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Dopaminergic activity is reduced in the prefrontal cortex and increased in the nucleus accumbens of rats predisposed to develop amphetamine self-administration
Brain Research, 567 (1991) 169-174 ~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/911503.50
169
BRES 24966
Dopaminergic activity is reduced in the prefrontal cortex and increased in the nucleus accumbens of rats predisposed to develop amphetamine self-administration Pier Vincenzo Piazza, Fran~oise Roug6-Pont, Jean Marie Demini~re, Martine Kharoubi, Michel Le Moal and Herv6 Simon Laboratoire de Psychobiologie des Comportements Adaptatifs 1NSERM U259, Universit# de Bordeaux 11, Bordeaux (France)
(Accepted 10 September 1991) Key words: Addiction; Self-administration; Amphetamine; Individual difference; Stress; Novelty; Dopamine; Frontal cortex; Nucleus aeeumbens; Striatum
Individual vulnerability to the reinforcing effects of drugs appear to be a crucial factor in the development of addiction in humans. In the rat, individuals at risk for psychostimulant self-administration (SA) may be identified from their locomotor reactivity to a stress situation such as exposure to a novel environment. Animals with higher locomotor responses to novelty (High Responders, HR) tend to acquire amphetamine SA, while animals with the lower responses (Low Responders, LR) do not. In this study, we examined whether activity of dopaminergic (DA) and serotoninergic (5-HT) systems differed between HR and LR animals. These transmitter systems are thought to be involved in the reinforcing effects of psychostimulants. Animals from both groups were sacrificed under basal conditions and after exposure for 30 or 120 min to a novel environment, and the DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-HT, and 5-hydroxyindolacetic acid (5-HIAA) contents were determined in the prefrontal cortex, nucleus accumbens and striatum. The HR rats displayed a specific neurochemical pattern: a higher DOPAC/DA ratio in the nucleus accumbens and striatum and a lower one in the prefrontal cortex. Furthermore, HR animals had lower overall 5-HT and 5-HIAA levels, corresponding to the mean of these compounds for the three structures studied over the three environmental conditions. Since similar DOPAC/DA ratios are found in animals in which the propensity to psychostimulants SA was induced by brain lesions or life events, an opposite pattern of dopaminergic activity in the prefrontal cortex (decrease) and in the ventral and dorsal striatum (increase) may be one of the neurobiological substrate of the predisposition to acquire amphetamine self-administration. The development of this difference may be influenced by interactions between dopaminergic and serotoninergic systems. These results suggest that potential therapeutic strategies for the treatment of addiction will need to take into account the functional heterogeneity of projection systems rather than concentrate on a single neurotransmitter. Clinical investigations of addictive behavior indicate that individual vulnerability to the reinforcing effects of drugs 4 has a large influence on the development of drugseeking behavior in humans 28. In a recent study, by using an intravenous self-administration (SA) acquisition paradigm 2°, we showed that rats exhibit a wide range of individual differences in their propensity to self-administer amphetamine 7. This individual predisposition may be predicted from the behavioral 31'32 and endocrinologica134 reactivity to stressfull environmental and pharmacological challenges. Animals with a high reactivity to novelty, defined as High Responders (HR) and having an activity score above the median of the group, acquire and maintain amphetamine self-administration (SA). Conversely, animals with a low reactivity to novelty, defined as Low Responders (LR) do not acquire SA. The H R rats also showed a higher locomotor response to amphetamine, and a prolonged corticosterone secretion in response to novelty than the L R animals (for review
see ref. 33). Furthermore, it was found that the H R rats had a lower affinity of central corticosteroids receptors 25, which may underlie the difference in endocrinological reactivity to novelty between the two groups 26. In the present series of experiments, we compared the functioning of mesencephalic dopaminergic and serotoninergic neurons of H R and L R animals by measuring the content of the neurotransmitter and its main metabolite in different anterior brain structures. These neurotransmitter systems were chosen for two main reasons. First, numerous lesional and pharmacological studies have indicated that the dopaminergic mesolimbic system is a principal neurobiological substrate for the psychostimulant reinforcing effect both during acquisition and maintenance of SA (for review see refs. 5, 15, 19). Second, serotoninergic activity is thought to modulate the reinforcing properties of psychostimulants 21'24'4°. H R and L R animals were compared to try to identify the neurobiological characteristics of rats with different
Correspondence: P.V. Piazza, INSERM U259, Rue Camille Saint Sa6ns, 33077 Bordeaux Cedex, France.
170 propensities for acquisition of psychostimulant SA without exposing the animals to these drugs. Comparisons made after testing for SA cannot discriminate between preexisting and drug-induced differences. Indeed, repeated exposure to psychostimulants may induce longlasting changes in the activity of the relevant neuronal substrates 36. As an index of dopaminergic and serotoninergic activity, the post-mortem contents of dopamine (DA), serotonin (5-HT), and their respective metabolites, 3,4dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA) of H R and LR rats were determined in the prefrontal cortex, nucleus accumbens and dorsal striatum under basal condition and after exposure to novelty. Male Sprague-Dawley rats (280-300 g b.wt.) were used. The animals were individually housed with ad libitum access to food and water. A constant dark light cycle (on 08.00, off 20.00 h) was maintained in the animal house, in which temperature (22 °C) and humidity were controlled. For this experiments, 60 rats were first tested for locomotor reactivity to novelty, and divided into 3 experimental groups (n -- 20 each), equals for activity scores. One week later all the rats were sacrificed for biochemical analysis of the 3 brain regions. The animals from the first group were sacrificed immediately after removal from their home cage (basal condition). The rats from the other two groups were sacrificed after 30 or 120 min of exposure to novelty. In each experimental group, the 8 rats with the highest activity scores were designated as High Responders (HR), while the 8 animals with lowest activity scores were designated as Low Responders (LR). The remaining 4 animals in each group were eliminated from the statistical analysis of the group study. Otherwise, for the correlative study all the animals were taken into account. The novel environment consisted of a circular corridor (10 cm wide and 70 cm in diameter). Four photoelectric cells placed at the perpendicular axes of this apparatus automatically recorded locomotion. The locomotor response was recorded over 10 min intervals for a period of 2 h. Animals were tested between 16.00 and 18.00 h. The score of each animal (number of photocell counts) cumulated over this period was used as an index of individual reactivity to the novel environment. For the biochemical assays, animals were sacrificed by decapitation, and the brains rapidly removed and frozen. The antero medial prefrontal cortex (PFC), the nucleus accumbens (ACC) and the dorsal striatum (STR) were dissected out of frozen brain slices of different thicknesses (PFC = 600/~m, ACC = 700/~m, STR = 900/~m) using a micropunching technique 3°. The following an-
teroposterior (AP) levels (from bregma) of the K6nig and Klippel atlas TM constituted the anterior surface of the slices: PFC, AP = 10.5 mm; ACC, AP = 9.9 mm; STR, AP = 7.9 ram. After tissue homogenization in 0.1 N perchloric acid and centrifugation, dopamine (DA), 3,4dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) contents were measured by high performance liquid chromatography (HPLC) with electrochemical detection 27. The chromatographic system consisted of a Milton Roy Constametric pump, a refrigerated automatic injector (CMA200 Carnegie Medicine), a precolumn (C18 cartridge, 30 mm x 4.6 mm i.d.) and C18 reverse phase column (Spherisorb ODS II 5 gm, 150 mm x 4.6 mm i.d.). The compounds of interest were detected using an amperometric detector (BAS LC4B), and chromatograms were recorded on an RC5A integrator (Shimadzu). Protein content of the punches was analyzed using the Micro BCA Protein assay reagent (Pierce). The results are expressed as ng/mg of protein. The behavioral and biochemical data from the H R and LR animals were compared by analysis of variance (ANOVA) for repeated measures. Student's t-test was employed for post hoc comparisons, and Pearson's test was used for the correlation analysis for each condition over all the groups. The locomotor activity displayed by the H R (n = 24) and the LR (n = 24) animals of the 3 experimental groups during the first exposure to novelty was: 422 --- 23 for LR and 843 - 41 for HR. Thus, H R rats showed a higher locomotor activity (F1,46 = 19.52, P < 0.001), but activities of both groups returned to the baseline after 2 h testing. For the two groups of animals sacrificed after exposure to novelty, locomotor activity during the first and second tests were highly correlated (r = 0.67 (P < 0.01) for 30 min novelty exposure, and r = 0.70 (P < 0.01) for 120 min). Dopamine content did not differ between the HRs and LRs in any of the structures studied, although the H R animals had a significant higher DOPAC content than the LR group (ANOVA Group effect F2,42 = 14.05, P < 0.001) (Table I). This difference was dependent on the structure studied (ANOVA Group x Structure interaction F2,84 = 11.39, P < 0.0001). The H R animals had less DOPAC in the cortex than the LR animals (ANOVA Group effect F1,42 = 2.99, P < 0.05), whereas the H R rats had more DOPAC in the nucleus accumbens (ANOVA Group effect F1,42 = 14.52, P < 0.001) and striatum (ANOVA Group effect F1,42 = 3.27, P < 0.05) than the LR animals. No group-novelty interaction was found in any of the structures studied. DOPAC/DA ratios differed between the H R and LR animals in the various brain structures (ANOVA Group
171 TABLE I
Dopamine and DOPAC content in High-Responder (HR) and Low Responder (LR) animals Basal: animals were sacrificed in basal condition. Novelty (30"): animals were sacrificed after 30 rain of exposure to novelty. Novelty (120"): animals were sacrificed after 120 rain of exposure to novelty.
DOPAC (ng/mg of protein)
Dopamine (ng/mg of protein) Basal Frontal cortex LR HR
Novelty (30 9
1.10 ± 0.094 1.33 ± 0.09
Accumbens LR HR
89.06 - 4.48 105.02 -+ 7.2
Striatum LR HR
90.36 ± 5.68 94.49 +- 6.35
Novelty (120")
Basal
Novelty (30") 1.05 ± 0.39 0.45 ± 0.04**
Novelty (120")
1.33 ±0.13 1.31 ± 0.17
1,46 ± 0.16 1.57 ± 0.27
0.32 ± 0.03 0.22 ± 0.03*
0.51 ± 0.14 0.53 ± 0.05
89.63 ± 7,15 94.74 ± 5,47
119.08 - 6.7 117.05 ± 10.4
15.42 ± 1.22 20,35 -- 1.02"*
13.82 ± 1.27 14.97 - 1.1
11.04 - 1.39 16.94 ± 1.59"*
129.69± 3.36 123.32± 4.64
146.85 - 6.5 141.29 ± 3.9
5.11 +- 0 . 6 0 5.87 ± 0.58
10.041± 0.55 10.02 ± 0.56
9.31 ± 0.5 11.18-+ 0.74*
*P < 0.05; **P < 0.01 (Student t-test).
x Structure interaction/72,84 = 3.42, P < 0.05) (Fig. 1). I n the cortex, the H R animals had a lower D O P A C / D A ratio than the L R group ( A N O V A G r o u p effect F1.42 = 2.89, P < 0.05), while in the nucleus accumbens ( A N O V A G r o u p effect FI,, 2 = 13.40, P < 0.001) and dorsal striatum ( A N O V A G r o u p effect F1,42 = 8.41, P < 0.01) the H R animals had a higher D O P A C / D A ratio than the L R group. No group-novelty interaction was
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Fig. 1. DOPAC/dopamine ratio of HR and LR animals in basal condition (Basal) and after 30 rnin [Novelty(30')] and 120 min [Novelty(120")] exposure to novelty. HR animals had a lower ratio in the prefrontal cortex (/71,42 = 2.89, P < 0.05) and a higher one in the nucleus aeeumbens (F1,42 = 13.40, P < 0.001) and dorsal striatum (F1,42 = 8.41, P < 0.01) *P < 0.05; **P < 0.01.
found in any of the structures studied. However, a post hoc comparison showed that these differences were significant: (i) in the cortex, u n d e r basal condition and after 30 rain exposure to novelty (P < 0.05); (ii) in the nucleus accumbens, u n d e r basal condition (P < 0.05) and after 120 rain exposure to novelty ( P < 0.01): (iii) in the striatum, after 120 min of exposure to novelty (P <
0.01). The correlative analysis extended the results of the group study. There was a significant correlation between the DOPAC content and the locomotorresponse to novelty. In this case, the main result was a positive correlation between the locomotor response to novelty and the DOPAC content in the nucleus accumbens in both the basal condition (r -- 0.54, P < 0.01) (Fig. 2A) and after exposure to novelty (after 30 min r = 0.39, P < 0.05; after 120 min r = 0.62, P < 0.01). There was a negative correlation between the DOPAC content in the prefrontal cortex and the locomotor response to novelty (basal r = -0.56, P < 0.01; after 30 rain r = -0.39, P < 0.05) (Fig. 2B), while in the striatum a positive correlation with locomotor response to novelty was only found after 120 rain exposure (r = 0.70, P < 0.01). Similar correlations were found between the DOPAC/DA ratio and the locomotor response to novelty: (i) cortex = basal, r = -0.59, P < 0.01; novelty 30 rain, r = -0.39, P < 0.05; (ii) accumhens = basal, r = 0.39, P < 0.05; novelty 30 rnin, r = -0.47, P < 0.05; novelty 120 rain, r = -0.67, P < 0.01; (iii) striatum = novelty 120 min, r = -0.75, P < 0.001. The H R animals had a lower overall content of 5-HT ( A N O V A G r o u p effect F1,42 = 5.66, P < 0.05) and 5 - H I A A ( A N O V A G r o u p effect F1,42 = 4.01, P < 0.05) than the L R animals in the various brain structures. No
172
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Fig. 2. Correlation between DOPAC content in basal condition and the locomotor response to novelty in the nucleus accumbens (A) and in the prefrontal cortex (B). The two parameters were positively correlated in the nucleus accumbens (r = 0.54, P < 0.01) and negatively correlated in the prefrontal cortex (r = -0.56, P < 0.01).
interactions between the groups and the different structures were found for either compound. Furthermore, the 5-HIAA/5-HT ratio did not differ between the two groups. The overall content, as calculated by ANOVA, corresponded to the mean of these compounds for the three structures studied over the three environmental conditions. The content of 5-HT in ng/mg of proteins was: LR = 6.5 - 0.3, H R = 5.7 - 0.23. The content of 5-HIAA in ng/mg of proteins was: LR = 5.6 --- 0.22, H R = 5 . 0 ± 0.15. A correlative analysis was not carded out for the serotoninergic system as the two groups only showed an overal group effect. Our results show that different predispositions to drug-seeking behavior are related to differences in the dopamine utilization in the mesocorticolimbic system. Subjects with a higher predisposition to acquire amphetamine self-administration (HR group) had a lower D O P A C / D A ratio in the cortex and a higher ratio in the nucleus accumbens and dorsal striatum than the less predisposed subjects (LR group). These differences were matched by changes in D O P A C content, although the two groups did not differ in dopamine content. H R animals also had less serotonin and 5-HIAA than the LR group over the 3 structures studied. The opposite difference in D O P A C content between H R and LR animals in the prefrontal cortex ( H R < LR) and the nucleus accumbens or dorsal striatum (HR > LR) may be understood in terms of the functional relationships of D A transmission in these structures. Biochemical studies have suggested that D A release in the prefrontal cortex exercises an inhibitory control on D A transmission in subcortical structures 19'22'39. For example, in vivo studies have shown that stimulation of D A transmission in the prefrontal cortex by local infusion of
D A agonists decreases D O P A C release in the nucleus accumbens 23. Conversely, inhibition of D A transmission in the cortex by local infusion of neuroleptics increases D O P A C release in the nucleus accumbens 2a. Furthermore, it has been shown that the up-regulation of striatal D 1 dopaminergic receptor induced by lesion of the nigrostriatal dopaminergic system is attenuated by a concomitant lesion of D A afferents to the prefrontal cortex 42. Thus the higher D A activity in the nucleus accumbens or striatum of the H R animals may stem from the lower D A activity in the prefrontal cortex. This notion is supported by the different temporal pattern in the response to novelty in these structures between the H R and LR animals Thus, the difference between the two groups appears first in cortex (30 min of novelty exposure) and then in the ventral striatum (120 min of novelty exposure). Differences in dopaminergic activity in the prefrontal cortex may account for the differences in behavioral reactivity to environmental and pharmacological challenges between the two groups. Indeed, selective lesion of D A afferents in the prefrontal cortex after local injection of 6 - O H D A has been reported to induce an increase in: i) stress-induced D A release in the nucleus accumbensS; ii) the locomotor response to novelty2; iii) the propensity to self-administer cocaine 38. These biochemical differences between the H R and LR animals may throw more light on the biological basis of drug-seeking behavior. Thus, a decrease in D A transmission in the cortex and an increase in the nucleus accumbens have been repeatedly associated with an enhanced propensity to self-administer psychostimulants. For example, 6 - O H D A lesion of the amygdala 6 or electrolytic destruction of the ventral raphe nuclei 4°, both of which induce this biochemical pattern 12'41, also increase
173 the vulnerability to self-administer amphetamine and the psychomotor effects of this drug. Furthermore, social isolation, which increases both the propensity to self-administer cocaine 37 and the locomotor response to novelty 9,t° and amphetamine 17, also induces the differential effects on dopaminergic activity in the cortex and nucleus accumbens 1. Finally, an opposite effect of D A receptor activation in the prefrontal cortex (inhibition) and in the nucleus accumbens (facilitation) has been described for forebrain self-stimulation 29. Thus, a lower dopaminergic activity in the prefrontal cortex and a higher one in the nucleus accumbens may be considered as a c o m m o n biological substrate of a spontaneous or experimentally induced predisposition to psychostimulant drug-seeking behavior. The lower 5-HT and 5 - H I A A contents observed in the H R animals may be attributed to reduced serotoninergic activity. This result is in line with the role ascribed to serotoninergic systems in psychomotor stimulant reinforcement. For example, injection of the serotoninergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) in the ventricles 24 and medial forebrain bundle ls has been shown to increase the rate of amphetamine SA. Recently L o h and Roberts 21 have shown that serotoninergic lesions also increase cocaine seeking behavior. Infusion of 5,7-DHT into the medial forebrain bundle or amygdala increases the break-point for cocaine SA in a progressive ratio schedule of reinforcement. This effect may be explained in terms of interactions between dopaminergic and serotoninergic systems. U n d e r certain experimental conditions, serotonin seems to exercise an inhib-
itory control on D A activity n-13, and a decrease in 5-HT transmission may drive the dopaminergic system to higher reactivity to psychostimulants. Indeed, 5-HT depletion also potentiates other psychostimulant effects, such as locomotor activity a and stereotypy 16. However, it has also been suggested that the direct action of psychostimulants on serotoninergic neurons may have an antagonist effect on the reinforcing properties of these drugs. The correlative study of Ritz et al. 38 indicated that the behavioral potency in self-administration of cocaine analogs was positively related to the affinity of the drug at the D A uptake site, whereas a negative relationship was observed with the binding potency at the 5-HT uptake site, In conclusion, our results suggest that an opposite pattern of dopaminergic activity in the prefrontal cortex (decrease) and in the nucleus accumbens (increase) is a possible neurobiological substrate of the predisposition to acquire amphetamine self-administration. This neurobiological profile may originate from interactions between dopaminergic and serotoninergic systems. These results suggest that strategies for the treatment of addiction should take into account not only the role of a specific neurotransmitter and its receptors, but also the functional heterogeneity of its projections and interactions between different neurochemical systems.
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This work was supported by rInstitut National de la Sant6 et de la Recherche M6dicale (INSERM) and l'Universit6 de Bordeaux II. We thank Isabelle Bathy for secretarial assistance.
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41
42
I.E Stolerman (Eds.), Behavioral Analysis of Drug Dependence, Academic Press, London, 1986, pp. 329-338. Olds, M.E., Enhanced dopamine receptor activation in accumhens and frontal cortex has opposite effects on medial forebrain bundle self-stimulation, Neuroscience 35 (1990) 313-325. Palkovits, M., Isolated removal of hypothalamic or other brain nuclei, Brain Research, 59 (1973) 449-450. Piazza, EV., Deminiere, J.M., Le Moal, M. and Simon, H., Factors that predict individual vulnerability to amphetamine self-administration, Science, 245 (1989) 1511-1513. Piazza, P.V., Deminiere, J.M., Maccari, S., Mormede, E, Le Moal, M. and Simon, H., Individual reactivity to novelty predicts probability of amphetamine self-administration, Behav. Pharmacol., 1 (1990) 339-345. Piazza, EV., Deminiere, J.M., Maccari, S., Le Moal, M., Mormede, E and Simon, H., Individual vulnerability to drugs self-administration: action of corticosterone on dopaminergic systems as a possible pathophysiological mechanism. In E Wilner and J. Scheel-Kruger (Eds.), The Mesolimbic Dopamine System: From Motivation to Action, Wiley, Chichester, 1991, pp. 473-495. Piazza, EV., Mac,carl, S., Deminiere, J,M., Le Moal, M., Mormede, E and Simon, H., Corticosterone levels determine individual vulnerability to amphetamine self-administration, Proc. Natl. Acad. Sci. U.S.A., 88 (1991) 2088-2092. Ritz, M.C., Lamb, R.J., Goldeberg, S.R. and Kuhar, M.J., Cocaine receptors on dopamine transporters are related to the self-administration of cocaine, Science, 237 (1987) 1219-1223. Robinson, T.E. and Becker, J.B., Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychosis, Brain Res. Rev., 11 (1986) 157-198. Sclienk, S., Lacelle, G., Gorman, K. and Amit, Z., Cocaine self-administration in rats influenced by environmental conditions: implications for the etiology of drug abuse, Neurosci. Lett., 81 (1987) 227-231. Schenk, S., Horger, B.A., Peltier, R. and Shelton, K., Supersensitivity to the reinforcing effects of cocaine following 6-hydroxydopamine lesions to the median prefrontal cortex in rats, Brain Research, 543 (1991) 227-235. Simon, H. and Le Moal, M., Mesencephalic dopaminergic neurons: role in the general economy of the brain, Ann. N.Y. Acad. Sci., 537, 1988, pp. 293-307. Simon, H., Stinus, L. and Le Moal, M., Effets de la 16sion des noyaux du rapli6 sur l'auto-administration de d-amphetamine chez le rat: augmentation considerable de l'appetence au toxique, CR Acad. Sci. (III), 290 (1980) 225-258. Simon, H., Taghzouti, K., Gozlan, H., Studler, J.M., Louilot, A., Herve, D., Olowinski, J., Tassin, J.P. and Le Moal, M., Lesion of dopaminergic terminals in the amygdala produces enhanced locomotor response to d-amphetamine and apposite changes in dopaminergic activity in prefrontal cortex and nucleus accumbens, Brain Research, 447 (1988) 335-340. Tassin, J.P., Herve, D., Vezina, P., Trovero, E, Blanc, G. and Glowinski, J., Relationships between mesocortical and mesolimbic dopamine neurons: functional correlates of D1 receptors heteroregulation. In P. Wilner and J. Scheel-Kruger (Eds.), The Mesolimbic Dopamine System." From Motivation to Action, Wiley, Chichester, 1991, pp. 175-196.
169
BRES 24966
Dopaminergic activity is reduced in the prefrontal cortex and increased in the nucleus accumbens of rats predisposed to develop amphetamine self-administration Pier Vincenzo Piazza, Fran~oise Roug6-Pont, Jean Marie Demini~re, Martine Kharoubi, Michel Le Moal and Herv6 Simon Laboratoire de Psychobiologie des Comportements Adaptatifs 1NSERM U259, Universit# de Bordeaux 11, Bordeaux (France)
(Accepted 10 September 1991) Key words: Addiction; Self-administration; Amphetamine; Individual difference; Stress; Novelty; Dopamine; Frontal cortex; Nucleus aeeumbens; Striatum
Individual vulnerability to the reinforcing effects of drugs appear to be a crucial factor in the development of addiction in humans. In the rat, individuals at risk for psychostimulant self-administration (SA) may be identified from their locomotor reactivity to a stress situation such as exposure to a novel environment. Animals with higher locomotor responses to novelty (High Responders, HR) tend to acquire amphetamine SA, while animals with the lower responses (Low Responders, LR) do not. In this study, we examined whether activity of dopaminergic (DA) and serotoninergic (5-HT) systems differed between HR and LR animals. These transmitter systems are thought to be involved in the reinforcing effects of psychostimulants. Animals from both groups were sacrificed under basal conditions and after exposure for 30 or 120 min to a novel environment, and the DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-HT, and 5-hydroxyindolacetic acid (5-HIAA) contents were determined in the prefrontal cortex, nucleus accumbens and striatum. The HR rats displayed a specific neurochemical pattern: a higher DOPAC/DA ratio in the nucleus accumbens and striatum and a lower one in the prefrontal cortex. Furthermore, HR animals had lower overall 5-HT and 5-HIAA levels, corresponding to the mean of these compounds for the three structures studied over the three environmental conditions. Since similar DOPAC/DA ratios are found in animals in which the propensity to psychostimulants SA was induced by brain lesions or life events, an opposite pattern of dopaminergic activity in the prefrontal cortex (decrease) and in the ventral and dorsal striatum (increase) may be one of the neurobiological substrate of the predisposition to acquire amphetamine self-administration. The development of this difference may be influenced by interactions between dopaminergic and serotoninergic systems. These results suggest that potential therapeutic strategies for the treatment of addiction will need to take into account the functional heterogeneity of projection systems rather than concentrate on a single neurotransmitter. Clinical investigations of addictive behavior indicate that individual vulnerability to the reinforcing effects of drugs 4 has a large influence on the development of drugseeking behavior in humans 28. In a recent study, by using an intravenous self-administration (SA) acquisition paradigm 2°, we showed that rats exhibit a wide range of individual differences in their propensity to self-administer amphetamine 7. This individual predisposition may be predicted from the behavioral 31'32 and endocrinologica134 reactivity to stressfull environmental and pharmacological challenges. Animals with a high reactivity to novelty, defined as High Responders (HR) and having an activity score above the median of the group, acquire and maintain amphetamine self-administration (SA). Conversely, animals with a low reactivity to novelty, defined as Low Responders (LR) do not acquire SA. The H R rats also showed a higher locomotor response to amphetamine, and a prolonged corticosterone secretion in response to novelty than the L R animals (for review
see ref. 33). Furthermore, it was found that the H R rats had a lower affinity of central corticosteroids receptors 25, which may underlie the difference in endocrinological reactivity to novelty between the two groups 26. In the present series of experiments, we compared the functioning of mesencephalic dopaminergic and serotoninergic neurons of H R and L R animals by measuring the content of the neurotransmitter and its main metabolite in different anterior brain structures. These neurotransmitter systems were chosen for two main reasons. First, numerous lesional and pharmacological studies have indicated that the dopaminergic mesolimbic system is a principal neurobiological substrate for the psychostimulant reinforcing effect both during acquisition and maintenance of SA (for review see refs. 5, 15, 19). Second, serotoninergic activity is thought to modulate the reinforcing properties of psychostimulants 21'24'4°. H R and L R animals were compared to try to identify the neurobiological characteristics of rats with different
Correspondence: P.V. Piazza, INSERM U259, Rue Camille Saint Sa6ns, 33077 Bordeaux Cedex, France.
170 propensities for acquisition of psychostimulant SA without exposing the animals to these drugs. Comparisons made after testing for SA cannot discriminate between preexisting and drug-induced differences. Indeed, repeated exposure to psychostimulants may induce longlasting changes in the activity of the relevant neuronal substrates 36. As an index of dopaminergic and serotoninergic activity, the post-mortem contents of dopamine (DA), serotonin (5-HT), and their respective metabolites, 3,4dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA) of H R and LR rats were determined in the prefrontal cortex, nucleus accumbens and dorsal striatum under basal condition and after exposure to novelty. Male Sprague-Dawley rats (280-300 g b.wt.) were used. The animals were individually housed with ad libitum access to food and water. A constant dark light cycle (on 08.00, off 20.00 h) was maintained in the animal house, in which temperature (22 °C) and humidity were controlled. For this experiments, 60 rats were first tested for locomotor reactivity to novelty, and divided into 3 experimental groups (n -- 20 each), equals for activity scores. One week later all the rats were sacrificed for biochemical analysis of the 3 brain regions. The animals from the first group were sacrificed immediately after removal from their home cage (basal condition). The rats from the other two groups were sacrificed after 30 or 120 min of exposure to novelty. In each experimental group, the 8 rats with the highest activity scores were designated as High Responders (HR), while the 8 animals with lowest activity scores were designated as Low Responders (LR). The remaining 4 animals in each group were eliminated from the statistical analysis of the group study. Otherwise, for the correlative study all the animals were taken into account. The novel environment consisted of a circular corridor (10 cm wide and 70 cm in diameter). Four photoelectric cells placed at the perpendicular axes of this apparatus automatically recorded locomotion. The locomotor response was recorded over 10 min intervals for a period of 2 h. Animals were tested between 16.00 and 18.00 h. The score of each animal (number of photocell counts) cumulated over this period was used as an index of individual reactivity to the novel environment. For the biochemical assays, animals were sacrificed by decapitation, and the brains rapidly removed and frozen. The antero medial prefrontal cortex (PFC), the nucleus accumbens (ACC) and the dorsal striatum (STR) were dissected out of frozen brain slices of different thicknesses (PFC = 600/~m, ACC = 700/~m, STR = 900/~m) using a micropunching technique 3°. The following an-
teroposterior (AP) levels (from bregma) of the K6nig and Klippel atlas TM constituted the anterior surface of the slices: PFC, AP = 10.5 mm; ACC, AP = 9.9 mm; STR, AP = 7.9 ram. After tissue homogenization in 0.1 N perchloric acid and centrifugation, dopamine (DA), 3,4dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) contents were measured by high performance liquid chromatography (HPLC) with electrochemical detection 27. The chromatographic system consisted of a Milton Roy Constametric pump, a refrigerated automatic injector (CMA200 Carnegie Medicine), a precolumn (C18 cartridge, 30 mm x 4.6 mm i.d.) and C18 reverse phase column (Spherisorb ODS II 5 gm, 150 mm x 4.6 mm i.d.). The compounds of interest were detected using an amperometric detector (BAS LC4B), and chromatograms were recorded on an RC5A integrator (Shimadzu). Protein content of the punches was analyzed using the Micro BCA Protein assay reagent (Pierce). The results are expressed as ng/mg of protein. The behavioral and biochemical data from the H R and LR animals were compared by analysis of variance (ANOVA) for repeated measures. Student's t-test was employed for post hoc comparisons, and Pearson's test was used for the correlation analysis for each condition over all the groups. The locomotor activity displayed by the H R (n = 24) and the LR (n = 24) animals of the 3 experimental groups during the first exposure to novelty was: 422 --- 23 for LR and 843 - 41 for HR. Thus, H R rats showed a higher locomotor activity (F1,46 = 19.52, P < 0.001), but activities of both groups returned to the baseline after 2 h testing. For the two groups of animals sacrificed after exposure to novelty, locomotor activity during the first and second tests were highly correlated (r = 0.67 (P < 0.01) for 30 min novelty exposure, and r = 0.70 (P < 0.01) for 120 min). Dopamine content did not differ between the HRs and LRs in any of the structures studied, although the H R animals had a significant higher DOPAC content than the LR group (ANOVA Group effect F2,42 = 14.05, P < 0.001) (Table I). This difference was dependent on the structure studied (ANOVA Group x Structure interaction F2,84 = 11.39, P < 0.0001). The H R animals had less DOPAC in the cortex than the LR animals (ANOVA Group effect F1,42 = 2.99, P < 0.05), whereas the H R rats had more DOPAC in the nucleus accumbens (ANOVA Group effect F1,42 = 14.52, P < 0.001) and striatum (ANOVA Group effect F1,42 = 3.27, P < 0.05) than the LR animals. No group-novelty interaction was found in any of the structures studied. DOPAC/DA ratios differed between the H R and LR animals in the various brain structures (ANOVA Group
171 TABLE I
Dopamine and DOPAC content in High-Responder (HR) and Low Responder (LR) animals Basal: animals were sacrificed in basal condition. Novelty (30"): animals were sacrificed after 30 rain of exposure to novelty. Novelty (120"): animals were sacrificed after 120 rain of exposure to novelty.
DOPAC (ng/mg of protein)
Dopamine (ng/mg of protein) Basal Frontal cortex LR HR
Novelty (30 9
1.10 ± 0.094 1.33 ± 0.09
Accumbens LR HR
89.06 - 4.48 105.02 -+ 7.2
Striatum LR HR
90.36 ± 5.68 94.49 +- 6.35
Novelty (120")
Basal
Novelty (30") 1.05 ± 0.39 0.45 ± 0.04**
Novelty (120")
1.33 ±0.13 1.31 ± 0.17
1,46 ± 0.16 1.57 ± 0.27
0.32 ± 0.03 0.22 ± 0.03*
0.51 ± 0.14 0.53 ± 0.05
89.63 ± 7,15 94.74 ± 5,47
119.08 - 6.7 117.05 ± 10.4
15.42 ± 1.22 20,35 -- 1.02"*
13.82 ± 1.27 14.97 - 1.1
11.04 - 1.39 16.94 ± 1.59"*
129.69± 3.36 123.32± 4.64
146.85 - 6.5 141.29 ± 3.9
5.11 +- 0 . 6 0 5.87 ± 0.58
10.041± 0.55 10.02 ± 0.56
9.31 ± 0.5 11.18-+ 0.74*
*P < 0.05; **P < 0.01 (Student t-test).
x Structure interaction/72,84 = 3.42, P < 0.05) (Fig. 1). I n the cortex, the H R animals had a lower D O P A C / D A ratio than the L R group ( A N O V A G r o u p effect F1.42 = 2.89, P < 0.05), while in the nucleus accumbens ( A N O V A G r o u p effect FI,, 2 = 13.40, P < 0.001) and dorsal striatum ( A N O V A G r o u p effect F1,42 = 8.41, P < 0.01) the H R animals had a higher D O P A C / D A ratio than the L R group. No group-novelty interaction was
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Fig. 1. DOPAC/dopamine ratio of HR and LR animals in basal condition (Basal) and after 30 rnin [Novelty(30')] and 120 min [Novelty(120")] exposure to novelty. HR animals had a lower ratio in the prefrontal cortex (/71,42 = 2.89, P < 0.05) and a higher one in the nucleus aeeumbens (F1,42 = 13.40, P < 0.001) and dorsal striatum (F1,42 = 8.41, P < 0.01) *P < 0.05; **P < 0.01.
found in any of the structures studied. However, a post hoc comparison showed that these differences were significant: (i) in the cortex, u n d e r basal condition and after 30 rain exposure to novelty (P < 0.05); (ii) in the nucleus accumbens, u n d e r basal condition (P < 0.05) and after 120 rain exposure to novelty ( P < 0.01): (iii) in the striatum, after 120 min of exposure to novelty (P <
0.01). The correlative analysis extended the results of the group study. There was a significant correlation between the DOPAC content and the locomotorresponse to novelty. In this case, the main result was a positive correlation between the locomotor response to novelty and the DOPAC content in the nucleus accumbens in both the basal condition (r -- 0.54, P < 0.01) (Fig. 2A) and after exposure to novelty (after 30 min r = 0.39, P < 0.05; after 120 min r = 0.62, P < 0.01). There was a negative correlation between the DOPAC content in the prefrontal cortex and the locomotor response to novelty (basal r = -0.56, P < 0.01; after 30 rain r = -0.39, P < 0.05) (Fig. 2B), while in the striatum a positive correlation with locomotor response to novelty was only found after 120 rain exposure (r = 0.70, P < 0.01). Similar correlations were found between the DOPAC/DA ratio and the locomotor response to novelty: (i) cortex = basal, r = -0.59, P < 0.01; novelty 30 rain, r = -0.39, P < 0.05; (ii) accumhens = basal, r = 0.39, P < 0.05; novelty 30 rnin, r = -0.47, P < 0.05; novelty 120 rain, r = -0.67, P < 0.01; (iii) striatum = novelty 120 min, r = -0.75, P < 0.001. The H R animals had a lower overall content of 5-HT ( A N O V A G r o u p effect F1,42 = 5.66, P < 0.05) and 5 - H I A A ( A N O V A G r o u p effect F1,42 = 4.01, P < 0.05) than the L R animals in the various brain structures. No
172
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Fig. 2. Correlation between DOPAC content in basal condition and the locomotor response to novelty in the nucleus accumbens (A) and in the prefrontal cortex (B). The two parameters were positively correlated in the nucleus accumbens (r = 0.54, P < 0.01) and negatively correlated in the prefrontal cortex (r = -0.56, P < 0.01).
interactions between the groups and the different structures were found for either compound. Furthermore, the 5-HIAA/5-HT ratio did not differ between the two groups. The overall content, as calculated by ANOVA, corresponded to the mean of these compounds for the three structures studied over the three environmental conditions. The content of 5-HT in ng/mg of proteins was: LR = 6.5 - 0.3, H R = 5.7 - 0.23. The content of 5-HIAA in ng/mg of proteins was: LR = 5.6 --- 0.22, H R = 5 . 0 ± 0.15. A correlative analysis was not carded out for the serotoninergic system as the two groups only showed an overal group effect. Our results show that different predispositions to drug-seeking behavior are related to differences in the dopamine utilization in the mesocorticolimbic system. Subjects with a higher predisposition to acquire amphetamine self-administration (HR group) had a lower D O P A C / D A ratio in the cortex and a higher ratio in the nucleus accumbens and dorsal striatum than the less predisposed subjects (LR group). These differences were matched by changes in D O P A C content, although the two groups did not differ in dopamine content. H R animals also had less serotonin and 5-HIAA than the LR group over the 3 structures studied. The opposite difference in D O P A C content between H R and LR animals in the prefrontal cortex ( H R < LR) and the nucleus accumbens or dorsal striatum (HR > LR) may be understood in terms of the functional relationships of D A transmission in these structures. Biochemical studies have suggested that D A release in the prefrontal cortex exercises an inhibitory control on D A transmission in subcortical structures 19'22'39. For example, in vivo studies have shown that stimulation of D A transmission in the prefrontal cortex by local infusion of
D A agonists decreases D O P A C release in the nucleus accumbens 23. Conversely, inhibition of D A transmission in the cortex by local infusion of neuroleptics increases D O P A C release in the nucleus accumbens 2a. Furthermore, it has been shown that the up-regulation of striatal D 1 dopaminergic receptor induced by lesion of the nigrostriatal dopaminergic system is attenuated by a concomitant lesion of D A afferents to the prefrontal cortex 42. Thus the higher D A activity in the nucleus accumbens or striatum of the H R animals may stem from the lower D A activity in the prefrontal cortex. This notion is supported by the different temporal pattern in the response to novelty in these structures between the H R and LR animals Thus, the difference between the two groups appears first in cortex (30 min of novelty exposure) and then in the ventral striatum (120 min of novelty exposure). Differences in dopaminergic activity in the prefrontal cortex may account for the differences in behavioral reactivity to environmental and pharmacological challenges between the two groups. Indeed, selective lesion of D A afferents in the prefrontal cortex after local injection of 6 - O H D A has been reported to induce an increase in: i) stress-induced D A release in the nucleus accumbensS; ii) the locomotor response to novelty2; iii) the propensity to self-administer cocaine 38. These biochemical differences between the H R and LR animals may throw more light on the biological basis of drug-seeking behavior. Thus, a decrease in D A transmission in the cortex and an increase in the nucleus accumbens have been repeatedly associated with an enhanced propensity to self-administer psychostimulants. For example, 6 - O H D A lesion of the amygdala 6 or electrolytic destruction of the ventral raphe nuclei 4°, both of which induce this biochemical pattern 12'41, also increase
173 the vulnerability to self-administer amphetamine and the psychomotor effects of this drug. Furthermore, social isolation, which increases both the propensity to self-administer cocaine 37 and the locomotor response to novelty 9,t° and amphetamine 17, also induces the differential effects on dopaminergic activity in the cortex and nucleus accumbens 1. Finally, an opposite effect of D A receptor activation in the prefrontal cortex (inhibition) and in the nucleus accumbens (facilitation) has been described for forebrain self-stimulation 29. Thus, a lower dopaminergic activity in the prefrontal cortex and a higher one in the nucleus accumbens may be considered as a c o m m o n biological substrate of a spontaneous or experimentally induced predisposition to psychostimulant drug-seeking behavior. The lower 5-HT and 5 - H I A A contents observed in the H R animals may be attributed to reduced serotoninergic activity. This result is in line with the role ascribed to serotoninergic systems in psychomotor stimulant reinforcement. For example, injection of the serotoninergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) in the ventricles 24 and medial forebrain bundle ls has been shown to increase the rate of amphetamine SA. Recently L o h and Roberts 21 have shown that serotoninergic lesions also increase cocaine seeking behavior. Infusion of 5,7-DHT into the medial forebrain bundle or amygdala increases the break-point for cocaine SA in a progressive ratio schedule of reinforcement. This effect may be explained in terms of interactions between dopaminergic and serotoninergic systems. U n d e r certain experimental conditions, serotonin seems to exercise an inhib-
itory control on D A activity n-13, and a decrease in 5-HT transmission may drive the dopaminergic system to higher reactivity to psychostimulants. Indeed, 5-HT depletion also potentiates other psychostimulant effects, such as locomotor activity a and stereotypy 16. However, it has also been suggested that the direct action of psychostimulants on serotoninergic neurons may have an antagonist effect on the reinforcing properties of these drugs. The correlative study of Ritz et al. 38 indicated that the behavioral potency in self-administration of cocaine analogs was positively related to the affinity of the drug at the D A uptake site, whereas a negative relationship was observed with the binding potency at the 5-HT uptake site, In conclusion, our results suggest that an opposite pattern of dopaminergic activity in the prefrontal cortex (decrease) and in the nucleus accumbens (increase) is a possible neurobiological substrate of the predisposition to acquire amphetamine self-administration. This neurobiological profile may originate from interactions between dopaminergic and serotoninergic systems. These results suggest that strategies for the treatment of addiction should take into account not only the role of a specific neurotransmitter and its receptors, but also the functional heterogeneity of its projections and interactions between different neurochemical systems.
1 Blanc, G., Herve, D., Simon, H., Lisoprawski, A., Glowinski, J. and Tassin, J.P., Response to stress of mesocortieo-frontal dopaminergic neurones in rats after long-term isolation, Nature, 284 (1980) 265-267. 2 Bnhser, M. and Sehmidt, W.J., 6-Hydroxydopamine lesions of the rat prefrontal cortex increases locomotor activity, impairs acquisition of delayed alternation task, but does not affect uninterrupted tasks in the radial maze, Behav. Brain Res., 37 (1990) 157-168. 3 Chow, H.L., Beck, C.H.M., p-Chlorophenylalanine and p-chloroamphetamine pretreatment of apomorphine challenged rats: effect in solitary and social behavior, Eur. J. Pharmacol., 22 (1984) 2801-2813. 4 de Wit, H., Uhlenhuth, E.H. and Johanson, C.E., Individual differences in the reinforcing and subjective effects of amphetamine and diazepam, Drug Alcohol Depend., 16 (1986) 341360. 5 Deminiere, J.M., Le Moal, M. and Simon, H., Catecholamine neuronal systems and (+) amphetamine administration in the rat. In M. Sandier (Ed.), Progress in Catecholamine Research, Liss, New York, 1988, pp. 489--494. 6 Deminiere, J.M., Taghzouti, K., Tassin, J.P., Le Moal, M. and Simon, H., Increased sensitivity to amphetamine and facilitation of amphetamine self-administration after 6-hydroxydopamine lesions of the amygdala, Psychopharmacology, 94 (1988) 232-236. 7 Deminiere, J.M., Piazza, P.V., Le Moai, M. and Simon, H.,
Experimental approach to individual vulnerability to psychostimulant addiction, Neurosci. Biobehav. Rev., 13 (1989) 141147. Deutch, A.Y., Clark, W.A. and Roth, R.H., Prefrontal cortical dopamine depletion enhances the responsiveness of mesolimbic dopamine neurons to stress, Brain Research, 521 (1990) 311315. Gentsch, C., Lichtsteiner, M. and Feet, H., Behavioral comparison between individually- and group-housed male rats: effects of novel environments and diurnal rhythm, Behav. Brain Res., 6 (1982) 93-100. Gentsch, C., Lichtsteiner, M., Frischknecht, H.R., Feer, H. and Siegfried, B., Isolation-induced locomotor hyperactivity and hypoalgesia in rats are prevented by handling and reversed by resocialization, Physiol. Behav., 43 (1988) 13-16. Hamon, M., Fattaccini, C.M., Adrien, J., Gallissot, M.C., Martin, P. and Gozlan, H., Alteration of central serotonin and dopamine turnover in rats treated with ipsapirone and other 5-hydroxytryptaminelA agonists with potential anxiolytic properties, J. Pharmacol. Exp. Ther., 246 (1988) 745-752. Herve, D., Simon, H., Blanc, G., Le Moal, M., Glowinski, J. and Tassin, J.P., Opposite changes in dopamine utilization in the nucleus aceumbens and the frontal cortex after electrolytic lesion of the median raphe in the rat, Brain Research, 216 (1981) 422-428. Kelland, M.D., Freeman, A.S. and Chiodo, L.A., Serotonergic afferent regulation of the basic physiology and pharmaco-
This work was supported by rInstitut National de la Sant6 et de la Recherche M6dicale (INSERM) and l'Universit6 de Bordeaux II. We thank Isabelle Bathy for secretarial assistance.
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