Department of Psychiatry, University of Oulu, FIN-90220 Oulu,
1 Department of Nuclear Medicine, University of Oulu, FIN-90220 Oulu, Department of Clinical Physiology and Nuclear Medicine, University of Helsinki, FIN-00029 HUS Helsinki,
2 Department of Pharmacology, University of Turku, FIN-20520 Turku,
3 Department of Forensic Psychiatry, Department of Neurophysiology, University of Kuopio, FIN-70210 Kuopio and
4 Department of Psychiatry, University of Turku, Turku PET Centre, FIN-20520 Turku, Finland
Received 21 September 2000; in revised form 28 November 2000; accepted 27 December 2000
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ABSTRACT |
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INTRODUCTION |
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Based on these results, we postulated that aspects of the genetic polymorphism of the D2 receptor gene of the dopaminergic system might reliably be examined through imaging of DAT densities in patients with alcohol dependence under controlled sobriety.
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MATERIALS AND METHODS |
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DAT density was determined by SPET using radioligand ß-CIT when 4 weeks of controlled sobriety had elapsed. SPET procedure (Laine et al., 1999a) and some DAT binding data have previously been presented (Laine et al., 1999a
,b
).
DNA isolation and TaqI A RFLP
For DNA analyses, blood samples were collected from each subject and frozen at 70°C in glass tubes. DNA was extracted from 10 ml samples of peripheral blood according to standard procedures (Vandenplas et al., 1984). Subjects were genotyped for TaqI A restriction fragment length polymorphism (RFLP) located in the 3' flanking region of the dopamine D2 receptor gene as described by Grandy et al. (1993). Polymerase chain reaction (PCR) was carried out on a total volume of 10 µl containing 1 x reaction buffer supplied with Pfu polymerase (Stratagene, La Jolla, CA, USA), 100 ng of genomic DNA, 40 pmol of each primer, 200 µM of each deoxynucleotide and 0.15 U of Pfu polymerase. PCR conditions were: denaturation at 94°C for 3 min followed by 35 cycles at 94°C for 45 s, 64°C for 45 s, 72°C for 45 s, and a final extension of 5 min at 72°C. The digested fragments, separated on a 3% agarose gel containing 0.5 µg/ml ethidium bromide, were then visualized and photographed. The A1 allele remained intact, whereas the A2 allele was cut into one 180 bp and one 130 bp piece.
Statistical analysis
The variables used in the statistical analysis were age, duration of the last drinking bout (days), daily amount of alcohol consumed during the last drinking bout (g/day), number of days of abstinence preceding the first SPET imaging, withdrawal symptom score of the Selected Severity Assessment scale (SSA) (Gross et al., 1973), MADRS during withdrawal and after 4 weeks of abstinence (Montgomery and Åsberg, 1979
), and the total amount of benzodiazepines (as diazepam equivalents) administered to the patient for detoxification.
Means and SD were used in descriptions of the continuous variables. Student's t-tests were used in two-tailed independent samples. To reconcile effect of age with DAT density (Volkow et al., 1996a), and to measure differences between the hemispheres, we used a repeated measures analysis of variance. Statistical analyses were completed using the Statistical Package for the Social Sciences (SPSS), version 6.1, for Microsoft Windows.
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RESULTS |
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DISCUSSION |
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The mechanism linking the A1 allele to increased DAT levels is unclear. In principle, low postsynaptic D2-receptor density among patients with the A1 allele may result in low net dopamine neurotransmission. DAT density is relatively stable, possibly reflecting the condition of the dopaminergic tract (Kuikka et al., 1995; Moody et al., 1996
; Scheffel et al., 1996
). In diseases like Parkinson's, a decrease of DAT density is indicative of cell losses (Menza et al., 1995
). In our earlier studies, we found increases in striatal DAT densities following remissions of alcohol withdrawal (Laine et al., 1999a
). Cocaine has previously been found to up-regulate DAT density by blocking dopamine transporters (Little et al., 1993
; Malison et al., 1998
). In a study involving rats, decreased dopamine flow did not decrease DAT density (Moody et al., 1996
; Scheffel et al., 1996
), and dopaminergic medication, used in Parkinson's disease, was not found to affect ß-CIT SPECT imaging (Ahlskog et al., 1999
; Innis et al., 1999
).
According to our observations, alcoholics with the A1/A2 allele are more depressed during acute withdrawal. Two of the patients were clinically depressed after 4 weeks of sobriety, suffering from either primary depression or long-lasting secondary depression. Alcoholics with higher recoveries in DAT densities during alcohol withdrawal had more severe depressive symptoms possibly because of fragility of their mood system (Laine et al., 1999b). This is also in line with the reported increase of DAT levels in depressed patients (Laasonen-Balk et al., 1999
).
We conclude that the DRD2 A1/A2 genotype can be associated with relatively higher ß-CIT binding to DAT in dopaminergic nerve terminals of alcohol-dependent patients than the A2/A2 genotype. One limitation of this study is the small sample size, which is unfortunate, but typical in studies of functional neuroimaging. The carriers of the A1 allele also displayed increased depressive symptoms during alcohol withdrawal, suggesting that this DRD2 gene variant (or a functional variation in linkage disequilibrium with the allele) can be associated with the symptom phenotype in alcohol withdrawal.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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American Psychiatric Association (1987) Diagnostic and Statistical Manual of Mental Disorders, 3rd edn, revised. American Psychiatric Association, Washington, DC.
Grandy, D. K., Zhang, Y. and Civelli, O. (1993) PCR detection of the TaqI A RFLP at the DRD2 locus. Human Molecular Genetics 2, 2197.[ISI][Medline]
Gross, M. M., Eastlyn, L. and Nagarajan, M. (1973) An improved quantitative system for assessing the acute alcoholic psychoses and related states (TSA and SSA). In Alcohol Intoxication and Withdrawal: Experimental Studies, Gross, M. M. ed., pp. 365376. Plenum Press, New York.
Innis, R. B., Marek, K. L., Sheff, K., Zoghbi, S., Castronuovo, J., Feigin, A. and Seibyl, J. B. (1999) Effect of treatment with l-dopa/carbidopa or l-selegiline on striatal dopamine transporter SPECT imaging with [123I]ß-CIT. Movement Disorders 14, 940946.[ISI][Medline]
Jönsson, E. G., Nothen, M. M., Grunhage, F., Farde, L., Nakashima, Y., Propping, P. and Sedvall, G. C. (1999) Polymorphisms in the dopamine D2 receptor gene and their relationships to striatal dopamine receptor density of healthy volunteers. Molecular Psychiatry 4, 290296.[ISI][Medline]
Koob, G. R. (1992) Drugs of abuse. Anatomy, pharmacology and function of reward pathways. Trends in Pharmacological Sciences 13, 177184.[ISI][Medline]
Kuikka, J. T., Tiihonen, J., Bergström, K. A., Karhu, J., Hartikainen, P., Viinamäki, H., Länsimies, E., Lehtonen, J. and Hakola, P. (1995) Imaging of serotonin and dopamine transporters in the living human brain. European Journal of Nuclear Medicine 22, 346350.[ISI][Medline]
Laasonen-Balk, T., Kuikka, J., Viinamäki, H., Husso-Saastamoinen, M., Lehtonen, J. and Tiihonen, J. (1999) Striatal dopamine transporter density in major depression. Psychopharmacology 144, 282285.[ISI][Medline]
Laine, T. P. J., Ahonen, A., Torniainen, P., Heikkilä, J., Pyhtinen, J., Räsänen, P., Niemelä, O. and Hillbom, M. (1999a) Dopamine transporters increase in human brain after alcohol withdrawal. Molecular Psychiatry 4, 189191.[ISI][Medline]
Laine, T. P. J., Ahonen, A., Räsänen, P. and Tiihonen, J. (1999b) Dopamine transporter availability and depressive symptoms during alcohol withdrawal. Psychiatry Research: Neuroimaging section 90, 153157.[Medline]
Laruelle, M., Gelernter, J. and Innis, R. B. (1998) D2 receptors binding potential is not affected by TaqI polymorphism at the D2 receptor gene. Molecular Psychiatry 3, 261265.[ISI][Medline]
Little, K. Y., Kirkman, J. A., Carrol, F. I., Clark, T. B. and Duncan, G. E. (1993) Cocaine use increases [3H]WIN 35428 binding sites in human striatum. Brain Research 628, 1725.[ISI][Medline]
Malison, R. T., Best, S. E., van Dyck, C. H., McCance, E. F., Wallace, E. A., Laruelle, M., Baldwin, R. M., Seibyl, J. P., Price, L. H., Kosten, T. R. and Innis, R. B. (1998) Elevated striatal transporters during acute cocaine abstinence as measured by [123I]ß-CIT SPECT. American Journal of Psychiatry 155, 832834.
Menza, M. A., Mark, M. H., Burn, D. J. and Brooks, D. J. (1995) Personality correlates of [18F]dopa striatal uptake: Results of positron-emission tomography in Parkinson's disease. Journal of Neuropsychiatry and Clinical Neurosciences 7, 176179.[Abstract]
Mittman, N., Mitter, S., Borden, E. K., Herrmann, N., Naranjo, C.A. and Shear, N. H. (1997) MontgomeryÅsberg severity gradations. American Journal of Psychiatry 154, 13201321.
Montgomery, S. A. and Åsberg, M. (1979) A new depression scale designed to be sensitive to change. British Journal of Psychiatry 134, 282289.
Moody, C. A., Granneman, J. G. and Bannon, M. J. (1996) Dopamine transporter binding in striatum and nucleus accumbens is unaltered following chronic changes in dopamine levels. Neuroscience Letters 217, 5557.[ISI][Medline]
Noble, E. P. (2000) Addiction and its reward process through polymorphism of the D2 dopamine receptor gene: a review. European Psychiatry 15, 7989.[Medline]
Noble, E. P., Blum, K., Ritchie, T., Montgomery, A. and Sheridan, P. J. (1991) Allelic association of the D2 dopamine receptor gene with receptor binding characteristics in alcoholism. Archives of General Psychiatry 48, 648654.[Abstract]
Pohjalainen, T., Rinne, J. O., Någren, K., Lehikoinen, P., Anttila, K., Syvälahti, E. and Hietala, J. (1998) The A1 allele of the human D2 dopamine receptor gene predicts low D2 receptor availability in healthy volunteers. Molecular Psychiatry 3, 256260.[ISI][Medline]
Scheffel, U., Steinert, C., Kim, S. E., Ehlers, M. D., Boja, J. W. and Kuhar, M. J. (1996) Effect of dopaminergic drugs on the in vivo binding of [3H] WIN 35,428 to central dopamine transporters. Synapse 23, 6169.[ISI][Medline]
Seibyl, J. P., Laruelle, M., van Dyck, C. H., Wallace, E., Baldwin, R. M., Zoghbi, S., Zea-Ponce, Y., Neumeyer, J. L., Charney, D. S., Hoffer, P. B. and Innis, R. B. (1996) Reproducibility of iodine-123-ß-CIT SPECT brain measurement of dopamine transporters. Journal of Nuclear Medicine 37, 222228.[Abstract]
Thompson, J., Thomas, N., Singleton, A., Piggot, M., Lloyd, S., Perry, E. K., Morris, C. M., Perry, R. H., Ferrier, I. N. and Court, J. A. (1997) D2 dopamine receptor gene (DRD2) TaqI A polymorphism: Reduced dopamine D2 receptor binding in the human striatum associated with the A1 allele. Pharmacogenetics 7, 479484.[ISI][Medline]
Vandenplas, S., Wiid, I., Grobler-Rabie, A., Brebner, K., Ricketts, M., Wallis, G. and Mathew, C. (1984) Blot hybridization analysis of genomic DNA. Journal of Medical Genetics 21, 164172.[Abstract]
Volkow, N. D., Ding, Y., Fowler, J. S., Wang, G., Logan, J., Catley, J., Hitzeman, R., Smith, G., Fields, S. D. and Gur, R. (1996a) Dopamine transporters decrease with age. Journal of Nuclear Medicine 37, 554559.[Abstract]
Volkow, N. D., Wang, G., Fowler, J. S., Logan, J., Hitzeman, R., Ding, Y-S., Pappas, N., Shea, C. and Piscani, K. (1996b) Decreases in dopamine receptors but not in dopamine transporters in alcoholics. Alcoholism: Clinical and Experimental Research 20, 15941588.[ISI][Medline]