Binding pattern of [35S]t-butylbicyclophosphorothionate is not altered following electroconvulsive shock treatment in rats

Single or repeated electroconvulsive shock (ECS) treatment-induced changes in [35S]t-butylbicyclophosphorothionate [( 35S]TBPS) binding patterns in specific regions, i.e., cerebral cortex, cerebellum, hippocampus, and striatum of rat brain were investigated. Specific [35S]TBPS binding in these brain regions was not altered following a single or repeated administration of ECS, nor was the inhibition of [35S]TBPS binding to GABA affected. These observations tend to suggest that the picrotoxin-site on the GABA receptor complex may not be directly involved in electroconvulsive shock.     

35S]TBPS binding sites are decreased in the colliculi of mice with a genetic predisposition to bicuculline-induced seizures

Several differences have been found in GABAergic function between the long sleep (LS) and short sleep (SS) mice which were genetically selected for different ethanol-induced sleeptimes, and it has been suggested that these differences may explain their differential ethanol sensitivity. However, these lines also differ in seizure susceptibility, a behavior which may also be mediated by GABAergic pathways. Thus, it is difficult to associate differences in GABA neurochemistry with either of these behaviors, particularly when only two selected lines are used. We measured differences in the density and affinity of the [35S]TBPS binding site on the GABAA receptor/Cl- ionophore complex in discrete brain areas; and in order to determine the relationship between receptor binding and behavioral differences, we included mice from 5 of the LS and SS recombinant inbred strains (LS x SS RI) in addition to mice from the LS and SS lines. [35S]TBPS binding in sagittal brain sections was analyzed by quantitative autoradiography, and the amount of binding differed depending on whether bicuculline was added to inhibit endogenous GABA binding. In the presence of bicuculline, the number of [35S]TBPS sites in SS mice was highest in the colliculi (4.5 +/- 0.5 pmol/mg protein), cerebellum (4.8 +/- 0.6 pmol/mg), hippocampus (3.2 +/- 0.7 pmol/mg) and cortex (2.9 +/- 0.3 pmol/mg). The Bmax was two-fold lower in both superior and inferior colliculi (IC) of LS mice. There were no differences between lines in Bmax in any other area and in Kd values in any area (58 +/- 4.0 nM).(ABSTRACT TRUNCATED AT 250 WORDS)     

gamma-Aminobutyric acid- and benzodiazepine-induced modulation of [35S]-t-butylbicyclophosphorothionate binding to cerebellar granule cells

t-Butylbicyclophosphorothionate (TBPS) is a bicyclophosphate derivative with potent picrotoxin-like convulsant activity that binds with high affinity and specificity to a Cl- channel-modulatory site of the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex. Using intact cerebellar granule cells maintained in primary culture, we have studied the modifications induced by GABA and diazepam on the ion channel-modulatory binding site labeled by [35S]TBPS. At 25 degrees C, and in a modified Locke solution, the [35S]TBPS specific binding, determined by displacing the radioligand with an excess (10(-4) M) of picrotoxin, was approximately 70% of the total radioactivity bound to the cells. [35S]TBPS specific binding was saturable with a Kd of approximately 100 nM, a Bmax of approximately 440 fmol/mg of protein, and a Hill coefficient of 1.18. Neither cerebellar astrocytes maintained in culture for 2 weeks nor a neuroblastoma cell line (NB-2A) exhibited any specific [35S]TBPS binding. Muscimol (0.3 to 5 microM) enhanced and bicuculline (0.1 to 5 microM) inhibited [35S]TBPS specific binding to intact cerebellar granule cells. The effect of muscimol and bicuculline on [35S]TBPS binding was noncompetitive. Muscimol (0.1 to 5 microM) reversed bicuculline inhibition in a dose-dependent fashion but failed to reverse picrotoxin-induced inhibition. [35S]TBPS binding was also modulated by benzodiazepine receptor ligands. The binding was increased by diazepam and decreased by 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methylester. Muscimol (0.05 microM) failed to reverse bicuculline inhibition in the absence of diazepam, but it became effective in the presence of 0.1 to 1 microM diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic selection for benzodiazepine ataxia produces functional changes in the gamma-aminobutyric acid receptor chloride channel complex

The gamma-aminobutyric acid (GABA) receptor-operated chloride channel complex was evaluated in mice selected for differential sensitivity to the ataxic effects of diazepam (diazepam-sensitive (DS) and diazepam-resistant (DR) lines). The ataxic effects of several drugs purported to produce some of their actions through the benzodiazepine-GABA receptor complex were examined using the rotarod test. The duration of impairment produced by diazepam, ethanol, 4,5,6,7-tetrahydroisoxazol[5,4-C]pyridine-3-ol (THIP) and phenobarbital was greater in the diazepam-sensitive than in the diazepam-resistant mice. In contrast, pentobarbital produced an equivalent duration of ataxia in the two lines. Muscimol-stimulated 36Cl- influx and the binding of [35S]t-butylbicyclophosphorothionate (TBPS) and [3H]flunitrazepam were measured using isolated brain membrane vesicles (microsacs). Depolarization-dependent 45Ca2+ uptake was measured in whole brain synaptosomes. Muscimol was a more potent stimulator of 36Cl- flux in the DS compared to the DR mice, although no difference between the lines was found in muscimol-stimulation of [3H]flunitrazepam binding. Flunitrazepam augmented the muscimol-stimulated 36Cl- uptake in the DS but not in the DR mice. However, no differences between the lines of mice were found in either density or affinity of [3H]flunitrazepam binding sites. Similarly, no differences in either the density or affinity of [35S]TBPS binding sites was found. Ethanol (10-45 mM) potentiated the muscimol-stimulation of 36Cl- in DS, with no effect in DR mice. However, ethanol inhibition of [35S]TBPS binding was equivalent in the two lines of mice. Pentobarbital produced an equal potentiation of the muscimol-stimulated 36Cl- flux in the two lines, but phenobarbital potentiated the muscimol-induced 36Cl- influx slightly more in DS mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Differential seizure sensitivities to picrotoxinin in two inbred strains of mice (DBA/2J and BALB/c ByJ): parallel changes in GABA receptor-mediated chloride flux and receptor binding

Two strains of mice were shown to possess a differential sensitivity to picrotoxinin-induced convulsions; picrotoxinin elicited both tonic and clonic seizures at lower doses in the DBA/2J (DBA) strain compared to the BALB/c ByJ (BALB) strain. Less protection of picrotoxinin-induced tonic seizures was afforded by pentobarbital in the DBA strain. Biochemical studies revealed that picrotoxin inhibited 36Cl- efflux from forebrain synaptoneurosomes only in the DBA strain. In addition, picrotoxin inhibited pentobarbital-induced 36Cl- efflux to a greater extent in the DBA strain. No differences were observed in the binding of [3H]muscimol or [35S]t-butylbicyclophosphorothionate (TBPS) to forebrain homogenates, while pentobarbital was a less potent inhibitor of [35S]TBPS binding in the DBA strain. These findings suggest a genetic basis for the behavioral differences in convulsant sensitivity as well as for the neurochemical differences in allosteric coupling between convulsant and depressant/anticonvulsant sites associated with the GABA receptor-gated Cl- channel.     

A ketogenic diet rescues the murine succinic semialdehyde dehydrogenase deficient phenotype

Succinic semialdehyde dehydrogenase (SSADH) deficiency is a heritable disorder of GABA degradation characterized by ataxia, psychomotor retardation and seizures. To date, there is no effective treatment for SSADH deficiency. We tested the hypothesis that a ketogenic diet (KD) would improve outcome in an animal model of SSADH deficiency, the SSADH knockout mouse (Aldh5a1-/-). Using a 4:1 ratio of fat to combined carbohydrate and protein KD we set out to compare the general phenotype, in vivo and in vitro electrophysiology and [35S]TBPS binding in both Aldh5a1-/- mice and control (Aldh5a1+/+) mice. We found that the KD prolonged the lifespan of mutant mice by >300% with normalization of ataxia, weight gain and EEG compared to mutants fed a control diet. Aldh5a1-/- mice showed significantly reduced mIPSC frequency in CA1 hippocampal neurons as well as significantly decreased [35S]TBPS binding in all brain areas examined. In KD fed mutants, mIPSC activity normalized and [35S]TBPS binding was restored in the cortex and hippocampus. The KD appears to reverse toward normal the perturbations seen in Aldh5a1-/- mice. Our data suggest that the KD may work in this model by restoring GABAergic inhibition. These data demonstrate a successful experimental treatment for murine SSADH deficiency using a KD, giving promise to the idea that the KD may be successful in the clinical treatment of SSADH deficiency.     

Complex interactions between pregnenolone sulfate and the t-butylbicyclophosphorothionate-labeled chloride ionophore in rat brain

Detailed studies of the interactions between the 'neuroactive' steroid, pregnenolone sulfate (PS), and the gamma-aminobutyric acid (GABA)/benzodiazepine receptor-linked chloride ionophore (GBRC) labeled by [35S]t-butylbicyclophosphorothionate ([35S]TBPS) in the rat brain reveal a site of action allosteric to the TBPS-labeled site as demonstrated by the ability of PS to accelerate TBPS-initiated dissociation of [35S]TBPS. In contrast to previous findings, PS modulates [35S]TBPS binding in a GABA 'agonist'-like fashion with micromolar potencies. The role of PS in the regulation of neuronal excitability through the GBRC is questioned, based upon the observation that brain concentrations of PS are 2-3 orders of magnitude less than that necessary for the modulation of chloride conductance in vitro.     

Dramatic increase in nigral t-[35S]butylbicyclophosphorothionate binding sites elicited by the degeneration of the striato-nigral GABAergic pathway: reversal by diazepam

In rats, the degeneration of the striato-nigral GABAergic pathway caused by the intrastriatal injection of kainic acid induced a marked decrease (65%) of GABA content and glutamic acid decarboxylase (GAD) activity and a dramatic increase (225%) in the binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a membrane preparation from the substantia nigra homolateral to the injected striatum. The increase in [35S]TBPS binding in the denervated substantia nigra was exclusively due to an increased density of binding sites (Bmax) with no change in the dissociation constant (kd). The enhancement in [35S]TBPS binding was almost completely reversed by the intraperitoneal administration of diazepam (3 mg/kg) to kainic acid-lesioned rats. Moreover, diazepam produced a significant decrease (30%) in the density of [35S]TBPS binding sites also in the sham-operated side. In contrast the 'in vitro' addition of the GABAA receptor antagonist bicuculline (1 microM) to the membrane preparation from the denervated substantia nigra further increased [35S]TBPS binding. These findings suggest the view that the increase of nigral [35S]TBPS binding is directly related to the inhibition in the function of nigral GABAergic synapses following the loss of the striato-nigral GABAergic pathway. Our results indicate that [35S]TBPS binding to brain structure is a potential tool to reveal alteration in the function of GABAA receptor complex elicited by physiological, pharmacological and pathological conditions.     

Aging: Effect on the interaction of ethanol and pentobarbital with the benzodiazepine-GABA receptor-ionophore complex

Benzodiazepine receptor binding and modulation by pentobarbital and ethanol was studied using the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate to solubilize the gamma-aminobutyric acid (GABA)-benzodiazepine receptor-ionophore complex from the brains of Fischer 344 rats of 3-4, 12-15 and more than 28 months of age. The affinity of the benzodiazepine binding site was significantly lower in the young rats compared to either the mature or senescent animals. However, no age-related changes in the maximum number of benzodiazepine binding sites or GABA concentrations occurred in the detergent extract. Pentobarbital produced practically identical dose-dependent enhancement of [3H]flunitrazepam specific binding in all 3 age groups. In contrast, ethanol between 0.1 and 200 microM failed to produce a dose-dependent effect on [3H]flunitrazepam specific binding in any age group. The effect of pentobarbital and ethanol on [35S]t-butyl-bicyclophosphorothionate [( 35S]TBPS) specific binding to the picrotoxinin binding site was examined in the above solubilized receptor/ionophore complex under the same binding conditions. Both sedative-hypnotics produced a dose-dependent decrease in [35S]TBPS specific binding. However, pentobarbital was over 10,000 times more potent. It appears that ethanol may not enhance [3H]flunitrazepam specific binding in this solubilized preparation because of its weak action at the picrotoxinin binding site.     

3H]Ethynylbicycloorthobenzoate ([3H]EBOB) binding in recombinant GABAA receptors

Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABAA receptor. While a few studies have examined the binding of [3H]EBOB in vertebrate brain tissue and insect preparations, none have examined [3H]EBOB binding in preparations that express known configurations of the GABAA receptor. We have thus examined [3H]EBOB binding in HEK293 cells stably expressing human alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and the effects of CNS convulsants on its binding. The ability of the CNS convulsants to displace the prototypical convulsant site ligand, [35S]TBPS, was also assessed. Saturation analysis revealed [3H]EBOB binding at a single site, with a K(d) of approximately 9 nM in alpha1beta2gamma2 and alpha2beta2gamma2 receptors. Binding of both [3H]EBOB and [35S]TBPS was inhibited by dieldrin, lindane, tert-butylbicycloorthobenzoate (TBOB), PTX, TBPS, and pentylenetetrazol (PTZ) at one site in a concentration-dependent fashion. Affinities were in the high nM to low microM range for all compounds except PTZ (low mM range), and the rank order of potency for these convulsants to displace [3H]EBOB and [35S]TBPS was the same. Low [GABA] stimulated [3H]EBOB binding, while higher [GABA] (greater than 10 microM) inhibited [3H]EBOB binding. Overall, our data demonstrate that [3H]EBOB binds to a single, high affinity site in alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and modulation of its binding is similar to that seen with [35S]TBPS. [3H]EBOB has a number of desirable traits that may make it preferable to [35S]TBPS for analysis of the convulsant site of the GABAA receptor.     

Allosteric modulation of t-[35S]butylbicyclophosphorothionate binding in rat brain by melatonin

In order to clarify melatonin's pharmacological interaction with central gamma-aminobutyric acid (GABA) receptors, its effects on the binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS), which specifically labels GABA-gated chloride channels, was examined in the rat brain. Saturation binding studies indicated that the effect of melatonin (500 microM) was due to a significant decrease in binding site density (Bmax) while the dissociation constant (Kd) was unchanged. The central-type benzodiazepine (BZ) receptor site antagonist Ro15-1788 (flumazenil) did not reverse the effect of melatonin but blocked the effect of diazepam, indicating that central-type BZ sites do not mediate the effects of melatonin. Since the ability to allosterically inhibit TBPS binding is characteristic of GABA-positive ligands, these findings provide further evidence that the pharmacological effects of melatonin involve enhancement of central GABAergic activity.     

Altered atypical coupling of gamma-aminobutyrate type A receptor agonist and convulsant binding sites in subunit-deficient mouse lines

We searched for subunit correlations for GABA(A) receptor-associated atypically GABA-insensitive [35S]TBPS binding. The homomeric beta3 subunit receptors could be excluded, as GABA-insensitive [35S]TBPS binding was present in beta3-/- mice. Localization of GABA-insensitive [35S]TBPS binding correlated best with those of delta, alpha4 and alpha6 subunit mRNAs. The amounts of GABA-insensitive [35S]TBPS binding components were increased in delta-/- mice, but dramatically reduced in alpha6-/- mice, suggesting a role for alpha6 but excluding delta subunits.     

[S]TBPS binding sites are decreased in the colliculi of mice with a genetic predisposition to bicuculline-induced seizures

Several differences have been found in GABAergic function between the long sleep (LS) and short sleep (SS) mice which were genetically selected for different ethanol-induced sleeptimes, and it has been suggested that these differences may explain their differential ethanol sensitivity. However, these lines also differ in seizure susceptibility, a behavior which may also be mediated by GABAergic pathways. Thus, it is difficult to associate differences in GABA neurochemistry with either of these behaviors, particularly when only two selected lines are used. We measured differences in the density and affinity of the [³⁵S]TBPS binding site on the GABA_A receptor/Cl⁻ ionophore complex in discrete brain areas; and in order to determine the relationship between receptor binding and behavioral differences, we included mice from 5 of the LS and SS recombinant inbred strains (LS × SS RI) in addition to mice from the LS and SS lines. [³⁵S]TBPS binding in sagittal brain sections was analyzed by quantitative autoradiography, and the amount of binding differed depending on whether bicuculline was added to inhibit endogenous GABA binding. In the presence of bicuculline, the number of [³⁵S]TBPS sites in SS mice was highest in the colliculi (4.5 ± 0.5 pmol/mg protein), cerebellum (4.8 ± 0.6 pmol/mg), hippocampus (3.2 ± 0.7 pmol/mg) and cortex (2.9 ± 0.3 pmol/mg). The B_max was two-fold lower in both superior and inferior colliculi (IC) of LS mice. There were no differences between lines in B_max in any other area and in K_d values in any area (58 ± 4.0 nM). When we compared [³⁵S]TBPS binding in the LS × SS RI strains, B_max values in IC were negatively correlated with latency to bicuculline-induced seizures but were not correlated with the duration of ethanol-induced sleeptime. These data support the hypothesis that differences in GABAergic neurochemistry in these mouse lines may not always be related to differences in ethanol sensitivity and point to GABAergic receptors in IC as possibly being involved in determining seizure sensitivity to bicuculline.     

Regional distribution and kinetics of three sites on the GABAA receptor: lack of effect of portacaval shunting.

The regional distribution of binding sites on the GABAA receptor and their kinetic parameters were measured by quantitative autoradiography in brains from normal rats and rats with a portacaval shunt, a model of portal systemic encephalopathy in which GABA neurotransmission may be altered. The ligands used were [3H]flunitrazepam (a benzodiazepine-site agonist), [3H]-Ro15-1788 (a benzodiazepine-site antagonist), [3H]muscimol (a GABA-site agonist), and [35S]t-butylbicyclophosphorothionate (35S-TBPS, a convulsant that binds to a site near the chloride channel). Some brains were analyzed by computerized image analysis and three-dimensional reconstruction. The regional distribution of binding of the benzodiazepines was very similar, but the patterns obtained with [3H]muscimol and [35S]TBPS were different in many areas, suggesting a heterogeneous distribution of several subtypes of the GABAA receptor. The kinetic parameters were determined in brain regions for [3H]flunitrazepam, [3H]Ro15-1788, and [3H]muscimol. For each ligand, the Kd showed a significant heterogeneity among brain regions (at least threefold), contrary to conclusions drawn from earlier studies. In portacaval shunted rats, binding of all four ligands was essentially unchanged from that in control rats, indicating that, if there was an abnormality in GABA neurotransmission during portal systemic shunting, it was not reflected by altered binding to the main sites on the GABAA receptor.     

Effects of Tyr-MIF-1 and MIF-1 at the GABAA receptor chloride channel site

The peptides Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and, to a lesser extent, MIF-1 (Pro-Leu-Gly-NH2) recently have been found to augment the effects of gamma-aminobutyric acid (GABA) on benzodiazepine receptor binding at the GABAA receptor complex. To assess their interaction with the chloride channel binding site on the GABAA receptor, we evaluated the effects of these two peptides on [35S]-t-butylbicyclophosphorothionate (TBPS) binding in mouse brain membranes. In cortex, neither peptide altered [35S]-TBPS binding over a broad dose range, but Tyr-MIF-1 significantly augmented displacement of radioligand binding by the GABA analog muscimol at peptide concentrations of 10(-10) to 10(-7) M; MIF-1 had little effect on muscimol displacement of [35S]-TBPS binding. In cerebellum and brainstem, neither peptide was active in altering muscimol displacement of binding. Thus, Tyr-MIF-1 augments the displacement of [35S]-TBPS binding by the GABA analog muscimol in mouse brain cortical membranes, indicating that this peptide enhances the effects of GABA at the chloride channel as well as at the benzodiazepine receptor.     

GABAA receptor blockers reverse the inhibitory effect of GABA on brain-specific [35S]TBPS binding

Thirteen substances previously reported to antagonize the electrophysiological effects of gamma-aminobutyric acid (GABA) on neurons also reversed the inhibitory effects of GABA on specific [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to sites on rat brain membranes in vitro with a rank-order of potencies similar to those found in electrophysiological systems (R 5135 greater than pitrazepin greater than bicuculline greater than SR 95103 greater than securinine) confirming the earlier conclusion that GABA inhibits [35S]TBPS binding by acting allosterically on physiologically relevant GABAA receptors. Pitrazepin is the most potent of a series of mono N-aryl piperazines that block GABAA receptors. The new aryl amino pyridazine GABA derivative SR 95531 was about 3-fold more potent than bicuculline and 39-fold more potent than the structurally related SR 95103. Four known GABA antagonists have the same rank orders of potencies as convulsants and as reversers of GABA's inhibitory action on [35S]TBPS binding (bicuculline greater than securinine greater than theophylline greater than caffeine). Reversal of GABA-induced suppression of [35S]TBPS binding provides a simple method for further characterizing GABAA receptors linked to TBPS binding sites, and facilitates identification of convulsants and novel, perhaps selective, GABA antagonists.     

Benzodiazepine receptors in fish brain: [3H]-flunitrazepam binding and modulatory effects of GABA in rainbow trout

We have identified and partially characterised benzodiazepine binding sites in whole brain membranes of male rainbow trout. In terms of Bmax and KD values trout brain receptors are remarkably similar to those in rat and human brain. The Hill coefficient was 0.98, indicating a single binding site. GABA (10(-4) M) was able to significantly elevate binding of [3H]-FNZ through a change in KD rather than Bmax. This effect was prevented by the GABA receptor antagonist bicuculline methiodide.     

Complex interactions between pregnenolone sulfate and the t-butylbicyclophosphorothionate-labeled chloride ionophore in rat brain

Detailed studies of the interactions between the ‘neuroactive’ steroid, pregnenolone sulfate (PS), and the γ-aminobutyric acid (GABA)/benzodiazepine receptor-linked chloride ionophore (GBRC) labeled by [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) in the rat brain reveal a site of action allosteric to the TBPS-labeled site as demonstrated by the ability of PS to accelerate TBPS-initiated dissociation of [³⁵S]TBPS. In contrast to previous findings, PS modulates [³⁵S]TBPS binding in a GABA ‘agonist’-like fashion with micromolar potencies. The role of PS in the regulation of neuronal excitability through the GBRC is questioned, based upon the observation that brain concentrations of PS are 2–3 orders of magnitude less than that necessary for the modulation of chloride conductance in vitro.     

Chronic benzodiazepine antagonist treatment and its withdrawal upregulates components of GABA-benzodiazepine receptor ionophore complex in cerebral cortex of rat

Effect of chronic administration of benzodiazepine (BZ) receptor antagonist Ro 15-1788 (flumazenil) (4 mg/kg once daily for 14 days) treatment and its withdrawal on locomotor activity, body temperature, and the binding pattern of receptor ligands that bind to GABA-BZ receptor ionophore complex in different regions of the brain of the rat was studied. Ro 15-1788 (x 14 d) increased the specific binding of [3H]ethyl-8-fluoro-5-6-dihydro-5-methyl-6-oxo-4H- imidazo[1,5 alpha][1,4]benzodiazepine-3-carboxylate [( 3H]Ro 15-1788), [3H]ethyl-8-azido-5-6-dihydro-5-methyl-6-oxo-4H- imidazo[1,5 alpha][1,4]benzodiazepine-3-carboxylate [( 3H]Ro 15-4513), [3H]flunitrazepam, and [35S]t-butylbicyclophosphorothionate [( 35S]TBPS) in cerebral cortex, and this increase in binding remained upregulated during the drug withdrawal at 24 h. The binding of [3H]Ro 15-1788 was also found significantly increased in the hippocampus, but not in cerebellum and striatum. The chronic Ro 15-1788 treatment did not alter the specific binding of [3H]GABA. Rosenthal analysis of the saturation isotherms indicated that the observed upregulation in the binding pattern of [3H]Ro 15-1788 and [3H]Ro 15-4513 in the cerebral cortex was due to an increase in the binding capacity (Bmax). The receptor affinity (Kd) was not changed. The withdrawal of Ro 15-1788 following its chronic administration also enhanced locomotor activity. However, no apparent change in body temperature was observed either due to chronic treatment or withdrawal. These data indicate that chronic Ro 15-1788 treatment and its withdrawal may produce an upregulation of subunits which bind the positive (benzodiazepines), negative (inverse agonist), and neutral (antagonist) ligands of benzodiazepine receptor.(ABSTRACT TRUNCATED AT 250 WORDS)