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Chronic morphine alters dopamine transporter density in the rat brain: Possible role in the mechanism of drug addiction
Neuroseience Letters, 163 (1993) 121 124 © 1993 Elsevier Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
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Chronic morphine alters dopamine transporter density in the rat brain" possible role in the mechanism of drug addiction Rabi Simantov Department o[Molecular Genetics and Viroh~gy. The Weizmann Institute q/Science. Rehovot 7¢5100. Lsrael
(Received 13 July 1993; Revised version received 3 September 1993:Accepted 3 September 1993) Key words.
Drugreinforcement: Opioid: Nucleus accumbens:Transporter
The effect of acute or chronic morphine treatment on dopamine transporters was studied with the selective transporter blocker [~H]GBR12935. Chronic, but not acute treatment of rats with morphine significantlydecreased the Bm~,~of [3H]GBRI2935binding to membranes from the anterior basal forebrain, that includes the nucleus accumbens, but had no such effect on binding to striatum membranes. No effect on the affinity (Kd) of the radioligand to either one of the two brain regions was observed. The selectivity of morphine interaction with the dopamine system was tested with a ligand that binds selectively to serotonin transporters, [~H]citalopram. Neither acute nor chronic morphine altered [~H]citalopram binding to the anterior basal forebrain, or the striatum membranes. It is suggested, therefore, that chronic morphine treatment has a long-lasting and selective effect on the activity of dopamine transporters in the dopaminergicreward pathway, but not in the striatum.
Compounds abused by humans may belong to different chemical groups such as opiates, stimulants (cocaine or D-amphetamine), alcohol or nicotine. It is conceivable, therefore, that upon entering the brain the first neurochemical effect of the various agents is different, though characteristic, for each compound. Yet, as they all are abused by humans, and induce reward and reinforcement, it is likely that they promote some c o m m o n secondary neurochemical changes. It is currently accepted that out of the major neurotransmitters of the brain, dopamine is the prime candidate for playing a key role in reward processes [3, 7, 9, 16, 18, 25]. As regard to opioids, many studies have shown their interaction with the dopamine system [1, 3, 5-10, 13, 14, 16 18, 21, 24 27]. Thus, cocaine, a dopamine uptake blocker [15, 22], increases several in vivo effects of morphine, including analgesia, whereas opiate antagonists, such as naloxone and naltrexone, can partially block the effect of increased dopamine levels, induced by D-amphetamine. Additional studies supporting the notion of the close relationship between the opioid and dopamine system in the mesolimbic pathway were obtained by lesion experiments; destruction of presynaptic dopaminergic structures in the nucleus accumbens blocked cocaine self-administration, and kainic acid application to this nucleus disrupts both cocaine and heroine self-administration [27]. The recent observations that morphine [6 8] or enkephalin [13] alter dopamine release in the stria-
tum and/or the nucleus accumbens prompted us to investigate the possibility that acute or chronic morphine treatment may modulate the activity of the key element controlling dopamine levels at the synapse, the dopamine transporter. Male albino Wistar rats (Weizmann Institute of Science, Department of Hormone Research colony) were housed 4 per cage with free access to laboratory food pellets and water. Colony lights were on tYom 05.00 to 19.00 h and the room was maintained at 24°C. Groups of four rats were injected intraperitoneally with morphine chloride (purchased from Teva, Petach-Tikva, Israel) as follows. Acutely treated rats were injected once with 40 mg/kg. Chronically treated rats were injected once daily for four consecutive days with 30, 40, 50 and 75 mg/kg. In both treatments an additional group of control rats was treated similarly with the vehicle (saline). Animals were decapitated 15 min after the acute treatment, or 24 h after the last injection of the chronic treatment, and the brains were dissected, frozen on dry-ice and maintained at -80°C. Behaviorally, animals treated with the single treatment of morphine showed the characteristic symptoms of an acute, high morphine dose, which lasts up to 2 3 h. No withdrawal symptoms were observed in the rats treated chronically with morphine, apparently because injections were made in 24-h intervals. The amount of tissue needed for the binding experiments dictated the way the brains were dissected: each brain was divided
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[3H-CITALOPRAM] ,nM Fig. 1. Scatchard analysis o f [3H]GBR 12935 (A) a n d [3H]citalopram (B) binding to rat anterior basal forebrain membranes; effect of chronic morphine treatment. Animals were injected for 4 consecutive days and analyzed as described in the text. Data are from a representative experiment, replicated 4 times.
sagitally into two halves, the striatum was separated and saved, and the thalamus, hypothalamus, midbrain, hippocampus, hindbrain, cerebellum and posterior + lateral cerebral cortex were removed. The anterior basal forebrain, which includes the nucleus accumbens, was then dissected and analyzed in parallel to the striatum. Though analysis of just the nucleus accumbens could be more informative and accurate, it was beyond the capabilities of the present study. It is important, therefore, to clarify that the anterior basal forebrain does contain other cortical dopaminergic target structures in addition to the nucleus accumbens, such as the olfactory tubercle. [3H]GBR12935 binding to crude membrane preparation was performed, as described [2, 4] with minor changes. Each tissue sample was weighted and homogenized for 10 s (Polytron, setting 4) in 3 ml ice-cold 0.32 M sucrose containing 10 mM sodium phosphate buffer pH 7.4 at 25°C. Five ml of the same buffer was added after the homogenization, and samples were centrifuged for 10 min at 27,000 x g. The pellets were suspended by Polytron (5 s) in 3 ml 50 mM Tris-HC1, pH 7.4 at 25°C,
containing 120 mM NaCI and 5 mM KCI. Five mt ot the same buffer was added, and samples were recentrifuged 10 min at 27,000 × g. The final pellet was suspended in the Tris-NaC1-KC1 buffer (Polytron, 10 s) at 5.5 mg wet tissue per ml. The same protocol has been used to determine the binding of [3H]citalopram. Binding was conducted with 0.45 ml of the tissue homogenate and 0.13 5.0 nM [3H]GBR12935 or 0.22-12.6 nM [3H]citalopram. Non-specific binding was determined with samples containing 10-5 M nomifensine or zimelidine, respectively. Samples were incubated at final volume of 0.5 ml for 30 rain at 25°C, filtered with the Brandel Cell Harvester, washed 4 times, dried and counted with 70% of efficiency. The results were presented as means + S.D., and statistical significance was determined by the Student's t-test. Fig. IA shows the result of [3H]GBR12935 binding to membranes prepared from the anterior basal tbrebrain of rats treated for four consecutive days with saline (control group) or morphine chloride. Scatchard analysis of this saturation binding experiment indicates that the chronic morphine treatment reduced the Bm,x of [3H]GBR12935 binding from 48.0 + 6.1 to 34.1 +4.3 pmol/g wet tissue (n = 5; P - 0.05), with no effect on the affinity of the ligand (K~ 4.6 + 0.8 and 4.1 + 0.1 nM for control and morphine-treated samples, respectively). It was of interest to find that the chronic morphine application had no effect on the binding of the selective serotonin transporter blocker [3H]citalopram to the same tissue (Fig. 1B). The Bin,x of [3H]citalopram binding to saline or morphine-treated rats was 24.4 + 2.1 and 24.6 + 2.9 pmol/g tissue, and the Kd 2.2 + 0.3 and 1.8 + 0.5 nM, respectively. High concentration of [3H]GBR12935 (5 nM) was used to test whether a single application of morphine chloride had any effect on the number of dopamine transporters. Fig. 2 shows that acute treatment with morphine has no effect on the anterior basal forebraim suggesting therefore that upon repeated treatment with this drug there is a change in the dopamine uptake machinery. As the anterior basal forebrain tested in the experiments described above contains the nucleus accumbens, the possibility that the alteration in dopamine transporters may be confined to brain regions involved in drug reinforcement and reward needs further analysis. Thus, the effect of chronic morphine application on a dopamine-rich region which has not been involved with drug abuse, the striatum, was determined (Fig. 2). It has been found that neither [3H]GBR12935 nor [3H]citalopram binding to striatal membranes were significantly affected by chronic morphine. Also, single treatment with morphine has no effect on the binding of these two transporter blockers to striatal membranes (Fig. 2).
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Fig. 2. [3H]GBR12935 and [3H]citalopram binding to anterior basal forebrain m e m b r a n e s or striatum m e m b r a n e s of rats treated acutely or chronically with morphine. A single concentration of the two ligands (5.0 and 12.6 n M , respectively) has been used in these experiments. The anterior basal forebrain o f chronically treated rats was reassayed with [SH]GBRI2935 as a reference to the other treatments and to confirm the results depicted in Fig. 1. A,C: anterior basal forebrain; B,D: striaturn. Data are mean + S.D. from three experiments. N o significant differences between saline and morphine-treated groups have been observed, except the indicated *, P < 0.05.
This study shows for the first time that morphine alters the binding of the selective dopamine transporter inhibitor [3H]GBR 12935 to rat brain membranes. The selectivity of this phenomenon was indicated by several criteria. (i) Decreased binding of the ligand was observed in chronic but not acutely treated animals. (ii) Morphine effect on dopamine transporters appears to be regionally confined to the anterior basal forebrain, as no effect on striatal transporters was observed. (iii) Neither acute nor chronic morphine treatment affect serotonin transporters in either the anterior basal forebrain or the striaturn. Also, serotonin transporters were not affected in the midbrain, occipital cortex and pons-medulla (data not shown). (iv) The chronic morphine treatment reduces the Bm,x of [3H]GBR12935 binding, namely the apparent number of transporter molecules, but has no effect on the affinity of the ligand to the protein. Taken together, the results suggest that chronic morphine application down regulates the expression of dopamine transporters in the anterior basal forebrain. As the nucleus accumbens is the richest dopaminergic structure in this brain region, down regulation of dopamine transporters apparently reflects adaptive changes associated with the rewarding and reinforcing effect of long-term exposure to the opioid drug. Yet, one cannot exclude the possibility that the difference between the anterior basal forebrain and striatum dopamine transporters may reflect some of the pharmacological and molecular differences between striatal and nucleus accumbens dopamine transporters [19, 20]. Over the last decade it has been established that opi-
ates increase the firing of ventral tegmental dopaminergic neurons [5, 10], enhance dopamine release in the nucleus accumbens [7], and facilitate various behavioral activities, including reward and reinforcement. Though the detailed cellular and molecular interactions between opioids and the mesolimbic dopamine pathways are still unclear, possibly due to the involvement of several opioid receptors such as/~, x and 5 (see refs. 5, 8 and 17), it is likely that the enhanced release of dopamine in the nucleus accumbens can stimulate a sequence of adaptive processes. In this context, it is of interest to recall the following observations. Studies in the seventies (e.g. ref. 3) have shown that rodents treated chronically with morphine are supersensitive to dopamine-like compounds, or to the dopamine precursor L-dopa. A more recent study from DiChiara laboratory [1] indicates that morphine withdrawal decreases the extracellular level of dopamine in the nucleus accumbens. Interestingly, when compared to acute injection, daily application ofenkephalin into the ventral tegmental area elevates the extracellular dopamine levels in the nucleus accumbens [13]. Thus, activation of/a, x or 5 receptors in the brain may have different and even opposite effects on dopamine release (e.g. refs. 6, 8, 17 and 24). It is, however, important to bear in mind that the systemic application of morphine [1], in contrast to the local injection of enkephalin to the nucleus accumbens [13], can initiate a cross-talk between opioid receptors (possibly ofx type) and presynaptic dopamine terminals. Lesion experiments with kainic acid and 6-hydroxydopamine indeed support the notion of dual interaction between opioid receptors and the dopamine reward pathway [27 and references included]. Though clear conclusions about the nature of the adaptive changes related to the dopamine transporter need further analysis, some information on this issue may be received from experiments with another rewarding and reinforcing drug [22] that enhances dopamine release [7], namely cocaine. Recent studies have shown that daily treatment of rats with cocaine produced persistent reduction in dopamine uptake capacity of the nucleus accumbens [11]. This important finding, which was not observed in the striatum, therefore raises the possibility that a long-term increase in dopamine release reduces dopamine uptake in a selective fashion, confined to the pathway involved in reward. Thus, though acute treatment with cocaine and morphine has a similar stimulatory effect on both the striatum and nucleus accumbens [7], the two brain regions differ in their response to chronic exposure to the two drugs. As opioids have dual effect on the ventral tegmental dopamine cells and nucleus accumbens terminals, possibly of opposite nature (see refs. 7, 8, 14, 17, 18 and 24), it will be interesting to analyze whether the nigl:ostriatal pathway differs in this
124
respect. It is also important to note that the opioid's effect on the ventral tegmental dopamine neurons is apparently indirect [23, 24], and thus involves other cells, such as GABA neurons. Further analysis of the changes in dopamine transporters, at the functional as well as molecular levels, may shed light on some of the key elements involved in the abuse of opiates and possibly other drugs. The study was supported by the Center for Neuroscience at the Weizmann Institute of Science, The AntiDrug Authority of Israel, and the Office of the Chief Scientist, Ministry of Health, Israel. 1 Acquas, E., Carboni, E. and DiChiara, G., Prolbund depression of mesolimbic dopamine release after morphine withdrawal in dependent rats, Eur. J. Pharmacol., 193 (1991) 133-134. 2 Anderson, P.H., Biochemical and pharmacological characterization of 3H GBRI2935 binding in vitro to rat striatal membranes: labeling of the dopamine uptake complex, J. Neurochem., 48 (1987) 1887-1896. 3 Attila, L.M. and Ahtee, L., Retardation of cerebral dopamine turnover after morphine withdrawal and its enhanced acceleration by acute morphine administration in rats, Naunyn-Schmiedeberg's Arch. Pharmacol., 327 (1984) 201 207. 4 Berger, E, Janowsky, A., Vocci, F., Skolnick, R, Schweri, M.M. and Paul, S.M., [3H]GBR-12935: a specific high affinity ligand for labeling the dopamine transport complex, Eur. J. Pharmacol., 107 (1985) 289--290. 5 Broekkamp, C.L.E., Phillips, A.G. and Cools, A.R., Stimulant effects of enkephalin microinjection into the dopaminergic A 10 area, Nature, 278 (1979) 560-562. 6 Chesselet, M.F., Cheramy, A., Reisine, T.D. and Glowinski, J., Morphine and ~-opiate agonists locally stimulate in vivo dopamine release in cat caudate nucleus, Nature, 291 (1981) 320-322. 7 DiChiara, G. and Imperato, A., Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats, Proc. Natl. Acad. Sci. USA, 85 (1988) 5274 5278. 8 DiChiara, G. and Imperato, A., Opposite effects of Mu and Kappa opiate agonists on dopamine release in the nucleus accumbens and in the dorsal caudate of freely moving rats, J. PharmacoI. Exp. Ther., 244 (1988) 1067 1080. 9 Fibiger, H.C., Drugs and reinforcement mechanisms: a critical review of the catecholamine theory, Annu. Rev. Pharmacol. Toxicol., 18 (1978) 37 56. 10 Gysling, K. and Wang, R.Y., Morphine-induced activation of AI0 dopamine neurons in the rat, Brain Res., 119 (1983) 119- 127. 11 Izenwasser, S. and Cox, B.M., Daily cocaine treatment produces a persistent reduction of [3H]dopamine uptake in vitro in rat nucleus accumbens but not in striatum, Brain Res., 531 (1990) 338 341.
12 Kalivas, EW. and Duffy, P., Effect of acute and daily cocaine treatment on extracellular dopamine in the nucleus accumbens. Synapse, 5 (1990) 48-58. 13 Kalivas, EW. and Duffy, E, Effect of acute and daily neurotensiL1 and enkephalin treatments on extracellular dopaminc in the nucleu~ accumbens, J. Neurosci., 10 (1990) 2940-2949. 14 Kalivas, EW., Widerlov, E., Stanley, D., Breese, G. and Prange. A.J., Enkephalin action on the mesolimbic system: a dopaminedependent and a dopamine-independent increase in locomotor activity, J. Pharmacol. Exp. Ther., 227 (1983) 229 237. 15 Kennedy, L.T. and Hanbauer, I., Sodium-sensitive cocaine binding to rat striatal membranes: possible relationship to dopamine uptake sites, J. Neurochem., 41 (1983) 172 178. 16 Koob, G.F. and Bloom, F.E., Cellular and molecular mechanisms of drug dependence, Science, 242 (19881 715-723. 17 Latimer, E.G., Duffy, E and Kalivas, EW., Mu opioid receptor involvement in enkephalin activation of dopamine neurons in the ventral tegmental area, J. Pharmacol. Exp. Ther., 241 (1987) 328 337. 18 Leone, E, Pocock, D. and Wise, R.A., Morphine-dopamine interaction: ventral tegmental morphine increases nucleus accumbens dopamlne release, Pharmacol. Biochem. Bebav., 39 ( 1991) 469-472. 19 Lew, R., Vaughan, R., Simantov, R., Wilson, A. and Kuhar, M.S., Dopamine transporters in the nucleus accumbens and the striatum have different apparent molecular weights, Synapse, 8 (1991) 152153. 20 Missale, C.L., Castellati, S., Gavoni, S., Spano, EF., Trabucchi, M. and Hanbauer, I., Dopamine uptake is differentially regulated in rat striatum and nucleus accumbens, J. Neurochem,, 45 (1985) 51-59. 21 Moleman, P., Effect of morphine on dopaminergic neurons in frontal cortex and striatum in rat, Life Sci., 19 (1976) 1277-1282. 22 Ritz, M.C., Lamb, R.J., Goldberg, S.R. and Kuhar, M.J., Cocaine receptors on dopamine transporters are related to self-administration of cocaine, Science, 237 (1987) 1219 1223. 23 Seutin, V., Franehimont, N., Massone, L. and Dreese, A., Comparison of the effect of morphine on locus coeruleus noradrenergic and ventral tegmental area dopaminergic neurons in vitro, Life Sci,, 46 (1990) 1879-1885. 24 Spanagel, R., Herz, A. and Shippenberg, T.S., Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway, Proc. Natl. Acad. Sci. USA, 89 (1992) 20462050. 25 Wise, R.A. and Bozarth, M.A., Action of drugs of abuse on brain reward system: an update with specific attention to opiates, Pharmacol. Biochem. Behav., 17 (1982) 239 243. 26 Wood, EL., Opioid regulation of CNS dopaminergic pathways: a review of methodology, receptor types, regional variations and species differences, Peptides, 4 (1983) 595-601. 27 Zito, K.A., Vickers, G. and Roberts, D.C.S., Disruption of cocaine and heroin self-administration following kainic acid lesions of the nucleus accumbens, Pharmacol. Biochem. Behav., 23 (1985) 1029 1036.
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NSL 10016
Chronic morphine alters dopamine transporter density in the rat brain" possible role in the mechanism of drug addiction Rabi Simantov Department o[Molecular Genetics and Viroh~gy. The Weizmann Institute q/Science. Rehovot 7¢5100. Lsrael
(Received 13 July 1993; Revised version received 3 September 1993:Accepted 3 September 1993) Key words.
Drugreinforcement: Opioid: Nucleus accumbens:Transporter
The effect of acute or chronic morphine treatment on dopamine transporters was studied with the selective transporter blocker [~H]GBR12935. Chronic, but not acute treatment of rats with morphine significantlydecreased the Bm~,~of [3H]GBRI2935binding to membranes from the anterior basal forebrain, that includes the nucleus accumbens, but had no such effect on binding to striatum membranes. No effect on the affinity (Kd) of the radioligand to either one of the two brain regions was observed. The selectivity of morphine interaction with the dopamine system was tested with a ligand that binds selectively to serotonin transporters, [~H]citalopram. Neither acute nor chronic morphine altered [~H]citalopram binding to the anterior basal forebrain, or the striatum membranes. It is suggested, therefore, that chronic morphine treatment has a long-lasting and selective effect on the activity of dopamine transporters in the dopaminergicreward pathway, but not in the striatum.
Compounds abused by humans may belong to different chemical groups such as opiates, stimulants (cocaine or D-amphetamine), alcohol or nicotine. It is conceivable, therefore, that upon entering the brain the first neurochemical effect of the various agents is different, though characteristic, for each compound. Yet, as they all are abused by humans, and induce reward and reinforcement, it is likely that they promote some c o m m o n secondary neurochemical changes. It is currently accepted that out of the major neurotransmitters of the brain, dopamine is the prime candidate for playing a key role in reward processes [3, 7, 9, 16, 18, 25]. As regard to opioids, many studies have shown their interaction with the dopamine system [1, 3, 5-10, 13, 14, 16 18, 21, 24 27]. Thus, cocaine, a dopamine uptake blocker [15, 22], increases several in vivo effects of morphine, including analgesia, whereas opiate antagonists, such as naloxone and naltrexone, can partially block the effect of increased dopamine levels, induced by D-amphetamine. Additional studies supporting the notion of the close relationship between the opioid and dopamine system in the mesolimbic pathway were obtained by lesion experiments; destruction of presynaptic dopaminergic structures in the nucleus accumbens blocked cocaine self-administration, and kainic acid application to this nucleus disrupts both cocaine and heroine self-administration [27]. The recent observations that morphine [6 8] or enkephalin [13] alter dopamine release in the stria-
tum and/or the nucleus accumbens prompted us to investigate the possibility that acute or chronic morphine treatment may modulate the activity of the key element controlling dopamine levels at the synapse, the dopamine transporter. Male albino Wistar rats (Weizmann Institute of Science, Department of Hormone Research colony) were housed 4 per cage with free access to laboratory food pellets and water. Colony lights were on tYom 05.00 to 19.00 h and the room was maintained at 24°C. Groups of four rats were injected intraperitoneally with morphine chloride (purchased from Teva, Petach-Tikva, Israel) as follows. Acutely treated rats were injected once with 40 mg/kg. Chronically treated rats were injected once daily for four consecutive days with 30, 40, 50 and 75 mg/kg. In both treatments an additional group of control rats was treated similarly with the vehicle (saline). Animals were decapitated 15 min after the acute treatment, or 24 h after the last injection of the chronic treatment, and the brains were dissected, frozen on dry-ice and maintained at -80°C. Behaviorally, animals treated with the single treatment of morphine showed the characteristic symptoms of an acute, high morphine dose, which lasts up to 2 3 h. No withdrawal symptoms were observed in the rats treated chronically with morphine, apparently because injections were made in 24-h intervals. The amount of tissue needed for the binding experiments dictated the way the brains were dissected: each brain was divided
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[3H-CITALOPRAM] ,nM Fig. 1. Scatchard analysis o f [3H]GBR 12935 (A) a n d [3H]citalopram (B) binding to rat anterior basal forebrain membranes; effect of chronic morphine treatment. Animals were injected for 4 consecutive days and analyzed as described in the text. Data are from a representative experiment, replicated 4 times.
sagitally into two halves, the striatum was separated and saved, and the thalamus, hypothalamus, midbrain, hippocampus, hindbrain, cerebellum and posterior + lateral cerebral cortex were removed. The anterior basal forebrain, which includes the nucleus accumbens, was then dissected and analyzed in parallel to the striatum. Though analysis of just the nucleus accumbens could be more informative and accurate, it was beyond the capabilities of the present study. It is important, therefore, to clarify that the anterior basal forebrain does contain other cortical dopaminergic target structures in addition to the nucleus accumbens, such as the olfactory tubercle. [3H]GBR12935 binding to crude membrane preparation was performed, as described [2, 4] with minor changes. Each tissue sample was weighted and homogenized for 10 s (Polytron, setting 4) in 3 ml ice-cold 0.32 M sucrose containing 10 mM sodium phosphate buffer pH 7.4 at 25°C. Five ml of the same buffer was added after the homogenization, and samples were centrifuged for 10 min at 27,000 x g. The pellets were suspended by Polytron (5 s) in 3 ml 50 mM Tris-HC1, pH 7.4 at 25°C,
containing 120 mM NaCI and 5 mM KCI. Five mt ot the same buffer was added, and samples were recentrifuged 10 min at 27,000 × g. The final pellet was suspended in the Tris-NaC1-KC1 buffer (Polytron, 10 s) at 5.5 mg wet tissue per ml. The same protocol has been used to determine the binding of [3H]citalopram. Binding was conducted with 0.45 ml of the tissue homogenate and 0.13 5.0 nM [3H]GBR12935 or 0.22-12.6 nM [3H]citalopram. Non-specific binding was determined with samples containing 10-5 M nomifensine or zimelidine, respectively. Samples were incubated at final volume of 0.5 ml for 30 rain at 25°C, filtered with the Brandel Cell Harvester, washed 4 times, dried and counted with 70% of efficiency. The results were presented as means + S.D., and statistical significance was determined by the Student's t-test. Fig. IA shows the result of [3H]GBR12935 binding to membranes prepared from the anterior basal tbrebrain of rats treated for four consecutive days with saline (control group) or morphine chloride. Scatchard analysis of this saturation binding experiment indicates that the chronic morphine treatment reduced the Bm,x of [3H]GBR12935 binding from 48.0 + 6.1 to 34.1 +4.3 pmol/g wet tissue (n = 5; P - 0.05), with no effect on the affinity of the ligand (K~ 4.6 + 0.8 and 4.1 + 0.1 nM for control and morphine-treated samples, respectively). It was of interest to find that the chronic morphine application had no effect on the binding of the selective serotonin transporter blocker [3H]citalopram to the same tissue (Fig. 1B). The Bin,x of [3H]citalopram binding to saline or morphine-treated rats was 24.4 + 2.1 and 24.6 + 2.9 pmol/g tissue, and the Kd 2.2 + 0.3 and 1.8 + 0.5 nM, respectively. High concentration of [3H]GBR12935 (5 nM) was used to test whether a single application of morphine chloride had any effect on the number of dopamine transporters. Fig. 2 shows that acute treatment with morphine has no effect on the anterior basal forebraim suggesting therefore that upon repeated treatment with this drug there is a change in the dopamine uptake machinery. As the anterior basal forebrain tested in the experiments described above contains the nucleus accumbens, the possibility that the alteration in dopamine transporters may be confined to brain regions involved in drug reinforcement and reward needs further analysis. Thus, the effect of chronic morphine application on a dopamine-rich region which has not been involved with drug abuse, the striatum, was determined (Fig. 2). It has been found that neither [3H]GBR12935 nor [3H]citalopram binding to striatal membranes were significantly affected by chronic morphine. Also, single treatment with morphine has no effect on the binding of these two transporter blockers to striatal membranes (Fig. 2).
123 DOPAMINE TRANSPORTERS
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Fig. 2. [3H]GBR12935 and [3H]citalopram binding to anterior basal forebrain m e m b r a n e s or striatum m e m b r a n e s of rats treated acutely or chronically with morphine. A single concentration of the two ligands (5.0 and 12.6 n M , respectively) has been used in these experiments. The anterior basal forebrain o f chronically treated rats was reassayed with [SH]GBRI2935 as a reference to the other treatments and to confirm the results depicted in Fig. 1. A,C: anterior basal forebrain; B,D: striaturn. Data are mean + S.D. from three experiments. N o significant differences between saline and morphine-treated groups have been observed, except the indicated *, P < 0.05.
This study shows for the first time that morphine alters the binding of the selective dopamine transporter inhibitor [3H]GBR 12935 to rat brain membranes. The selectivity of this phenomenon was indicated by several criteria. (i) Decreased binding of the ligand was observed in chronic but not acutely treated animals. (ii) Morphine effect on dopamine transporters appears to be regionally confined to the anterior basal forebrain, as no effect on striatal transporters was observed. (iii) Neither acute nor chronic morphine treatment affect serotonin transporters in either the anterior basal forebrain or the striaturn. Also, serotonin transporters were not affected in the midbrain, occipital cortex and pons-medulla (data not shown). (iv) The chronic morphine treatment reduces the Bm,x of [3H]GBR12935 binding, namely the apparent number of transporter molecules, but has no effect on the affinity of the ligand to the protein. Taken together, the results suggest that chronic morphine application down regulates the expression of dopamine transporters in the anterior basal forebrain. As the nucleus accumbens is the richest dopaminergic structure in this brain region, down regulation of dopamine transporters apparently reflects adaptive changes associated with the rewarding and reinforcing effect of long-term exposure to the opioid drug. Yet, one cannot exclude the possibility that the difference between the anterior basal forebrain and striatum dopamine transporters may reflect some of the pharmacological and molecular differences between striatal and nucleus accumbens dopamine transporters [19, 20]. Over the last decade it has been established that opi-
ates increase the firing of ventral tegmental dopaminergic neurons [5, 10], enhance dopamine release in the nucleus accumbens [7], and facilitate various behavioral activities, including reward and reinforcement. Though the detailed cellular and molecular interactions between opioids and the mesolimbic dopamine pathways are still unclear, possibly due to the involvement of several opioid receptors such as/~, x and 5 (see refs. 5, 8 and 17), it is likely that the enhanced release of dopamine in the nucleus accumbens can stimulate a sequence of adaptive processes. In this context, it is of interest to recall the following observations. Studies in the seventies (e.g. ref. 3) have shown that rodents treated chronically with morphine are supersensitive to dopamine-like compounds, or to the dopamine precursor L-dopa. A more recent study from DiChiara laboratory [1] indicates that morphine withdrawal decreases the extracellular level of dopamine in the nucleus accumbens. Interestingly, when compared to acute injection, daily application ofenkephalin into the ventral tegmental area elevates the extracellular dopamine levels in the nucleus accumbens [13]. Thus, activation of/a, x or 5 receptors in the brain may have different and even opposite effects on dopamine release (e.g. refs. 6, 8, 17 and 24). It is, however, important to bear in mind that the systemic application of morphine [1], in contrast to the local injection of enkephalin to the nucleus accumbens [13], can initiate a cross-talk between opioid receptors (possibly ofx type) and presynaptic dopamine terminals. Lesion experiments with kainic acid and 6-hydroxydopamine indeed support the notion of dual interaction between opioid receptors and the dopamine reward pathway [27 and references included]. Though clear conclusions about the nature of the adaptive changes related to the dopamine transporter need further analysis, some information on this issue may be received from experiments with another rewarding and reinforcing drug [22] that enhances dopamine release [7], namely cocaine. Recent studies have shown that daily treatment of rats with cocaine produced persistent reduction in dopamine uptake capacity of the nucleus accumbens [11]. This important finding, which was not observed in the striatum, therefore raises the possibility that a long-term increase in dopamine release reduces dopamine uptake in a selective fashion, confined to the pathway involved in reward. Thus, though acute treatment with cocaine and morphine has a similar stimulatory effect on both the striatum and nucleus accumbens [7], the two brain regions differ in their response to chronic exposure to the two drugs. As opioids have dual effect on the ventral tegmental dopamine cells and nucleus accumbens terminals, possibly of opposite nature (see refs. 7, 8, 14, 17, 18 and 24), it will be interesting to analyze whether the nigl:ostriatal pathway differs in this
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respect. It is also important to note that the opioid's effect on the ventral tegmental dopamine neurons is apparently indirect [23, 24], and thus involves other cells, such as GABA neurons. Further analysis of the changes in dopamine transporters, at the functional as well as molecular levels, may shed light on some of the key elements involved in the abuse of opiates and possibly other drugs. The study was supported by the Center for Neuroscience at the Weizmann Institute of Science, The AntiDrug Authority of Israel, and the Office of the Chief Scientist, Ministry of Health, Israel. 1 Acquas, E., Carboni, E. and DiChiara, G., Prolbund depression of mesolimbic dopamine release after morphine withdrawal in dependent rats, Eur. J. Pharmacol., 193 (1991) 133-134. 2 Anderson, P.H., Biochemical and pharmacological characterization of 3H GBRI2935 binding in vitro to rat striatal membranes: labeling of the dopamine uptake complex, J. Neurochem., 48 (1987) 1887-1896. 3 Attila, L.M. and Ahtee, L., Retardation of cerebral dopamine turnover after morphine withdrawal and its enhanced acceleration by acute morphine administration in rats, Naunyn-Schmiedeberg's Arch. Pharmacol., 327 (1984) 201 207. 4 Berger, E, Janowsky, A., Vocci, F., Skolnick, R, Schweri, M.M. and Paul, S.M., [3H]GBR-12935: a specific high affinity ligand for labeling the dopamine transport complex, Eur. J. Pharmacol., 107 (1985) 289--290. 5 Broekkamp, C.L.E., Phillips, A.G. and Cools, A.R., Stimulant effects of enkephalin microinjection into the dopaminergic A 10 area, Nature, 278 (1979) 560-562. 6 Chesselet, M.F., Cheramy, A., Reisine, T.D. and Glowinski, J., Morphine and ~-opiate agonists locally stimulate in vivo dopamine release in cat caudate nucleus, Nature, 291 (1981) 320-322. 7 DiChiara, G. and Imperato, A., Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats, Proc. Natl. Acad. Sci. USA, 85 (1988) 5274 5278. 8 DiChiara, G. and Imperato, A., Opposite effects of Mu and Kappa opiate agonists on dopamine release in the nucleus accumbens and in the dorsal caudate of freely moving rats, J. PharmacoI. Exp. Ther., 244 (1988) 1067 1080. 9 Fibiger, H.C., Drugs and reinforcement mechanisms: a critical review of the catecholamine theory, Annu. Rev. Pharmacol. Toxicol., 18 (1978) 37 56. 10 Gysling, K. and Wang, R.Y., Morphine-induced activation of AI0 dopamine neurons in the rat, Brain Res., 119 (1983) 119- 127. 11 Izenwasser, S. and Cox, B.M., Daily cocaine treatment produces a persistent reduction of [3H]dopamine uptake in vitro in rat nucleus accumbens but not in striatum, Brain Res., 531 (1990) 338 341.
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