Intraaccumbens injections of substance P, morphine and amphetamine: effects on conditioned place preference and behavioral activity

Brain Research 790 Ž1998. 185–194

Research report

Intraaccumbens injections of substance P, morphine and amphetamine: effects on conditioned place preference and behavioral activity b ˚ S. Schildein a , A. Agmo , J.P. Huston a , R.K.W. Schwarting a

a,)

Institute of Physiological Psychology I, and Center for Biological and Medical Research, Heinrich-Heine-UniÕersitat UniÕersitatsstr. 1, ¨ of Dusseldorf, ¨ ¨ 40225 Dusseldorf, Germany ¨ b Laboratoire d’Ethologie et de Psychophysiologie, Faculte` de Sciences et Techniques, UniÕersite´ de Tours, Tours, France Accepted 13 January 1998

Abstract The nucleus accumbens of the rat plays a critical role in behavioral activation and appetitive motivation. Within the nucleus accumbens, the shell subarea may be especially relevant, since this site is anatomically related to other brain areas that are considered to play a critical role in the processing of motivation. We investigated the behavioral effects of local drug treatments aimed at the shell of the nucleus accumbens and tested the indirect dopamine agonist D-amphetamine, the opiate agonist morphine, and the neurokinin substance P. These substances are known to exert positive reinforcing effects, and can affect behavioral activity; effects that are physiologically closely related to the nucleus accumbens and its inputs and outputs. Our results show that unilateral microinjections of amphetamine Ž1.0 m g, 10.0 m g. into the shell of the nucleus accumbens dose-dependently stimulated behavioral activity Žlocomotion, rears, sniffing., and led to conditioned place preference. Furthermore, the effect of amphetamine on place preference was negatively related to the psychomotor stimulant action on rears. Morphine injections Ž5.0 m g. also stimulated behavioral activity and elicited contraversive turning, but were ineffective with respect to place preference. Finally, the neuropeptide substance P, injected in a dose range of 0.1–10.0 ng, had no significant behavioral effects. These findings are discussed with respect to the role of dopaminergic, peptidergic and cholinergic mechanisms in the nucleus accumbens. It is suggested that dopamine, opiates, and neurokinins in the shell of the nucleus accumbens are differentially involved in mediating behavioral activity and appetitive motivation. q 1998 Elsevier Science B.V. Keywords: Reward; Reinforcement; Addiction; Turning; Locomotion; Rearing; Grooming; Neurokinins; Opiates; Psychostimulants; Ventral striatum; Rat

1. Introduction The nucleus accumbens ŽNAcc., which receives a mesolimbic dopamine ŽDA. input from the ventral tegmental area ŽVTA., is thought to serve as an interface between motivational stimuli and relevant behavioral action Žfor review, see Ref. w83x.. Substantial evidence for this hypothesis has been obtained from the study of psychostimulants and opioid drugs of addiction, like amphetamine and morphine, which can exert reinforcing effects, and which affect behavioral activity Žsee Ref. w85x.. Various studies have shown that NAcc and VTA are highly effective sites for these drugs to be self-administered w30,80x, to produce conditioned place preference w9,69,77x, and to modify conditioned responding w74x. Neurochemically, these actions have been related to the mesolimbic DA system: its activ)

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ity is usually increased by these drugs w62x, and lesions or pharmacological blockade of DA function can impair or block the drugs’ effect on measures of reinforcement and behavioral activity Žfor reviews, see Refs. w13,45x.. Apart from psychostimulants and opiates, the neurokinin substance P ŽSP. has also been shown to exert reinforcing effects Žfor review, see Ref. w35x.. Thus, conditioned place preference was obtained with systemic and intracerebral microinjections of SP. With respect to central administrations, injections of nanogram amounts into the nucleus basalis magnocellularis ŽNBM., the medial septum, and the lateral hypothalamus were found to be effective w24,25,33,34,70x. Neurochemically, the reinforcing effects of SP have been related to DA function in the NAcc, since systemic treatment with SP and central injections into the area of the NBM or the VTA were found to increase accumbal DA release w5,7,19x. In contrast to these findings, it is not known whether injections of SP into the NAcc itself might also be effec-

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tive to induce place preference. Since the NAcc is rich in several neurokinin mechanisms, an involvement in reinforcement and behavioral activity might be expected for several reasons: first, the NAcc shows dense immunoreactivity for SP w86x, and is very rich in SP receptors, which are mainly located on cholinergic interneurons w22,37,42,49,50x. Second, the major efferent projection of the NAcc to VTA and substantia nigra uses SP Žbesides GABA. as its neuromodulator. Third, this SPergic projection is anatomically and functionally related to the DAergic input to the NAcc w18,19,39,41x. The present study was performed to investigate whether direct microinjections of amphetamine, morphine, or SP into the NAcc have acute effects on behavior, and whether they are effective to induce conditioned place preference. All injections were aimed at the shell of the NAcc. This subarea is anatomically related to the limbic system and is thought to play a prominent role in motivational functions w62x. Furthermore, the shell contains compartments that are neurochemically characterized by dense SP, DA and ChAT staining w78,86x Žfor review, see Ref. w56x.. The ‘corral’ place preference apparatus w26x was used, in which reinforcing effects of systemic or cerebral SP w7,24,25x, and also of systemic morphine w26x have repeatedly been shown. In addition to conditioning, acute drug effects can be measured with this method that allows to investigate possible relationships between acute behavioral activation like in locomotor activity and motivational processes. Since unilateral injections were used, the possible induction of behavioral asymmetries could also be gauged. Such asymmetries, like in turning, can be expected in the case of unilateral manipulations of the NAcc w10,52,59,64x, and have specifically been related to its DAergic input Žfor review, see Refs. w53,66x..

2. Materials and methods 2.1. Subjects Male Wistar rats ŽJanvier, France. weighing between 250 and 300 g were housed under standard conditions under a 12-h light:12-h dark regime with food ŽAltromin, Lage. and water continuously available. Behavioral observations were performed during the light cycle between 09:00 and 17:00 h. The animals were weighed and handled daily for 5 min beginning one week prior to the start of the experiments. 2.2. Surgery The animals were anaesthetized with a mixture of 0.4 mlrkg xylazinhydrochloride ŽRompun w , Bayer, Germany. and 0.9 mlrkg ketaminhydrochloride ŽKetavet w , Upjohn, Germany.. Through a hole in the skull a stainless steel cannula Ži.d.: 0.24 mm, o.d.: 0.46 mm; Plastics One. was

unilaterally implanted aimed at the shell of the nucleus accumbens Žcoordinates relative to bregma, anterior: 0.7 mm, lateral: 1.0 mm, ventral: 7.0 mm; according to Ref. w54,55x.. The guide cannulae were secured in place with 2 skull screws and dental cement. Removable stainless-steel stylets ŽPlastics One. were placed in the guide cannulae. 2.3. Drug treatment All substances were dissolved in phosphate-buffered physiological saline, which served as the vehicle in all experiments ŽPBS; NaH 2 PO4 H 2 O and NaHPO4 , pH 6.5.. The following drugs and doses were used: Substance P ŽSP1-11 , Peninsula Labs, USA. 0.1 ng, 1.0 ng and 10.0 ng, morphine sulfate ŽMerck, Germany. 5.0 m g, and Damphetamine sulfate ŽMerck, Germany. 1.0 m g and 10.0 m g. All substances were injected unilaterally in a volume of 0.5 m lr30 s. Control animals received injections of vehicle. The infusion cannula Ži.d.: 0.10 mm, o.d.: 0.20 mm, Plastics One. was attached via vinyl tubing ŽPlastics One. to a 10-m l syringe ŽHamilton Products. mounted on a microinjection pump ŽWorld Precision Instruments; model SP101i syringe pump.. After injection, the infusion cannula remained in place for an additional 30 s. During the infusion, the subjects were hand-held. 2.4. Apparatus Place preference conditioning was performed using a circular open field Ž83 cm in diameter. with white walls Ž45 cm high. and a smooth brass floor Žfor details see Ref. w25x.. Two crossed black lines marked four different quadrants of equal size and identical floor and wall texture. During conditioning Žday 2., these quadrants were separated by transparent plexiglass barriers. Discriminative visual and tactile cues were absent within the open field. Spatial orientation was provided by surrounding visual stimuli, such as the ceiling of the experimental room, or the video camera placed 150 cm above the open field. The experimental room was illuminated by a 25-W bulb. Masking noise Ž68 dB. was provided by a noise generator. The animals’ behavior was recorded via the video camera and registered outside the experimental chamber with a computer system. After each test, the apparatus was cleaned with 0.1 mol acetic acid solution and was dried carefully. 2.5. BehaÕioral procedure The experiment was performed on three consecutive days consisting of a baseline Žday 1., a conditioning Žtreatment, day 2. and a test trial Žday 3.. Each trial lasted 15 min. For baseline testing, the animal was placed into the center of the open field and had free access to the entire apparatus. The time spent in and the number of entries into each of the four quadrants were scored. Thereby, the four quadrants could be ranked according to

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the degree of spontaneous preference in each animal. One of the two quadrants with intermediate ranks of preference was selected by chance for conditioning Žtermed the treatment quadrant; Žsee Refs. w24,25x.. For conditioning on day 2, the plexiglass barriers were inserted into the open field. This forced the rat to remain in its respective treatment quadrant. Five minutes before being placed into the treatment quadrant, the rat received an intracerebral injection of either morphine Ž5.0 m g., amphetamine Ž1.0 m g, 10.0 m g., SP Ž0.1 ng, 1.0 ng, 10.0 ng., or vehicle. During the subsequent 15 min in the treatment quadrant, the following behavioral measures were taken in terms of frequency and duration: locomotion Žperiods of movement of all four limbs., rears and grooming. Finally, the possible occurrence of behavioral asymmetries was scored using an observational method. Here, quarter- and half-turns ipsi- and contralateral to the side of injection were scored. For the test trial Žday 3., the plexiglass barriers were removed and the animals had free access to the corral. Again, as during baseline, the time spent in and the entries into each of the four quadrants were scored.

Statistical analyses were performed using non-parametric tests. Between group comparisons were made with two-tailed Mann–Whitney U-tests. Whenever multiple comparisons were performed, the level of significance Ž0.05. was corrected using the Bonferroni method Ža) s arn; n s number of tests.. The Wilcoxon matched signed-ranks tests were used to evaluate differences within groups.

2.6. Histological and statistical eÕaluation

3.2. Acute drug effects: locomotion, rears, grooming

The animals were deeply anaesthetized with 1.0 ml Nembutal w for transcardial perfusion. After one week of post-fixation, the brains were cut into 50 m m slices and stained with Cresyl violet.

These effects were tested after drug injection on experimental day 2, i.e., during 15 min in the treatment quadrant of the ‘corral’ apparatus. In general, this analysis showed that measuring behavior in terms of duration was more

3. Results 3.1. Histological eÕaluation Only data from rats with correctly placed cannulae were analysed ŽFig. 1.. Data of six animals had to be excluded, because their injection sites were not located in the shell area of the nucleus accumbens. The histological analysis provided no indications for systematic differences in the injection sites of the different treatment groups.

Fig. 1. Coronal sections from the atlas of Paxinos and Watson w55x showing the areas of cannula tip placement in the shell of the nucleus accumbens. The figure summarizes the tip placements from all animals. In the individual animals, cannula were situated either only in the left or only in the right hemisphere. ŽA. gives injection sites for amphetamine Žfilled diamonds. and substance P Žopen circles., and ŽB. gives injection sites for morphine Žfilled circles. and vehicle Žopen squares..

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Table 1 Durations Žin seconds; means"S.E.M.. of rearing, locomotor activity, and grooming behavior in animals that had received unilateral injections of either amphetamine Ž1.0 m g, 10.0 m g., morphine Ž5.0 m g., or substance P Ž0.1 ng, 1.0 ng, 10.0 ng. into the shell of the nucleus accumbens Rears

Locomotion

Grooming

Amphetamine 1.0 m g Ž ns10. 212.60 ) "25.24 175.18 ) "17.10 179.16"29.15 148.03"16.47 241.05"47.25 10.0 m g Ž ns10. 188.60"34.06 Vehicle Ž ns8. 129.83"20.83 106.85"14.08 274.27"33.72 Morphine 5.0 m g Ž ns10. 96.36 "18.22 Vehicle Ž ns10. 127.24"15.35

240.26 ) "51.09 158.42"51.08 104.86"14.19 268.87"48.13

Substance P 0.1 ng Ž ns10. 1.0 ng Ž ns10. 10.0 ng Ž ns10. Vehicle Ž ns 7.

133.80"20.82 143.78"21.37 172.07"12.51 133.95"22.34

131.16"13.36 142.03"23.17 153.51"12.78 141.00"22.02

285.45"29.97 273.21 "40.59 235.59 "14.09 241.52 "34.40

In parallel to each drug treatment, controls were run, injected with vehicle. Asterisks indicate significant differences from vehicle-injected animals according to Mann–Whitney U-test.

sensitive to indicate drug effects than measuring behavior in terms of frequency. Therefore, data analysis and presentation was confined to the former measure. This behavioral analysis showed that acute behavioral activity was affected by injections of amphetamine and morphine, but not SP ŽTable 1.. 3.2.1. Amphetamine Compared to their controls ŽMann–Whitney U-tests, a ) s 0.025., animals injected with the low dose of amphetamine Ž1.0 m g. spent significantly more time locomoting Ž p s 0.006., whereas the higher dose Ž10.0 m g, p s 0.142. did not affect this measure significantly. In addition to horizontal locomotion, the time spent rearing ŽTable 1. was also increased by the low Ž1.0 m g, p s 0.021., but not by the high dose of amphetamine Ž10.0 m g, p s 0.289.. Since the lack of effect after the higher dose of amphetamine may have been due to increased stereotypies, we checked the animals for the occurrence of such stereotypies. This analysis showed that the injections of amphetamine into the nucleus accumbens shell led to sniffing behavior, whereas other stereotypies could not consistently be observed. Quantitatively, vehicle-injected animals spent 138.3 " 11.2 s sniffing Žmean " S.E.M.., whereas animals injected with 1.0 m g amphetamine spent 266.0 " 18.7 s, and those injected with 10.0 m g spent 313.3 " 39.8 s sniffing. Statistical analysis ŽMann–Whitney U-test. yielded that the durations of sniffing did not differ between the two doses of amphetamine Ž p s 0.496., but that both doses led to more sniffing than in controls Ž1.0 m g, p - 0.001; 10.0 m g, p s 0.003.. Finally, the low dose of amphetamine tendentiously decreased the time spent grooming; however, this effect did not reach statistical significance Ž1.0 m g, p s 0.041; a ) s 0.025..

3.2.2. Morphine Animals injected with 5.0 m g of morphine spent significantly more time locomoting Ž p s 0.010. compared to their controls ŽMann–Whitney U-tests, a s 0.050.. The durations of grooming and rears did not differ from controls. 3.2.3. Substance P Neither dose of SP significantly affected the time spent locomoting Ž0.1 ng, p s 0.922; 1.0 ng, p s 0.626; 10.0 ng, p s 0.205.. The durations of rearing and grooming were also not significantly different from vehicle-injected controls ŽMann–Whitney U-tests, a ) s 0.017.. 3.3. Acute drug-effects: behaÕioral asymmetries Indications for specific acute drug effects were also obtained when measuring turns away or towards the side of unilateral injections in the shell of the NAcc: animals injected with 5.0 m g of morphine showed more turns contra- than ipsiversive to the side of injection during the 15-min testing period Ž p s 0.037; Wilcoxon matchedpaired signed-ranks test; Fig. 2.. This effect was probably due to a contraversive increase, since the number of contraversive Ž p s 0.015; Mann–Whitney U-test., but not that of ipsiversive turns Ž p s 0.130. was higher than that of vehicle-injected animals. In contrast to morphine, neither dose of amphetamine Ž1.0 m g, p s 0.053; 10 m g, p s 0.333. nor SP Ž0.1 ng, p s 0.722; 1.0 ng, p s 0.241; 10.0 ng, p s 0.154. led to differences between ipsi- and

Fig. 2. The number of turns Žmeans"S.E.M.. during an observational period of 15 min. Turns are given as either contra- Žblack symbols. or ipsiversive Žgrey symbols. to the side of unilateral injections into the nucleus accumbens. The rats had received either amphetamine Ž1.0 m g, 10.0 m g., morphine Ž5.0 m g., substance P Ž0.1 ng, 1.0 ng, 10.0 ng., or vehicle. For group sizes, see Table 1. Asterisk indicates difference Ž p- 0.05. between ipsi- and contraversive turns according to Wilcoxon matched-paired signed rank test.

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contraversive turns ŽWilcoxon matched-paired signed-ranks test.. Compared to controls, the highest dose of SP tendentiously enhanced contraversive turning Ž p s 0.079.. 3.4. Conditioned place preference In the conditioned place preference paradigm, a possible reinforcing effect of drug treatment is tested in the undrugged state Žtest day 3., and it is defined as an increase in time spent in that compartment of the ‘corral’ apparatus where the drug treatment had been experienced before. In the case of injections into the shell of the NAcc, such effects were registered in animals that had been injected with amphetamine, but not morphine, or SP ŽFig. 3.. Furthermore, place preference after amphetamine was dose-dependent, since the effect reached statistical significance Ž a ) s 0.017. only with the higher dose Ž10.0 m g, p s 0.001 vs. vehicle; Mann–Whitney U-test., whereas the lower dose yielded only a tendency Ž1.0 m g p s 0.033.. Additional comparisons between the two doses showed that the conditioning effect with the 10.0 m g, dose was larger than that of the 1.0 m g dose Ž p s 0.015.. The preference induced by the higher dose was due to increases in time spent in the treatment quadrant, whereas the number of entries into it was not affected in any of the groups Ždata not shown; all p-values) 0.05.. When comparing these conditioning effects Žday 3. with the acute drug effects Žday 2., it was found that the degree of place preference was negatively correlated with the stimulant effects of the higher amphetamine dose Ž10.0 m g.: the lesser amphetamine acutely enhanced rearing time Žthe behavior which has a strong link to explorative activ-

Fig. 4. Scatter-plot depicting the individual relationships between the psychomotor stimulant action Žduration of rears in seconds. of 1.0 m g or 10.0 m g of amphetamine Žtriangles. injected into the shell of the nucleus accumbens; vehicle controls are shown for comparison Žopen circles.. Rearing behavior was analyzed acutely after drug injection in the treatment quadrant, whereas the degree of place preference was measured as the time spent Žin seconds. in the same quadrant on the subsequent day. Furthermore, a linear regression was fitted to the amphetamine data.

ity., the stronger the degree of place preference on the subsequent day ŽSpearman correlation coefficient: 10.0 m g, r s y0.60, p s 0.033; 1.0 m g, r s y0.19, p s 0.30, vehicle: r s y0.19, p s 0.33; one-tailed. Žsee Fig. 4.. Furthermore, rearing was correlated with sniffing. This effect was evident for both doses of amphetamine Ž10.0 m g, r s 0.61, p s 0.030; 1.0 m g, r s 0.65, p s 0.021; one-tailed.. The time spent locomoting was not correlated with place preference. In contrast to amphetamine, no indications for conditioned place preference were found with morphine Ž p s 0.406., nor any of the three doses of SP Ž0.1 ng, p s 0.922; 1.0 ng, p s 0.558; 10.0 ng, p s 0.922; Fig. 3..

4. Discussion

Fig. 3. Test for conditioned place preference Žgray bars.: Time in seconds ŽmeanqS.E.M.. spent in the former treatment quadrant, that is, where the animals had experienced drug treatments on the preceding day. The rats, which were now tested in the drug-free state, had received either amphetamine Ž1.0 m g, 10.0 m g., morphine Ž5.0 m g., substance P Ž0.1 ng, 1.0 ng, 10.0 ng., or vehicle into the shell of the nucleus accumbens. Behavior during the initial baseline test Žblack bars. is also shown for comparison. For group sizes see Table 1. )) p- 0.01 according to Mann–Whitney U-test for treatment vs. vehicle.

Here, we behaviorally investigated three intracerebral drug treatments that are known to exert positively reinforcing effects, and which can affect locomotor activity, namely the DA agonist amphetamine, the opiate agonist morphine, and the neurokinin SP. We injected them into the shell of the nucleus accumbens; first, since this brain area is thought to play a critical role in reinforcement and behavioral activation; second, because this brain area is very rich in DAergic, opioidergic, and SPergic elements; and third, since the behavioral effects of these drug treatments have at least partly been attributed to these mechanisms. Our results show, that with respect to place preference, only amphetamine had positive reinforcing effects; this effect was dose-dependent and was negatively related to the drug’s acute stimulatory effects on behavior. In contrast, intraaccumbens injections of morphine did not lead to conditioned place preference, but enhanced locomotor ac-

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tivity acutely and led to turning away from the side of injection. Finally, injections of SP also did not lead to conditioned place preference, and were largely without effect on acute behavior. From these findings, it can be concluded that the known reinforcing effects of systemically injected SP or morphine are not mediated by direct actions in the NAcc—in contrast to the reinforcing actions of the DA agonist amphetamine. Furthermore, it can be concluded that the acute behavioral activation induced by pharmacological stimulation in the NAcc can be dissociated from effects on measures of reinforcement. 4.1. BehaÕioral effects of D-amphetamine The present data support previous reports where similar doses of D-amphetamine injected into the NAcc were found to enhance locomotion and rears with peak increases within 30 min after injection w12,44,60,71,76x. In the present study, the lower dose was more effective than the higher dose to enhance rears and locomotion during the observational period of 15 min after injection. These increases were paralleled by decreased grooming behavior, which seems to reflect the incompatibility of grooming with locomotion. It is known that the behavioral pattern elicited by amphetamine can shift with increasing drug doses, since lower doses are more effective to induce locomotor activity and rears, whereas stereotypies are more likely with higher doses. Thus, it may be argued that the lack of locomotor activation observed with the higher dose here may have been due to increases in stereotypic behavior Žsniffing, head waving, etc... Such an explanation, however, is rather unlikely, since the stereotyped response that we observed after injections into the shell of the NAcc, namely increased sniffing, did not differ between the two doses of amphetamine. So far, increased locomotion, rears and sniffing were usually obtained after systemic or bilateral NAcc injections of amphetamine w16,20,36x. Other studies showed that unilateral injections are sufficient to enhance locomotor activity w8x. Interestingly, the injection sites in that latter study seem to have been located mainly in the shell subarea, whereas the former studies did not differentiate between core and shell. Since the stimulatory effects of D-amphetamine are known to be dependent on DA mechanisms within the NAcc w76x, it can be concluded that stimulating DA in the NAcc shell of one hemisphere is sufficient for the psychomotor stimulant action of this drug. In contrast to enhancing locomotion, rears and sniffing, unilateral amphetamine injections into the NAcc did not lead to behavioral asymmetry, which supports previous findings with a similar dose range w51x. Others, however, have reported contraversive asymmetries after Damphetamine w10x, DA receptor agonists w46,47,52,64x, and non-DAergic drugs w51,59,65x. These effects were not due to solvent spread into the overlying neostriatum. Compared

to the previous study with amphetamine w10x, the present lack of asymmetry is not due to differences in drug dosage or time of testing after injection, but rather to the actual site of injection within the NAcc: contraversive asymmetry after amphetamine was observed with centrally placed Žcore. injections Ž10., which we injected into the shell. Therefore, it can be assumed that DAergic mechanisms differ between the subcompartments of the NAcc, since increasing DA release in the shell Žin contrast to the core. may not be sufficient to affect behavior asymmetrically Žbut see Refs. w46,47x.. With respect to place preference, the present results on amphetamine are well in accord with previous studies that have shown that the NAcc is critically involved in its positive reinforcing effects. Thus, directly injecting this drug into the NAcc is highly effective to produce conditioned place preference w9,29,69x. This effect of amphetamine is closely linked to DAergic mechanisms within the NAcc, since it can be effectively blocked by lesions or pharmacological manipulations of DA function w31,48,69x. Furthermore, amphetamine in reinforcing doses potently increases DA levels in the NAcc, especially in its shell w62x, and reinforcing actions can also be obtained with direct stimulation of accumbal DA receptors w81x. These effects were usually obtained with bilateral injections of 10.0 m g doses w29x, and were measured in preference paradigms Žtwo- or three-compartment boxes., which require repeated pairings of drug injections and exposures to the conditioning compartments. Our data show that a single and unilateral injection of D-amphetamine into the shell is effective to establish place preference. In support of our previous studies, these data point at the usefulness of the corral apparatus, since this paradigm is sensitive to gauge place preference using only one intracerebral drug treatment w7,25,26x. This fact is especially advantageous, since it minimizes tissue damage due to repeated brain penetrations. Furthermore, since the same doses of amphetamine used in other preference paradigms were also effective in the case of the corral method, it can be assumed that the results obtained with the different paradigms are comparable. Our results with amphetamine address the potential relationship between behavioral activation and reinforcement w85x. It has been suggested that the behaviorally activating properties of a drug like amphetamine can induce place preference by increasing the familiarity with the treatment quadrant during conditioning w73x. In the present study, however, the reinforcing action of amphetamine was apparently dissociable from the drug’s general stimulatory effects: the dose of amphetamine that was less effective with respect to place preference had the stronger effect on locomotion and rearing behavior, whereas the higher dose, which was more effective with respect to reinforcement, had only weak effects on rears and locomotion activity. Amphetamine is thought to affect behavioral activation and reward by increasing the extra-

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cellular availability of DA, which then acts on DA receptors. From our results, it can be suggested that the reinforcing effects of amphetamine require either stimulation of more DA receptors than those necessary for behavioral activation, or that a different population of DA receptors is necessary for the reinforcing effects, for example, those with lower affinity. 4.2. BehaÕioral effects of morphine One prominent acute response elicited after unilateral injections of 5.0 m g morphine into the shell of the NAcc was enhanced locomotion. Similar effects can be induced by systemic injections of morphine, and these effects have been suggested to be due to an action in the VTA Žfor review, see Ref. w14x.. With respect to injections into the NAcc, previous studies using morphine and related opioids have been rather controversial, since either no effects, decreases, or increases were obtained w4,11,16,28,40,57,71x. These inconsistencies may be due to different sites of injection within the NAcc: thus, previous work has shown that stimulation of opiate receptors within the shell was necessary for morphine injections to be behaviorally stimulating w15x, which is supported by the present results. Together, it can be concluded that a direct action in the NAcc may contribute to the locomotor effects elicited by systemic injections of morphine. Our injections of morphine into the NAcc not only enhanced locomotion, but also induced contraversive turning behavior. In contrast, amphetamine, although applied at the same accumbal site, enhanced locomotion, but had no effects on turning. This finding indicates that the turning response after morphine was not due to a DA-enhancing action of this drug; thus, it supports the view that Žat least some of. the behavioral effects of opiate infusions into the NAcc can occur independent of its DA function w40,57x. Alternatively, one can assume that the turning response might have resulted from stimulation of m-receptors w1x, which are primarily located on the output neurons of the NAcc w15,68,75x. These outputs belong to the GABA and enkephalinergic efferents of the NAcc which project to the ventral pallidum and ventral mesencephalon Žfor review see Ref. w56x.. Interestingly, unilateral stimulation of opiate receptors in these projection sites can also induce contraversive turning w32,79,84x. In contrast to enhancing locomotion and turning, morphine injected into the shell of the NAcc did not lead to conditioned place preference Žsee also Ref. w67x.. Others, however, reported conditioned place preference with the same dose of morphine w77x. Regarding opiate injections into the NAcc, such inconsistencies are not restricted to the measure of place preference, since previous work with other conditioning paradigms has also provided controversial results w38,54,58x. One critical factor here might be the site of experimental manipulation Žcore vs. shell.. Thus, in the case of place preference, detailed anatomical mapping

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using different doses of morphine within the NAcc will be needed to actually clarify this issue. Independent of this aspect, our results show that the same site within the NAcc, which was negative with respect to morphine, proved to be positive with respect to amphetamine and place preference. In general, it is assumed that a strong DAergic impact within the NAcc is important for the reinforcing and addictive effects of drugs Žfor review see Ref. w13x.. Since morphine is clearly less potent than amphetamine to stimulate DA release in the NAcc, the differential contribution of DA might have been the critical mechanism for our differential results on place preference. However, compared to the core, especially, the shell seems to be sensitive with respect to the DA-stimulating effects of morphine; therefore, a reinforcing effect should be expected in case of injections into the shell. However, and as already outlined above, opiate injections into the NAcc, rather than acting via the DAergic input, may be mainly effective by direct actions on output neurons Žlike the motor-related ventral pallidum., which was probably manifested here as enhanced locomotion and contraversive turning. Even more, morphine’s action on these output neurons may override the impact of other inputs to the NAcc, which are otherwise important to associate a reward with a specific environment Žlike from amygdala, hippocampus, or cortex w11x.. Such a mechanism might explain why morphine did not lead to place preference. Amphetamine, in contrast, acts by enhancing DA release. This DAergic signal does not only affect the outputs of the NAcc, but can also amplify the other incoming signals to the Nacc that are probably relevant in situations like conditioned response and place preference. 4.3. BehaÕioral effects of substance P Previous studies using systemic and intracerebral injections of SP have reported effects on locomotor activity, grooming and conditioned place preference w17,27x. With respect to intraparenchymal injections, especially, the VTA and the NBM proved to be effective w25x, and in both cases, the effects on locomotion and place preference were related to activations of DA in the NAcc w5,7,19,61x. The NAcc itself, especially its shell w78,86x, is rich in SP and its receptors; furthermore, local administration of SP can enhance DA activity there w39x. Taken together, effects on acute behavior and place preference might be expected with NAcc-injections of SP; however, the present unilateral injections of SP in doses of 0.1–10.0 ng were behaviorally rather inconspicuous. Several aspects have to be considered here: first, the dose range and the unilateral injection procedure used should not have been critical, since they have repeatedly been shown to be behaviorally effective in other brain sites w7,25x. Alternatively, one might assume that SPergic mechanisms in the shell of the NAcc play a different functional role than those in the NBM or VTA. One possibility might

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be that stimulation of SPergic receptors within the shell of the NAcc is, in itself, behaviorally only weakly effective, but may become critically involved in case of DA-stimulated behavioral activation. Evidence for this conclusion is derived from studies which showed that injecting antibodies against SP into the NAcc were found to block amphetamine-induced behavioral activity w18x, or that administration of neurokinin A enhanced amphetamine-induced locomotion w76x. These effects may have been due to the stimulation of NK-3 receptors that are located on the DAergic afferents in the NAcc w82x, whereas SP binds preferentially to the NK-1 subtype. The NK-1 receptors, on the other hand, are located mainly on cholinergic interneurons of the NAcc w22x, and firing probability of these striatal interneurons is enhanced by local application of SP w2x. Functionally, the activity of these interneurons has been related to learning w23x, and to mechanisms of aversive motivation w6,63x. The possible role of accumbal SP in learning and memory has not yet been investigated in detail, except for one study which showed that pre-retention treatments can affect passive avoidance behavior w21x. With respect to aversive motivation, on the other hand, it has been suggested that enhanced cholinergic release in the NAcc is related to aversive stimulation w6,63x. Interestingly, not only the release of DA w5,18,39,43x, but also that of ACh is enhanced by local application of SP w3,43x. Since striatal ACh is thought to act in a counterbalanced fashion with DA Žfor review, see Ref. w72x., the agonistic effects of SP on ACh might have masked a possible activational and reinforcing effect induced by the concurrent DAergic stimulation via SP-receptors located on the DAergic input. These possible relationships should be addressed in future studies, for example, by investigating the behavioral effects of agonists and antagonists that are specific for the different NK-receptor subtypes. Taken together, our results show that unilateral microinjections of amphetamine into the shell of the NAcc dosedependently stimulated behavioral activity, and led to conditioned place preference. The effect of amphetamine on place preference seemed to be negatively related to the psychomotor stimulant action. Morphine injections also stimulated behavioral activity, and elicited contraversive turning, but were ineffective with respect to place preference. Finally, SP injections were behaviorally inconspicuous. These findings indicate that DA, opiates, and neurokinins in the shell of the NAcc are differentially involved in mediating behavioral activity and appetitive motivation.

Acknowledgements R.K.W. Schwarting is a Heisenberg fellow of the Deutsche Forschungsgemeinschaft. This work was sup-

ported by grant Schw 559r2-1 from the Deutsche Forschungsgemeinschaft.

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