Substantia Nigra γ-Aminobutyric Acid Receptors in Huntington''s Disease

The specific binding of [3H]gamma-aminobutyric acid (GABA) to nigral GABA receptors has been studied in postmortem brains from controls and patients with Huntington's disease (HD). A specific increase in the number of high-affinity binding sites for [3H]GABA was observed in HD patients, analogous to changes observed in rat substantia nigra [3H]GABA binding after striatal kainic acid (KA) lesion. The results provide further support for the striatal KA lesion in the rat as an animal model of HD. The implications of the results for the proposed therapeutic potential of GABA agonists in HD are discussed.     

Trimethyltin-induced loss of NMDA and kainate receptors in the rat brain

Summary Adult rats exposed acutely to trimethyltin (TMT) manifest a number of behavioral alterations, in conjunction with neuronal degeneration in the limbic system. In the present study, changes in³H-TCP binding to N-methyl-D-aspartate (NMDA) receptors and³H-kainic acid (KA) binding to kainate receptors were studied by autoradiographic methods following TMT exposure (8 mg/kg, i.p.) in adult Sprague Dawley rats. No significant alterations were found at 4 hours after exposure. An extensive loss of³H-TCP and³H-KA binding was seen in the hilar region of the CA3 field at 2 and 12 weeks after TMT exposure. Also, the³H-TCP binding was decreased in piriform cortex and in striatum. Thus, TMT exposure leads to a major and regional selective loss of NMDA and kainate receptors in the limbic system, alterations that may be involved in the neuropathology and behavioral sequelae of TMT toxicity.Abbreviations TMT trimethyltin - NMDA N-methyl-D-aspartate - KA Kainic acid - TCP N-(1-2-thienylcyclohexyl)-3,4-piperidine     

Analysis of 3H-kainic acid binding to brain membranes in rats and frogs

It has been shown that H3-kainic acid (3H-KA) specifically binds with membrane preparations from various parts of rat brain or whole frog brain. The saturation isotherms of 3H-KA binding revealed the presence of two sites with a high and low affinity. An exception was for rat cerebellum where Scatchard analysis showed but one low affinity site. The density of 3H-KA binding sites in frog brain was 5 to 10 times higher than in rat brain. Among the drugs studied, KA itself, L-glutamate and folic acid were the most potent inhibitors of specific binding. Methyltetrahydrofolate, quinolinic acid, kynurenine, GABA, taurine, L-aspartate were ineffective in this respect. The kinetic analysis of the binding data in the presence or absence of L-glutamate and folic acid showed, however, that these drugs inhibited 3H-KA binding in a noncompetitive manner. In the light of these findings L-glutamate or folate cannot be considered as endogenous ligands for hypothetic "kainate receptors".     

Increase in Striatal [3H]Muscimol Binding Following Intrastriatal Injection of Kainic Acid: A Denervation Supersensitivity Phenomenon

The effect of intrastriatal microinjection of kainic acid (KA) on specific binding of [³H]muscimol to the particulate fractions obtained from corpus striatum (CS), globus pallidus (GP), substantia nigra (SN), and cerebral cortex (CC) was examined. Seven days after the unilateral intrastriatal microinjection of KA, the amount of specifically bound [³H]muscimol was significantly increased at the injected site, whereas no significant alteration of [³H]muscimol binding was found in GP, SN, or CC. Scatchard analysis of striatal binding revealed that microinjection of KA significantly increased the affinity (K_D) of GABA receptors on the injected (lesioned) side of the CS without affecting the total number of binding sites (B_max) therein. This significant increase in [³H]muscimol binding, however, was eliminated by pretreating particulate fractions from the CS with Triton X-100, a non-ionic detergent. No statistically significant difference in amounts of [³H]muscimol binding was detected when the preparations from the KA-treated and non-treated CS were preincubated with 0.05% Triton X-100, respectively. Scatchard analysis using CS preparations treated with 0.05% Triton X-100 revealed that the affinity of the GABA receptor was increased by treatment with Triton X-100, while the total number of binding sites (B_max) was unchanged by this treatment. These results suggest that neuronal degeneration produced by KA in vivo and pretreatment of particulate preparations with Triton X-100 in vitro may increase the amount of specifically bound [³H]muscimol to CS preparations by a similar molecular mechanism.     

Change in the charactertistics of 3H-spiperone binding to rat striatal membranes after acute chlorpromazine administration: effects of buffer washing of membranes.

After intraperitoneal injections of ³H-spiperone into the rat, brain membrane preparations retain the majority of the radioactivity even after several buffer washes. With ³H-spiperone as ligand, dissociation constants were significantly elevated and maximum binding unchanged in rat striatal membranes after acute intraperitoneal injection of chlorpromazine (14 mg/kg). It is suggested that in studies of post-mortem brains of schizophrenics that contain neuroleptics specific ³H-spiperone binding will be lowered by competition from residual drug in membrane preparations and valid comparisons of ³H-spiperone binding to preparations from control and schizophrenic brains can only be made if maximum binding values are determined.     

Changes in Excitatory Amino Acid Receptor Binding in the Intact and Decorticated Rat Neostriatum Following Insulin-Induced Hypoglycemia

An involvement of excitatory amino acid (EAA) transmitter-receptor interactions in the development of hypoglycemia-induced neuronal damage has been suggested. We report here on the binding to EAA receptors in the rat caudate nucleus and cerebral cortex, during and following severe insulin-induced hypoglycemia with an isoelectric EEG of 10 or 30 min duration. The binding of alpha-[3H]amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [( 3H]AMPA) to quisqualate receptors, [3H]kainic acid (KA) to kainate receptors, and [3H]glutamate to N-methyl-D-aspartate (NMDA)-sensitive sites was determined by quantitative autoradiography. During EEG isoelectricity, AMPA binding was reduced by approximately 40%, which could represent quisqualate receptor desensitization. One hour following glucose-induced recovery, AMPA binding was no longer different from control level. As the recovery period was prolonged to 1 or 4 weeks, AMPA binding decreased. The decrease was more pronounced in the dorsolateral than in the ventromedial part of the striatum. This correlates with the distribution of neuronal damage, and probably reflects loss of receptor binding sites due to cell death. During the period of EEG silence there was a tendency toward an increase in NMDA displaceable glutamate binding. Following 4 weeks of recovery, binding to NMDA receptors was significantly decreased. Glutamate binding to NMDA-sensitive sites was remarkably resistant to neuronal necrosis and was not significantly different from control values in the dorsolateral caudate 1 week following the hypoglycemic coma. No changes in KA binding were found until 1 week posthypoglycemia, when a significant reduction in binding was noted in the lateral striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of long-term L-dopa administration on the dopaminergic and cholinergic (muscarinic) receptors of striatum in 6-hydroxydopamine lesioned rats

L-Dihydroxyphenylalanine (L-Dopa) (200 mg/kg/day) was administered for 30 days to the rats whose nigrostriatal dopamine pathway was lesioned unilaterally with 6-hydroxydopamine and the receptor binding of ³H-spiperone and ³H-quinuclidinyl benzilate (³HQNB) was measured in the dopaminergic and muscarinic cholinergic receptors of the striatum. ³H-spiperone binding increased by 73% and ³HQNB binding decreased by 14% in the lesioned side when compared to the control side of L-Dopa-non-treated rats. ³H-spiperone binding was measured in the lesioned sides of L-Dopa-treated and L-Dopa-non-treated rats and was found to have decreased by 21% in the former. In the control side of the L-Dopa-treated lesioned rats, however, ³H-spiperone binding increased by 27% when compared to the opposite striatum of the same rats. ³HQNB binding in the lesioned side of L-Dopa-treated rats was not significantly different from that of the control side statistically. These results suggest that changes in functional equilibrium between the dopaminergic and cholinergic mechanisms influence the muscarinic cholinergic receptors and that supersensitivity of dopamine receptors after lesion of the nigrostriatal pathway also remains after long-term L-Dopa treatment.     

Kainic acid binding to membranes of striatal neurons.

The specific binding of [³H]-kainic acid to membrane fragments of rat striatum was examined. The specific binding was found to be saturable and of high affinity. The dissociation constant was about 71 nM, while the apparent maximal number of receptor sites was 254 fmoles/mg protein. [³H]-Kainic acid binding was effectively competed by both unlabeled kainic acid and glutamate, Lesions of the striatum by stereotaxic injection of 5 nmoles of kainic acid reduced the density of [³H]-kainic acid binding sites by half, without affecting their affinity. Lesions of the cortico-striatal afferents, however, did not affect the binding of [³H]-kainic acid, although sodium-dependent glutamate uptake was reduced by 30%. It is concluded that [³H]-kainic acid binds to a population of receptors localized on neurons of the caudate-putamen.     

Effects of intrastriatal kainic acid injection on [3H]dopamine metabolism in rat striatal slices: evidence for postsynaptic glial cell metabolism by both the type A and B forms of monoamine oxidase

Abstract: Intrastriatal injections of kainic acid (KA) were utilized to investigate the cellular localization of postsynaptic dopamine (DA) metabolism by type A and B monoamine oxidase (MAO) in rat striatum. At 2 days postinjection, maximal degeneration of cholinergic and γ-aminobutyric acid (GABA)ergic neurons was observed and found to be associated with a significant decrease in both type A and B MAO activity. However, over the next 8-day period, when only the process of gliosis appeared to be occurring, a selective return to control of type B MAO activity was seen. When the metabolism of [³H]DA (10^?7 M) was examined in 8-day KA-lesioned rat striatal slices, an increase in [³H]dihydroxyphenylacetic acid (DOPAC) and [³H]homovanillic acid (HVA) formation was observed. The KA-induced elevation of [³H]DOPAC formation (but not [³H]HVA) was abolished by the DA neuronal uptake inhibitor nomifensine. This is consistent with earlier findings suggesting that HVA is formed exclusively within sites external to DA neurons. Experiments with clorgyline and/or deprenyl revealed that the relative roles of type A and B MAO in striatal DA deamination remained unchanged following KA (90% deamination by type A MAO) even though total deamination was substantially enhanced. At high concentrations of [³H]DA (10^?5 M), deamination by type B MAO could be increased to 30% of the total MAO activity; however, this was observed in both control and KA-lesioned striata. These results suggest that KA-sensitive neurons contain type A and/or type B MAO. Moreover, whereas these neurons may metabolize DA, a major portion of postsynaptic DA deamination appears to occur within glial sites of rat striatal tissue. Furthermore, glial cells would appear to contain functionally important quantities of both type A and B MAO.     

GABA synthesis by cultured fibroblasts obtained from persons with Huntington's disease.

Abstract— A consistent observation in particular regions of brains of persons having died with Huntington's disease (HD) is a reduction in the concentration of γ-aminobutyric acid (GABA) and a decrease in the activity of its synthetic enzyme, glutamate decarboxylase (EC 4.1.4.15). GABA levels are also reduced in HD cerebrospinal fluids. This study suggests that skin fibroblasts obtained from persons with HD can be used to study their GABA system. A rapid and specific assay for [¹⁴C]glutamate– [¹⁴C]GABA based on Aminex A-7 chromatography has been developed. Cell monolayers and homogenates of HD cells convert [¹⁴C]glutamate to [¹⁴C]GABA. GABA synthesis by HD cell homogenates is pyridoxal dependent and is inhibited by 1 mm -aminooxyacetic acid. GABA synthesis by HD and control cell homogenates also show the same thermal sensitivity as rat brain GAD. When compared to non-HD human cells the HD cells reveal disturbances in the non-neuronal GABA metabolic pathway. Concentrated HD cell homogenates synthesize approx 3 times the amount of GABA as control cells. When diluted both extracts made similar amounts of GABA. Synthesis of GABA by HD cell homogenates is not inhibited by cysteine sulfinate. Decarboxylation of glutamate in these cells is therefore most likely due to glutamate decarboxylase and not cysteine sulfinate decarboxylase. HD cells in monolayer also synthesize 3 times the amount of GABA as compared to control cells. In addition, glutamate upake is altered in HD cells. This report indicates there may be a different pattern of enzyme regulation between HD and control cells.     

Effects of bicuculline on [3H]SR 95531 binding in discrete regions of rat brains

Effects of bicuculline in vitro, and acute and chronic treatment of a subconvulsive dose of bicuculline on [³H]SR 95531 binding to discrete regions of rat brains were studied in Sprague-Dawley rats. Scatchard analysis of the binding isotherms exhibited two populations of binding sites for [³H]SR 95531 in frontal cortex, cerebellum, striatum and substantia nigra. The apparent K_D for high-affinity sites was significantly increased in the frontal cortex and cerebellum in the presence of bicuculline (1 M) with no change in B_max. In contrast, the apparent affinity for low-affinity sites was not altered in the presence of bicuculline in these regions, whereas the B_max was significantly decreased in the cerebellum. Following acute (2 mg/kg, i.p.) or chronic (2 mg/kg, i.p. for 10 days) bicuculline treatment, [³H]SR 95531 binding was also investigated in various regions of brains. The acute bicuculline treatment did not affect the [³H]SR 95531 binding in any of the regions studied. In contrast, apparent affinity for [³H]SR 95531 was significantly decreased in low-affinity sites of all regions studied in rats treated chronically with bicuculline. The B_max values of high and low-affinity sites were significantly increased in the cerebellum with no change in the frontal cortex, striatum and substantia nigra. The present study demonstrates that chronic bicuculline treatment decreases apparent affinity of [³H]SR 95531 binding whereas the treatment increases apparent affinity of [³H]SR 95531 and [³H]muscimol binding in the cerebellum may be due to true up-regulation of GABA binding sites, involving increased de novo synthesis of receptor protein. These results also suggest that properties of cerebellar GABA_A receptors are different from those in other regions.Abbreviations used GABA -aminobutyric acid - FC frontal cortex - CB cerebellum - ST striatum - SN substantia nigra     

3H-kainic acid binding: relevance for evaluating the neurotoxicity of kainic acid.

Binding of ³H-kainic acid (KA) to brain membranes is saturable (K_D = 72 nM) and displays markes specificity for compounds that bear relation to excitatory amino acids. Its regional distribution correlates roughly with the regional neurotoxicity of in situ KA injections. Striatal kainate lesions, but not interruption of striatal afferents, reduce striatal KA binding by 36%. This decrease is due to a loss in the number of neuronal binding sites without affecting the affinity for the ligand. It is suggested that ³HKA binding to neuronal elements may be a valuable instrument for studies of the mechanism of neuronal degeneration caused by KA.     

Discrimination of Multiple [3H]5-Hydroxytryptamine Binding Sites by the Neuroleptic Spiperone in Rat Brain

Certain neuroleptic drugs, such as spiperone and (+) butaclamol, can discriminate between two populations of [³H]5-hydroxytryptamine ([³H]5-HT) binding sites in rat brain. The butyrophenone neuroleptic spiperone shows the greatest selectivity for these two binding sites, having at least a 3000-fold difference between its dissociation constants (2-12 nM versus 35,000 nM) for the high- and low-affinity sites, respectively. Inhibition of [³H]5-HT binding by spiperone in rat frontal cortex and corpus striatum yields distinctly biphasic inhibition curves with Hill slopes significantly less than unity. Results from nonlinear regression analysis of these inhibition studies were consistent with a two-site model in each brain region. In the frontal cortex the high-affinity neuroleptic sites comprised about 60% of the total [^3/H]5-HT binding sites whereas in the corpus striatum they accounted for only 20% of the sites. Furthermore, saturation studies of [³H]5-HT binding assayed in the absence or presence of 1 μM-spiperone (a concentration that completely blocks the high-affinity site while having minimal activity at the low-affinity site) reveal a parallel shift in the Scatchard plot with no change in the dissociation constant of [³H]5-HT, but a significant decrease (64% in frontal cortex or 28% in corpus striatum) in the number of specific binding sites. These observations are consistent with the existence of at least two populations of [³H]5-HT binding sites having a differential regional distribution in rat brain.     

Direct binding of 3H-lisuride to adrenergic and serotonergic receptors

The characteristics of the specific binding of ³H-lisuride hydrogen maleate (³H-LHM) to homogenates of rat striatum and bovine frontal cortex tissue were investigated. In rat striatum 50% of ³H-LHM binding was inhibited potently by spiperone and haloperidol indicating a component of ³H-LHM binding to D₂ dopamine receptors. Specific ³H-LHM binding was detected in rat striatum after selective blockade of the D₂ dopamine component indicating specific ³H-LHM binding to other striatal sites. In bovine frontal cortex clonidine and serotonin competition curves for specific ³H-LHM binding included high affinity phases indicating alpha₂ adrenergic and high affinity serotonergic components of binding. Blockade of the adrenergic component by 10^?7M clonidine resulted in the specific ³H-LHM binding exhibiting distinctly serotonergic characteristics. Conversely, blockade of the serotonergic component by 2 × 10^?7M serotonin resulted in the specific ³H-LHM binding exhibiting distinct alpha₂ receptor characteristics. These data demonstrate the specific binding of ³H-LHM to alpha₂ adrenergic receptors, to a high affinity serotonin site, and to D₂ dopamine receptors.     

Dopamine receptor binding: Specificity,localization and regulation by ions and guanyl nucleotides

³H-Spiroperidol labels dopamine receptors in rats striatum but in frontal cortex and hippocampus ³H-spiroperidol labels serotonin receptors. The agonists ³H-ADTN and ³H-apomorphine label rat striatal dopamine receptors. Comparison with calf striatal binding indicates a species difference in ³H-apomorphine binding. Drug displacement and lesion studies suggest that in the rat ³H-apomorphine labels two distinct dopamine receptors, one associated with the dopamine-sensitive adenylate cyclase and the other with presynaptic dopamine receptors also labeled by ³H-spiroperidol. Whereas divalent cations increase specific dopamine receptor binding of ³H-agonists and ³H-antagonists, ³H-agonist binding is selectively decreased by some guanyl nucleotides.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

Brain neurotransmitter receptors after long-term haloperidol: dopamine, acetylcholine, serotonin, alpha-noradrenergic and naloxone receptors.

Since long-term neuroleptic therapy is known to alter brain dopaminergic sensitivity, we tested the effects of chronic haloperidol administration (10 mg/kg/day for over 3 weeks) on the amount of the dopamine receptors (using ³H-apomorphine and ³H-haloperidol) in various regions of the rat brain. To test whether the changes in dopamine receptors were selectively produced, we also assayed acetylcholine receptors (with ³H-quinuclidinyl benzilate or ³H-QNB), alpha-noradrenergic receptors (with ³H-WB-4101), ³H-serotonin receptors and ³H-naloxone receptors.The specific binding of ³H-haloperidol increased significantly by 34% in the striatum and by 45% in the mesolimbic region after long-term haloperidol. The specific binding of ³H-apomorphine also increased significantly by 77% in the striatum and 55% in the mesolimbic area. Although there was a small significant increase of 20% in specific ³H-serotonin binding in the striatum, no such increment occurred in the hippocampus or the cerebral cortex. No significantly different binding occurred for the other ³H-ligands in these brain regions except for a 13% increase in alpha-noradrenergic binding in the cerebral cortex. These results indicate that long-term haloperidol treatment produces rather selective increases in dopamine/neuroleptic receptors, without much change in 4 other types of receptors. Such relatively selective increments in these receptors may be the basis of dopaminergic supersensitivity (e.g. tardive dyskinesia) after long-term haloperidol.     

Endogenous Dopamine (Da) Modulates [3H]spiperone Binding in Vivo in Rat Brain

Abstract

[³H]spiperone (SPI) binding in vivo, biochemical parameters and behavior were measured after modulating DA levels by various drug treatments. DA releasers and uptake inhibitors increased SPI binding in rat striatum. In other brain areas, the effects were variable; but only the pituitary remained unaffected. Surprisingly, nomifensine decreased SPI binding in frontal cortex. The effects of these drugs were monitored by measuring DA, serotonin (5–HT) and their metabolites in the same rats. The increased SPI binding in striatum was parallel to the locomotor stimulation with the following rank order: amfonelic acid > nomifensine > D-amphetamine ± methylphenidate > amineptine > bupropion. Decreasing DA levels with reserpine or alpha-methyl-para-tyrosine reduced SPI binding by 45% in striatum only when both drugs were combined. In contrast, reserpine enhanced SPI binding in pituitary. Thus, the amount of releasable DA seems to modulate SPI binding characteristics. It is suggested that in vivo, DA receptors are submitted to dynamic regulation in response to changes in intrasynaptic concentrations of DA.     

Activity of Ionotropic Glutamate Receptors in Retinal Cells: Effect of Ascorbate/Fe2+-Induced Oxidative Stress

Abstract: The effect of oxidative stress induced by the oxidant pair ascorbate/Fe²⁺ on the activity of ionotropic glutamate receptors was studied in cultured chick retina cells. The release of [³H]GABA and the increase of the intracellular free Na⁺ concentration ([Na⁺]_i), evoked by glutamate receptor agonists, were used as functional assays for the activity of the receptors. The results show that the maximal release of [³H]GABA evoked by kainate (KA; ～20% of the total) or AMPA (～11% of the total) was not different in control and peroxidized cells, whereas the EC₅₀ values determined for peroxidized cells (33.6 ± 1.7 and 8.0 ± 2.0 µM for KA and AMPA, respectively) were significantly lower than those determined under control conditions (54.1 ± 6.6 and 13.0 ± 2.2 µM for KA and AMPA, respectively). The maximal release of [³H]GABA evoked by NMDA under K⁺ depolarization was significantly higher in peroxidized cells (7.5 ± 0.5% of the total) as compared with control cells (4.0 ± 0.2% of the total), and the effect of oxidative stress was significantly reduced by a phospholipase A₂ inhibitor or by fatty acid-free bovine serum albumin. The change in the intracellular [Na⁺]_i evoked by saturating concentrations of NMDA under depolarizing conditions was significantly higher in peroxidized cells (8.9 ± 0.6 mM) than in control cells (5.9 ± 1.0 mM). KA, used at a subsaturating concentration (35 µM), evoked significantly greater increases of the [Na⁺]_i in peroxidized cells (11.8 ± 1.7 mM) than in control cells (7.1 ± 0.8 mM). A saturating concentration (150 µM) of this agonist triggered similar increases of the [Na⁺]_i in control and peroxidized cells. Accordingly, the maximal number of binding sites for (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate ([³H]MK-801) was increased after peroxidation, whereas the maximal number of binding sites for [³H]KA was not affected by oxidative stress. These data suggest that under oxidative stress the activity of the ionotropic glutamate receptors is increased, with the NMDA receptor being the most affected by peroxidation.     

Endogenous modulator of dopamine receptor(s)

An endogenous modulator(s) of the dopamine receptor(s) in bovine and rat brain striatum was detected by demonstrating that water extracts of the striatum inhibited [³H]apomorphine binding. This modulator(s) was partially purified by methanol extraction and then successive ion exchange chromatographies on SP-Sephadex C-25 and QAE-Sephadex A-25, and gel chromatography on Sephadex G-25. The partially purified (about 1,500-fold) modulator was a fluorescamine-positive substance, Mr = 500 1000, which was heat-stable (95°C, 10 min), and was destroyed by acid- and alkali-treatment, but not by treatments with various peptidases. The modulator inhibited binding of the dopamine agonist, [³H]apomorphine non-competitively, but did not inhibit binding of the dopamine antagonist, [³H]spiroperidol. Direct injection of the modulator into rat brain striatum depressed apomorphine-induced locomotor activity. Moreover the modulator inhibited dopamine-sensitive adenylate cyclase activity. These findings indicate that the modulator acts at a site(s) other than the ligand binding site of the dopamine receptor(s) and modulates the activities of dopamine agonists.