Comparative evaluation of [3H]WIN 35428 and [3H]GBR 12935 as markers of dopamine innervation density in brain.

WIN 35428 and GBR 12935, two uptake blocker ligands of the membrane transporter for dopamine (DA), were evaluated as quantitative markers of DA innervation density in CNS tissue. From alternate rat brain slices respectively processed for either light microscope or film autoradiography, counts of DA axon terminals (varicosities) labeled by uptake/storage of [3H]DA were matched with densitometric measurements of the specific binding of [3H]WIN 35428 and [3H]GBR 12935 in the same anatomical areas. The relation between the two parameters was examined in 1) the normal cingulate cortex; 2) the neostriatum severely DA-denervated by unilateral intramesencephalic injections of 6-hydroxydopamine; and 3) the neostriatum, partly DA-reinnervated by an intrastriatal graft of fetal mesencephalic neurons after prior 6-hydroxydopamine lesion. For technical reasons, the hyperdense DA innervation of normal striatum was not amenable to such correlative testing. Data were subjected to multilevel analysis. Specific [3H]WIN binding at 37 degrees C was tightly and linearly correlated with the number of DA varicosities over the full range of DA innervation densities tested. The regression lines for intact cortex and for DA-denervated as well as DA-reinnervated neostriatum had the same slope and crossed the ordinate near zero. In contrast, [3H]GBR 12935 binding at 37 degrees C showed no correlation with the number of DA varicosities. A linear correlation could be obtained after incubation with [3H]GBR 12935 at 4 degrees C in the presence of ZnSO4, but the intercept of this regression line remained significantly above zero at origin, indicating extraneous binding to non-DA transporter sites. Providing that the hyperdense DA innervation of the normal neostriatum does not generate a particular problem in vivo as it does in vitro. WIN 35428, but not GBR 12935, might satisfy the selectivity and sensitivity requirements of a quantitative marker of DA innervation density for eventual use in positron emission tomographic studies.

Evaluation of three transporter ligands as quantitative markers of serotonin innervation density in rat brain.

Direct counting of axon terminals (varicosities) labeled by uptake/storage of a tritiated monoamine provides a means to test radioligands of the corresponding membrane transporter as quantitative markers of regional monoamine innervation density in brain tissue. In autoradiographs from alternate rat brain slices, counts of [3H]5-HT-labeled axon terminals were matched with densitometric measurements of the specific binding of tritiated cyanoimipramine (CYI), citalopram (CITAL), and 6-nitroquipazine (6-NTQ), under conditions of hypo-, normo-, or hyper-5-HT innervation of the neostriatum. A total of 267 pairs of data were subjected to a multilevel analysis (iterative generalized least square procedure). With all three ligands, there was a linear relationship between the density of 5-HT innervation and the density of specific binding and no change in the slope of the regression lines as a function of 5-HT innervation density. Thus, none of these ligands gave any sign of down- or up-regulation of the 5-HT transporter consequent to 5-HT hypo- or hyper-innervation. The regression lines for CYI and CITAL were not significantly different from one another and crossed the ordinate near zero, whereas the regression line for 6-NTQ was less steep and had a higher intercept with the ordinate. In addition, the dispersion of values around the regression line (residuals) was lower with CYI and CITAL than 6-NTQ. It was concluded that both CYI and CITAL may serve as quantitative markers of 5-HT innervation density, at least in vitro, whereas 6-NTQ demonstrates a certain lack of specificity and sensitivity. Further work will be needed to assess the potential of CYI and CITAL for positron emission tomographic studies of living brain. Such empirical testing should also be applicable for screening radioligands of the dopamine or the noradrenaline transporters.

Quantification of the serotonin hyperinnervation in adult rat neostriatum after neonatal 6-hydroxydopamine lesion of nigral dopamine neurons.

Light microscope autoradiography after uptake and storage of tritiated serotonin (5-HT) in brain slices was used to count 5-HT axon terminals (varicosities) in the 5-HT-hyperinnervated neostriatum of adult rats subjected to neonatal 6-hydroxydopamine treatment and age-matched, normal controls. After correction for incomplete autoradiographic exposure and for section thickness, the results were expressed in millions of varicosities per mm3 of tissue. Control values ranged from 4.8 in the rostral to 6.3 in the caudal neostriatum (5.8 at intermediate level), for an average of 5.6. The corresponding values in 5-HT-hyperinnervated tissue ranged from 9.7 to 7.7 (8.8 at intermediate level), for an average of 8.7 and increases of 102%, 52% and 22% above control in the rostral, intermediate and caudal neostriatum, respectively (average increase of 55%). These data confirmed the predilection of the 5-HT hyperinnervation for the rostral neostriatum and demonstrated its presence in the caudal neostriatum also.

Validation of the transporter ligand cyanoimipramine as a marker of serotonin innervation density in brain.

UNLABELLED: Radiolabeled ligands of monoamine transporters have already been used to visualize cerebral monoamine innervation by tissue autoradiography and by PET or SPECT in vivo. METHODS: A sampling technique was developed to allow for both the autoradiographic counting of serotonin (5-HT) axonal varicosities, labeled by uptake and storage of [3H]5-HT, and the measurement of the binding of [3H]cyanoimipramine ([3H]CYI), a specific 5-HT transporter ligand, in adjacent slices of adult rat neostriatum. The experiments were conducted in normal, decreased (after 5,7-dihydroxytryptamine lesions in adults) or increased (after 6-hydroxydopamine lesions in neonates) states of neostriatal 5-HT innervation. RESULTS: In normal tissue, the regional density of [3H]CYI binding faithfully reproduced rostrocaudal variations in the number of [3H]5-HT-labeled axonal varicosities. Pairs of values from all three experimental groups showed a highly significant linear correlation (r = 0.93) between the density of [3H]CYI binding and the number of 5-HT varicosities per cubic millimeter of tissue. The intercept of the regression line was close to zero; this confirmed the selectivity of the ligand. CONCLUSION: Under drug-free conditions, specific [3H]CYI binding is a good quantitative index of 5-HT innervation density in brain tissue and is not significantly up- or downregulated on 5-HT denervation or hyperinnervation. When it is adequately labeled, such a ligand might therefore be appropriate to quantify regional 5-HT innervation in vivo by PET or SPECT. The present approach should also be useful to select ligands to quantify 5-HT and monoamine systems.

Regulation of serotonin type 2 (5-HT2) and beta-adrenergic receptors in rat cerebral cortex following novel and classical antidepressant treatment.

Most antidepressant therapies require an initial period of sustained use before therapeutic effects are observed; the literature suggests that this may be due to alterations in either serotonergic or noradrenergic systems. To further explore the mechanism of action of antidepressants, the effect of 21-day drug treatment to rats on serotonin type 2 and beta-adrenergic receptors in fronto-parietal hemicortices was evaluated. Because the same brain was used to measure concomitant changes in both receptors, confounding effects due to inter-animal variability are reduced. The effect of a classical antidepressant drug, desmethylimipramine, was compared with two compounds which have more recently been used to treat depression, ie., adinazolam, a tirazolobenzodiazepine, and buspirone, a serotonin type 1A partial agonist, both of which possesses combined anxiolytic and antidepressant effects. The anxiolytic drug diazepam, a benzodiazepine devoid of antidepressant properties, was used as an active control treatment. Membrane binding studies showed that the maximal binding of [125I]cyanopindolol to beta-adrenoceptors was significantly decreased only by desmethylimipramine treatment. On the other hand, adinazolam and buspirone, as well as desmethylimipramine, decreased the maximal binding of [125I]7-amino-8-iodo-ketanserin binding to serotonin type 2 receptors. Diazepam was without effect on either receptor. These results suggest that down-regulation of serotonin type 2 receptors may be a common element in the mechanism of action of antidepressant therapies.

Decreased density of peripheral benzodiazepine binding sites on platelets of currently drinking, but not abstinent alcoholics.

Using the isoquinoline carboxamide derivative, PK 11195 as selective ligand, the binding properties of peripheral benzodiazepine binding sites were compared on platelets of alcoholics and non-alcoholic, healthy controls. When compared to controls, alcoholics during prolonged ethanol consumption show a significant reduction in the density of platelet [3H]PK 11195 binding sites. However, following abstention from alcohol, the density of these sites is similar to controls. These data suggest that a reduction in the density of peripheral benzodiazepine binding sites on platelets may be a biochemical index of prolonged ethanol use and indicate a possible role for these sites in mediating the chronic effects of alcohol.

Coexistence of central and peripheral benzodiazepine binding sites in the human pineal gland.

The pineal gland and particularly its major hormone, melatonin, may participate in several physiological functions, including sleep promotion, anticonvulsant activity and the modulation of biological rhythms and affective disorders. These effects may be related to an interaction with benzodiazepine receptors, which have been demonstrated to be present in the pineal gland of several species including man. The present study examined the characteristics of benzodiazepine binding site subtypes in the human pineal gland, using [3H]flunitrazepam and [3H]PK 11195 as specific ligands for central and peripheral type benzodiazepine binding sites respectively. Scatchard analysis of [3H]flunitrazepam binding to pineal membrane preparations was linear, indicating the presence of a single population of sites. Clonazepam and RO 15-1788, which have a high affinity for central benzodiazepine binding sites, were potent competitors for [3H]flunitrazepam binding in the human pineal, whereas RO 5-4864 had a low affinity for these sites. Analyses of [3H]PK 11195 binding to pineal membranes also revealed the presence of a single population of sites. RO 5-4864, a specific ligand for peripheral benzodiazepine binding sites was the most potent of the drugs tested in displacing [3H]PK 11195, whereas clonazepam and RO 15-1788 were weak inhibitors of [3H]PK 11195 binding to pineal membranes. Overall, these results demonstrate, for the first time, the coexistence of peripheral and central benzodiazepine binding sites in the human pineal gland.

Muscarinic, benzodiazepine, GABA, chloride channel and other binding sites in frontal cortex in hepatic coma in man.

Alterations in several neurotransmitter systems in brain have been implicated in the pathophysiology of hepatic coma (HC). Studies on human autopsy material are few. We investigated 3H-quinuclidinylbenzilate (QNB), 3H-spiperone, 3H-imipramine, 3H-PN-200-110, 3naloxone, 3H-flunitrazepam, 3H-muscimol, 35S-t-butylbicyclophosphothionate and 3H-cyclohexyladenosine binding sites in frontal cortex from seven patients with HC and five controls. The density of 3H-QNB binding sites was significantly decreased and the affinity slightly increased in HC. The functional significance of these selective changes in muscarinic receptor binding sites is unclear. Further studies evaluating cholinergic function in HC are indicated. Acute studies in animals point to an increase in GABA and BZ binding sites in HC. The present results show that the BZ/GABA-receptor-chloride-ionophore complex is unchanged in HC in man. Serotonergic (5HT-2), adenosine (A-1), imipramine (5HT uptake sites), opiate (naloxone) and calcium channel antagonist binding sites are unchanged in HC.

Platelet 3H-imipramine binding distinguishes depression from Alzheimer dementia.

Platelet 3H-imipramine binding and serotonin uptake were studied simultaneously in normal subjects and in depressed, parkinsonian and Alzheimer's disease patients to investigate the usefulness of these variables in the diagnosis of depression in the elderly. Whereas Vmax of platelet serotonin uptake was significantly reduced in all patient groups compared to age matched normal subjects, the density of 3H-imipramine binding was reduced in depressed patients only. The lower Bmax values in depressed patients was independent of patient age. These data suggest that platelet 3H-imipramine binding may be a useful laboratory index which discriminates depression from dementia in the elderly.

Comparative potencies of calcium channel antagonists and antischizophrenic drugs on central and peripheral calcium channel binding sites.

Dihydropyridines are potent agents on [3H]nitrendipine binding sites in heart and brain membranes. Like the phenylalkylamines, they are slightly more active on heart than on brain [3H]nitrendipine binding sites. On the other hand, the diphenylalkylamines, the diphenylpiperazines and the antischizophrenic drugs of the diphenylbutylpiperdine type are more potent on brain [3H]nitrendipine binding sites. The findings suggest tissue heterogeneity of [3H]nitrendipine binding sites and the possible development of diphenylbutylpiperidine-diphenylbutylpiperazine analogues that could selectively act on brain calcium channel antagonist binding sites.

Imipramine treatment differentially affects platelet 3H-imipramine binding and serotonin uptake in depressed patients.

Uptake of serotonin and 3H-imipramine binding in platelets of depressed patients were investigated simultaneously with changes in clinical state. Both Vmax for serotonin uptake and Bmax for 3H-imipramine binding were significantly lower in unmedicated depressed patients with respect to normal subjects. Successful treatment with imipramine led to a significant increase in Bmax for 3H-imipramine binding, without significant change in Vmax for serotonin uptake. Bmax values increased to the normal range following complete, rather than partial clinical improvement. These data indicate that successful antidepressant treatment may increase the density of 3H-imipramine binding sites on platelets by a process which is independent of the uptake of serotonin.

Unique [3H]tryptamine binding sites in rat brain: distribution and pharmacology.

[3H]Tryptamine binding in rat brain is widely distributed with highest densities in the cortex, striatum and hippocampus. This binding is stereospecific and is potently displaced by tryptamine analogues and beta-carbolines. Phenethylamines also possess a weaker activity in displacing [3H]tryptamine. The strict structural requirements for binding to this site substantiate its unique character and suggest that it may represent a tryptamine receptor in the CNS.

Lack of association between [3H]imipramine binding sites and uptake of serotonin in control, depressed and schizophrenic patients.

Uptake of serotonin and [3H]imipramine binding were studied in parallel in the platelets of control, depressed and schizophrenic patients. In the depressed patient group, uptake of serotonin was consistently reduced while [3H]imipramine binding was only decreased in a number of these patients. In the schizophrenic group, uptake of serotonin was reduced to 62% of control with no changes in [3H]imipramine binding being observed. These data demonstrate a clear dissociation between uptake of serotonin and [3H]imipramine binding sites in human platelets. The possible functional role of these [3H]imipramine binding sites remains to be determined.