphine and D-amphetamine on SS must necessarily be regarded as tentative in the absence of further supporting evidence. Regardless of the neuropharmacological mechanisms involved, these results

116 indicate that SS from closely adjacent b r a i n regions m a y nonetheless manifest differential sensitivity to various pharmacological treatments. In particular, the possibility that the SN region may thus be pharmacologically differentiated would suggest caution in the interpretation o f previous pharmacological or n e u r o a n a t o m i c a l investigations of SN SS. A t h o r o u g h pharmacological characterization of these sites would help to indicate which o f the neural systems which mediate SS may be responsible for the rewarding effects of morphine, D-amphetamine and other drugs of abuse. ACKNOWLEDGEMENTS A t the time that this research was performed, Jeffrey L i e b m a n was sponsored by a Post-doctoral Research Fellowship from the N a t i o n a l Institute of D r u g Abuse. David S. Segal is the recipient of N a t i o n a l Institute of M e n t a l Health Career Scientist A w a r d MH-70183-03. This research was supported by U S P H S G r a n t DA-01568-01. We gratefully acknowledge the assistance of Susan McGehee with these experiments.

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