Measurement of the striatal dopamine transporter density and heterogeneity in type 1 alcoholics using human whole hemisphere autoradiography.

Dopaminergic mechanisms are involved in the positive reinforcing and addicting effects of alcohol. Positron emission tomography (PET) and single photon emission tomography (SPET) studies have indicated alterations in striatal dopamine transporters (DAT) and in presynaptic dopamine (DA) function in alcoholics, although also contradictory results have been reported. Normal variations in blood flow, metabolism, and receptor densities are apparently important to brain function. Such variations are known to decrease during pathophysiological processes, such as epilepsy, whereas normal receptor distributions are broadly heterogenous. We evaluated the densities and heterogeneities of striatal DAT in 8 adult-onset, Cloninger type I alcoholics and 10 controls using [125I]N-(3-iodoprop-2E-enyl)-2beta-carbomethoxy-3beta- (4'-methylphenyl)nortropane ([125I]PE2I) as a ligand for human postmortem whole hemisphere autoradiography, which provided high resolution images of the brain when compared with in vivo PET and SPET. The mean density and heterogeneity of DAT were markedly lower in the alcoholics. A significant linear correlation existed between DAT density and heterogeneity, as well as between DAT densities in the nucleus accumbens and in the dorsal striatum (caudate and putamen) in alcoholics, but not consistently in controls. The observed low DAT density and heterogeneity in the dorsal striatum suggest that type 1 alcoholics may have a dysfunctional DA system. These data indicate that human whole hemisphere autoradiography with the analysis of binding heterogeneity may be a relevant tool to measure pathological processes in the brain.

Dopamine transporter in vitro binding and in vivo imaging in the brain.

Much research interest has lately been focused on the dopamine transporter function in brain. Recent findings indicate that dopamine reuptake is more like a highly regulated than a constitutive determinant of dopamine clearance. Positron emission tomography (PET) and single-photon emission tomography (SPET) offer unique methods to study dopamine transporter function. Results from in vivo PET and SPET studies correspond well with in vitro studies performed on post mortem human brain tissue. Considering some of the variances between in vitro and in vivo receptor binding phenomena it may be that the role of a compound to alter binding to monoamine uptake sites in vitro does not indicate its potential to affect monoamine transporters after administration in vivo. This discrepancy may be better understood taking into account recent studies indicating the possibility of a rapid regulation of transporter function and surface expression. Furthermore, the dopamine transporter is a fruitful target for CNS drug discovery. Fundamental nature of drug actions in vivo may be studied using demonstrated in vitro and in vivo imaging methods.

Dopamine D(2)/D(3)-receptor and transporter densities in nucleus accumbens and amygdala of type 1 and 2 alcoholics.

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 D(2)/D(3) receptors and transporters in the nucleus accumbens and amygdala of post-mortem human brains by using [(125)l]epidepride and [(125)I]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 [(125)I]epidepride to DA D(2)/D(3) receptors was 20% lower in the nucleus accumbens and 41% lower in the amygdala, and [(125)I]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.

Characterization of gamma-aminobutyrate type A receptors with atypical coupling between agonist and convulsant binding sites in discrete brain regions.

Gamma-ainobutyric acid type A (GABA(A)) receptor ionophore ligand t-[35S]butylbicyclophosphorothionate ([35S]TBPS) was used in an autoradiographic assay on brain cryostat sections to visualize and characterize atypical GABA-insensitive [35S]TBPS binding previously described in certain recombinant GABA(A) receptors and the cerebellar granule cell layer. Picrotoxinin-sensitive but 1-mM GABA-insensitive [35S]TBPS binding was present in the rat cerebellar granule cell layer, many thalamic nuclei, subiculum and the internal rim of the cerebral cortex, amounting in these regions up to 6% of the basal binding determined in the absence of exogenous GABA. Similar binding properties were detected also in human and chicken brain sections. Like the GABA-sensitive [35S]TBPS binding, GABA-insensitive binding was profoundly decreased by pentobarbital, pregnanolone, loreclezole and Mg2+. The binding was reversible and apparently dependent on Cl- ions. Localization of the GABA-insensitive [35S]TBPS binding was not identical to that of high-affinity [3H]muscimol binding and diazepam-insensitive [3H]Ro 15-4513 binding, two previously established receptor subtype-dependent binding heterogeneities in the rat brain. The present study reveals a component of the GABA-ionophore enriched in the thalamus and cerebellar granule cells, possibly representing poorly desensitized or desensitizing receptors.

Specific binding and laterality of human extrastriatal dopamine D2/D3 receptors in late onset type 1 alcoholic patients.

Late onset type 1 alcoholism has been suggested to be associated with decreased dopaminergic transmission. Our hypothesis was that late onset type 1 alcoholics have also abnormal extrastriatal dopamine D(2)/D(3) receptor distribution. We performed binding, heterogeneity and laterality analysis of extrastriatal and striatal dopamine D(2)/D(3) receptors in nine late onset male alcoholics and in 12 age-matched healthy males. A radioligand, [(123)I]epidepride was used in high resolution single-photon emission tomography (SPET). Specific binding of epidepride in the left temporal pole was significantly (P<0.05) lower in type 1 alcoholics (0.74+/-0.14 ml/ml) than in controls (0.89+/-0.14 ml/ml). In alcoholics, there was no normal left-to-right asymmetry of the temporal cortical heterogeneity of epidepride distribution observed in control males (0.89+/-0.19 vs. 1.10+/-0.19; P<0.05). The results suggest that the specific binding of dopamine D(2)/D(3) receptors in late type 1 alcoholics is decreased and its laterality in the temporal brain is altered from normal.

Dopamine-transporter density in nucleus accumbens of type-1 alcoholics.

We have shown that alcoholic patients have a lower number of dopamine transporters in the nucleus accumbens, which mediates the rewarding effects of addictive drugs. Thus, certain dopaminergic agents may be beneficial in the treatment of alcohol withdrawal and in the long-term treatment of alcoholism with selective use.

Iodine-123 labelled PE2I for dopamine transporter imaging: influence of age in healthy subjects.

The iodine-123 labelled selective ligand N-(3-iodoprop-2E-enyl)-2beta-carbomethoxy-3beta-(4-methylphe nyl)nortr opane ([(123)I]PE2I) has been developed and has been shown to be suitable for single-photon emission tomography imaging of the dopamine transporter. In this study the influence of age on ligand binding was investigated in 16 healthy males with an age range of 23- 75 years. Single-photon emission tomography (SPET) imaging was performed with a triple-headed gamma camera. A simplified reference region model, in which the input function was derived from the non-displaceable cerebellar compartment, was used to calculate the volume of distribution in the striatum. The volume of distribution was shown to decline with age (-0.4%/year; P<0.005). The results were in agreement with in vivo and in vitro findings of a decline in dopamine transporter binding with age. The findings confirm the suitability of [(123)I]PE2I for SPET imaging in clinical routine but emphasize the necessity of using age-matched controls in patient studies.

Effects of repeated low dose administration and withdrawal of haloperidol on sexual behaviour of male rats.

Neuroleptics are known to cause anhedonia and attenuate sexual behaviour at therapeutic doses in humans. These effects are assumed to result from the dopamine antagonism of the drugs. It has been observed that a mixed dopamine D1/D2 antagonist, haloperidol, may cause a reduction in the number of intromissions required to achieve ejaculation. On the other hand, dopamine antagonists are considered unable to modify sexual behaviour once the copulatory sequence is initiated. In this study, male rats received low doses of haloperidol (30 or 60 microg/kg) before the investigation of sexual behaviour in five consecutive days and the mating test was repeated after withdrawal periods of four and five days. Haloperidol dose-dependently reduced intromission frequency, and this effect was maintained for four days after withdrawal. Ejaculation latency was reduced in all groups, including controls. The results indicate that at low doses haloperidol dose-dependently reduces intromission frequency, and the effect of a repeated dosage may persist several days after cessation of medication.

Imaging the structure of the striatum: a fractal approach to SPECT image interpretation.

The spatial pattern of striatal dopamine transporter density in the living human brain was tested by duplicate SPECT scans with [123I]PE2I, [123I]beta-CIT or [123I]beta-CIT-FP and striatal phantom measurements. The resolution-dependent spatial variation was calculated by the fractal analysis of SPECT images. This variation, which depends on the size of the region of interest, was described by the spatial dispersion i.e. the standard deviation of the count densities divided by the mean density. In each sub-region, the observed and methodological dispersions were computed, and the resulting spatial dispersion was calculated. The methodological dispersion is caused by the imaging resolution, flood field non-uniformity, count density, scatter, reconstruction errors and partial volume effects, whereas the spatial dispersion is based on the cerebral heterogeneity of the dopamine transporter density. Recognition of the normal variation in heterogeneity is important in evaluation of the striatal dopamine transporter density between controls and patients suffering from various neuropsychiatric disorders.

Pharmacokinetics and dosimetry of iodine-123 labelled PE2I in humans, a radioligand for dopamine transporter imaging.

The iodine-123 labelled selective ligand N-(3-iodoprop-2E-enyl)-2-beta-carbomethoxy-3beta-(4-methylphenyl) nortropane ([123I]PE2I) was evaluated as a probe for in vivo dopamine transporter imaging in the human brain. Six healthy subjects were imaged with a high-resolution single-photon emission tomography scanner. Striatal radioactivity peaked at 1 h after injection. The background radioactivity was low. The volume of distribution in the striatum was 94+/-24 ml/ml. The results were compared with those of [123I]beta-CIT imaging. There was no significant uptake of [123I]PE2I in serotonin-rich regions such as the midbrain, hypothalamus and anterior gingulus, suggesting that in vivo binding is specific for the dopamine transporter. One main polar metabolite of [123I]PE2I was found in plasma, and the parent plasma concentration decayed rapidly. Radiation exposure to the study subject is 0.022+/-0.004 mSv/MBq (effective dose). The preliminary results suggest that [123I]PE2I is a selective SPET ligand for imaging striatal dopamine transporter density.

Changes in GABA-benzodiazepine receptor complex and in peripheral benzodiazepine receptors in male mice after copulation.

We studied the effect of copulation on GABA and benzodiazepine (BZD) receptors in the male mouse. After copulation, there was an 18% increase in the in vitro number of [3H]muscimol binding sites in frontal cortex. No changes were observed in central BZD binding sites labelled either in vivo by [3H]flunitrazepam or in vitro (in olfactory bulbs and in frontal cortex) by [3H]flumazenil, but further in vitro studies demonstrated that the GABA-stimulated [3H]flunitrazepam binding was reduced in both frontal cortex and olfactory bulbs. Copulation increased the number of peripheral BZD binding sites labelled by 3H-Ro 5-4864 in olfactory bulbs by 22% and in heart by 36%, but not in frontal cortex or in testes. The changes of GABA/BZD and peripheral BZD receptors in mouse suggest that the GABAergic system may be affected by copulation.

3H-atipamezole binding sites in mouse cerebral cortex: possible involvement of alpha 2-adrenoceptors in sexual behavior.

Atipamezole is a new specific alpha 2-adrenoceptor antagonist. In this study, first, the presence of specific 3H-atipamezole binding sites in the sagittal and coronal sections of mouse brain was established using autoradiography. In vitro experiments with mouse cerebral cortex membranes indicated that d-medetomidine, a new alpha 2-adrenoceptor agonist structurally related to atipamezole, displaces labelled atipamezole more potently than noradrenaline. The saturation isotherm with d-medetomidine demonstrated high affinity binding with the apparent number of binding sites KD 1.36 nM and 760 fmol/mg, respectively. In the next series of experiments male mice were sacrificed immediately after copulation and cerebral cortex 3H-atipamezole and 3H-flumazenil binding was studied. Oxymetazoline and prazosin are known to label preferably alpha 2A and alpha 2B subtypes of alpha 2-adrenoceptors. Therefore, parallelly with noradrenaline both these compounds were used to determine non-specific binding of 3H-atipamezole. When noradrenaline or oxymetazoline were used as displacing agents copulation caused a significant increase of 3H-atipamezole binding sites. No significant changes were observed when prazosin was used. 3H-Flumazenil binding remained unchanged by copulation. The up-regulation of 3H-atipamezole binding sites indicates that not only alpha 2-adrenoceptors in the periphery but also in the CNS may participate in the regulation of sexual behavior. Moreover, in regulation of sexual behavior central alpha 2-adrenoceptors may be more important than benzodiazepine receptors.