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Increased brain dopamine D4-like binding after chronic ethanol is not associated with behavioral sensitization in mice
Alcohol 37 (2005) 99–104
Increased brain dopamine D4-like binding after chronic ethanol is not associated with behavioral sensitization in mice Isabel Marian Hartmann Quadrosa, Jose Nascimento Nobregab, Debora Cristina Hipolidea, Maria Lucia Oliveira Souza-Formigonia,* a
Psychobiology Department, Federal University of Sa˜o Paulo (UNIFESP), Rua Botucatu 862, 1 . andar, 04023-062 Sa˜o Paulo, SP, Brazil b Neuroimaging Research Section, Centre for Addiction and Mental Health, 250 College Street, Toronto, ON M5T 1R8, Canada Received 1 September 2005; received in revised form 30 November 2005; accepted 1 December 2005
Abstract Dopaminergic D4 receptors have been hypothesized to be involved in neuropsychiatric disorders and substance abuse. In mice, repeated ethanol administration may induce behavioral sensitization, a phenomenon of increased sensitivity to the drug’s stimulant properties. This study aimed to analyze brain D4 receptors binding in mice with different levels of behavioral sensitization to ethanol. Male Swiss mice received 2.2 g/kg ethanol (n 5 64) or saline (n 5 16) intraperitoneally daily for 21 days and were weekly tested for locomotor activity and for blood ethanol levels. According to the locomotor scores presented across test days, ethanol-treated mice were classified as ‘‘sensitized’’ or ‘‘nonsensitized.’’ Twenty-four hours after the last administration, mice were sacrificed and brains were processed for autoradiography. Brain D4 binding was assessed by quantitative autoradiography using [3H]nemonapride 1 raclopride in three groups: salinetreated controls (n 5 10), ethanol-sensitized (n 5 11), and ethanol-nonsensitized (n 5 9) mice. Both sensitized and nonsensitized mice showed higher D4 binding densities than saline-treated controls in the posterior caudate–putamen and the olfactory tubercle ( p ! .02), but only sensitized mice presented higher D4 binding than controls at the lateral septal nucleus ( p ! .02). However, there were no differences between sensitized and nonsensitized mice in any of the brain regions analyzed. Furthermore, sensitized and nonsensitized mice presented similar blood ethanol levels during the treatment. The higher D4 binding levels observed in both ethanol-treated subgroups (sensitized and nonsensitized) suggest that chronic ethanol treatment may induce upregulation of D4 receptors in specific brain regions. However, this mechanism does not seem to be associated with the differential ability to develop behavioral sensitization to ethanol in mice. Ó 2005 Elsevier Inc. All rights reserved. Keywords: [3H]Nemonapride; Dopamine; Striatum; Lateral septal nucleus; Blood ethanol levels
1. Introduction Dopamine D4 receptors, a subtype of the D2 family of dopaminergic receptors, have been associated with several psychiatric disorders, including drug dependence and alcoholism (Tarazi & Baldessarini, 1999). A putative involvement of D4 receptor gene in the genetic basis of alcoholism and associated disorders has also been proposed, although compelling evidence for this involvement is still lacking (George et al., 1993; Hutchison et al., 2002; Lusher et al., 2001; Paterson et al., 1999; Sander et al., 1997). A recent report has also suggested that antagonism of D4 receptors may be relevant for the treatment of alcoholism, because olanzapine, a drug with significant D4 antagonistic activity, reduced
* Corresponding author. Tel.: 155-11-2149-0155; fax: 155-11-55725092. E-mail address: [email protected] (M.L.O. Souza-Formigoni). 0741-8329/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2005.12.001
basal and alcohol-induced craving levels in a sample of heavy social drinkers (Hutchison et al., 2003). In animal models, D4 receptor knockout mice have been shown to be hypersensitive to the acute stimulant effects of ethanol, cocaine, and methamphetamine (Drago et al., 1998; Katz et al., 2003; Kruzich et al., 2004; Rubinstein et al., 1997). Furthermore, coadministration of clozapine, a D4 receptor antagonist, blocked ethanol-induced locomotor stimulation in mice, although it did not affect some of ethanol’s motivational properties (Thrasher et al., 1999). However, the role of D4 receptors in the chronic effects of ethanol has not yet received proper attention. One of the few available studies reported no alterations in brain D4 gene expression in rats after chronic ethanol consumption (Eravci et al., 1997). Under some circumstances, repeated administration of ethanol and other drugs of abuse may lead to behavioral sensitization, that is, potentiation of drug-induced
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psychomotor activation (Masur et al., 1986; Phillips et al., 1997). This neuroadaptive phenomenon has been hypothesized to reflect sensitization to the motivational properties of drugs of abuse, which could contribute to drug addiction and to an increased vulnerability to drug abuse (Newlin & Thomson, 1991; Robinson & Berridge, 1993). Selective blockade of D4 receptors was shown to block the development of behavioral sensitization to amphetamine, suggesting that D4 receptors may also be important in the neuroadaptive processes associated with behavioral sensitization (Feldpausch et al., 1998). On the other hand, D4 knockout mice present sensitized responses to amphetamine, suggesting a complex role for D4 receptors in amphetamine sensitization (Kruzich et al., 2004). In previous studies we showed that ethanol-induced behavioral sensitization was specifically associated with increased D2 receptor binding in striatal areas (Souza-Formigoni et al., 1999), whereas D1 receptor and dopamine transporter binding were unaltered in ethanol-sensitized mice (Quadros et al., 2002). This study tested the hypothesis that D4 receptor binding levels might be specifically altered in mice sensitized to the stimulant effects of ethanol. 2. Materials and methods 2.1. Animals Eighty male mice from the Federal University of Saoˆ Paulo (UNIFESP) colony, originally derived from the Swiss Webster line, were housed in groups of 20 in plastic cages, with free access to food and water. They were kept in a temperature-controlled colony room (22 6 1 C), with lights on between 0700 and 1900 h. Mice were approximately 90 days old at the beginning of the experiment. All animal procedures were carried out in accordance with the National Institutes of Health Principles of Laboratory Animal Care (1985), and were approved by the local UNIFESP Ethics Committee in Research (CEP #352/00). 2.2. Locomotor activity tests Mice were individually tested in Opto-Varimex activity cages (Columbus Instruments, Columbus, OH), which detect locomotion by interruptions of horizontal photoelectric beams. All animals were initially subjected to a 15-min test session, without any drug treatment, for basal activity assessment. Two groups, equated in terms of basal activity scores, were then assigned to daily ethanol (2.2 g/kg, 15% wt/vol in 0.9% NaCl, given intraperitoneally, n 5 64) or saline treatment (n 5 16), which started 48 h after the basal test and lasted for 21 days. Locomotor activity was measured for 15 min immediately after injections on days 1, 7, 14, and 21. All injections of ethanol or saline were given in the test room, to which animals were taken at least 1 h before injections and activity testing. All procedures were carried out in the afternoon (between 1300 and 1700 h).
2.3. Determination of blood ethanol levels with gas chromatography After each locomotor activity test (on days 1, 7, 14, and 21), each mouse was set in a restrainer and two samples of 20-ml tail blood were collected with pipettes and put into 20-ml glass vials containing 1 ml of 0.02% (vol/vol) n-propanol (internal standard for the gas chromatography analyses). All tail blood samples were collected at 20 (61) min after administration. Saline-treated mice went through the same procedure, except that the tail blood samples were not analyzed. Ethanol concentrations were determined in each sample by gas chromatography, using a Shimadzu 14B Gas Chromatographer equipped with a Carbowax 20 M column, headspace and flame ionizing detection (FID), detector. 2.4. Quantitative autoradiography of brain D4 receptors Mice were sacrificed 24 h after the last activity test. Brains were quickly removed, frozen over dry ice, and stored at 280 C. Serial 20-mm coronal sections were cut on a Hacker–Bright cryostat at 220 C and collected onto glass slides at 0.3-mm intervals, through the longitudinal extent of the brain. Slides were returned to 280 C storage until the day of the assays. [3H]-YM 09151-2 (nemonapride) autoradiographic assays followed the procedures of Tarazi et al. (1997) with minor modifications. Briefly, slices were brought to room temperature and then preincubated in 50 mM Tris–HCl buffer (pH 5 7.4) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2 for 60 min at room temperature. Sections were then incubated for 60 min in buffer containing 1 nM [3H]nemonapride (20.3 Ci/mmol, NEN Dupont), at room temperature. To avoid binding to sites other than D4 receptors, the incubation solution also contained 300 nM unlabeled raclopride, 0.5 mM guanidine, and 0.1 mM pindolol. An additional set of slides was incubated in the presence of 10 mM sulpiride, for determination of nonspecific binding. Sections were then washed in buffer and allowed to dry at room temperature. Slides were exposed to [3H] Hyperfilm (Amersham), in tungsten cassettes together with calibrated standards for 4 weeks. Films were developed and densitometric analyses performed using an M2 MCID system (Imaging Research, St. Catharines, Ontario). Anatomical regions were defined according to the Franklin and Paxinos (1997) atlas. The final binding value for each animal in each brain region was averaged from bilateral measures of approximately three to five sections, performed by an investigator who was unaware of group membership of the samples. 3. Results 3.1. Ethanol-induced locomotor sensitization To verify whether D4 receptor binding alterations would be specifically associated with the development of ethanol
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sensitizationdand not simply to chronic ethanol treatmentdsubgroups of ethanol-treated mice were classified according to their stimulant response to ethanol on day 21, as previously described (Quadros et al., 2002; SouzaFormigoni et al., 1999). Sensitized (n 5 11) and nonsensitized (n 5 9) mice included in the autoradiographic analyses were consistently in the upper (sensitized) or lower (nonsensitized) 30% of the locomotor scores distribution on days 7, 14, and 21. The statistical analyses below only present behavioral data from mice that were used in the subsequent autoradiographic study (11 sensitized, 9 nonsensitized, and 10 randomly selected saline-treated mice), as shown in Fig. 1. When locomotor activity scores from sensitized, nonsensitized, and saline-treated mice were compared using a twoway analysis of variance (ANOVA) with repeated measures, there was a significant effect of group (F(2,27) 5 165.77, p ! .0001), test (F(4,108) 5 35.66, p ! .0001), and a group 3 test interaction (F(8,108) 5 37.26, p ! .0001). Duncan’s new multiple range tests for the group factor indicated that sensitized mice were statistically different from both nonsensitized and control mice ( p ! .001), whereas the nonsensitized group was similar to the saline controls. Separate one-way ANOVAs for each test day revealed significant group differences at the acute test [F(2,27) 5 10.47, p ! .01], on days 7 [F(2,27) 5 84.56, p ! .0001], 14 [F(2,27) 5 113.78, p ! .0001], and 21 [F(2,27) 5 88.42, p ! .0001], as shown in Fig. 1. Duncan’s new multiple range tests indicated that ethanol-sensitized animals presented higher activity levels than the other two groups on every test day ( p ! .001), except for the basal activity test.
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Within-group analyses were carried out for each subgroup across the 21-day treatment, using one-way ANOVAs with repeated measures. Sensitized mice showed significantly higher activity scores on day 21 than on all preceding activity tests [F(4,40) 5 47.65, p ! .01], whereas for nonsensitized mice only the acute response was significantly lower than locomotor scores displayed in all other tests [F(4,32) 5 11.25, p ! .001]. Saline-treated mice seemed to display habituation across treatment: scores on the basal activity test were higher than scores in all other activity tests [F(4,36) 5 35.93, p ! .001].
3.2. Blood ethanol nonsensitized mice
levels
in
ethanol-sensitized
or
Blood ethanol levels (BELs) were not different in sensitized and nonsensitized mice in any of the time points analyzed. Mean BELs 6 S.E.M. (in mg/ml) for sensitized and nonsensitized subgroups were, respectively, 1.30 6 0.09 versus 1.37 6 0.15 (acute test); 1.48 6 0.07 versus 1.61 6 0.08 (day 7); 1.68 6 0.08 versus 1.65 6 0.08 (day 14); and 1.60 6 0.06 versus 1.54 6 0.11 (day 21). A two-way ANOVA detected no significant effects of group or group 3 test interaction ( p O .3), but only a significant effect of test day [F(3,48) 5 7.76, p ! .001]. Duncan’s multiple range tests showed that BELs at the acute test were significantly lower than in any other tests ( p ! .01). No significant correlation was detected between BELs and locomotor activity in any test day (Pearson’s r values ranged from 2.23 to 1.35 across tests).
Fig. 1. Locomotor activity scores (means 1 S.E.M.) of ethanol-treated mice classified as sensitized (n 5 11) and nonsensitized (n 5 9), and of the salinetreated group (n 5 10), across the 21-day treatment. *Significantly higher activity levels than nonsensitized and saline-treated mice ( p ! .04); #significantly higher activity levels than saline-treated mice ( p ! .05); $significantly higher activity levels than the own group scores in every other activity test ( p ! .01).
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3.3. [ H]Nemonapride binding to dopamine D4 receptors As shown in Table 1, separate ANOVAs indicated significant differences between the three groups in the olfactory tubercle (F2,27 5 4.65, p ! .02), the posterior caudate– putamen (F2,27 5 4.39, p ! .03), and the lateral septal nucleus (F2,27 5 4.62, p ! .02). Post hoc comparisons using Duncan’s new multiple range test indicated that [3H]nemonapride binding levels were significantly increased in both the sensitized and the nonsensitized groups when compared to saline controls at the olfactory tubercle and the posterior caudate–putamen ( p ! .03). Fig. 2 shows illustrative autoradiographs from one mouse at different brain levels. In the lateral septal nucleus, sensitized mice presented significantly higher D4 binding levels than controls ( p ! .01), whereas nonsensitized mice displayed just a trend ( p ! .08). There were no significant D4 binding differences between sensitized and nonsensitized groups in any of the brain regions analyzed. To assess the general effect of ethanol treatment on D4 binding levelsdregardless of behavioral sensitization levelsddata from both sensitized and nonsensitized subgroups were collapsed (n 5 20) and compared with data from saline-treated controls, as shown in Table 1. Student’s t tests for each brain structure showed that the 21-day ethanol treatment induced significant increases in D4 binding levels in several brain regions, such as the caudate–putamen (dorsolateral, ventromedial, and posterior divisions), the lateral septal nucleus, and the olfactory tubercle ( p ! .05). There was also a trend towards increased D4 binding levels in ethanol-treated mice at the core of nucleus accumbens and other subdivisions of caudate–putamen (anterior, ventrolateral, and dorsomedial; p ! .08). Supporting the absence of association between D4-like binding and ethanol-induced sensitization, [3H]nemonapride binding levels did not correlate with locomotor activity
levels on day 21 in any of the brain regions analyzed in the 20 ethanol-treated mice used in the autoradiographic study (Pearson’s r values between 1.2 and 2.2). For the 10 saline-treated mice, similar analyses revealed a single significant correlation in the olfactory tubercle (Pearson’s r 5 2.75, p ! .02).
4. Discussion The main finding of this study was that dopamine D4 receptor binding levels were increased in mice submitted to a 21-day ethanol treatment in striatal and limbic regions. However, the observed increases in D4 binding do not seem to be particularly associated with the development of behavioral sensitization to ethanol’s stimulant effect, because there were no differences in D4 binding levels between ‘‘sensitized’’ and ‘‘nonsensitized’’ mice. Furthermore, sensitized and nonsensitized mice did not present different BELs across treatment, suggesting that systemic pharmacokinetic factors do not seem to account importantly for the individual susceptibility to develop ethanol sensitization, as suggested by other authors (Broadbent & Harless, 1999; Lessov & Phillips, 1998; Phillips et al., 1995). Amphetamine sensitization and associated neuroadaptations have been reported to be prevented by the coadministration of a D4 antagonist, suggesting that D4 receptor activation would be required for the development of behavioral and neurochemical sensitization to amphetamine (Feldpausch et al., 1998). However, the occurrence of D4 binding alterations in prosencephalic regions does not seem to underlie this phenomenon, because D4 binding densities were not modified in amphetamine-sensitized rats (Zhang et al., 2000). Our results with ethanol sensitization seem to agree with those presented by Zhang et al. (2000), because we did not observe an association between ethanol
Table 1 [3H]Nemonapride binding in different brain regions after 21 days of ethanol treatmenta Ethanol Nonsensitized (n 5 9)
Sensitized (n 5 11)
All animalsb (n 5 20)
3.08 6 0.39 4.77 6 0.32 3.48 6 0.33 4.39 6 0.33
3.31 6 0.48 *6.15 6 0.41 4.35 6 0.52 4.79 6 0.44
3.75 6 0.42 *6.40 6 0.47 4.69 6 0.49 5.36 6 0.66
3.55 6 0.31 *6.28 6 0.31 4.54 6 0.35 5.10 6 0.41
4.98 6 0.33 5.01 6 0.55 7.09 6 0.64 2.68 6 0.24 10.80 6 0.77 5.10 6 0.49
5.66 6 0.67 6.32 6 0.61 8.65 6 0.78 3.50 6 0.42 12.87 6 0.83 *6.85 6 0.61
7.03 6 0.72 6.15 6 0.55 8.83 6 0.60 4.02 6 0.44 12.63 6 0.83 *7.30 6 0.58
6.41 6 0.51 6.23 6 0.39 *8.75 6 0.47 *3.78 6 0.31 12.74 6 0.57 *7.10 6 0.41
2.42 6 0.28 0.76 6 0.14
2.33 6 0.29 1.10 6 0.14
2.49 6 0.23 *1.31 6 0.12
2.42 6 0.18 *1.22 6 0.09
Saline (n 5 10) Olfactory bulb Olfactory tubercle n. Accumbensdcore n. Accumbensdshell Caudate–putamen Anterior Dorsomedial Dorsolateral Ventromedial Ventrolateral Posterior Ventral tegmental area Lateral septal nucleus
*Significantly higher than saline-treated controls ( p ! .02, in bold). a Values are means 6 S.E.M. in pmol/g tissue. b Combined data from sensitized and nonsensitized subgroups.
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activation of these receptors seems to be necessary for ethanol-induced locomotor stimulation in mice (Cohen et al., 1997; Liljequist et al., 1981; Thrasher et al., 1999). Furthermore, D4 knockout mice are hyperreactive to ethanol stimulation and to other drugs (Drago et al., 1998; Rubinstein et al., 1997), confirming the involvement of these receptors in ethanol-induced behavioral activation. In this study, repeated ethanol treatment induced increased D4 binding levels in different areas of the caudate–putamen, and also in the olfactory tubercle and the lateral septal nucleus. These brain regions are all part of the nigrostriatal and mesolimbic dopamine pathways, which are known to be involved in both the motivational and stimulating properties of ethanol (Di Chiara, 1993; Koob et al., 1998; Phillips & Shen, 1996). Therefore, the observed alterations in D4 binding densities may putatively be of behavioral and neurochemical relevance, although they seem to be unrelated to the individual susceptibility to develop ethanol sensitization. In rats, chronic ethanol consumption (for 2 or 9 months) did not induce significant alterations in D4 messenger RNA expression levels (Eravci et al., 1997). However, in the latter study, rats were kept abstinent from ethanol ingestion for 1 month before they were sacrificed for brain receptor expression assays (Eravci et al., 1997), whereas in this study mice were ‘‘abstinent’’ for only 24 h before sacrifice. Therefore, it seems possible that eventual neuroadaptations in D4 receptor functioning induced by repeated ethanol administration may be time dependent, and the behavioral relevance and duration of these neuroadaptations remain to be determined. In previous studies, we reported increased binding densities of D2 receptors in ethanol-sensitized mice (Souza-Formigoni et al., 1999), whereas neither D1 receptor nor the dopamine transporter binding levels were associated with ethanol sensitization (Quadros et al., 2002). Therefore, it seems that the D2-like family of dopamine receptorsd including D2 and D4 receptorsdis more susceptible to ethanol-induced neuroadaptations than other dopaminergic binding sites. Although increases in D2 binding densities were particularly associated with ethanol sensitization, an upregulation of D4 receptors in specific brain regions seemed to be induced by chronic ethanol treatment, regardless of the manifestation of ethanol-induced sensitization in mice. Fig. 2. Digitized images illustrating the binding pattern of [3H]nemonapride to dopamine D4 receptors. Olf bulb (gl) 5 olfactory bulb, glomerular layer; CPu 5 caudate–putamen; CPu-P 5 caudate–putamen, posterior division; Acc 5 nucleus accumbens; Olf tubercle 5 olfactory tubercle.
sensitization and D4 receptor binding densities. However, in this study we did observe a general effect of repeated ethanol administration on D4 binding densities, suggesting that D4 receptor plasticity processes may be more sensitive to ethanol than to amphetamine treatment. Although no pharmacological studies have analyzed the role of dopamine D4 receptors in ethanol sensitization, the
Acknowledgments This work was supported by funds from AFIP (Associac¸a˜o Fundo de Incentivo a Psicofarmacologia), FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo), and CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico). References Broadbent, J., & Harless, W. E. (1999). Differential effects of GABA(A) and GABA(B) agonists on sensitization to the locomotor stimulant effects of ethanol in DBA/2 J mice. Psychopharmacology 141, 197–205.
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Cohen, C., Perrault, G., & Sanger, D. J. (1997). Evidence for the involvement of dopamine receptors in ethanol-induced hyperactivity in mice. Neuropharmacology 36(8), 1099–1108. Di Chiara, G. (1993). Searching for the hidden order in chaos. Commentary on Kalivas et al., ‘‘The pharmacology of neural circuitry of sensitization to psychostimulants’’. Behav Pharmacol 4, 335–337. Drago, J., Padungchaicot, P., Accili, D., & Fuchs, S. (1998). Dopamine receptors and dopamine transporter in brain function and addictive behaviors: insights from targeted mouse mutants. Dev Neurosci 20, 188–203. Eravci, M., Grosspietsch, T., Pinna, G., Schulz, O., Kley, S., Bachmann, M., Wolffgramm, J., Gotz, E., Heyne, A., Meinhold, H., & Baumgartner, A. (1997). Dopamine receptor gene expression in an animal model of ‘behavioral dependence’ on ethanol. Brain Res Mol Brain Res 50, 221–229. Feldpausch, D. L., Needham, L. M., Stone, M. P., Althaus, J. S., Yamamoto, B. K., Svensson, K. A., & Merchant, K. M. (1998). The role of dopamine D4 receptor in the induction of behavioral sensitization to amphetamine and accompanying biochemical and molecular adaptations. J Pharmacol Exp Ther 286, 497–508. Franklin, K., & Paxinos, G. (1997). The Mouse Brain in Stereotaxic Coordinates. New York: Academic Press. George, S. R., Cheng, R., Nguyen, T., Israel, Y., & O’Dowd, B. F. (1993). Polymorphisms of the D4 dopamine receptor alleles in chronic alcoholism. Biochem Biophys Res Commun 196(1), 107–114. Hutchison, K. E., McGeary, J., Smolen, A., Bryan, A., & Swift, R. M. (2002). The DRD4 VNTR polymorphism moderates craving after alcohol consumption. Health Psychol 21(2), 139–146. Hutchison, K. E., Wooden, A., Swift, R. M., Smolen, A., McGeary, J., Adler, L., & Paris, L. (2003). Olanzapine reduces craving for alcohol: a DRD4 VNTR polymorphism by pharmacotherapy interaction. Neuropsychopharmacology 28(10), 1882–1888. Katz, J. L., Chausmer, A. L., Elmer, G. I., Rubinstein, M., Low, M. J., & Grandy, D. K. (2003). Cocaine-induced locomotor activity and cocaine discrimination in dopamine D4 receptor mutant mice. Psychopharmacology 170(1), 108–114. Koob, G. F., Roberts, A. J., Schulteis, G., Parsons, L. H., Heyser, C. J., Hyytia, P., Merlo-Pich, E., & Weiss, F. (1998). Neurocircuitry targets in ethanol reward and dependence. Alcohol Clin Exp Res 22, 3–9. Kruzich, P. J., Suchland, K. L., & Grandy, D. K. (2004). Dopamine D4 receptor-deficient mice, congenic on the C57BL/6J background, are hypersensitive to amphetamine. Synapse 53(2), 131–139. Lessov, C. N., & Phillips, T. J. (1998). Duration of sensitization to the locomotor stimulant effects of ethanol in mice. Psychopharmacology 135, 374–382. Liljequist, S., Berggren, U., & Engel, J. (1981). The effect of catecholamine receptor antagonists on ethanol-induced locomotor stimulation. J Neural Transm 50(1), 57–67. Lusher, J. M., Chandler, C., & Ball, D. (2001). Dopamine D4 receptor gene (DRD4) is associated with Novelty Seeking (NS) and substance abuse: the saga continues. Mol Psychiatry 6(5), 497–499.
Masur, J., Souza, M. L. O., & Zwicker, A. P. (1986). The excitatory effect of ethanol: absence in rats, no tolerance and increased sensitivity in mice. Pharmacol Biochem Behav 24, 1225–1228. Newlin, D. B., & Thomson, J. B. (1991). Chronic tolerance and sensitization to alcohol in sons of alcoholics. Alcohol Clin Exp Res 15, 399– 405. Paterson, A. D., Sunohara, G. A., & Kennedy, J. L. (1999). Dopamine D4 receptor gene: novelty or nonsense? Neuropsychopharmacology 21(1), 3–16. Phillips, T. J., Huson, M., Gwiazdon, C., Burkhart-Kasch, S., & Shen, E. H. (1995). Effects of acute and repeated ethanol exposures on the locomotor activity of BXD recombinant inbred mice. Alcohol Clin Exp Res 19, 269–278. Phillips, T. J., Roberts, A., & Lessov, C. (1997). Behavioral sensitization to ethanol: genetics and the effects of stress. Pharmacol Biochem Behav 57(3), 487–493. Phillips, T. J., & Shen, E. H. (1996). Neurochemical bases of locomotion and ethanol stimulant effects. Int Rev Neurobiol 39, 243–282. Quadros, I. M. H., Nobrega, J. N., Hipolide, D. C., De Lucca, E. M., & Souza-Formigoni, M. L. O. (2002). Differential propensity to ethanol sensitization is not associated with altered binding to D1 receptors or dopamine transporters in mouse brain. Addict Biol 7, 291–299. Robinson, T. E., & Berridge, K. C. (1993). The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res Brain Res Rev 18, 247–291. Rubinstein, M., Phillips, T. J., Bunzow, J. R., Falzone, T. L., Dziewczapolski, G., Zhang, G., Fang, Y., Larson, J. L., McDougall, J. A., Chester, J. A., Saez, C., Pugsley, T. A., Gershanik, O., Low, M. J., & Grandy, D. K. (1997). Mice lacking dopamine D4 receptors are supersensitive to ethanol, cocaine, and methamphetamine. Cell 90, 991–1001. Sander, T., Harms, H., Dufeu, P., Kuhn, S., Rommelspacher, H., & Schmidt, L. G. (1997). Dopamine D4 receptor exon III alleles and variation of novelty seeking in alcoholics. Am J Med Genet 74(5), 483–487. Souza-Formigoni, M. L. O., De Lucca, E. M., Hipo´lide, D. C., Enns, S. C., Oliveira, M. G. M., & Nobrega, J. N. (1999). Sensitization to ethanol’s stimulant effect is associated with localized increases in brain D2 receptor binding. Psychopharmacology 146, 262–267. Tarazi, F. I., & Baldessarini, R. J. (1999). Dopamine D4 receptors: significance for molecular psychiatry at the millennium. Mol Psychiatry 4, 529–538. Tarazi, F. I., Kula, N. S., & Baldessarini, R. J. (1997). Regional distribution of dopamine D4 receptors in rat forebrain. Neuroreport 8, 3423– 3426. Thrasher, M. J., Freeman, P. A., & Risinger, F. O. (1999). Clozapine’s effects on ethanol’s motivational properties. Alcohol Clin Exp Res 23, 1377–1385. Zhang, K., Tarazi, F. I., & Baldessarini, R. J. (2000). Dopamine D(4) receptors in rat forebrain: unchanged with amphetamine-induced behavioral sensitization. Neuroscience 97, 211–213.
Increased brain dopamine D4-like binding after chronic ethanol is not associated with behavioral sensitization in mice Isabel Marian Hartmann Quadrosa, Jose Nascimento Nobregab, Debora Cristina Hipolidea, Maria Lucia Oliveira Souza-Formigonia,* a
Psychobiology Department, Federal University of Sa˜o Paulo (UNIFESP), Rua Botucatu 862, 1 . andar, 04023-062 Sa˜o Paulo, SP, Brazil b Neuroimaging Research Section, Centre for Addiction and Mental Health, 250 College Street, Toronto, ON M5T 1R8, Canada Received 1 September 2005; received in revised form 30 November 2005; accepted 1 December 2005
Abstract Dopaminergic D4 receptors have been hypothesized to be involved in neuropsychiatric disorders and substance abuse. In mice, repeated ethanol administration may induce behavioral sensitization, a phenomenon of increased sensitivity to the drug’s stimulant properties. This study aimed to analyze brain D4 receptors binding in mice with different levels of behavioral sensitization to ethanol. Male Swiss mice received 2.2 g/kg ethanol (n 5 64) or saline (n 5 16) intraperitoneally daily for 21 days and were weekly tested for locomotor activity and for blood ethanol levels. According to the locomotor scores presented across test days, ethanol-treated mice were classified as ‘‘sensitized’’ or ‘‘nonsensitized.’’ Twenty-four hours after the last administration, mice were sacrificed and brains were processed for autoradiography. Brain D4 binding was assessed by quantitative autoradiography using [3H]nemonapride 1 raclopride in three groups: salinetreated controls (n 5 10), ethanol-sensitized (n 5 11), and ethanol-nonsensitized (n 5 9) mice. Both sensitized and nonsensitized mice showed higher D4 binding densities than saline-treated controls in the posterior caudate–putamen and the olfactory tubercle ( p ! .02), but only sensitized mice presented higher D4 binding than controls at the lateral septal nucleus ( p ! .02). However, there were no differences between sensitized and nonsensitized mice in any of the brain regions analyzed. Furthermore, sensitized and nonsensitized mice presented similar blood ethanol levels during the treatment. The higher D4 binding levels observed in both ethanol-treated subgroups (sensitized and nonsensitized) suggest that chronic ethanol treatment may induce upregulation of D4 receptors in specific brain regions. However, this mechanism does not seem to be associated with the differential ability to develop behavioral sensitization to ethanol in mice. Ó 2005 Elsevier Inc. All rights reserved. Keywords: [3H]Nemonapride; Dopamine; Striatum; Lateral septal nucleus; Blood ethanol levels
1. Introduction Dopamine D4 receptors, a subtype of the D2 family of dopaminergic receptors, have been associated with several psychiatric disorders, including drug dependence and alcoholism (Tarazi & Baldessarini, 1999). A putative involvement of D4 receptor gene in the genetic basis of alcoholism and associated disorders has also been proposed, although compelling evidence for this involvement is still lacking (George et al., 1993; Hutchison et al., 2002; Lusher et al., 2001; Paterson et al., 1999; Sander et al., 1997). A recent report has also suggested that antagonism of D4 receptors may be relevant for the treatment of alcoholism, because olanzapine, a drug with significant D4 antagonistic activity, reduced
* Corresponding author. Tel.: 155-11-2149-0155; fax: 155-11-55725092. E-mail address: [email protected] (M.L.O. Souza-Formigoni). 0741-8329/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2005.12.001
basal and alcohol-induced craving levels in a sample of heavy social drinkers (Hutchison et al., 2003). In animal models, D4 receptor knockout mice have been shown to be hypersensitive to the acute stimulant effects of ethanol, cocaine, and methamphetamine (Drago et al., 1998; Katz et al., 2003; Kruzich et al., 2004; Rubinstein et al., 1997). Furthermore, coadministration of clozapine, a D4 receptor antagonist, blocked ethanol-induced locomotor stimulation in mice, although it did not affect some of ethanol’s motivational properties (Thrasher et al., 1999). However, the role of D4 receptors in the chronic effects of ethanol has not yet received proper attention. One of the few available studies reported no alterations in brain D4 gene expression in rats after chronic ethanol consumption (Eravci et al., 1997). Under some circumstances, repeated administration of ethanol and other drugs of abuse may lead to behavioral sensitization, that is, potentiation of drug-induced
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psychomotor activation (Masur et al., 1986; Phillips et al., 1997). This neuroadaptive phenomenon has been hypothesized to reflect sensitization to the motivational properties of drugs of abuse, which could contribute to drug addiction and to an increased vulnerability to drug abuse (Newlin & Thomson, 1991; Robinson & Berridge, 1993). Selective blockade of D4 receptors was shown to block the development of behavioral sensitization to amphetamine, suggesting that D4 receptors may also be important in the neuroadaptive processes associated with behavioral sensitization (Feldpausch et al., 1998). On the other hand, D4 knockout mice present sensitized responses to amphetamine, suggesting a complex role for D4 receptors in amphetamine sensitization (Kruzich et al., 2004). In previous studies we showed that ethanol-induced behavioral sensitization was specifically associated with increased D2 receptor binding in striatal areas (Souza-Formigoni et al., 1999), whereas D1 receptor and dopamine transporter binding were unaltered in ethanol-sensitized mice (Quadros et al., 2002). This study tested the hypothesis that D4 receptor binding levels might be specifically altered in mice sensitized to the stimulant effects of ethanol. 2. Materials and methods 2.1. Animals Eighty male mice from the Federal University of Saoˆ Paulo (UNIFESP) colony, originally derived from the Swiss Webster line, were housed in groups of 20 in plastic cages, with free access to food and water. They were kept in a temperature-controlled colony room (22 6 1 C), with lights on between 0700 and 1900 h. Mice were approximately 90 days old at the beginning of the experiment. All animal procedures were carried out in accordance with the National Institutes of Health Principles of Laboratory Animal Care (1985), and were approved by the local UNIFESP Ethics Committee in Research (CEP #352/00). 2.2. Locomotor activity tests Mice were individually tested in Opto-Varimex activity cages (Columbus Instruments, Columbus, OH), which detect locomotion by interruptions of horizontal photoelectric beams. All animals were initially subjected to a 15-min test session, without any drug treatment, for basal activity assessment. Two groups, equated in terms of basal activity scores, were then assigned to daily ethanol (2.2 g/kg, 15% wt/vol in 0.9% NaCl, given intraperitoneally, n 5 64) or saline treatment (n 5 16), which started 48 h after the basal test and lasted for 21 days. Locomotor activity was measured for 15 min immediately after injections on days 1, 7, 14, and 21. All injections of ethanol or saline were given in the test room, to which animals were taken at least 1 h before injections and activity testing. All procedures were carried out in the afternoon (between 1300 and 1700 h).
2.3. Determination of blood ethanol levels with gas chromatography After each locomotor activity test (on days 1, 7, 14, and 21), each mouse was set in a restrainer and two samples of 20-ml tail blood were collected with pipettes and put into 20-ml glass vials containing 1 ml of 0.02% (vol/vol) n-propanol (internal standard for the gas chromatography analyses). All tail blood samples were collected at 20 (61) min after administration. Saline-treated mice went through the same procedure, except that the tail blood samples were not analyzed. Ethanol concentrations were determined in each sample by gas chromatography, using a Shimadzu 14B Gas Chromatographer equipped with a Carbowax 20 M column, headspace and flame ionizing detection (FID), detector. 2.4. Quantitative autoradiography of brain D4 receptors Mice were sacrificed 24 h after the last activity test. Brains were quickly removed, frozen over dry ice, and stored at 280 C. Serial 20-mm coronal sections were cut on a Hacker–Bright cryostat at 220 C and collected onto glass slides at 0.3-mm intervals, through the longitudinal extent of the brain. Slides were returned to 280 C storage until the day of the assays. [3H]-YM 09151-2 (nemonapride) autoradiographic assays followed the procedures of Tarazi et al. (1997) with minor modifications. Briefly, slices were brought to room temperature and then preincubated in 50 mM Tris–HCl buffer (pH 5 7.4) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2 for 60 min at room temperature. Sections were then incubated for 60 min in buffer containing 1 nM [3H]nemonapride (20.3 Ci/mmol, NEN Dupont), at room temperature. To avoid binding to sites other than D4 receptors, the incubation solution also contained 300 nM unlabeled raclopride, 0.5 mM guanidine, and 0.1 mM pindolol. An additional set of slides was incubated in the presence of 10 mM sulpiride, for determination of nonspecific binding. Sections were then washed in buffer and allowed to dry at room temperature. Slides were exposed to [3H] Hyperfilm (Amersham), in tungsten cassettes together with calibrated standards for 4 weeks. Films were developed and densitometric analyses performed using an M2 MCID system (Imaging Research, St. Catharines, Ontario). Anatomical regions were defined according to the Franklin and Paxinos (1997) atlas. The final binding value for each animal in each brain region was averaged from bilateral measures of approximately three to five sections, performed by an investigator who was unaware of group membership of the samples. 3. Results 3.1. Ethanol-induced locomotor sensitization To verify whether D4 receptor binding alterations would be specifically associated with the development of ethanol
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sensitizationdand not simply to chronic ethanol treatmentdsubgroups of ethanol-treated mice were classified according to their stimulant response to ethanol on day 21, as previously described (Quadros et al., 2002; SouzaFormigoni et al., 1999). Sensitized (n 5 11) and nonsensitized (n 5 9) mice included in the autoradiographic analyses were consistently in the upper (sensitized) or lower (nonsensitized) 30% of the locomotor scores distribution on days 7, 14, and 21. The statistical analyses below only present behavioral data from mice that were used in the subsequent autoradiographic study (11 sensitized, 9 nonsensitized, and 10 randomly selected saline-treated mice), as shown in Fig. 1. When locomotor activity scores from sensitized, nonsensitized, and saline-treated mice were compared using a twoway analysis of variance (ANOVA) with repeated measures, there was a significant effect of group (F(2,27) 5 165.77, p ! .0001), test (F(4,108) 5 35.66, p ! .0001), and a group 3 test interaction (F(8,108) 5 37.26, p ! .0001). Duncan’s new multiple range tests for the group factor indicated that sensitized mice were statistically different from both nonsensitized and control mice ( p ! .001), whereas the nonsensitized group was similar to the saline controls. Separate one-way ANOVAs for each test day revealed significant group differences at the acute test [F(2,27) 5 10.47, p ! .01], on days 7 [F(2,27) 5 84.56, p ! .0001], 14 [F(2,27) 5 113.78, p ! .0001], and 21 [F(2,27) 5 88.42, p ! .0001], as shown in Fig. 1. Duncan’s new multiple range tests indicated that ethanol-sensitized animals presented higher activity levels than the other two groups on every test day ( p ! .001), except for the basal activity test.
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Within-group analyses were carried out for each subgroup across the 21-day treatment, using one-way ANOVAs with repeated measures. Sensitized mice showed significantly higher activity scores on day 21 than on all preceding activity tests [F(4,40) 5 47.65, p ! .01], whereas for nonsensitized mice only the acute response was significantly lower than locomotor scores displayed in all other tests [F(4,32) 5 11.25, p ! .001]. Saline-treated mice seemed to display habituation across treatment: scores on the basal activity test were higher than scores in all other activity tests [F(4,36) 5 35.93, p ! .001].
3.2. Blood ethanol nonsensitized mice
levels
in
ethanol-sensitized
or
Blood ethanol levels (BELs) were not different in sensitized and nonsensitized mice in any of the time points analyzed. Mean BELs 6 S.E.M. (in mg/ml) for sensitized and nonsensitized subgroups were, respectively, 1.30 6 0.09 versus 1.37 6 0.15 (acute test); 1.48 6 0.07 versus 1.61 6 0.08 (day 7); 1.68 6 0.08 versus 1.65 6 0.08 (day 14); and 1.60 6 0.06 versus 1.54 6 0.11 (day 21). A two-way ANOVA detected no significant effects of group or group 3 test interaction ( p O .3), but only a significant effect of test day [F(3,48) 5 7.76, p ! .001]. Duncan’s multiple range tests showed that BELs at the acute test were significantly lower than in any other tests ( p ! .01). No significant correlation was detected between BELs and locomotor activity in any test day (Pearson’s r values ranged from 2.23 to 1.35 across tests).
Fig. 1. Locomotor activity scores (means 1 S.E.M.) of ethanol-treated mice classified as sensitized (n 5 11) and nonsensitized (n 5 9), and of the salinetreated group (n 5 10), across the 21-day treatment. *Significantly higher activity levels than nonsensitized and saline-treated mice ( p ! .04); #significantly higher activity levels than saline-treated mice ( p ! .05); $significantly higher activity levels than the own group scores in every other activity test ( p ! .01).
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3.3. [ H]Nemonapride binding to dopamine D4 receptors As shown in Table 1, separate ANOVAs indicated significant differences between the three groups in the olfactory tubercle (F2,27 5 4.65, p ! .02), the posterior caudate– putamen (F2,27 5 4.39, p ! .03), and the lateral septal nucleus (F2,27 5 4.62, p ! .02). Post hoc comparisons using Duncan’s new multiple range test indicated that [3H]nemonapride binding levels were significantly increased in both the sensitized and the nonsensitized groups when compared to saline controls at the olfactory tubercle and the posterior caudate–putamen ( p ! .03). Fig. 2 shows illustrative autoradiographs from one mouse at different brain levels. In the lateral septal nucleus, sensitized mice presented significantly higher D4 binding levels than controls ( p ! .01), whereas nonsensitized mice displayed just a trend ( p ! .08). There were no significant D4 binding differences between sensitized and nonsensitized groups in any of the brain regions analyzed. To assess the general effect of ethanol treatment on D4 binding levelsdregardless of behavioral sensitization levelsddata from both sensitized and nonsensitized subgroups were collapsed (n 5 20) and compared with data from saline-treated controls, as shown in Table 1. Student’s t tests for each brain structure showed that the 21-day ethanol treatment induced significant increases in D4 binding levels in several brain regions, such as the caudate–putamen (dorsolateral, ventromedial, and posterior divisions), the lateral septal nucleus, and the olfactory tubercle ( p ! .05). There was also a trend towards increased D4 binding levels in ethanol-treated mice at the core of nucleus accumbens and other subdivisions of caudate–putamen (anterior, ventrolateral, and dorsomedial; p ! .08). Supporting the absence of association between D4-like binding and ethanol-induced sensitization, [3H]nemonapride binding levels did not correlate with locomotor activity
levels on day 21 in any of the brain regions analyzed in the 20 ethanol-treated mice used in the autoradiographic study (Pearson’s r values between 1.2 and 2.2). For the 10 saline-treated mice, similar analyses revealed a single significant correlation in the olfactory tubercle (Pearson’s r 5 2.75, p ! .02).
4. Discussion The main finding of this study was that dopamine D4 receptor binding levels were increased in mice submitted to a 21-day ethanol treatment in striatal and limbic regions. However, the observed increases in D4 binding do not seem to be particularly associated with the development of behavioral sensitization to ethanol’s stimulant effect, because there were no differences in D4 binding levels between ‘‘sensitized’’ and ‘‘nonsensitized’’ mice. Furthermore, sensitized and nonsensitized mice did not present different BELs across treatment, suggesting that systemic pharmacokinetic factors do not seem to account importantly for the individual susceptibility to develop ethanol sensitization, as suggested by other authors (Broadbent & Harless, 1999; Lessov & Phillips, 1998; Phillips et al., 1995). Amphetamine sensitization and associated neuroadaptations have been reported to be prevented by the coadministration of a D4 antagonist, suggesting that D4 receptor activation would be required for the development of behavioral and neurochemical sensitization to amphetamine (Feldpausch et al., 1998). However, the occurrence of D4 binding alterations in prosencephalic regions does not seem to underlie this phenomenon, because D4 binding densities were not modified in amphetamine-sensitized rats (Zhang et al., 2000). Our results with ethanol sensitization seem to agree with those presented by Zhang et al. (2000), because we did not observe an association between ethanol
Table 1 [3H]Nemonapride binding in different brain regions after 21 days of ethanol treatmenta Ethanol Nonsensitized (n 5 9)
Sensitized (n 5 11)
All animalsb (n 5 20)
3.08 6 0.39 4.77 6 0.32 3.48 6 0.33 4.39 6 0.33
3.31 6 0.48 *6.15 6 0.41 4.35 6 0.52 4.79 6 0.44
3.75 6 0.42 *6.40 6 0.47 4.69 6 0.49 5.36 6 0.66
3.55 6 0.31 *6.28 6 0.31 4.54 6 0.35 5.10 6 0.41
4.98 6 0.33 5.01 6 0.55 7.09 6 0.64 2.68 6 0.24 10.80 6 0.77 5.10 6 0.49
5.66 6 0.67 6.32 6 0.61 8.65 6 0.78 3.50 6 0.42 12.87 6 0.83 *6.85 6 0.61
7.03 6 0.72 6.15 6 0.55 8.83 6 0.60 4.02 6 0.44 12.63 6 0.83 *7.30 6 0.58
6.41 6 0.51 6.23 6 0.39 *8.75 6 0.47 *3.78 6 0.31 12.74 6 0.57 *7.10 6 0.41
2.42 6 0.28 0.76 6 0.14
2.33 6 0.29 1.10 6 0.14
2.49 6 0.23 *1.31 6 0.12
2.42 6 0.18 *1.22 6 0.09
Saline (n 5 10) Olfactory bulb Olfactory tubercle n. Accumbensdcore n. Accumbensdshell Caudate–putamen Anterior Dorsomedial Dorsolateral Ventromedial Ventrolateral Posterior Ventral tegmental area Lateral septal nucleus
*Significantly higher than saline-treated controls ( p ! .02, in bold). a Values are means 6 S.E.M. in pmol/g tissue. b Combined data from sensitized and nonsensitized subgroups.
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activation of these receptors seems to be necessary for ethanol-induced locomotor stimulation in mice (Cohen et al., 1997; Liljequist et al., 1981; Thrasher et al., 1999). Furthermore, D4 knockout mice are hyperreactive to ethanol stimulation and to other drugs (Drago et al., 1998; Rubinstein et al., 1997), confirming the involvement of these receptors in ethanol-induced behavioral activation. In this study, repeated ethanol treatment induced increased D4 binding levels in different areas of the caudate–putamen, and also in the olfactory tubercle and the lateral septal nucleus. These brain regions are all part of the nigrostriatal and mesolimbic dopamine pathways, which are known to be involved in both the motivational and stimulating properties of ethanol (Di Chiara, 1993; Koob et al., 1998; Phillips & Shen, 1996). Therefore, the observed alterations in D4 binding densities may putatively be of behavioral and neurochemical relevance, although they seem to be unrelated to the individual susceptibility to develop ethanol sensitization. In rats, chronic ethanol consumption (for 2 or 9 months) did not induce significant alterations in D4 messenger RNA expression levels (Eravci et al., 1997). However, in the latter study, rats were kept abstinent from ethanol ingestion for 1 month before they were sacrificed for brain receptor expression assays (Eravci et al., 1997), whereas in this study mice were ‘‘abstinent’’ for only 24 h before sacrifice. Therefore, it seems possible that eventual neuroadaptations in D4 receptor functioning induced by repeated ethanol administration may be time dependent, and the behavioral relevance and duration of these neuroadaptations remain to be determined. In previous studies, we reported increased binding densities of D2 receptors in ethanol-sensitized mice (Souza-Formigoni et al., 1999), whereas neither D1 receptor nor the dopamine transporter binding levels were associated with ethanol sensitization (Quadros et al., 2002). Therefore, it seems that the D2-like family of dopamine receptorsd including D2 and D4 receptorsdis more susceptible to ethanol-induced neuroadaptations than other dopaminergic binding sites. Although increases in D2 binding densities were particularly associated with ethanol sensitization, an upregulation of D4 receptors in specific brain regions seemed to be induced by chronic ethanol treatment, regardless of the manifestation of ethanol-induced sensitization in mice. Fig. 2. Digitized images illustrating the binding pattern of [3H]nemonapride to dopamine D4 receptors. Olf bulb (gl) 5 olfactory bulb, glomerular layer; CPu 5 caudate–putamen; CPu-P 5 caudate–putamen, posterior division; Acc 5 nucleus accumbens; Olf tubercle 5 olfactory tubercle.
sensitization and D4 receptor binding densities. However, in this study we did observe a general effect of repeated ethanol administration on D4 binding densities, suggesting that D4 receptor plasticity processes may be more sensitive to ethanol than to amphetamine treatment. Although no pharmacological studies have analyzed the role of dopamine D4 receptors in ethanol sensitization, the
Acknowledgments This work was supported by funds from AFIP (Associac¸a˜o Fundo de Incentivo a Psicofarmacologia), FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo), and CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico). References Broadbent, J., & Harless, W. E. (1999). Differential effects of GABA(A) and GABA(B) agonists on sensitization to the locomotor stimulant effects of ethanol in DBA/2 J mice. Psychopharmacology 141, 197–205.
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Cohen, C., Perrault, G., & Sanger, D. J. (1997). Evidence for the involvement of dopamine receptors in ethanol-induced hyperactivity in mice. Neuropharmacology 36(8), 1099–1108. Di Chiara, G. (1993). Searching for the hidden order in chaos. Commentary on Kalivas et al., ‘‘The pharmacology of neural circuitry of sensitization to psychostimulants’’. Behav Pharmacol 4, 335–337. Drago, J., Padungchaicot, P., Accili, D., & Fuchs, S. (1998). Dopamine receptors and dopamine transporter in brain function and addictive behaviors: insights from targeted mouse mutants. Dev Neurosci 20, 188–203. Eravci, M., Grosspietsch, T., Pinna, G., Schulz, O., Kley, S., Bachmann, M., Wolffgramm, J., Gotz, E., Heyne, A., Meinhold, H., & Baumgartner, A. (1997). Dopamine receptor gene expression in an animal model of ‘behavioral dependence’ on ethanol. Brain Res Mol Brain Res 50, 221–229. Feldpausch, D. L., Needham, L. M., Stone, M. P., Althaus, J. S., Yamamoto, B. K., Svensson, K. A., & Merchant, K. M. (1998). The role of dopamine D4 receptor in the induction of behavioral sensitization to amphetamine and accompanying biochemical and molecular adaptations. J Pharmacol Exp Ther 286, 497–508. Franklin, K., & Paxinos, G. (1997). The Mouse Brain in Stereotaxic Coordinates. New York: Academic Press. George, S. R., Cheng, R., Nguyen, T., Israel, Y., & O’Dowd, B. F. (1993). Polymorphisms of the D4 dopamine receptor alleles in chronic alcoholism. Biochem Biophys Res Commun 196(1), 107–114. Hutchison, K. E., McGeary, J., Smolen, A., Bryan, A., & Swift, R. M. (2002). The DRD4 VNTR polymorphism moderates craving after alcohol consumption. Health Psychol 21(2), 139–146. Hutchison, K. E., Wooden, A., Swift, R. M., Smolen, A., McGeary, J., Adler, L., & Paris, L. (2003). Olanzapine reduces craving for alcohol: a DRD4 VNTR polymorphism by pharmacotherapy interaction. Neuropsychopharmacology 28(10), 1882–1888. Katz, J. L., Chausmer, A. L., Elmer, G. I., Rubinstein, M., Low, M. J., & Grandy, D. K. (2003). Cocaine-induced locomotor activity and cocaine discrimination in dopamine D4 receptor mutant mice. Psychopharmacology 170(1), 108–114. Koob, G. F., Roberts, A. J., Schulteis, G., Parsons, L. H., Heyser, C. J., Hyytia, P., Merlo-Pich, E., & Weiss, F. (1998). Neurocircuitry targets in ethanol reward and dependence. Alcohol Clin Exp Res 22, 3–9. Kruzich, P. J., Suchland, K. L., & Grandy, D. K. (2004). Dopamine D4 receptor-deficient mice, congenic on the C57BL/6J background, are hypersensitive to amphetamine. Synapse 53(2), 131–139. Lessov, C. N., & Phillips, T. J. (1998). Duration of sensitization to the locomotor stimulant effects of ethanol in mice. Psychopharmacology 135, 374–382. Liljequist, S., Berggren, U., & Engel, J. (1981). The effect of catecholamine receptor antagonists on ethanol-induced locomotor stimulation. J Neural Transm 50(1), 57–67. Lusher, J. M., Chandler, C., & Ball, D. (2001). Dopamine D4 receptor gene (DRD4) is associated with Novelty Seeking (NS) and substance abuse: the saga continues. Mol Psychiatry 6(5), 497–499.
Masur, J., Souza, M. L. O., & Zwicker, A. P. (1986). The excitatory effect of ethanol: absence in rats, no tolerance and increased sensitivity in mice. Pharmacol Biochem Behav 24, 1225–1228. Newlin, D. B., & Thomson, J. B. (1991). Chronic tolerance and sensitization to alcohol in sons of alcoholics. Alcohol Clin Exp Res 15, 399– 405. Paterson, A. D., Sunohara, G. A., & Kennedy, J. L. (1999). Dopamine D4 receptor gene: novelty or nonsense? Neuropsychopharmacology 21(1), 3–16. Phillips, T. J., Huson, M., Gwiazdon, C., Burkhart-Kasch, S., & Shen, E. H. (1995). Effects of acute and repeated ethanol exposures on the locomotor activity of BXD recombinant inbred mice. Alcohol Clin Exp Res 19, 269–278. Phillips, T. J., Roberts, A., & Lessov, C. (1997). Behavioral sensitization to ethanol: genetics and the effects of stress. Pharmacol Biochem Behav 57(3), 487–493. Phillips, T. J., & Shen, E. H. (1996). Neurochemical bases of locomotion and ethanol stimulant effects. Int Rev Neurobiol 39, 243–282. Quadros, I. M. H., Nobrega, J. N., Hipolide, D. C., De Lucca, E. M., & Souza-Formigoni, M. L. O. (2002). Differential propensity to ethanol sensitization is not associated with altered binding to D1 receptors or dopamine transporters in mouse brain. Addict Biol 7, 291–299. Robinson, T. E., & Berridge, K. C. (1993). The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res Brain Res Rev 18, 247–291. Rubinstein, M., Phillips, T. J., Bunzow, J. R., Falzone, T. L., Dziewczapolski, G., Zhang, G., Fang, Y., Larson, J. L., McDougall, J. A., Chester, J. A., Saez, C., Pugsley, T. A., Gershanik, O., Low, M. J., & Grandy, D. K. (1997). Mice lacking dopamine D4 receptors are supersensitive to ethanol, cocaine, and methamphetamine. Cell 90, 991–1001. Sander, T., Harms, H., Dufeu, P., Kuhn, S., Rommelspacher, H., & Schmidt, L. G. (1997). Dopamine D4 receptor exon III alleles and variation of novelty seeking in alcoholics. Am J Med Genet 74(5), 483–487. Souza-Formigoni, M. L. O., De Lucca, E. M., Hipo´lide, D. C., Enns, S. C., Oliveira, M. G. M., & Nobrega, J. N. (1999). Sensitization to ethanol’s stimulant effect is associated with localized increases in brain D2 receptor binding. Psychopharmacology 146, 262–267. Tarazi, F. I., & Baldessarini, R. J. (1999). Dopamine D4 receptors: significance for molecular psychiatry at the millennium. Mol Psychiatry 4, 529–538. Tarazi, F. I., Kula, N. S., & Baldessarini, R. J. (1997). Regional distribution of dopamine D4 receptors in rat forebrain. Neuroreport 8, 3423– 3426. Thrasher, M. J., Freeman, P. A., & Risinger, F. O. (1999). Clozapine’s effects on ethanol’s motivational properties. Alcohol Clin Exp Res 23, 1377–1385. Zhang, K., Tarazi, F. I., & Baldessarini, R. J. (2000). Dopamine D(4) receptors in rat forebrain: unchanged with amphetamine-induced behavioral sensitization. Neuroscience 97, 211–213.