¹Department of Forensic Psychiatry, Niuvanniemi Hospital, University of Kuopio, FIN-70240 Kuopio, Finland

²Department of Pharmacology and Toxicology, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland

³Department of Clinical Neuroscience, Psychiatry Section, Karolinska Institute and Hospital, S-17176 Stockholm, Sweden

⁴Department of Clinical Physiology and Nuclear Medicine, Kuopio University Hospital, PO Box 1777, FIN-70211 Kuopio, Finland

⁵Department of Forensic Medicine, University of Oulu, Kajaanintie 52 D, FIN-90220, Oulu, Finland

⁶Department of Psychiatry, University of Oulu, Peltolantie 5, FIN-90210, Oulu, Finland

⁷Department of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland

⁸MAP-Medical Technologies Oy, PO Box 9, FIN-00251 Helsinki, Finland

Correspondence to: E Tupala MD, University of Kuopio, Department of Forensic Psychiatry, Niuvanniemi Hospital, FIN-70240 Kuopio, Finland. E-mail: erkki.tupala@niuva.fi

Alcohol acts through mechanisms involving the brain neurotransmitter dopamine (DA) with the nucleus accumbens as the key zone for mediating these effects. We evaluated the densities of DA D₂/D₃ receptors and transporters in the nucleus accumbens and amygdala of post-mortem human brains by using [¹²⁵l]epidepride and [¹²⁵I]PE2I as radioligands in whole hemispheric autoradiography of Cloninger type 1 and 2 alcoholics and healthy controls. When compared with controls, the mean binding of [¹²⁵I]epidepride to DA D₂/D₃ receptors was 20% lower in the nucleus accumbens and 41% lower in the amygdala, and [¹²⁵I]PE2I binding to DA transporters in the nucleus accumbens was 39% lower in type 1 alcoholics. These data indicate that dopaminergic functions in these limbic areas may be impaired among type 1 alcoholics, due to the substantially lower number of receptor sites. Our results suggest that such a reduction may result in the chronic overuse of alcohol as an attempt to stimulate DA function. Molecular Psychiatry (2001) 6, 261-267.

alcohol-induced disorders; ethanol-induced nervous system disorders; dopamine; autoradiography; human brain; receptors; transporters

Introduction

Converging evidence suggests that stimulatory drugs of abuse, including alcohol, act through mechanisms involving the brain neurotransmitter dopamine (DA), and the neural systems which it innervates, with the nucleus accumbens as the key mesolimbic zone for mediating these effects.^1,2,3 Alcohol dose dependently stimulates the release of DA in the nucleus accumbens,⁴ and withdrawal from alcohol has been shown to be associated with reduced levels of dopamine in this same region.⁵ The amygdala is another dopamine-rich brain region which has close connections with the nucleus accumbens and recently has received increasing interest in the context of alcohol and other drug abuse.^6,7,8 Alcoholism characterized by late-onset, social dependency and anxiety (ie, type 1) has been suggested to be associated with an underlying dopaminergic deficit. This is in contrast to the observed serotonergic deficit in early-onset type 2 alcoholism, which is characterized by novelty seeking, impulsive behavior and social hostility.⁹ Preliminary results from positron emission tomographic (PET) and single photon emission tomographic (SPET) studies have indicated alterations in either dopamine transporter (DAT) densities or presynaptic DA function in the striatum of type 1 alcoholics^10,11,12 and in unclassified (type 1 vs type 2) alcoholics,¹³ although contradictory results have also been reported in a study of unclassified alcoholics.¹⁴ Other articles have reported low DA D₂ receptor densities in the striatum of unclassified alcoholics^14,15,16 and endocrinological measurements have shown decreased DA mediated activity among unclassified alcoholics.^{17,18,19,20,21}

The radioligand [¹²⁵I]epidepride ((S)-N-((1-ethyl-2-pyrrolidinyl)methyl)-5-iodo-2,3, dimethoxybenzamine) has a very high affinity for DA D₂ receptors, with very low non-specific binding, and has been widely used to label DA D₂ receptors in the human brain.^22,23 It also binds to DA D₃-receptors, where it has been estimated that approximately 34% of the sites labelled by [¹²⁵I] epidepride in the nucleus accumbens are D₃-like receptors, as opposed to the nucleus caudatus, where only a very small percentage of the DA receptors are D₃-like.²² [¹²⁵I]PE2I ((E)-N-(3-iodoprop-2E-enyl)-2-carbomethoxy-3-(4'-methylphenyl) nortropane) is a novel radioligand ligand having high selectivity and affinity for DAT,^24,25,26 and has been previously used for imaging DAT both in vitro and in vivo.^24,25,26,27 It has 30- and >60-fold selectivities in vitro over serotonin and noradrenalin transporters, respectively,^25,26 which makes it far superior to older cocaine derivatives like -CIT, frequently used in previous in vivo studies. Such radioligands in human post-mortem whole hemispheric autoradiography provide high resolution images from different brain levels, to compare with in vivo studies (ie, PET and SPET) and enable a more detailed study of various structures.^{23,27,28,29,30}

Because of the relatively low spatial resolution of in vivo imaging methods, it has been difficult to differentiate nucleus accumbens from the dorsal striatum, and earlier results have indicated changes in the nigrostriatal system (ie, substantia nigra, nucleus caudatus, putamen, globus pallidus), which has been implied in the control of movement and may not be as important as the mesolimbic system in the context of addiction.^{10,12,13,14,15} Aside from our preliminary results, regarding DAT in the nucleus accumbens of seven type 1 alcoholics and healthy controls,³⁰ there are no other reports on DA D₂/D₃ receptor and DAT densities in the nucleus accumbens or amygdala of human alcoholics. The aim of this study was to compare the DA D₂/D₃ receptor and transporter densities in the nucleus accumbens and amygdala in alcoholics classified as type 1 or 2, according to Cloninger,⁹ and healthy controls. Whole hemispheric autoradiography was used in the present study to evaluate DA binding sites in both the nucleus accumbens and amygdala from the same sections. Only type 1 alcoholics had significantly lower numbers of pre- and post-synaptic DA receptor binding sites in these limbic areas, which supports Cloninger's neurogenetic model of alcoholic subtypes, where type 1 alcoholism is associated with a dopaminergic deficit, and type 2 is not.

Materials and methods

Brain sampling

Human brains used were obtained for clinical necropsy by the Department of Forensic Medicine, University of Oulu, Finland, and this portion of the study was approved by the Ethics Committee of the University of Oulu and the National Institute of Medicolegal Affairs, Helsinki, Finland. The brains were removed, cleaned of the dura, and divided at the midsagittal plane. The left hemisphere was placed with the midsagittal plane on a glass-plate before freezing (-75°C). Medical records on the cause of death, previous diseases and medical treatments were collected. A postmortem analysis for drugs, including alcohol, and the normal necropsy protocol were also performed. Gross neuropathological evaluation for the right hemisphere was performed during necropsy, and a more detailed neuroanatomical evaluation was made on the left hemisphere sections used in the study from an adjacent Nissl stained section. None of the study hemispheres exhibited damage or neuroanatomical abnormalities.

Diagnostics

Diagnoses were made by two physicians, independently of each other. Mental disorders were coded according to DSM-III-R diagnostic criteria and alcoholics were subclassified as type 1 or type 2 according to Cloninger.⁹ The kappa coefficient of diagnostic agreement was 0.9; ie, one type 2 alcoholic included in the study was diagnosed as a type 1 alcoholic by the second diagnostician.³¹ Otherwise diagnoses were unanimous. Alcoholics were selected after alcohol dependence or harmful use was evident, based on autopsy results, medical records and anamnestic data. The most important criteria for defining the two groups of alcoholics were early onset (before the age of 25) of alcohol abuse and documented, severe antisocial behavior among type 2 alcoholics. Controls were selected when autopsy or anamnestic data revealed no indications of harmful substance misuse. Subjects having psychotic disorders or any diseases (such as epilepsy), or using medication that affected the central nervous system (such as neuroleptics or antidepressants), were excluded.

Study subjects

All 27 cases were white Caucasians. The study groups consisted of nine type-1 alcoholics in the [¹²⁵I]epidepride binding study (seven males, two females; mean age 52.7 years; post-mortem delay 11.9 ± 4.5 h; mean ± SD), eight type 1 alcoholics in the [¹²⁵I]PE2I binding study (seven males, one female; mean age 52.9 years; post mortem delay 12.0 ± 4.8 h; mean ± SD), and eight type 2 alcoholics (males; mean age 34.6 years; post mortem delay 14.1 ± 3.4 h; mean ± SD) and 10 controls who were free of psychiatric diagnosis in both the [¹²⁵I]epidepride and [¹²⁵I]PE2I study (eight males, two females; mean age 53.5 years; post-mortem delay 14.8 ± 9.2 h; mean ± SD). From one female type 1 alcoholic only DA D₂/D₃-receptors were measured, due to methodological limitations. Eight of the nine type 1 alcoholics had alcohol in their blood at the time of death, and one alcoholic (included in both ligand binding studies) had an abstinence period of 10 h. One of the controls had a small amount of alcohol in his blood at the time of death (0.036%). Two of the type 1 alcoholics had traces of diazepam in their blood samples. All type 2 alcoholics had alcohol in their blood at the time of death, three had traces of benzodiazepines and one was positive for cannabinoids. All subjects died of sudden causes. Nine controls died because of myocardial infarction and one due to aorta dissecation. The causes of death in the type 1 alcoholic group were pneumonia (2), lethal ethanol intoxication (2), myocardial infarction (2), suicide by hanging (1) and subdural hematoma on the right side (1). The causes of death in the type 2 alcoholic group were suicide by hanging (3), knife wound (2), gunshot wound (1), rupture of heart due to a car accident (1) and cardiac death (1).

Cryosectioning

The object glasses (size 100 ´ 220 ´ 1.0-1.2 mm) were washed in a laboratory dishwasher, dipped in ethanol for 15 min, dried and then dipped briefly in gelatine.²⁹ The glass plates were then dried overnight at room temperature under a cover to protect against dust. For cryosectioning, the glass plate was removed from the frozen hemisphere, which was then put into a specially designed rubber box with a specimen holder, with the midsagittal plane of the hemisphere facing up.²⁹ A semi-liquid gel (approximate concentration 5 g l^-1, 5°C) of carboxymethylcellulose (CMC) was added while carefully checking the horizontal level of the midsagittal surface. The positioning of the hemisphere to obtain parallel sections to the bicommisural line of Talairach was achieved by adjusting the hemisphere parallel to a ruler over the upper margin of the anterior and lower margin of the posterior commissura and the surface of the cryostat specimen holder (the cutting plane). Once the hemisphere-specimen holder-CMC block was totally frozen, the rubber box was removed.

Serial sectioning of the block was performed by a heavy-duty cryomicrotome (LKB 2250, LKB, Stockholm, Sweden), and the temperature of the hemisphere-CMC block was allowed to obtain a cutting temperature (-15°C optimal) before cryosectioning. The brain was sectioned into 100-m horizontal (canto-meatal) sections. A thin tissue paper was put on each section, after which a transparent tape (3M, type 810, St Paul, MN, USA) was fastened to the block by gentle rubbing. Tissue sections cut with the cryotome adhered to the tissue paper, and were transferred to the gelatinized glass plates using a soft paintbrush. The tape and paper were carefully removed, with the sections thawed onto the glass plates. The sections were allowed to air dry before they were stored with dehydrating agents at -25°C until use. The nucleus accumbens was defined as starting at the level where the caput nucleus caudatus and putamen unite, and sectioned through (from dorsal to ventral). The median accumbens sections located 2-4 mm ventrally from the uppermost edge of the amygdala were chosen for the study (see Figure 1 in Ref 27 for a frontal view). Because this region of interest's area was small, we decided to measure the visible structure as a whole without trying to distinguish separate nuclei. The amygdala could not be matched with the median accumbens' levels in one type 1 alcoholic and in one control.

Autoradiography

[¹²⁵I]Epidepride and [¹²⁵I]PE2I were obtained from MAP Medical Technologies, Helsinki, Finland. The radiolabelling was performed using stannylated precursors with [¹²⁵I]Nal, according to standard methods.^23,27 -CIT (2-carbomethoxy-3-(4-iodophenyl)tropane) was obtained from the Department of Chemistry, Kuopio University, Finland. (+)-Butaclamol was obtained from RBI, Natick, MA, USA. The study protocols were based on affinity constants and receptor occupancies from former autoradiographic and ligand binding studies with these same ligands,^23,27 and the study concentrations for both ligands were chosen based on their K_d values. Incubation time to reach equilibrium was chosen to be 1 h according to earlier studies with these same ligands.^22,23,26,27

The first series of sections (eight controls, seven type 1 alcoholics, six type 2 alcoholics) were incubated in a solution (volume 2.5 l) containing approximately 24 pM of [¹²⁵I]epidepride (the incubation concentration) in Tris-HCl buffer, pH 7.4, 0.05 M, containing 0.1% (weight/volume) ascorbic acid, 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, and 1 mM MgCl₂. (+)-Butaclamol (15 M) was used as a specific displacer to determine nonspecific binding. Washing was performed in cold buffer for 3 ´ 10 min, followed by a brief cold wash by dipping the sections into distilled water. The sections were dried for 10 min with a gentle stream of warm air and then for 3 h at room temperature before exposure to radiation-sensitive film (³H-Hyperfilm, Amersham, Bucks, UK) for 5 days before development (developer, Kodak D19; fixation, Kodak Fixer 3000). The autoradiograms were analyzed by computerized densitometry, using a Mikrotek Scanmaker E6 connected to an Osborne PC Pentium II. Software packages included Adobe Photoshop 4.01 (Mountain View, CA, USA) and Scion Image for Windows, version Beta 3 b (Frederick, MD, USA). The resulting pixel values of the binding data were mathematically transformed by an exponential calibration equation into relative radioactivity values by the use of ¹²⁵I-calibrating scales (cat.no.RPA 523L, Amersham), and the background was subtracted. All analyses were made blind to the clinical classification of the subjects. An adjacent section from the respective level was stained with cresyl violet (Nissl staining) to serve as an anatomical correlate to the autoradiography.

Autoradiography with [¹²⁵I]PE2I, using -CIT as a displacer for the first series of sections (seven controls, seven type 1 alcoholics, six type 2 alcoholics), was essentially performed as described above. The incubation concentration of [¹²⁵I]PE2I was approximately 20 pM and the displacing concentration of -CIT was 2 M. The second autoradiographic series (two controls, two type 1 alcoholics and two type 2 alcoholics with [¹²⁵I]epidepride; three controls, one type 1 alcoholic and two type 2 alcoholics with [¹²⁵I]PE2I) was made according to the same protocol, but only in an incubation volume of 750 ml.

Statistical analyses

The univariate analysis of co-variance (ANCOVA) with the experiment series (either first or second) as a covariant was used for comparing the mean values of [¹²⁵I]epidepride and [¹²⁵I]PE2I binding in each group. Because type 2 alcoholics were substantially younger than the two other groups, and a negative correlation between brain dopamine transporter and DA D₂/D₃ receptor binding with age has been well documented,^{14,32,33,34,35,36} age was also included as a covariant in univariate ANCOVA, followed by Bonferroni's test for multiple comparisons. A P-value less than 0.05 was considered significant. The Kappa coefficent of agreement was calculated with regard to the matching of diagnoses between two diagnosticians,³¹ where a value over 0.75 is considered excellent.³⁷

Results

[¹²⁵I]Epidepride bound with high intensity in the nucleus accumbens, indicating high densities of DA D₂/D₃-receptors (Figure 1). Total binding in the amygdala amounted to about 5.8% of that found in the nucleus accumbens. Extremely low [¹²⁵I]epidepride binding was detected in the cerebellum (<0.1% of accumbens), and [¹²⁵I]epidepride did not bind or accumulate in the white matter. Displacement by the D₂/D₃-dopamine receptor antagonist (+)-butaclamol (15 M) completely (>95%) inhibited specific binding in the nucleus accumbens and amygdala (data not shown). [¹²⁵I]PE2I bound with high intensity in the nucleus accumbens, indicating a high DAT density in this structure (Figure 2). No specific binding was detected in the amygdala. [¹²⁵I]PE2I binding in the nucleus accumbens was completely (>95%) displaced by -CIT (2 M), a DAT inhibitor and cocaine derivative (data not shown). [¹²⁵I]PE2I slightly accumulated in the white matter of both cerebrum and cerebellum (Figure 2), possibly due to its relatively high lipophilicity,²⁷ and the addition of -CIT did not affect this accumulation. This non-specific binding accounted for approximately 30% of the specific binding in the nucleus accumbens, and no differences were found between the groups in this regard (data not shown).

The mean binding of [¹²⁵I]epidepride (DA D₂/D₃-receptors) in the nucleus accumbens was 19.7% lower in the type 1 alcoholic group (5.08 ± 1.09 fmol mg^-1; mean ± SD; P = 0.007) when compared with the control group (6.33 ± 0.39 fmol mg^-1) (Figure 3) and 41.0% lower in the amygdala in the type 1 alcoholic group (0.23 ± 0.14 fmol mg^-1; mean ± SD; P = 0.029) when compared with the control group (0.39 ± 0.10 fmol mg^-1) (Figure 4). [¹²⁵I]PE2I-binding (DAT) was reduced by 39.1% in the nucleus accumbens of type 1 alcoholic group (2.12 ± 0.89 fmol mg^-1; P = 0.001) when compared with the control group (3.48 ± 0.74 fmol mg^-1) (Figure 5). In the type 2 alcoholic group the mean binding of [¹²⁵I]epidepride was slightly lower in the nucleus accumbens (5.77 ± 0.66 fmol mg^-1) and in the amygdala (0.31 ± 0.12 fmol mg^-1) as well as [¹²⁵I]PE2I-binding in the nucleus accumbens (3.01 ± 0.49 fmol mg^-1), but none of these results reached statistical significance (Figures 3-5).

Binding to the pre-synaptic (DAT) and post-synaptic (DA D₂/D₃ receptor) sites had a tendency towards positive correlation in the type 1 alcoholics (r = 0.53, P = 0.18) and in the control group (r = 0.50, P = 0.14), but in the type 2 alcoholics the tendency was negative (r = - 0.47, P = 0.24). Results from autoradiography with [¹²⁵I]epidepride binding to DA D₂ receptors on the striatal level will be reported separately.

Discussion

We observed marked reductions in both DAT and DA D₂/D₃ receptor binding potentials in the nucleus accumbens and a reduction in DA D₂/D₃ receptor binding in the amygdala (where no DAT binding was detected) among type 1, but not among type 2 alcoholics. There are several human^{10,12,13,14,15,16} and animal^38,39,40 studies reporting altered dopamine D₂-like receptor and/or transporter densities (B_max) and unaltered affinities (K_d)^{14,15,16, ,38,39,40} among alcoholics or alcohol preferring animals, and no reports have found changes in K_d. Therefore, we chose ligand concentration to be close to K_d, in order to optimize binding dynamics. By increasing the concentration to eg two or three times, the K_d value would have resulted in much higher non-specific binding, which could have disturbed the detection of differences between the groups. Furthermore, considering the aforementioned reports, we still had reasonable basis to assume that possible differences in binding would reflect the difference in binding site density (B_max) and not in the binding affinity (K_d).

Our results are consistent with previous reports from in vivo studies on striatal DAT,^10,12,13 although one PET study¹⁴ did not find a reduction in the DAT of unclassified alcoholics. However, only five alcoholics were evaluated in that study, which might explain the negative result. In previous PET studies,^14,15 variations in striatal DA D₂ receptor densities had no substantial correlation with the period of alcohol abstinence, suggesting some independence of this parameter from withdrawal. Regarding DAT, a previous study¹³ found considerably lower densities in the striatum of alcoholics after admission for detoxification, where DAT densities returned to levels of healthy controls after a 4-week abstinence. In the aforementioned study, about one half of the alcoholics had a history of criminal offences (P Laine, Dept of Psychiatry, University of Oulu, personal communication), which is indicative of type 2 alcoholism. One previous SPET study found normal DAT densities among type 2 alcoholics¹⁰ after a minimum abstinent period of 2 months, while type 1 alcoholics had considerably lower densities, as observed in our study. These results suggest some pathological difference between type 1 and type 2 alcoholics, which is reflected by the stark difference between the receptor binding profiles in the present study as well, where alcoholic subjects were intoxicated at the time of death.

Human genetic studies imply that the DA D₂ receptor gene allele A1 is associated with alcoholism.^41,42 However, there have now been numerous studies specifically addressing the issue of an association between alleles at DRD2 and alcoholism, which have been divided into those supporting the association^41,42 and those maintaining the contrary view.^43,44,45,46 PET and functional studies have indicated some evidence to suggest that decreased DAT and DA D₂ receptor densities in type 1 alcoholics are not merely a consequence of recent alcohol abuse but, at least to some extent, a trait marker for alcoholism.^12,19 Thus, our findings may reflect some genetic or developmental variability that is related to addiction vulnerability. Further studies are needed to clarify the extent of this apparent deficit in DA receptor densities in alcoholics, and if they are more related to variations in neurodevelopment or chronic alcohol exposure, and to what extent these variables apply to DA D₂/D₃ receptors and DAT.

A decrease in DA D₂/D₃ receptors should, in effect, result in an attenuation of DA activity. Endocrinological measurements, in response to DA agonists, have also been performed in unclassified alcoholics as a means of assessing brain tuberoinfundibular DA function. The growth hormone response to the DA agonist apomorphine is decreased in abstinent alcoholics,^17,18 and those alcoholics showing a persistent, attenuated response to apomorphine are more vulnerable to relapse,^20,21 which supports an association between low levels of DA neurotransmission and addiction potential. There is also evidence to suggest that a reduced DA D₂ receptor function in alcohol-dependent men is not only a state marker of residual heavy drinking, but also a more permanent trait marker.^12,19

In conclusion, our results support Cloninger's neurogenetic model of alcoholic subtypes, which suggests that type 1 alcoholism is associated with a dopaminergic deficit, while type 2 is not. Additionally these data suggest that type 1 alcoholics could benefit from drugs that enhance dopaminergic function, perhaps in an analogous way to what was recently demonstrated in an animal study using DA D₃ partial agonists to treat cocaine craving and reduce vulnerability to relapse.⁴⁷ In human type 1 alcoholics such drugs could help to restore sub-optimal levels of dopaminergic activity by reducing both the craving for alcohol in abstinence and the euphoria subsequent to alcohol's release of dopamine in the nucleus accumbens, thus providing a negative reinforcement for relapse. This may not be the case among type 2 alcoholics however, and alcoholic patients must be classified before such treatment strategies are applied.

Acknowledgements

We thank Pirjo Halonen, MSc for her excellent assistance with the statistical analyses, Professor Jouko Vepsäläinen for providing the -CIT used in the displacement studies and Kari Karkola, MD, PhD for providing two brains for the study. Radiolabelling of [¹²⁵I]PE2I was performed under the EUREKA Dopimag-program and was financially supported by TEKES. This study was supported by the Finnish Cultural Foundation of Northern Savo and the Swedish Medical Research council (11640).

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Figure 1 Color coded autoradiograms of human post-mortem brain sections showing the DA D₂/D₃ binding of [¹²⁵I]epidepride in type 1 alcoholic (a), type 2 alcoholic (b) and control (c) brains. An adjacent section from each brain was stained with cresyl violet (Nissl staining) to serve as an anatomical correlate (d). Nacc; nucleus accumbens, Am; amygdala. The number of the brain and the level of the section from the vertex are indicated on the panels.

Figure 2 Color coded autoradiograms of human post-mortem brain sections showing the DAT binding of [¹²⁵I]PE2I. See legend for details.

Figure 3 Scatter plot showing the binding of [¹²⁵I]epidepride to DA D₂/D₃ receptors in the nucleus accumbens of alcoholics and controls (m indicates the mean value).

Figure 4 Scatter plot showing the binding of [¹²⁵I]epidepride to DA D₂/D₃ receptors in the amygdala of alcoholics and controls (m indicates the mean value).

Figure 5 Scatter plot showing the binding of [¹²⁵I]PE2I to DAT in the nucleus accumbens of alcoholics and controls (m indicates the mean value).