Development of a micromethod to study the Na-independentl-[H]glutamic acid binding to rat striatal membranes. II. Effects of selective striatal lesions and deafferentations

An apparent single class of Na⁺-independentl-[³H]glutamate (l-[³H]Glu) binding sites was biochemically and pharmacologically identified on rat striatal tissue. The K_d value was 1.75 μM and the B_max3.89 nmol/g protein. In order to further elucidate the putative physiological role of these binding sites and to valid our binding assay, experiments were conducted to determine the anatomical location of thel-[³H]Glu binding sites in the striatum. Local injection of the neurotoxin, kainic acid into the striatum caused degeneration of target cells in the structure followed by an important decrease (-37%) in the number of these binding sites, with no significant change in the affinity constant. Lesions of the cortical frontal and parietal areas projecting to the striatum via putative glutamatergic fibers led, on the other hand, after 3 weeks to one month, to a significant increase (+23%) in the number ofl-[³H]Glu binding sites. The K_d value does not significantly change after decortication. Finally, suppression either of the nigrostriatal dopaminergic input or of the partially cholinergic thalamostriatal tract do not affect [³H]Glu binding to striatal tissue. These results suggest that about 40% of our [³H]Glu binding sites are located on striatal target cell sensitive to the neurotoxic effect of kainic acid. Therefore, they seem to be partly postsynaptic. The existence of a relation between these binding sites and the corticostriatal glutamatergic input was shown by the development of a supersensitivity response after suppression of this cortical input. These sites might therefore constitute one of the receptor subclass for Glu linked to the excitatory action of the corticostriatal afferent. Since degeneration of either the nigrostriatal dopaminergic or the thalamostriatal fibres did not affect the binding ofl-[³H]Glu characterized in our study, the binding sites will probably not occur at the presynaptic level on these nerve terminals in the striatum.     

Biochemical and functional alterations of central GABA receptors during chronic estradiol treatment

The characteristics of GABA and benzodiazepine receptors were examined in the hippocampus, striatum and cerebral cortex of female rats at various times (up to 9 months) after the subcutaneous implantation of an estradiol pellet (10 mg). A significant decrease in the B_max of the high-affinity binding of [³H]muscimol to membranes from these 3 regions was detected as soon as one week after the implantation. Although the characteristics of the high-affinity binding of [³H]flunitrazepam remained unaffected during the whole treatment, the stimulatory effect of GABA (and muscimol) on this binding was significantly reduced by estrogenization. The changes in GABA receptor binding appeared functionally relevant since the elevation of striatal acetylcholine levels normally induced by the peripheral administration of muscimol (5 mg/kg) was significantly lower in estradiol-treated than in control female rats.In contrast to that observed in intact female rats, the implantation of estradiol in hypophysectomized animals did not affect the characteristics of [³H]muscimol binding to hippocampal, striatal and cortical membranes. [³H]muscimol binding was also unchanged in female rats implanted with estradiol and treated chronically with bromocriptine for 3 weeks. Since both hypophysectomy and the chronic administration of bromocriptine suppressed the hyperprolactinemia normally induced by estrogenization, the down-regulation of central GABA receptors very likely involved prolactin in intact animals implanted with 17-β-estradiol.     

Localization of [H]N-propylnorapomorphine binding in mouse striatum

The anatomic localization of specific striatal [³H]N-propylnorapomorphine ([³H]PNA) binding was determined in male C57BL/6J mice. Striatal [³H]PNA binding was of high affinity and sensitive to guanine nucleotides. Frontal cortical ablation did not alter striatal [³H]PNA binding, but reduced [³H]spiperone binding by 36%. Kainic acid reduced and 6-hydroxydopamine elevated [³H]PNA binding. A combined frontal cortical ablation and striatal kainic acid lesion was similar to that of kainate alone. These data are consistent with a localization of [³H]PNA binding sites on neurons intrinsic to the mouse striatum.     

Decrease in δ-opioid receptor density in rat brain after chronic [d-Ala,d-Leu]enkephalin treatment

Chronic treatment of Sprague-Dawley rats with [d-Ala²,d-Leu⁵]enkephalin (DADLE) resulted in the development of tolerance to the antinociceptive effect of this opioid peptide. When opioid receptor binding was measured, time-dependent decreases in [³H]diprenorphine binding to the P₂ membranes prepared from the cortex, midbrain and striatum were observed. Scatchard analysis of the saturation binding data revealed a decrease in B_max values and no change in the K_d values of [³H]diprenorphine binding to these brain regions, indicative of down-regulation of the receptor. This reduction in the opioid receptor binding activities could be demonstrated to be due to the DADLE effect on the δ-opioid receptors in these brain regions. When [³H]DADLE binding was carried out in the presence of morphiceptin, a significant reduction in the δ-opioid receptor binding was observed in all brain areas tested. μ-Opioid receptor binding decrease was observed only in the striatum after 5 days of DADLE treatment. Additionally, the onset of δ-opioid receptor decrease in the midbrain area was rapid, within 6 h of the initiation of the chronic DADLE treatment. Thus, analogous to previous observations in which chronic etorphine treatment preferentially reduced μ-opioid receptor binding, chronic DADLE treatment preferentially reduced δ-opioid receptor binding activity.     

Frontal decortication decreases the affinity ofN-(1-[2-thienyl]cyclohexyl)[H]piperidine binding to rat striatum Frontal decortication; Striatum

Bilateral ablation or transection of the corticostriatal pathways of the rat causede a reduction inN-(1-[2-thienyl]cyclohexyl)-[³H]piperidine ([³H]TCP) binding to the striatum at 5–7, but not 3 and 14–28, days postlesion with a persistent decrease in striatal glutamate content 3–28 days after the decortication. This reduction was found to be due to an increase inK_d without changes inB_max of the [³H]TCP binding. The present binding suggest that interruption of striatal glutamatergic transmission following frontal decortication may produce a temporal reduction in the affinity of phencyclidine receptor in th striatum.     

Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors

This review summarizes studies designed to label and characterize mammalian synaptic receptors for glutamate, aspartate and related acidic amino acids using in vitro ligand binding techniques. The binding properties of the 3 major ligands employed — l-[³H]glutamate, l-[³H] aspartate and [³H]kainate —are described in terms of their kinetics, the influence of ions, pharmacology, molecular nature, localization and physiological/pharmacological function. In addition, the binding characteristics are described of some new radioligands — [³H]AMPA, l-[³H]cysteine sulphinate, l-[³⁵S]cysteate, d-[³H]as-partate, d,l-[³H]APB, d-[³H]APV and d,l-[³H]APH. Special emphasis is placed on recent findings which allow a unification of the existing binding data, and detailed comparisons are made between binding site characteristics and the known properties of the physiological/pharmacological receptors for acidic amino acids. Through these considerations, a binding site classification is suggested which differentiates 5 different sites. Four of the binding site subtypes are proposed to correspond to the individual receptor classes identified in electrophysiological experiments; thus, A1 = NMDA receptors; A2 = quis-qualate receptors; A3 = kainate receptors; A4 = l-APB receptors; the fifth site is proposed to be the recognition site for a Na⁺-dependent acidic amino acid membrane transport process. An evaluation of investigations designed to elucidate regulatory mechanisms at acidic amino acid binding sites is made; hypotheses such as the Ca²⁺-activated protease hypothesis of long-term potentiation are assessed in terms of the new binding site/receptor classification scheme, and experiments are suggested which will clarify and expand this exciting area in the future.     

Development of a micromethod to study the Na-independentl-[H]glutamic acid binding to rat striatal membranes. I. Biochemical and pharmacological characterization

A micromethod was developed to measure the Na⁺-independentl-[³H]glutamic acid (Glu) binding to rat striatal membranes by using slightly purified membranes from very small tissue amounts, ranging from 0.2 to 0.5 mg wet tissue. The specific binding reached equilibrium in about 30 min incubation at 37 °C and was shown to be partly reversible. Scatchard's analysis of saturation data suggests the presence on striatal membranes of an apparent single homogeneous population of Na⁺-independent binding sites with K_d value 1.75 μM and B_max 3.89 nmol/g protein. Hill's plot of these data was linear, with slope not significantly different from unity, indicating the absence of cooperative interactions. Cl⁻ and Ca²⁺ ions were shown to severely influence thel-[³H]Glu binding to striatal tissue. Maximal activating effects were obtained in the presence of both ions, although Cl⁻ alone was shown to have a powerful stimulating action on the binding. Pharmacological studies suggested, however, the presence of at least two subpopulations of binding sites which bound quisqualic acid as well as ibotenic acid with differential affinities.l-aspartic acid andl-serine-O-sulfate were shown to be potent inhibitors of thel-[³H]Glu binding whendl-2-amino-4-phosphonobutyric acid (dl-APB) and glutamic acid diethylester (GDEE) competed with the binding but only at high concentrations. N-methyl-d-aspartic acid (NMDA),dl-2-amino-5-phosphonovaleric acid (dl-APV),d-α-aminoadipate (d-αAA) and kainic acid were shown not to significantly influence the binding of Glu to striatal membranes     

Differential labeling of dopamine and sigma sites by [H]nemonapride and [H]raclopride in postmortem human brains

The difference between the binding of [³H]nemonapride and [³H]raclopride has been used to quantify dopamine D₄ receptors in postmortem schizophrenic brain studies. Recent work, however, has suggested that at least part of the differential between [³H]nemonapride and [³H]raclopride binding may represent σ rather than D₄ receptor sites. We applied the nemonapride-raclopride subtraction method to postmortem, non-schizophrenic human striatum to examine the variation in dopaminergic receptor binding labeled by these ligands. Variation in σ receptor binding labeled by [³H]nemonapride was studied in frontal cortex, striatum and cerebellum. Specific binding was defined by sulpiride (dopamine receptor ligand), PPAP (σ receptor ligand) and haloperidol (mixed dopaminergic/σ agent), respectively. Haloperidol defined a combination of sites, which were approximately the sum of the dopaminergic and σ components defined by sulpiride and PPAP, respectively. Significant inter-individual variation in the amount of specific binding for dopaminergic and σ receptor sites was observed. However, no significant nor consistent observation of striatal dopamine D₄ receptors or D₄-like binding sites was observed in the striatum even though two independent sets of tissues, with different dissections were used. The inconsistencies in some previous postmortem studies appear to be at least partially explained by the inclusion of both σ and dopaminergic components in [³H]nemonapride binding and the inherent high inter-individual variability of the different components.     

l-[H]Glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

The anatomical distribution ofl-[³H]glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding ofl-[³H]glutamate is accounted for the presence of 3 distinct binding sites when measured in the absence of Ca²⁺, Cl⁻ and Na⁺ ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labeled byd-[³H]2-amino-5-phosphonopentanoate (d-[³H]AP5), [³H]kainate ([³H]KA) and [³H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([³H]AMPA) which are thought to be selective ligands for the N-methyl-d-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively.     

Neurotransmitter receptor localizations: Brain lesion induced alterations in benzodiazepine, GABA, β-adrenergic and histamine H1-receptor binding

Selective neuronal lesions have been utilized in efforts to localize binding sites in rat brain for β-adrenergic, γ-aminobutyric acid (GABA), histamine H₁ and benzodiazepine receptors. The various receptors respond differentially to lesions both in extent of change and in time course. After kainate lesions in the corpus striatum, benzodiazepine receptors are depleted up to 45% at 45–78 days but are unaffected after 7 days. By contrast striatal GABA receptors are increased at 7 days but depleted at later times. Thus both striatal benzodiazepine and GABA receptors appear to be associated at least in part with intrinsic neurons.In the cerebellum both benzodiazepine and GABA receptors are reduced in kainate treated rats and in Nervous mice, mutants which lack Purkinje cells. The most pronounced dissimilarity between benzodiazepine and GABA receptors occurs in Weaver mice, which selectively lack granule cells and display a 60% reduction in GABA receptors but a 40% augmentation in benzodiazepine receptors. A major portion of cerebellar GABA receptors, therefore, appear to be localized to granule cells.Striatal β-adrenergic receptors are reduced following intrastriatal kainate injections but are unaffected by cerebral cortex ablation, suggesting an association with intrinsic neurons but not with axon terminals of the corticostriate pathway. While intraventricular injections of 6-hydroxydopamine enhance [³H]dihydroalprenolol binding to β-adrenergic receptors in the cerebral cortex and hippocampus, such binding is not augmented in the corpus striatum, brain stem, midbrain or thalamus-hypothalamus by this treatment. Moreover, medial forebrain bundle lesions, which destroy ascending adrenergic neurons, fail to alter cerebral cortical or striatal β-adrenergic receptors. Thus denervation-elicited increase in β-adrenergic receptors vary with brain region and the type of denervating lesion.Histamine H₁-receptors are the most resistant of all to neuronal lesions. In the corpus striatum [³H]mepyramine binding is unaffected by cerebral cortex ablation, nigral injections of 6-hydroxydopamine or brain stem hemisection. In the hippocampus, medial forebrain bundle lesions, intrahippocampal kainate injection, and fimbria and fornix transection largely fail to alter [³H]mepyramine binding. Accordingly, a major portion of these receptors may be associated with nonneuronal elements such as glia or blood vessels.     

Quantitative localization of [H]TCP binding in rat brain by light microscopy autoradiography

The anatomical localization of phencyclidine (PCP)/σ-opiate receptors in rat brain was determined by quantitative light microscopy autoradiography using the new ligand N-(1-[2-thienyl]cyclohexyl)[³H]TCP). TCP is a potent analog of PCP which possesses a higher affinity for PCP/σ-opiate receptor than those PCP itself. The higest of [³H]TCP binding was detected in the hippocampus. Intermediate levels were found in frontal cortex, striatum, amygdala and cerebellum. Specific [³H]TCP binding was undetectable in anterior commissure and corpus callosum. The distribution pattern of [³H]TCP binding sites is similar to the pattern obtained with [³H]PCP but more sharply defined. On the basis of its greater potency and specificity, [³H]TCP may prove superior to [³H]PCP as a molecular probe for the study of brain sigma opiate/phencyclidine receptors.     

Pharmacological and anatomical evidence of selective μ, δ, and χ opioid receptor binding in rat brain

While the distribution of opioid receptors can be differentiated in the rat central nervous system, their precise localization has remained controversial, due, in part, to the previous lack of selective ligands and insensitive assaying conditions. The present study analyzed this issue further by examining the receptor selectivity of [³H]DAGO (Tyr-d-Ala-Gly-MePhe-Gly-ol), [³H]DPDPE (2-d-penicillamine-5-d-penicillamine-enkephalin), [³H]DSLET (Tyr-d-Ser-Gly-Phe-Leu-Thr) and [³H](−)bremazocine, and their suitability in autoradiographically labelling selective subpopulations of opoiod receprtors in rat brain. The results from saturation, competitions, and autoradiographic experiments indicated that the three opioid receptor subtypes can be differentiated in the rat brain and that [³H]-DAGO and [³H]DPDPE selectively labelled μ and δ binding sites, respectively. In contrast, [³H]DSLET was found to be relatively non-selective, and labelled both μ and δ sites. [³H]Bremazocine was similarly non-selective in the absence of μ and δ ligands and labelled all three opioid receptor subtypes. However, in the presence of 100 nM DAGO and DPDPE, concentrations sufficient to saturate the μ and δ sites, [³H]bremazocine did label χ sites selectively. The affinity [³H]bremazocine binding sites showed a unique distribution with relatively dense χ labelling in the hypothalamus and median eminence, areas with extremely low μ and δ binding. These results point to the selectivity, under appropriate conditions, of [³H]DAGO, [³H]DPDPE and [³H]bremazocine and provide evidence for the differential distribution of μ, δ, and χ opioid receptors in rat brain.     

Loss of cortical GABA uptake sites in Alzheimer's disease

Summary [³H]Nipecotic acid bound to membranes of human brain in a saturable, reversible manner which was totally dependent on the presence of sodium ions. The potencies of compounds in inhibiting the specific binding of [³H] nipecotic acid were closely correlated with their potencies in inhibiting the neuronal uptake of [³H]GABA. Compounds selective for GABA receptors were inactive. [³H]Nipecotic acid appears to label neuronal high affinity GABA uptake sites. The binding of [³H]nipecotic acid was substantially reduced in the temporal cortex of brains from subjects with Alzheimer-type dementia, but not in other brain regions. It is concluded that some loss of GABA terminals occurs in this disease.     

Characterization of [3H]nemonapride binding to mouse brain dopamine D2 receptors assessedin vivo andex vivo for metabolic modeling in PET studies

Summary We characterized [³H]nemonapride ([³H]NEM, [³H]YM-09151-2) binding to dopamine D₂ receptors. In mice given [³H]NEM with and without sulpiride, thein vivo specific binding of the [³H]NEM to the D₂ receptors in the striatum was assessed: SB_{in vivo-1}, striatal uptake minus cerebellar uptake; and SB_{in vivo-2}, uptake in the control mice minus uptake in the sulpiride-treated mice. Tissue homogenates were divided into cytosol and the membrane binding fraction (MB). When the MB was incubatedin vitro with sulpiride, the dissociated and nondissociated fractions were defined as theex vivospecific binding (SB_{ex vivo}) and theex vivo nonspecific binding (NB_{ex vovo}), respectively. HPLC revealed that most of the radioactivity in the MB was [³H]NEM, whereas metabolites were found in the cytosol. In the striatum, the SB_{ex vivo} increased with time (50% of the total tissue uptake at 60 min), and was equivalent to the SB_{in vivo-2}. The SB_{in vivo-1} was comparable to the MB. In the cerebral cortex and cerebellum, the SB_{ex vivo} decreased with time and the SB_{ex vivo}/free [³H]NEM ratios were smaller than those in the striatum.     

Evidence for loss of brain [H]spiroperidol and [H]ADTN binding sites in rabbit brain with aging

[³H]Spiroperidol and [³H]2-amino-6,7-dihydroxyl-1,2,3,4,-tetrahydronaphthalene hydrochloride (ADTN) binding were measured in various central nervous system regions of 5 month and 5.5 year old rabbits. In striatum, young animals had a 38% higher number of [³H]spiroperidol binding sites and a 140% higher number of [³H]ADTN binding sites than did the older animals. In frontal cortex and anterior limbic cortex there were respectively 42% and 26% more [³H]spiroperidol binding sites in the young animals. There was no change in the binding site number or affinity for [³H]spiroperidol in retina with aging. Pharmacological characterization demonstrated that [³H]spiroperidol binds to a dopamine receptor in striatum and to a serotonin receptor in cortex.     

Regional differences in the enhancement by GABA of [H]zolpidem binding to ω1 sites in rat brain membranes and sections

The potential heterogeneity in the allosteric coupling between GABA and ω₁ binding sites within the native GABA_A receptor complex has been evaluated in the rat by measuring the enhancement by GABA of [³H]zolpidem binding to ω₁ site in membranes from three rat brain structures (neocortex, cerebellum and hippocampus) and brain sections. The maximal stimulatory effect of GABA was significantly higher (265 ± 47%) in cortical membranes than in cerebellar (165 ± 48%) or in hippocampal (118 ± 17%) membranes. These differences are not due either to the presence of different amounts of residual GABA in the membrane preparations or to the labeling, in presence of GABA, of binding sites other than ω₁ since: (1) the pharmacological properties of the [³H]zolpidem binding sites were similar in the three regions; (2) the degree of allosteric enhancement was unrelated to the relative proportion of ω₁ sites in each structure; and (3) GABA did not increase the B_max for [³H]zolpidem. Regional differences in the effect of 100 μM GABA on [³H]zolpidem binding were also observed by quantitative autoradiography. Regions where the strongest (3–4-fold) effects of GABA in [³H]zolpidem binding were observed included the substantia nigra, lateral geniculate body, olfactory tubercule and red nucleus. A large increase in [³H]zolpidem binding was also demonstrated in the cingulate and frontoparietal cortices with higher effects in deep (4.2-fold) rather than in superficial layers (3.3-fold). Heterogeneous subregional increases in [³H]zolpidem binding in the presence of GABA were quantified within the cerebellum, hippocampus and superior colliculus. In the cerebellum the effect of this neurotransmitter was larger in the molecular (3.8-fold) than in the granular (2.2-fold) layer. In the hippocampus the effect of GABA was also heterogeneous with larger increases in CA1 and CA2 fields (3.5-fold) than in CA3 field (2.2-fold) and dentate gyrus (2.5-fold). Finally in the deep layers of the superior colliculus GABA stimulation of [³H]zolpidem binding was greater than the superficial layer. In the other structures examined the GABA-induced increase in [³H]zolpidem binding was less than 3-fold. The smallest stimulations were quantified in the entorhinal cortex (2.1-fold), amygdala (2.4-fold) and nucleus accumbens (1.7-fold). These results suggest that [³H]zolpidem sites are associated to, at least, two GABA_A receptor subtypes that can be differentiated by their allosteric interaction between GABA and [³H]zolpidem sites.     

Regional distribution and ionic requirement of Cl/Ca-activated and Cl/Ca-independent glutamate receptors in rat brain

Recent studies have shown that Cl⁻ and Ca²⁺ ions increase [³H]glutamate binding to rat forebrain synaptic plasma membranes by expressing a new class of glutamate receptors. We examined the regional distribution of these two classes of glutamate binding sites and further characterized their ionic requirements. Significant differences in both Cl⁻/Ca²⁺-independent (basal) and Cl⁻/Ca²⁺-activated receptors, as well as the ratios of these two receptor classes were observed among different areas of the CNS. Cl⁻ and Ca²⁺ appeared to act synergistically, with Cl⁻ ion an absolute requirement for Ca²⁺ stimulation, in expressing these additional binding sites. Ca²⁺ alone did not affect glutamate binding.     

dl-2-[3,4-H]Amino-4-phosphonobutyrate binding sites in the rat hippocampus: distribution and possible physiological role

Binding sites for the nove, glutamate-like radioliganddl-2-[3,4-³H]amino-4-phosphonobutyrate (dl-[³H]APB) on rat hippocampal synaptic membranes were identified and characterised. The existence of a single, saturable population of binding sites was demonstrated. These appeared to be indistinguishable, in terms of their pharmacological profile and ionic dependence, from those described previously in the striatum and whole brain. The distribution of these sites was also examined using a number of discrete neuronal lesions. A majority of sites (approx. 55%) were located on dentate gyrus granule cells. Smaller populations appeared to be situated on perforant path terminals and on pyramidal cells. However,l-APB was found to be ineffective as an inhibitor of basal and potassium evokedd-[³H]aspartate release from hippocampal slices. A presynaptic location can therefore presumably be ruled out. The likely postsynaptic location ofdl-[³H]APB-binding sites in the hippocampus suggests that this site may be involved in synaptic neurotransmission. This possibility is discussed with regard to electrophysiological data concerning the synaptic pharmacology of neuronal connections within the hippocampus.     

Autoradiographic distribution of α2 adrenoceptors, NAIBS, and 5-HT1A receptors in human brain using [H]idazoxan and [H]rauwolscine

The regional distribution of [³H]idazoxan and [³H]rauwolscine was studied autoradiographically in human brain. [³H]Idazoxan binds with high affinity to α₂ adrenoceptors as well as to non-adrenergic sites (NAIBS). [³H]Rauwolscine, besides binding to α₂ adrenoceptors, also binds to 5-HT_1A receptors. Both radioligands labelled the same population of α₂ adrenoceptors, defined as the epinephrine-displaceable binding component. The highest densities of α₂ adrenoceptors occur in the leptomeninges, cerebral cortex and claustrum; lower densities were visualised in the basal ganglia, thalamus, pons, substantia nigra, cerebellum and medulla oblongata; no α₂ adrenoceptors were detected in amygdala and nucleus ruber. NAIBS were present in all the examined brain areas, with the highest densities found in the basal ganglia and substantia nigra. The finding that certain brain regions, such as the amygdala, contained NAIBS but no detectable α₂ adrenoceptors, suggests that the binding sites are independent from each other. The regional distribution of 5-HT_1A receptors labelled by [³H]rauwolscine is in agreement with previous studies using [³H]8-OH-DPAT.     

α2-Adrenoreceptors in rat brain are decreased after long-term tricyclic antidepressant drug treatment

After two weeks of twice-daily administration of amitriptyline to rats, the binding of [³H]clonidine to presynaptic α₂-adrenoreceptors was decreased in membranes isolated from 5 areas of the rat brain. After one day of treatment, binding did not differ from saline treated controls. In vitro, a high concentration of amitriptyline caused a competitive inhibition of [³H]clonidine binding but did not alter the number of binding sites. The decrease in the number of α₂-adrenoreceptor binding sites after two weeks of amitriptyline treatment would explain the subsensitivity of these receptors which occurs after prolonged administration of antidepressant drugs.