Differential modulation of etazolate or pentobarbital enhanced [H]muscimol binding by benzodiazepine agonists and inverse agonists

Flunitrazepam, a benzodiazepine agonist increases, and DMCM, an inverse agonist decreases the stimulation by etazolate or pentobarbital of [³H]muscimol binding to membranes of rat cerebral cortex. Ro 15–1788 has no marked effects but antagonizes the action of both flunitrazepam and DMCM. The investigation of several drugs acting on benzodiazepine receptors on etazolate enhancement of [³H]muscimol binding suggests that their receptor interaction reflects a spectrum from agonists to inverse agonists.     

Adrenalectomy prevents the stress-induced decrease in in vitro [H]Ro15-1788 binding to GABAA benzodiazepine receptors in the mouse

The effect of a single or repeated swim stress on in vivo benzodiazepine receptor binding to various brain regions in adrenalectomized and sham-operated (control) mice was assessed using the benzodiazepine receptor antagonist, [³H]Ro15-1788. In sham-operated mice the binding of [³H]Ro15-1788 to benzodiazepine receptors was reduced in the hippocampus and hypothalamus (single or repeated stress) and cerebral cortex (repeated swim stress) compared to non-stressed mice. In contrast, no alterations in [³H]Ro15-1788 binding were observed in any brain region in adrenalectomized mice after either single or repeated swim stress. These data suggest that an intact hypothalamic-pituitary-adrenal axis is required for the stress-induced decrease in benzodiazepine receptor occupancy measured using the in vivo binding method.     

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)     

Positron emission tomography study of brain benzodiazepine receptors in Friedreich's ataxia

Central type benzodiazepine receptors were studied in 9 patients with Friedreich's ataxia and 12 healthy subjects using positron emission tomography (PET) and [11C]Ro 15-1788, a specific antagonist of the central type benzodiazepine receptors, as radioligand. A standard PET procedure was used in 5 patients and 8 controls to obtain brain kinetics of the total binding of the radioligand. The remaining subjects were intravenously injected with a saturating dose of unlabeled Ro 15-1788, 30 minutes after the tracer injection, to determine the nondisplaceable binding of [11C]Ro 15-1788. A semi-quantitative method was used to quantify the [11C]Ro 15-1788 data. None of the quantification indices in the cerebellar hemispheres, or in the other brain areas investigated, was significantly modified in patients with Friedreich's ataxia. These findings suggest that brain benzodiazepine receptors are unaffected in Friedreich's ataxia.     

Visualization of alpha5 subunit of GABAA/benzodiazepine receptor by 11C Ro15-4513 using positron emission tomography

Although [(11)C]Ro15-4513 and [(11)C]flumazenil both bind to the central benzodiazepine (BZ) receptors, the distributions of the two ligands are not identical in vivo. Moreover, the in vivo pharmacological properties of [(11)C]Ro15-4513 have not been thoroughly examined. In the present study, we examined the pharmacological profile of [(11)C]Ro15-4513 binding in the monkey brain using positron emission tomography (PET). [(11)C]Ro15-4513 showed relatively high accumulation in the anterior cingulate cortex, hippocampus, and insular cortex, with the lowest uptake being observed in the pons. Accumulation in the cerebral cortex was significantly diminished by the BZ antagonist flumazenil (0.1 mg/kg, i.v.), but not that in the pons. Using the pons as a reference region, the specific binding of [(11)C]Ro15-4513 in most of the cerebral cortex including the limbic regions clearly revealed two different affinity sites. On the other hand, specific binding in the occipital cortex and cerebellum showed only a low affinity site. Zolpidem with affinity for alpha1, alpha2, and alpha3 subunits of GABA(A)/BZ receptor fully inhibited [(11)C]Ro15-4513 binding in the occipital cortex and cerebellum, while only about 23% of the binding was blocked in the anterior cingulate cortex. Diazepam with affinity for alpha1, alpha2, alpha3, and alpha5 subunits inhibited the binding in all brain regions. Since Ro15-4513 has relatively high affinity for the alpha5 subunit in vitro, these in vivo bindings of [(11)C]Ro15-4513 can be interpreted as the relatively high accumulation in the fronto-temporal limbic regions representing binding to the GABA(A)/BZ receptor alpha5 subunit.     

Relationships between benzodiazepine receptors, impairment of GABAergic transmission and convulsant activity of beta-CCM: a PET study in the baboon Papio papio.

Central type benzodiazepine receptors were studied in vivo by positron emission tomography in brain areas of 2 different groups of the baboon Papio papio: non-photosensitive (group 1) and those with an allylglycine-induced decrease in GABA-mediated inhibition (group 2). Further, a naturally photosensitive Papio papio (+3 level of photosensitive response) was compared to both groups. Regional brain binding of the specific benzodiazepine receptor ligand, [11C]Ro 15-1788, was not significantly different between groups 1 and 2. In addition, the data from the naturally photosensitive Papio papio did not seem to differ markedly from groups 1 and 2 either. Pharmacological effects of increasing doses of beta-CCM (0.05-3 mg/kg i.v.) and regional benzodiazepine receptor occupancy by the drug were simultaneously studied using electroencephalographic activity recording and positron emission tomography. A positive correlation was observed between the degree of photosensitivity of the baboon and sensitivity to the action of beta-CCM, with increasing convulsant efficacy of beta-CCM in going from group 1 to the naturally photosensitive baboon, then to group 2. Dose-related displacement curves of [11C]Ro 15-1788 binding by beta-CCM revealed that reduction in brain GABA concentration did not modify the inhibitory potency of beta-CCM on [11C]Ro 15-1788 binding in cerebral cortex. This suggests a lack of detectable in vivo allosteric effects of GABA on beta-CCM binding during beta-CCM-induced seizures. Thus, a given dose of beta-CCM displayed increasing pharmacological potency in going from baboons with the lowest photosensitivity to those with the highest, whereas benzodiazepine receptor occupancy by beta-CCM was similar in the cerebral cortex of the different baboons. Conversely, a given level of convulsant activity of beta-CCM was related to a different benzodiazepine receptor occupancy by the drug, depending on the photosensitivity of Papio papio. A given dose of a drug may, thus, have a different pharmacological potency when occupying the same number of receptors, depending on the physiopathological state of the subject.     

Central Benzodiazepine/γ-Aminobutyric AcidA Receptors in Idiopathic Generalized Epilepsy: An [11C]Flumazenil Positron Emission Tomography Study

Summary: Purpose: Previous [¹¹C]flumazenil (FMZ) positron emission tomography (PET) investigations in patients with idiopathic generalized epilepsy (IGE) have demonstrated nonsignificant global cortical decreases in central benzodiazepine γ-aminobutyric acid, (GABA_A) receptor (cBZR) binding or focal decreases in the thalamus and increases in the cerebellar nuclei with no changes in cerebral cortex. We previously reported lower [¹¹C]FMZ binding in cerebral cortex of IGE patients treated with valproate (VPA) than in cerebral cortex of controls. We now report high-resolution three-dimensional [¹¹C]FMZ PET studies in a larger number of subjects using an improved method to detect differences in cBZR between IGE patients and controls and a more powerful longitudinal design to determine the functional effect of VPA.
Methods: We compared parametric images of [¹¹C]FMZ volume of distribution (FMZVD) in 10 IGE patients before and after addition of VPA and in 20 normal subjects.
Results: Mean FMZVD was significantly higher in the cerebral cortex (11%, p = 0.009), thalamus (14%, p = 0.018), and cerebellum (15%, p = 0.027) of the 10 IGE patients as compared with that of 20 normal controls. Using statistical parametric mapping, no significant areas of focal abnormality of FMZVD were detected. Addition of VPA was not associated with a significant change in mean FMZVD in any brain area.
Conclusions: Our finding of increased FMZVD in IGE could reflect microdysgenesis or a state of cortical hyperexcitability. Our data suggest that short-term VPA therapy does not affect the number of available cBZR in patients with IGE.     

Effect of sedative drugs upon receptor binding in vivo.

Osamu Inoue, Kaoru Kobayashi and Tetsuya Suhara: Effect of Sedastive Drugs upon Receptor Binding In Vivo. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1992, 16(6): 783–789.

1. 1. Acute treatment with pentobarbital (PB), ethanol and flunitrazepam significantly decreased ³H-SCH 23390 binding in mouse striatum in a dose dependent manner. In contrast, no significant alterations in ³H-SCH 23390 binding in the cerebral cortex have been observed in mice treated with these sedative and hypnotic drugs.

2. 2. Flumazenil (Ro15-1788) reversed the effect of flunitrazepam suggesting the reduction in dopamine D₁ receptor binding in the striatum was mediated via GABA-Bz-Cl channel complex.

3. 3. Using kinetic analysis, it was found that such changes in dopamine D₁ receptor binding in vivo were mainly due to changes in rates of ligand-receptor binding in vivo.

4. 4. Other non-site-specific drugs such as propanol and buthanol also decreased ³H-SCH 23390 binding in vivo, depending on their lipophilicities. These results indicated that micro-environmental factors surrouwding receptors, including cellmembranes seem to have important roles in receptor binding in vivo.

5. 5. Both PB and flunitrazepam decreased muscarinic acetylcholine receptor binding in mouse cortex, striatum, hippocampus and other regions. Together with the fact that PB also altered ³H-Ro15-1788 binding in vivo, this suggested global changes in micro-environmental factors may occur due to these sedative drugs. In vivo qauntitative analysis of neuroreceptors with positron emission tomography (PET) seems to have some potencies to reveal the neurochemical base of benzodiazepine dependence.

Remarkable selectivity of the in vivo binding of [3H]Ro15‐4513 to α5 subtype of benzodiazepine receptor in the living mouse brain

To evaluate the binding characteristics of [³H]Ro15‐4513 with the central benzodiazepine (BZ) receptor, inhibition experiments of [³H]Ro15‐1788 and [³H]Ro15‐4513 were performed both in vitro and in vivo, using two BZ ligands, flunitrazepam (FNP), and ethyl‐β‐carboline‐3‐carboxylate (β‐CCE). FNP inhibited the binding of [³H]Ro15‐1788 and [³H]Ro15‐4513 in a dose‐dependent manner in the mouse cerebral cortex, hippocampus, and cerebellum, both in vitro and in vivo. β‐CCE also inhibited the binding of [³H]Ro15‐1788 and [³H]Ro15‐4513 in all the aforementioned brain regions in vitro. However, in vivo, β‐CCE inhibited the binding of [³H]Ro15‐4513 in the cerebral cortex and cerebellum, but not in the hippocampus, even at an injected dose of up to 1mg/kg. In contrast, more than 50% of the in vivo binding of [³H]Ro15‐1788 was inhibited by 1 mg/kg of β‐CCE in all regions. The time‐activity curve of [³H]Ro15‐4513 in the hippocampus also showed no alteration of the peak uptake between the control group and 0.3 mg/kg of β‐CCE coinjected group. These results indicated that the binding characteristics of [³H]Ro15‐4513 with the BZ receptor differed markedly between the in vitro and in vivo condition, and the selectivity of [³H]Ro15‐4513 binding to α5 subtype of BZ receptor in the mouse brain seemed to be remarkable under the in vivo condition. Synapse 64:928–936, 2010. © 2010 Wiley‐Liss, Inc.     

Sensitization of stress-induced feeding in rats repeatedly exposed to brief restraint: the role of corticosterone

The effects of 5-500 μM concentrations of neutral ammonium salts on the binding of ligands to components of the GABA_A receptor complex were investigated. [³H]Flunitrazepam binding to the benzodiazepine receptor was enhanced by ammonium (10–500 μM), but not sodium tartrate with EC₅₀ = 98 μM and E_max = 31%. Further increasing ammonium tartrate concentrations (500–2500 μM) decreased [³H]flunitrazepam binding to control levels. The ammonium tartrate-induced increase in [³H]flunitrazepam binding was manifested as a 50% decrease in K_d. Furthermore, GABA increased the potency of ammonium tartrate in enhancing [³H]flunitrazepam binding by 63%. [³H]Ro 15-1788 and [³H]Ro 15-4513 binding to the benzodiazepine receptor was not significantly enhanced by ammonium tartrate (E_max ≈ 13%). Ammonium tartrate also increased, then decreased the binding of 500 nM [³H]muscimol to the GABA_A receptor (EC₅₀ = 52 μM, E_max = 30%) in a concentration-dependent manner, but had no effect on [³H]SR 95-531 binding (E_max < 16%). The ammonium tartrate-induced alterations in [³H]muscimol binding were demonstrated in saturation assays as the loss of the high affinity binding site and a 27% increase in the _Bmax of the low affinity binding site. These results indicate that ammonia biphasically enhances, then returns ligand binding to both the GABA and benzodiazepine receptor components of the GABA_A receptor complex to control levels in a barbiturate-like fashion. This suggests that ammonia may enhance GABAergic neurotransmission at concentrations commonly encountered in hepatic failure, an event preceding the suppression of inhibitory neuronal function observed at higher ( > 1 mM) ammonia concentrations. This increase in GABAergic neurotransmission is consistent with the clinical picture of lethargy, ataxia and cognitive deficits associated with liver failure and congenital hyperammonemia.     

Benzodiazepine receptors in human brain: characterization, subcellular localization and solubilization

1. Benzodiazepine receptors have been characterized in human brain mainly using [3H]-Ro 15-1788 and [3H]-flunitrazepam. Both ligands present a very high affinity for the receptor sites (Kd values of 0.56 and 1.53 nM respectively). 2. GABA enhanced the affinity of [3H]-flunitrazepam and [3H]-diazepam, but not that of [3H]-Ro 15-1788 and [3H]-methyl-beta-carboline 3-carboxylate for their specific binding sites as well in cerebral as in cerebellar human cortex. 3. Subcellular distribution of the benzodiazepine receptors revealed a main synaptosomal localization in human cerebral cortex, cerebellum and striatum. 4. Solubilized benzodiazepine receptors were obtained using 0.5% sodium deoxycholate and were characterized with [3H]-Ro 15-1788. The solubilized receptors are still coupled to GABA receptors since the [3H]-flunitrazepam specific binding was enhanced in the presence of micromolar concentrations of GABA.     

Ro 15-1788 suppresses the development of kindling through the benzodiazepine receptor

beta-Carboline (norharman) has been shown to produce kindled seizures when given systemically for long periods of time. The expression of the kindled seizure activity can be blocked by ligands of the benzodiazepine receptor suggesting the receptor as a site of vulnerability in the kindling mechanism. The present data show that Ro 15-1788, a receptor antagonist, suppresses the development of kindled seizures as demonstrated by the delayed appearance of each behavioral stage and the decreased severity of symptoms within each stage. Animals treated with Ro 15-1788 still expressed lower behavioral stages when Ro 15-1788 was eliminated from the trials indicating that this compound suppresses the very process of kindling itself and not just the expression of the kindled seizures. Ro 15-1788 given with norharman causes an increase in Bmax of [3H]flunitrazepam binding to the benzodiazepine receptor in cortex. It is not known if this increase is instrumental in lowering the kindling rate.     

In vivo labeling of central benzodiazepine receptors with the partial inverse agonist [3H]Ro 15-4513

Ro 15-4513 is an imidazobenzodiazepine and a partial inverse agonist at the central benzodiazepine receptors (BZDr). It has been shown to antagonize behavioral and biochemical effects of ethanol. In vivo binding of [3H]Ro 15-4513 was evaluated in mouse brain. After intravenous injection [3H]Ro 15-4513 was readily taken up by the brain and distributed to brain areas enriched in benzodiazepine receptors. Binding was specific for central BZDr, saturable and reversible. A high degree of specific binding, relative to non-specific binding, was achieved. Analysis of dissociation kinetics revealed that [3H]Ro 15-4513 was retained significantly longer in hippocampus compared to other brain regions. In view of the known distribution of benzodiazepine receptor subtypes, this suggests that, in vivo, [3H]Ro 15-4513 has a higher affinity for benzodiazepine receptors type II and may explain quantitative differences in the regional distribution of this ligand compared to the antagonist [3H]Ro 15-1788. We conclude from these studies that Ro 15-4513 is a suitable ligand for in vivo studies of benzodiazepine receptors. Labeled with a positron-emitting isotope, it could be used with positron emission tomography to study BZDr in man under a variety of conditions.     

The shark GABA-benzodiazepine receptor: further evidence for a not so late phylogenetic appearance of the benzodiazepine receptor

Whilst the brain-specific benzodiazepine receptor has been assumed to show a late evolutionary appearance, we present evidence for the presence of a central benzodiazepine binding site in sharks, which shows a high affinity for [3H]Ro 15-1788. However, the receptor density and the affinities of several benzodiazepine receptor ligands are lower than in mammals, thus presumably explaining why the benzodiazepine binding sites had previously escaped detection in elasmobranchs. Additionally, radio- and immunohistochemistry were performed to localize the radioligand binding sites and the antigenic sites of the shark gamma-aminobutyric acid (GABA)-benzodiazepine receptor. In cerebellum, the granular layer reveals a high density of [3H]muscimol binding sites. The immunoreaction obtained with the beta-subunit-specific monoclonal antibody bd-17 seemingly parallels the distribution of high-affinity GABA binding sites. In contrast, [3H]Ro 15-1788 binding sites are evenly distributed in the molecular and granular layers, thus the results are similar to those previously described for rat cerebellum. Apparently, the respective distributions in this brain region are well conserved throughout vertebrate evolution.     

PET findings in a brain abscess associated with a silent atrial septal defect

Brain abscesses are classical complications of congenital heart disease (CHD) in children and adolescents. This association is rarely observed in adults. We report a 46-year-old man presenting a fronto-parietal abscess associated with an asymptomatic atrial septal defect. Positron emission tomography (PET) study revealed high uptake of l-[methyl-¹¹C]methionine ([¹¹C]methionine) and 2-[¹⁸F]fluoro-2-deoxy-d-glucose (FDG) around the brain abscess. We suggest (1) to exclude a silent cardiac malformation in the presence of a cerebral abscess of unknown source occurring in adults; (2) to consider the diagnosis of brain abscess in cases of high uptake of [¹¹C]methionine and FDG in relation to a brain lesion.     

Benzodiazepine-GABAA Receptor Binding During Absence Seizures

Summary: The role of benzodiazepine (BZD)-γ-aminobutyric acid, (GABA) receptors in the pathogenesis of absence seizures is uncertain. In this study, we examined the effect of absence seizures on the binding of flumazenil to the BZD binding site of the GABA, receptor. Five patients with idiopathic generalized epilepsy (IGE) were studied at rest and during absence seizures with [¹¹C]flumazenil and positron emission tomography (PET). Normalized regional cerebral time-activity curves from the resting and ictal scans were compared with each other and with computed simulations showing the effects of changes in cerebral blood flow (CBF) and [¹¹Clfluma-zenil binding. No evidence was found for a change in [¹¹C]flumazenil binding with absence seizures. This result, together with those of a recent study showing no abnormality of [¹¹C]flumazenil binding interictally in patients with childhood and juvenile absence epilepsy (JAE) does not support a primary role for the BZD binding site of the GABA, receptor in the pathogenesis of absence seizures.     

Benzodiazepine receptor binding of nonbenzodiazepines in vivo: Alpidem, zolpidem and zopiclone

Several classes of nonbenzodiazepine compounds, including imidazopyridines such as alpidem and zolpidem and cyclopyrrolones, e.g., zopiclone, have effects similar to benzodiazepines and may act at the benzodiazepine receptor in brain. We characterized the binding of these compounds to the benzodiazepine site in three brain regions using specific uptake of the high-affinity ligand [3H]Ro15-1788 (flumazenil). For alpidem, benzodiazepine binding was decreased in cortex and hippocampus with increasing drug dose. For zolpidem, receptor binding was reduced in cortex without a dose-response effect and no effect was observed on cerebellar binding. Zopiclone did not alter binding except for a decrease in binding at the lowest dose evaluated and an increase in binding above control at the highest dose. These data corroborate prior studies indicating that the imidazopyridines appear to act at the benzodiazepine receptor, but do not support receptor subtype selectivity of zolpidem. The limited effect of zopiclone except for increased binding at high doses is also consistent with prior studies suggesting that zopiclone acts at a site distinct from the benzodiazepine receptor.     

Autoradiographic localization of alpha5 subunit-containing GABAA receptors in rat brain

Multiple subtypes of GABAA receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of alpha5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [3H]L-655,708 and [3H]Ro15-1788, an alpha5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [3H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where alpha5 receptors accounted for 20-35% of total [3H]Ro15-1788 binding sites. Low levels of [3H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [3H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.     

[11C]Flumazenil Positron Emission Tomography Visualizes Frontal Epileptogenic Regions

Summary: Presently available noninvasive methods correctly localize epileptogenic regions in only ε50% of patients with frontal lobe epilepsy (FLE). Earlier studies have shown that temporal lobe epileptogenic regions may be identified readily by positron emission tomography (PET) measurements of regional benzodiazepine (BZD) receptor binding. We tested the specific applicability of this method in patients with FLE. Six patients with frontal partial seizures and 7 healthy men were investigated with PET and the BZD receptor ligand [¹¹C]flumazenil. All patients had magnetic resonance (MR) brain scans. The independent assessment of seizure–onset region was based on seizure semiology, intra– and extracranial EEG and, in 4 cases, also on [¹⁸F]fluorodeoxyglucose (FDG) PET. The epileptic focus/seizure-generating region was correctly identified by [¹¹C]flumazenil PET in all patients. This region was characterized by a significant reduction in BZD receptor density. The area with reduced BZD receptor density was better delimited than the corresponding hypometabolic region, which was observed in 50% of patients investigated with [¹⁸F]FDG–PET. MRI was normal in 5 patients. Visualization of BZD receptors with [¹¹C]flumazenil PET appears to be a promising approach for noninvasive identification of frontal lobe epileptogenic regions.     

Benzodiazepine-GABAA Receptors in Idiopathic Generalized Epilepsy Measured with [11C]Flumazenil and Positron Emission Tomography

Summary The neurochemical basis of absence seizures and the mechanism of their suppression by valproate (VPA) are uncertain. We used positron emission tomography (PET) to determine whether an abnormality of [¹¹C]flumazenil binding to benzodiazepine (BZD)-GABA_A receptors exists in patients with childhood and juvenile absence epilepsy and to examine the effects of VPA on [¹¹C] flumazenil binding. The regional cerebral volume of distribution ( V _d) of [¹¹C]flumazenil in patients not treated with VPA was not different from that in normal controls; V _d was lower in patients treated with VPA, and the number of receptors available for binding was significantly reduced in such patients as compared with normal controls. There was no evidence of a primary abnormality of the BZD-GABA, receptor in childhood and juvenile absence epilepsy (CAE/JAE), but the data suggest that treatment with VPA is associated with a reduction in [¹¹C]flumazenil binding that may be relevant to its mode of action in CAE/JAE.