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Molecular Psychiatry (2000) 5, 664–672  2000 Macmillan Publishers Ltd All rights reserved 1359-4184/00 $15.00 www.nature.com/mp

ORIGINAL RESEARCH ARTICLE

Dopamine D1 receptor protein is elevated in nucleus accumbens of human, chronic methamphetamine users JN Worsley1, A Moszczynska1, P Falardeau2, KS Kalasinsky3, G Schmunk4, M Guttman1, Y Furukawa1, L Ang5, V Adams6, G Reiber7, RA Anthony7, D Wickham8 and SJ Kish1 1

Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, Ontario, Canada M5T 1R8; 2Neuroscience Unit, Centre de Recherche du CHUQ, Laval University, PQ Canada G1V 4G2; 3Division of Forensic Toxicology, Armed Forces Institute of Pathology, Washington, DC 20306, USA; 4Office of the Santa Clara County Medical Examiner-Coroner, San Jose, CA 95128–4702, USA; 5Department of Pathology (Neuropathology), Sunnybrook Hospital, Toronto, Ontario, Canada M4N 3M5; 6Office of the Hillsborough County Medical Examiner, Tampa, FL 33602, USA; 7 Northern California Forensic Pathology, Sacramento, CA 95825, USA; 8Clark County Medical Examiner Office, Vancouver, WA 98660, USA Animal data have long suggested that an adaptive upregulation of nucleus accumbens dopamine D1 receptor function might underlie part of the dependency on drugs of abuse. We measured by quantitative immunoblotting protein levels of dopamine D1 and, for comparison, D2 receptors in brain of chronic users of methamphetamine, cocaine, and heroin. As compared with the controls, brain dopamine D1 receptor concentrations were selectively increased (by 44%) in the nucleus accumbens of the methamphetamine users, whereas a trend was observed in this brain area for reduced protein levels of the dopamine D2 receptor in all three drug groups (ⴚ25 to ⴚ37%; P < 0.05 for heroin group only). Our data support the hypothesis that aspects of the drug-dependent state in human methamphetamine users might be related to increased dopamine D1 receptor function in limbic brain. Molecular Psychiatry (2000) 5, 664–672. Keywords: methamphetamine; cocaine; heroin; dopamine; nucleus accumbens; dopamine D1 receptor; dopamine D2 receptor; addiction

Introduction The mechanism by which chronic exposure to drugs of abuse produces a state of drug dependence in some human users of the drugs is unknown. As psychostimulant (methamphetamine, cocaine) and opiate drugs (heroin) all have the capacity to increase synaptic levels of the neurotransmitter dopamine in limbic brain,1 it has been suggested that the addicted state is consequent to abnormal functioning of one or more dopamine receptor systems consequent to prolonged exposure of the drugs.2,3 Much attention has been focused on the dopamine D1 and D2 receptor subtypes, which are defined by their ability to stimulate or inhibit, adenylyl cyclase, respectively. However, the relative contribution of each receptor to the acute and chronic actions of drugs of abuse is still not understood. Electrophysiological and biochemical animal data indicate that some aspects of the drug-dependent state might be explained by supersensitivity of one or more components of the dopamine D1 receptor-cAMP sig-

Correspondence: SJ Kish, PhD, Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ontario, Canada M5T 1R8. E-mail: StephenFKish얀CAMH.net Received 16 December 1999; revised and accepted 17 April 2000

naling system.4–9 These findings, taken together with the demonstration that dopamine D1 agonist compounds can prevent relapse to drug-seeking behaviour in rodents10 have provided an experimental platform for the selection of the D1 receptor as a clinical target for therapeutic intervention in human drug users. To establish whether chronic exposure to drugs of abuse might upregulate levels of the brain dopamine D1 receptor in humans, we measured concentrations of dopamine D1 and, for comparison, dopamine D2 receptors in postmortem brain of chronic users of methamphetamine, cocaine, and heroin. Levels of the dopamine receptors in striatum (caudate, putamen, nucleus accumbens) and extra-striatal brain regions were determined using a quantitative immunoblotting procedure which employed antibodies specific to both receptor types. In this regard, the use of a Western blotting procedure allows greater specificity for specific dopamine receptor subtypes than radioligand binding procedures, which are not specific for dopamine receptor subtypes, and which might allow binding to other receptors (eg, serotonin).11,12 This procedure also allows for the detection of multimeric forms of receptors which might not be resolved by radioligand binding techniques.13 We report that concentrations of the dopamine D1 receptor are selectively increased in the nucleus accumbens of chronic users of methamphetamine.

Dopamine D1 receptors in human methamphetamine users JN Worsley et al

Subjects and methods Brain dissection Postmortem brain material from a total of 14 controls, 12 users of methamphetamine, 11 users of cocaine, and nine users of heroin was obtained from medical examiner offices in the US and Canada using a standardized protocol. The mean ages and postmortem time (interval between death and freezing of the brain) between the drug abuse and matched control groups did not differ significantly (one way analysis of variance [ANOVA]; P ⬎ 0.05) (Table 1). At autopsy, one half-brain was fixed in formalin fixative whereas the other half was immediately frozen at −80°C until biochemical analysis. Samples of cardiac blood were obtained from all of the drug users and from the control subjects for drug screening. Scalp hair samples for drug analyses could be obtained from 12 of the 14 control subjects, seven of the 12 methamphetamine users, seven of the 11 cocaine users, and eight of the nine heroin users. Levels of drugs of abuse in blood and other bodily fluids were measured by the local medical examiner whereas drug analyses in brain and hair samples were conducted at the Armed Forces Institute of Pathology (KK; Washington, DC, USA). Control subjects for comparison with drug users Autopsied brain was obtained from 14 neurologically normal subjects who died from a variety of causes (gunshot wound to chest [1], trauma [3], cardiovascular disease [8], leukemia [1], and drowning [1]). All control subjects tested negative for drugs of abuse in blood, autopsied brain, and, in the 12 cases for which hair was available, sequential hair samples. Drug users The subjects for the cocaine, methamphetamine, and heroin groups were selected from a large group of potential cases who met the following criteria: (1) presence of methamphetamine, or cocaine or metabolite benzoylecgonine, or heroin metabolites (6-acetylmorTable 1 Age and postmortem time (interval between death and freezing of the brain) for control subjects, drug users, and patients with Huntington’s disease Subjects (n)

Age (years)

Young controls (14) Methamphetamine (12) Cocaine (11) Heroin (9)

34.6 32.5 32.8 37.8

Older controls (10) Huntington’s disease (10)

74.6 ± 8.5 73.1 ± 7.0

± ± ± ±

2.7 2.6 1.9 1.7

Postmortem time (h) 13.8 14.8 18.8 13.1

± ± ± ±

1.7 2.0 1.9 2.1

12.6 ± 6.6 12.8 ± 7.3

Values represent mean ± SE. No significant differences were observed amongst the young control and drug user groups (P ⬎ 0.05, one way analysis of variance) or between the older control and Huntington’s disease groups (P ⬎ 0.05, Student’s two-tailed t-test) for either age or postmortem time.

phine, morphine, or morphine glucuronide), on toxicology screens in blood or urine, autopsied brain (see below), and, if available, scalp hair; (2) absence of other drugs of abuse in bodily fluids with the exception of ethanol; (3) evidence from the case records of use of the primary drug for at least one year prior to death; and (4) absence of neurological illness or, at autopsy, brain pathology unrelated to use of the drug. Most of the potential subjects were rejected because of a known history of significant polydrug abuse or the presence of other drugs of abuse in blood or brain at autopsy. Alcohol was known to have been used by eight of the 11 cocaine users and all nine of the heroin users as evidenced by review of the case records and by presence in bodily fluids of ethanol or cocaethylene (the transesterification product of cocaine and ethanol) in brain. Ethanol was not detected in bodily fluids of any of the methamphetamine users. With the exception of one cocaine user in which cocaine was detected in urine but not blood, all of the other users of cocaine, methamphetamine, and heroin, tested positive for the drug of abuse in blood and brain, suggesting that each subject had used the drug during the 72 hours preceeding death. Clinical information was obtained by the medical examiners from the medical examiner investigator, police, and hospital case reports using a questionnaire format and through structured telephone interviews with the next of kin. Extensive clinical, toxicological, and neuropathological data have been previously published for the 12 users of methamphetamine,14 11 cocaine users15 and nine users of heroin.16 Drug intoxication was considered to be a primary or secondary contributory cause of death for nine methamphetamine users, eight cocaine users, and all nine users of heroin.

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Patients with Huntington’s disease and matched controls Autopsied brain was also obtained from 10 clinically and neuropathologically confirmed patients with Huntington’s disease and 10 normal age-matched control subjects. The mean ages and postmortem times of the two groups (see Table 1) did not differ significantly (P ⬎ 0.05, Student’s two-tailed t-test). Brain dissection for neurochemical analyses For the brain dissection for neurochemical analysis, cerebral cortical subdivisions were excised according to Brodmann classification. Following dissection of the cerebral cortical subdivisions, approximately 2.5 mmthick coronal sections of the brain were taken for dissection of subcortical brain areas using the Atlas of Riley.17 A sample of the caudate (intermediate portion of slice 4) and putamen (intermediate portion of slice 7) was taken as described.18 A sample of the caudal nucleus accumbens was excised as described.19 Although the ‘core’ and ‘shell’ subdivisions of the nucleus accumbens observed in rodent brain have yet to be defined absolutely in the human, this caudal portion of the nucleus accumbens probably contains both core and shell portions.20,21 Molecular Psychiatry


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Quantitative immunoblotting Protein concentrations of the dopamine D1 and D2 receptors were determined by quantitative blot immunolabeling. Brain samples were homogenized in 100 volumes (w/v) Tris-HCl buffer (pH 7.5, 50 mM at 4°C) using a Brinkman polytron and centrifuged at 35 000 × g for 15 min. The resultant pellet was suspended in 100 volumes of buffer and again centrifuged at 35000 × g for 15 min. The supernatant was discarded and the pellet was suspended in Tris-HCl buffer (pH 7.5, 50 mM, containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 5 mM EDTA, and 1 mM MgCl2) and protein levels were determined. Samples were then centrifuged at 12 000 × g, and the supernatant discarded, and the pellet was then resuspended in 10% sodium dodecyl sulfate (SDS, 50 ␮l) and left at 25°C for 30 min, and diluted with 50% glycerol (50 ␮l) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (25 ␮l; 62.5 mM Tris-HCl, pH 8, 10% glycerol, 2% SDS, 5% ␤-mercaptoethanol), and left at room temperature for an additional 30 min. The samples (15–30 ␮g protein) were subjected to SDS-PAGE in 1.5 mm-thick, 12% minigels and transferred overnight (32 V) electrophoretically to polyvinylidene difluoride (PVDF) membranes using a wet transfer tank. PVDF membranes were rinsed briefly in deionized water and blocked with 10% nonfat dry milk in Tris-buffered saline (TBS) for 30 min at 25°C, rinsed three times in TBS-T (TBS with 0.1% Tween-20), and subsequently incubated with either a primary rat monoclonal antibody specific to the human dopamine D1 receptor (Research Biochemicals International, Natick, MA, USA),22–24 or a rabbit polyclonal antibody raised against a fusion peptide corresponding to amino acids 212–311 of the human D2 short receptor25,26 in plastic boxes in TBS containing 10% non fat dried milk for 1 h at 25°C. The PVDF membranes were rinsed with TBS-T, incubated with secondary antibody (anti-rat IgG, horseradish peroxidase-linked whole antibody from goat for the D2 antibody [Sigma, St Louis, MO, USA]) containing 10% non fat dried milk for 1 h at 25°C in plastic bags and washed in TBS-T at 25°C. Bound antibody was detected by enhanced chemiluminescence (ECL, Amersham, Canada Ltd, Oakville, ON, Canada) and visualized by exposure to autoradiographic film (Hyperfilm, Amersham). Quantification was performed using a computer-based imaging device (MCID, St Catherine’s, Ontario, Canada). Five concentrations of tissue standard (5–30 ␮g) consisting of normal human putamen were run on each gel, together with eight samples, and a standard curve (optical density units vs ␮g of tissue standard protein) was plotted for each blot. The amount of dopamine D1 and D2 receptor protein in each lane was calculated by interpolation from the standard curve and expressed as ␮g protein tissue standard ␮g−1 protein of the sample. Protein concentration was determined by the modified Bradford assay according to the instructions of the manufacturer (BioRad Laboratories, Hercules, CA, USA). For simplicity, the terms ‘protein’ or receptor ‘concentration’ or ‘level’ will be used to describe dopamine D1 and dopamine

Molecular Psychiatry

D2 receptor-like immunoreactivity. Variances (coefficient of variation) within and amongst blots for the D1 receptor protein were 3.8% and 9.5%, respectively, whereas those for the D2 receptor protein were 5.6% and 5.4%, respectively. Cells expressing the D1 (Sf9), D3 (CCL 11.3 mouse fibroblasts), and D5 (Sf9) receptor subtypes were obtained from Research Biochemicals International, Natick, MA, USA. Sf9 cells expressing the D2 long and D2 short receptors and HEK-293 cells expressing the D4.4 receptor were gifts from Drs Hyman Niznik and Hubert Van Tol, respectively (Centre for Addiction and Mental Health, Toronto, Canada). Statistical analyses A Student’s two-tailed t-test was employed for comparisons between two groups whereas a one way analysis of variance followed by the Least Significant Difference test was used for comparisons involving more than two groups (P ⬍ 0.05 criterion for all comparisons).

Results Characteristics of cloned and native dopamine D1 and D2 receptor protein In Sf9 cells transfected with the human D1 receptor, dopamine D1 protein immunoreactivity was detected as a broad band centered at approximately 50 kDa with a minor band (possibly corresponding to a dimer) at approximately 100 kDa (see Figure 1). Upon prolonged exposure of the blot, a faint band at approximately 200 kDa could also be observed in the D1-transfected Sf9 cells. No immunoreactivity to the dopamine D1 antibody could be detected in cells transfected with the dopamine D2 long, D2 short, D3, D4, or D5 receptors (Figure 1). In Sf9 cells transfected with the human dopamine D2 receptor subtypes, the dopamine D2 receptor antibody detected a broad band at approximately 50 kDa and 45 kDa in the cells transfected with the dopamine D2 long and D2 short receptors, respectively, with minor bands at 90 kDa and 85 kDa, respectively. Upon prolonged exposure of the blots, a faint band at approximately 175 kDa could be detected. No immunoreactivity to the dopamine D2 antibody could be detected in cells transfected with the dopamine D1, D3, D4, or D5 receptors (Figure 1). In striatum from normal human brain, dopamine D1 receptor protein was detected as a broad band of approximately 70–80 kDa whereas that from cerebral cortical brain areas migrated with a slightly lower molecular weight of approximately 65–75 kDa (Figure 2, panel 1). In some samples, prolonged exposure of the blots would reveal a minor band at approximately 100 kDa. In human striatum, dopamine D2 receptor protein was detected as a faint, very broad band (which could not be easily quantitated), spanning approximately 70–125 kDa when the electrophoresis proceeded, until the gel front reached the full length of the resolution gel (Figure 2, Panel IIb). However, when


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Figure 1 Immunoblots (representative of three independent experiments) demonstrating specificity of the dopamine D1 (Panel I) and D2 (Panel II) antibodies in crude membranes of cells expressing the dopamine D1 (Sf9; 0.02 ␮g protein), D2 long (D2L; Sf9; 0.1 ␮g protein), D2 short (D2S; Sf9; 0.1 ␮g protein), D3 (CCL1.3 mouse fibroblasts; 0.1 ␮g protein), D4 (HEK-293; 20 ␮g protein), and D5 (Sf9; 0.1 ␮g protein) receptor subtypes. Panel I: Dopamine D1 receptor immunoreactivity is observed only in dopamine D1-transfected cells, with a major band centered at 50 kDa and a minor band at 100 kDa. Upon prolonged exposure of the blot a faint band at approximately 200 kDa could be observed. Panel II: Dopamine D2 receptor immunoreactivity is observed only in cells transfected with the dopamine D2 long and D2 short receptors with broad bands centered at 50 kDa and 45 kDa, respectively, and minor bands at 90 kDa and 85 kDa, respectively. Upon prolonged exposure a faint band at approximately 175 kDa could be observed.

Figure 2 Representative immunoblot of dopamine D1 (Panel I) and D2 (Panel II) receptors in human brain membranes. Panel I: A band of dopamine D1 receptor immunoreactivity in putamen (15 ␮g protein) and cerebral cortical Brodmann area 10 (30 ␮g protein) from a 47-year-old male control subject is observed at 70–80 kDa and 65–75 kDa, respectively. In some samples prolonged exposure of the blot would reveal a minor band at approximately 100 kDa. Panel II: Dopamine D2 receptor immunoreactivity in caudate nucleus (20 ␮g protein) of two male control subjects (aged 46 and 48 years) is detected as a faint non-quantifiable broad band of approximately 70–125 kDa when the electrophoresis proceeded until the gel front reached the full length of the resolution gel (long run, IIb) and as a dense quantifiable band when the gel front was run to 50% of the length of the resolving gel (short run, lla). Upon prolonged exposure a faint band of molecular weight of less than 30 kDa could be observed in some samples.

the gel front was run to only about 50% of the length of the resolving gel a dense band of immunoreactivity could be observed and quantitated (Figure 2, Panel IIa). A faint band of D2 immunoreactivity of less than 30 kDa could also be detected in some samples. The glycoprotein nature of both the dopamine D1 and D2 receptors could be demonstrated as exposure of striatal homogenates to deglycosylating enzymes decreased the apparent molecular weight of the proteins to approximately 50 kDa and 45 kDa, respectively (n = 3 experiments; data not shown). As shown in Figure 3, concentrations of dopamine D1 protein in human brain were highest in the striatum, with lower levels in the substantia nigra, globus pallidus subdivisions and cerebral cortex. No detectable dopamine D1 protein could be detected in the cerebellar cortex or in the diencephalon or hippocampus,

although a faint band of immunoreactivity, which could not be quantitated, could be observed in the pulvinar nucleus of the thalamus and in the dentate gyrus and Ammon’s horn of the hippocampus. High levels of the dopamine D2 protein could be detected in striatum, with low to moderate levels in the subdivisions of the globus pallidus and the pars compacta of the substantia nigra (Figure 4). No dopamine D2 receptor immunoreactivity could be detected in any of the other examined areas. Dopamine receptor protein in Huntington’s disease Dopamine receptor levels were also measured in brain of patients with Huntington’s disease, a disorder previously characterised by striatal degeneration of dopamine D1 and D2-containing neurones.27–29 As compared with the age-matched control group, striatal Molecular Psychiatry


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Figure 3 Regional distribution of dopamine D1 receptor protein in brain of three male control subjects (aged 40, 46 and 48 years). Values represent mean ± SE. Abbreviations: CN, caudate nucleus; PUT, putamen; NACS, nucleus accumbens; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; VENT, ventral globus pallidus; GPE, globus pallidus external; GPI, globus pallidus internal; AOL, medial olfactory area (one subject only examined); C.CX, cerebellar cortex; HYPTH, hypothalamus; MDTH, medial dorsal thalamus; NL, nucleus lateralis of thalamus; NPM, medial pulvinar nucleus of the thalamus; GH, hippocampal gyrus; GD, dentate gyrus; CNA, Ammon’s horn; Cerebral cortical Brodmann (B) areas: 7a (parietal), 10 (frontal), 17 (occipital), 21 (temporal), 24 (cingulate), 25 (parolfactory). Inset: Representative immunoblot of brain dopamine D1 receptor protein in selected brain areas of a male 40-year-old control subject. Lanes: C, caudate nucleus; P, putamen; N, nucleus accumbens; S, substantia nigra pars compacta; Pe, globus pallidus externa; Pi, globus pallidus interna; H, hypothalamus; Tmd, medial dorsal thalamus; Tnl, nucleus lateralis of thalamus; cerebral cortical Brodmann areas 10 (frontal), 17 (occipital), 21 (temporal); Ce, cerebellar cortex. Lanes C to Pi, 10 ␮g protein; Lanes H to Ce, 25 ␮g protein.

(putamen) protein levels of dopamine D1 and D2 receptors were significantly reduced by 54% and 31% respectively (Table 2) in the patients with Huntington’s disease, whereas no significant differences were observed for the dopamine D1 receptor in the frontal cortex (Brodmann area 10; P ⬎ 0.05; dopamine D2 receptor protein could not be detected in human cerebral cortex).

Discussion

Dopamine receptor protein in drug users As shown in Table 3 and in a representative blot (Figure 5), dopamine D1 receptor protein levels were normal in all examined cerebral cortical and striatal areas of the drug users with the exception of increased (+44%) concentration of the receptor in the nucleus accumbens of the methamphetamine users. Striatal protein levels of the dopamine D2 receptor were normal in the three drug user groups. In the nucleus accumbens, however, concentrations of the dopamine D2 receptor were modestly reduced by 25–37% in all three drug user groups with the differences statistically significant for the heroin users (P ⬍ 0.05). No statistically significant correlations (Pearson) were observed between age or postmortem time and dopamine D1 or D2 receptor protein levels in any brain area of the control or drug user subjects (P ⬎ 0.05).

Dopamine receptors in normal human brain The specificity in human brain of the dopamine receptor antibodies was confirmed by demonstration that the dopamine D1 and D2 antibodies bound selectively to the respective cloned human receptors and that, on Western blots of human brain, the immunoreactivity in deglycosylated samples was of similar molecular weight to that of the cloned receptors. Consistent with earlier studies employing radioligand binding procedures,27–29 concentrations of both dopamine receptors were reduced in striatum of patients with Huntington’s disease, a disorder characterized in part by severe loss of dopamine D1 and D2 receptor-containing neurones in this brain area. Dopamine D1 and D2 receptor immunoreactivity was, as expected from radioligand binding studies in human brain (see Hall et al and references therein),30 highly enriched in the sub-


The major finding of our investigation is that concentration of dopamine D1 receptor protein is selectively increased in nucleus accumbens of chronic human methamphetamine users. These neurochemical data provide direct support for the involvement of the dopamine D1 receptor in the actions of methamphetamine in human brain.


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Figure 4 Regional distribution of the dopamine D2 receptor in brain of three male control subjects (aged 40, 46 and 48 years). Values represent mean ± SE. Abbreviations: CN, caudate nucleus; PUT, putamen; NACS, nucleus accumbens; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; VENT, ventral globus pallidus; GPE, globus pallidus external; GPI, globus pallidus internal; AOL, medial olfactory area (one subject only examined); C.CX, cerebellar cortex; HYPTH, hypothalamus; MDTH, medial dorsal thalamus; NL, nucleus lateralis of thalamus; NPM, medial pulvinar nucleus of the thalamus; GH, hippocampal gyrus; GD, dentate gyrus; CNA, Ammon’s horn; Cerebral cortical Brodmann (B) areas: 7a (parietal), 10 (frontal), 17 (occipital), 21 (temporal), 24 (cingulate), 25 (parolfactory). Inset: Representative immunoblot of brain dopamine D2 receptor protein in selected brain areas of a male 40-year-old control subject. Lanes: C, caudate nucleus; P, putamen; N, nucleus accumbens; S, substantia nigra pars compacta; Pe, globus pallidus externa; Pi, globus pallidus interna; H, hypothalamus; Tmd, medial dorsal thalamus; Tnl, nucleus lateralis of thalamus; Cerebral cortical Brodmann areas 10 (frontal), 17 (occipital), 21 (temporal); Ce, cerebellar cortex. Lanes C to Pi, 10 ␮g protein; Lanes H to Ce, 25 ␮g protein. Table 2 Brain dopamine D1 and D2 receptor protein levels in autopsied human brain of control subjects and in patients with Huntington’s disease Dopamine receptor

Brain region

Controls (10)

Huntington’s disease (10)

D1

Putamen area 10

1.07 ± 0.17 0.04 ± 0.01

0.49 ± 0.07* 0.05 ± 0.01

D2

Putamen

1.20 ± 0.13

0.83 ± 0.07*

Values (␮g protein tissue standard ␮g−1 protein) represent mean ± SE with number of subjects shown in parentheses. Abbreviation: area 10 (Brodmann subdivision of frontal cortex). Five concentrations of tissue standard (5–30 ␮g) consisting of normal human putamen or cortex were run on each gel, together with eight samples, and a standard curve (optical density units vs ␮g of tissue standard protein) was plotted for each blot. The amount of dopamine D1 and D2 receptor protein in each lane was calculated by interpolation from the standard curve and expressed as ␮g protein tissue standard ␮g−1 protein of the sample. *P ⬍ 0.05; Student’s two-tailed t-test.

divisions of the striatum. The lack of detectable dopamine D2 receptor immunoreactivity in extra-striatal areas of brain such as the cerebral cortex in which very low levels of the receptor have previously been

reported,30 could be explained by the relative insensitivity of the Western blotting procedure in brain areas of low receptor number and/or the possible lack of total specificity of the probes employed in radioligand binding studies. Dopamine receptors in brain of drug users Previous investigation of dopamine D1 and D2 receptors in human drug users appears to be limited to radioligand binding studies of the dopamine D1 receptor in cocaine users31 and of the dopamine D2 receptor in users of cocaine31–33 and in users of heroin.34 Our observation of normal striatal dopamine D1 and D2 receptors in human cocaine users is consistent with similar postmortem findings using radioligand binding procedures.31,33 Volkow and coworkers reported positron emission tomography (PET) data indicating decreased concentration of the striatal dopamine D2 receptor in users of either cocaine32 or heroin,34 whereas in our investigation a statistically significant dopamine D2 receptor reduction (heroin) or trend for a reduction (cocaine) was limited to the nucleus accumbens, a striatal subdivision which, because of its small size, could not have been examined separately in the PET investigations. In our study, dopamine receptor changes were limited to the nucleus accumbens, a brain area which has been implicated, more than any other, in the mechMolecular Psychiatry


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Table 3

Brain dopamine D1 and D2 receptor protein levels in autopsied human brain of control subjects and in drug users

Dopamine receptor

Brain area

Controls (14) ± ± ± ± ± ± ±

D1

Caudate Putamen Nacs Area 9 Area 10 Area 22 Area 39

0.92 0.99 1.14 0.21 0.24 0.18 0.19

0.10 0.17 0.12 0.02 0.02 0.02 0.02

D2

Caudate Putamen Nacs

0.99 ± 0.11 1.10 ± 0.14 1.28 ± 0.10

MA (12) 1.00 1.14 1.64 0.17 0.24 0.19 0.17

± ± ± ± ± ± ±

0.18 0.20 0.15* 0.02 0.02 0.02 0.02

1.18 ± 0.16 1.23 ± 0.21 0.96 ± 0.12

Cocaine (11) 0.98 1.31 1.23 0.16 0.23 0.18 0.14

± ± ± ± ± ± ±

0.11 0.32 0.17 0.03 0.03 0.02 0.01

0.95 ± 0.14 1.06 ± 0.14 0.97 ± 0.14

Heroin (9) 0.88 0.81 0.94 0.16 0.20 0.15 0.14

± ± ± ± ± ± ±

0.14 0.12 0.10 0.03 0.03 0.02 0.01

0.92 ± 0.15 1.01 ± 0.14 0.81 ± 0.10*

Values (␮g protein tissue standard ␮g−1 protein) represent mean ± SE with number of subjects shown in parentheses. Abbreviations: MA, methamphetamine; Nacs, nucleus accumbens; Brodmann areas 9 and 10 (prefrontal cortical subdivisions), 22 (temporal cortex), 39 (parietal cortex). Five concentrations of tissue standard (5–30 ␮g) consisting of normal human putamen or cortex were run on each gel, together with eight samples, and a standard curve (optical density units vs ␮g of tissue standard protein) was plotted for each blot. The amount of dopamine D1 and D2 receptor protein in each lane was calculated by interpolation from the standard curve and expressed as ␮g protein tissue standard ␮g−1 protein of the sample. *Mean nucleus accumbens dopamine D1 and D2 receptor levels are significantly different from mean levels in the control group (P ⬍ 0.05; one way analysis of variance followed by Least Significant Difference comparison test).

Figure 5 Representative immunoblot of dopamine D1 (Panel I) and D2 (Panel II) receptors in nucleus accumbens of an individual control (C) subject and users of heroin (H), methamphetamine (MA), and cocaine (COC) run in duplicate.

anism of action of drugs of abuse.1–3,6,7,19 We found a modest reduction in levels of the dopamine D2 receptor in nucleus accumbens in all three drug abuse groups which was statistically significant for the heroin group. This finding can be interpreted as a compensatory downregulation of receptor number due to chronic overstimulation of the dopamine D2 receptor by drugs which all increase synaptic levels of dopamine.1 Unlike the decreased levels of the dopamine D2 receptor, concentrations of the dopamine D1 receptor were altered only in the methamphetamine group, and the levels of this receptor actually increased in the nucleus accumbens. In the experimental animal literature no clear pattern of changes in striatal dopamine receptor number has been reported in rodents exposed to methamphetamine or amphetamine. In this regard, Robinson and Becker35 reviewed a total of 24 investigations (pre-1986), employing radioligands with some selectivity for the dopamine D2 family of receptors, in Molecular Psychiatry

which dopamine receptor binding was found to be up (n = 4), down (n = 11) or unchanged (n = 9). In more recent animal studies using relatively selective dopamine D1 receptor radioligands, striatal dopamine D1 receptor number has been reported to be unchanged36–41 or reduced.42,43 Decreased striatal dopamine D1 receptor levels have been observed in rodents who had received a high dose of methamphetamine sufficient to cause damage to dopamine nerve terminals.44,45 Similarly, in a preliminary study, we found a statistically non-significant trend for lower dopamine D1 receptor concentration in striatum (the rodent counterpart of the human caudate and putamen; −15%) and nucleus accumbens (−31%) of rats killed 3 h after the last of 10 daily injections of methamphetamine (20 mg kg−1; Moszczynska, Worsley, and Kish, unpublished observations). However, our observation that striatal dopamine D1 receptor concentration was not reduced in human users of the drug does not support


the notion, that, at least in the subjects of our study, methamphetamine is toxic to striatal dopamine D1containing neurones. In principle, the above-normal concentration of the dopamine D1 receptor in the methamphetamine users could have been a pre-existing abnormality which might represent a predisposing factor for the drug abuse condition. Alternatively, this increase could be consequent to an agonal state event present in the group of drug users (eg, hyperthermia) but not in the control subjects. More likely, however, the receptor increase was an adaptive phenomenon related to the ability of methamphetamine to indirectly affect dopamine receptor activity by release of dopamine from dopamine nerve terminals. Previously we reported that striatal and nucleus accumbens dopamine levels are low in the methamphetamine users examined in the present investigation,14 a neurochemical change which could be due to the acute dopamine-depleting effects of the drug at high doses. Although increased dopamine D1 receptor number might, in this context, be explained as a form of receptor supersensitivity consequent to decreased receptor occupancy by dopamine, we did not observe any correlation between nucleus accumbens dopamine and dopamine D1 receptor levels (data not shown). We suggest that the increased dopamine D1 receptor concentration could be part of a sensitization-type, positive feedback phenomenon occurring as a result of prolonged dopaminergic stimulation by the psychostimulant. An increase in brain receptor number following chronic exposure to a psychostimulant drug in humans, although somewhat counterintuitive, is not without precedent, as indicated by previous findings in the human literature of elevated concentrations of the striatal dopamine D346 and the ␮47 and ␬48 opioid receptors in some chronic cocaine users. The explanation for our finding that dopamine D1 receptors were elevated in the users of methamphetamine, but not in users of cocaine or heroin (drugs which also increase synaptic dopamine concentration) is unclear. However assuming that this process is, in fact, dopamine-related, this might be due to a more marked dopaminergic stimulation caused by methamphetamine, in the users selected for our study, than that caused by either cocaine or heroin. The special selectivity of the nucleus accumbens vs other similarly dopamine receptor-rich areas of the striatum (caudate, putamen) could be explained either by a higher responsiveness of the nucleus accumbens dopamine D1 receptor system to upregulation or by a more marked dopaminergic stimulation in the human caused by methamphetamine in this subdivision of the striatum. The above-normal responsiveness of the human nucleus accumbens to neurochemical changes caused by drugs of abuse is also indicated by our previous finding that levels of the inhibitory G protein, G␣i, are selectively decreased in the nucleus accumbens striatal subdivision of chronic methamphetamine users.19 In this regard, decreasing the inhibitory influence of G␣i might potentiate any overactivity of the dopamine D1

receptor-linked adenylyl cyclase system caused by increased receptor number. As mentioned in the Introduction, it has long been hypothesized, on the basis of experimental animal findings, that supersensitivity of the dopamine D1 receptor system explains some behavioural aspects of the state of drug dependence. However, in this animal literature, there appears to be no consensus yet as to whether this supersensitivity is more likely to reflect drug craving or, conversely, drug tolerance (see Self et al and references therein).49 Thus, clinical trials of pharmacological agents in the treatment of human psychostimulant users are likely to employ both dopamine D1 receptor agonist and antagonist strategies. The clinical relevance of our brain biochemical findings is related to the extent to which increased dopamine D1 receptor concentration of the magnitude observed in our study affects dopamine receptor function and drug seeking behaviour in human methamphetamine users. In conclusion, we found that in a group of human chronic methamphetamine users dopamine D1 receptor levels are moderately increased in a small subdivision of the striatum, the nucleus accumbens, considered to be involved in the mechanism of action of drugs of abuse. These data provide additional support to the hypothesis that overactivity of the dopamine D1 receptor system might underlie some of the features of dependence on psychostimulant drugs.

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Acknowledgements This study was supported by US NIH NIDA No. DA 07182 to SJK.

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