Benzodiazepine Receptors In Brain Research Articles

In vivo mapping of brain benzodiazepine receptor changes by positron emission tomography after focal ischemia in the anesthetized baboon.

Recent reports have shown an increase in specific binding (in vitro) of [3H]PK 11195 to peripheral-type benzodiazepine receptors in both experimental animals and humans, reflecting a glial/macrophagic reaction within and around focal ischemic insults. We have evaluated by positron emission tomography the time course of changes in brain uptake in vivo of 11C-labeled PK 11195 and flumazenil (an antagonist of central benzodiazepine receptors) as indirect and direct markers of neuronal loss, respectively, after focal cerebral ischemia. Ten anesthetized baboons were submitted to sequential positron emission tomography studies between day 1 and day 91 after unilateral middle cerebral artery occlusion. The studies consisted of successive assessments, in the same positron emission tomography session, of [11C]PK 11195, [11C]flumazenil, cerebral blood flow, and oxygen consumption; late computed tomographic scans were obtained to map the approximate contours of infarction and to define a concentric peri-infarct area. We found a significant time-dependent increase in [11C]PK 11195 uptake in the peri-infarcted area, maximum at 20 to 40 days after occlusion. In contrast, there was a time- and perfusion-independent significant decrease in [11C]flumazenil uptake in the infarcted area, stable from day 2 onward, and already present in one baboon at day 1. Challenge studies with saturating doses of cold ligands confirmed that these changes represented alterations in specific binding. [11C]Flumazenil uptake was not affected in hypometabolic (but apparently noninfarcted, ie, deafferented) cortical areas. The delayed and apparently transient increases in [11C]PK 11195 specific uptake in vivo presumably represent glial/macrophage reaction; the marked depression in [11C]flumazenil specific binding, which appears selective for synaptic damage, is both precocious and sustained and thus may be better suited for the early assessment of ischemic damage in humans.

Intracerebroventricular injection of naloxone blocks melatonin-dependent brain [3H]flunitrazepam binding.

The pineal hormone melatonin modulates the brain benzodiazepine binding sites and its circadian rhythm. In the present study the effects of intracerebroventricular (i.c.v.) administration of naloxone (10-20 ng), alone or in association with melatonin and/or beta-endorphin, on [3H]flunitrazepam ([3H]FNZ) binding to the rat cerebral cortex of hypophysectomized rats was investigated. Melatonin (10-20 ng), beta-endorphin (10-20 ng), and melatonin + beta-endorphin (10-20 ng of each compound) all increased [3H]FNZ binding to a similar extent and in a dose-related manner. The effects of melatonin (10 ng) on [3H]FNZ binding were prevented by simultaneous injection with the specific opioid antagonist naloxone. Naloxone also blocks, although to a lesser extent, the effects of beta-endorphin and of melatonin + beta-endorphin injections. Moreover, naloxone blocks the hypophysectomy-dependent increase in [3H]FNZ binding. These results implicate the modulation of melatonin-dependent changes on brain benzodiazepine receptors by opioid peptides.

Modeling Analysis of [11C]Flumazenil Kinetics Studied by PET: Application to a Critical Study of the Equilibrium Approaches

The multi-injection modeling approach was used for the in vivo quantitation of benzodiazepine receptors in baboon brain using positron emission tomography (PET) and [11C]flumazenil (RO 15-1788) as a specific ligand. The model included three compartments (plasma, free, and bound ligand) and five parameters (including the benzodiazepine receptor concentration). The plasma concentration after correction for the metabolites was used as the input function. The experimental protocol consisted of four injections of labeled and/or unlabeled ligand. This protocol allows the evaluation, from a single experiment, of the five model parameters in various regions of interest. For example, in the temporal cortex, the concentration of receptor sites available for binding (B'max) and the equilibrium dissociation constant (Kd) were estimated to be 70 +/- 15 pmol/ml and 15.8 +/- 2.2 nM, respectively. The validity of the equilibrium approach, which is the most often used quantitation method, has been studied from simulated data calculated using these model parameters. The equilibrium approaches consist of reproducing in PET studies the experimental conditions that permit the use of the usual in vitro methods such as Scatchard analysis. These approaches are often open to criticism because of the difficulty of defining the notion of equilibrium in in vivo studies. However, it appears that the basic relation of Scatchard analysis is valid over a broader range of conditions than those normally used, such as the requirement of a constant bound/free ratio. Simulations showed that the values of the receptor concentration (B'max) and the equilibrium dissociation constant (Kd) found using Scatchard analysis are always underestimated. These simulations also suggest an explanation concerning the dependency of B'max and Kd on the time point employed for the Scatchard analysis, a phenomenon found by several authors. To conclude, we propose new protocols that allow the estimation of the B'max and Kd parameters using a Scatchard analysis but based on a protocol including only one or two injections. These protocols being entirely noninvasive, it thus becomes possible to investigate possible changes in receptor density and/or affinity in patients.

Effect of sertraline treatment on benzodiazepine receptors in the rat brain.

In this paper we describe the modification of benzodiazepine (BDZ) binding sites in the rat brain after different times of treatment with the 5-hydroxytryptamine-(5HT) uptake blocker sertraline. We investigated the effect of 8, 15 and 30 days sertraline treatment (10 mg/kg/day, i.p.) on 3 H-flunitrazepam binding sites. In order to describe the anatomical site of action of the drug, the experiment has been carried out by means of quantitative receptor autoradiography. After 8 days of sertraline treatment, an increase of BDZ receptor density is found in the olfactory tubercle. This effect is reversed at 15 and 30 days. At 15 days of treatment, an increase is found in the anterior cingulate cortex. This increase is still present after 30 days of treatment. At 30 days of treatment, we also found an increase of BDZ receptor density in the frontoparietal motor cortex and in the septal nuclei. The Scatchard plots obtained from the saturation experiments indicate that this increase of the receptor density is due to an increase of both the receptor number and affinity. All the other investigated areas are unaffected by the sertraline treatment. The possible neurochemical basis of these BDZ receptor regulation by sertraline and its influence in the therapeutical profile are discussed.

Portal-systemic encephalopathy: a disorder of neuron-astrocytic metabolic trafficking.

Portal-systemic encephalopathy (PSE) is a major neuropsychiatric complication of chronic liver disease. Neuropathologic evaluation of brain tissue from cirrhotic patients who died in hepatic coma reveals astrocytic (rather than neuronal) changes referred to as Alzheimer type II astrocytosis. Evidence to date suggests that Alzheimer type II astrocytosis is the result of ammonia neurotoxicity. Exposure of cultured astrocytes to concentrations of ammonia equivalent to those encountered in brain in experimental PSE results in Alzheimer type II astrocytosis as well as changes in other astrocytic parameters such as glial fibrillary acidic protein and glycogen metabolism. A second characteristic of PSE is the appearance of increased densities of 'peripheral-type' benzodiazepine receptors (PTBRs) in both brain and peripheral tissues. In brain, PTBRs are highly localized on astrocytic outer mitochondrial membranes. Experimental PSE resulting from portacaval anastomosis in the rat results in increased densities of PTBRs in brain and in increased expression of the endogenous PTBR ligand octadecaneuropeptide in nonneuronal elements. It is suggested that the PTBR and its endogenous ligands could mediate the astrocytic response to chronic exposure to ammonia in PSE. Astrocytic changes in PSE are accompanied by disruption of neuron-astrocytic metabolic trafficking. In particular, reuptake of the neurotransmitter glutamate into the perineuronal astrocyte is inhibited in PSE resulting in glutamatergic synaptic dysregulation and potentially compromised astrocytic energy metabolism. Astrocytic metabolism of monoamine neurotransmitters may also be increased as a result of increased activities of monoamine oxidase MAOB.

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]Ro 15-1788 (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.

In vivo binding of [ 11C]Ro15-4513 in human brain measured with PET

Ro15–4513, an azide derivative of benzodiazepine antagonist flumazenil (Ro15–1788), and Ro15–1788 were labelled with carbon 11. Sequential PET scans following injection of [ 11C]Ro15–4513 or [ 11C]Ro15–1788 into normal male healthy volunteers were measured, and kinetic analysis using pons as a reference region was performed. [ 11C]Ro15–4513 was highly accumulated in frontal cortex, temporal cortex, hippocampus and relatively lower accumulation in occipital cortex, whereas almost homogeneous distribution of Ro15–1788 throughout cortex area was seen. The kinetic analysis revealed that such differences of regional distribution in brain between two labelled ligands were mainly due to the regional difference of the dissociation rate constants in vivo ( k 4). [ 11C]Ro15–4513 may be a useful tool for the in vivo study of benzodiazepine receptors in human brain.

Chronic ethanol consumption and withdrawal affects mitochondrial benzodiazepine receptors in rat brain and peripheral organs

Ethanol administration to rats for 30 days resulted in a significant decrease (−28%; P< 0.05) in the density of mitochondrial benzodiazepine receptors (MBR) in the olfactory bulb. The reduction in [ 3H]PK 11195 binding persisted 24 hr after cessation of alcohol and had returned to normal values when measured 4 days later. Alterations were confined to this brain region and were not detected in the cerebral cortex, cerebellum or hippocampus. [ 3H]PK 11195 binding was elevated in the liver (100%; P<0.01), heart (43%; P< 0.01) and testis (27%; P<0.05) 30 days following ethanol consumption and this persisted for 1 and 4 days after abrupt withdrawal. A transitory decrease (−20%; P<0.05) in MBR density was observed in the adrenal gland following 30 days of alcohol ingestion, but disappeared during withdrawal. The alterations in these receptors may be relevant to the cellular damage or dysfunction induced by chronic exposure to ethanol.

Central and peripheral benzodiazepine receptors in rat brain and platelets: effects of treatment with diazepam and clobazam

Tolerance to the effects of benzodiazepines (BZ) may be mediated by changes in benzodiazepine receptors (BZRs). Peripheral BZRs (in brain and platelets) and central BZRs (in brain) were measured in rats following intraperitoneal administration of diazepam and clobazam each for 4 and 12 days. BZRs were measured by binding assays using [ 3H] PK 11195 (peripheral) and [ 3H] flunitrazepam (central) as radioligands. Diazepam, but not clobazam, increased peripheral BZR numbers in platelets (both P < 0.005), but not in brain, after 4 and 12 days' treatment compared with appropriate controls. Neither drug altered central BZR affinities or numbers in rat brain. BZ effects on peripheral BZRs in platelets cannot be extrapolated to predict changes in brain receptors, either peripheral or central.

Ligand-dependent effects of ethanol and diethylether at brain benzodiazepine receptors

The GABAA receptor chloride channel complex interacts with various categories of sedatives, including the benzodiazepines, and possibly ethanol and volatile general anesthetics. Thus, specific binding of tritiated derivatives of a benzodiazepine antagonist, flumazenil, and an agonist, flunitrazepam, to rat brain membrane fragments was monitored at equilibrium in the presence and absence of anesthetizing concentrations of ethanol and diethylether. Ethanol produced a concentration-dependent inhibition of [3H]flumazenil binding, which was not reversed by the GABAA receptor competitive antagonist bicuculline, but had no effect on [3H]flunitrazepam binding. Both ethanol and diethylether decreased the affinity of the benzodiazepine site for [3H]flumazenil. These data indicate that ethanol and diethylether have GABA-independent effects at the benzodiazepine sites of the GABAA receptor. These findings are inconsistent with a two-state functional model of the benzodiazepine site and, instead, support a model containing a specific, antagonist-favored conformation.

Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: Characterization of receptor binding sites and receptor assay for benzodiazepine drugs

A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.

Binding of strychnocarpine and related β-carbolines to brain receptors in vitro

The affinity of strychnocarpine and related β-carbolines for serotonin, benzodiazepine, tryptamine, opiate and GABA receptors in rat brain was studied. Strychnocarpine showed a low to very low affinity for all the receptors tested. The weak binding to tryptamine receptors might explain part of the tremorigenic effects found earlier in the in vivo studies.

Central benzodiazepine receptors in human brain: estimation of regional B max and K D values with positron emission tomography

Studies of central benzodiazepine receptors in the human brain in vivo are now possible using positron emission tomography (PET) and [ 11C]flumazenil. With the aim of measuring B max and K d in brain regions, we used a two-injection [ 11C]flumazenil (at high and low specific radioactivity, respectively) pseudo-equilibrium paradigm to evaluate, in seven unmedicated healthy volunteers, the relative merits of three ‘reference’ structures (pons, hemispheric white matter and corpus callosum) in which the free radioligand concentration in brain tissue was estimated 15–40 min after i.v. injection of the radioligand. By means of high-resolution PET, the B max and K d were calculated for each subject in 18 gray matter structures, based on a two-point Scatchard plot. We found that the use of the corpus callosum as reference often resulted in spurious B max and K d values. The pons was the best reference structure because it provided satisfactory B max values (closest to in vitro data) and most consistent K d values, and was the region easiest to sample on PET images. The pattern of regional B max was consistent with that expected from in vitro studies, with values highest in the cerebral cortex, intermediate in the cerebellum, and lowest in the striatum and the thalamus. The K d values were uniform among regions and were consistent with earlier in vitro and in vivo data. This work documents the feasibility of estimating B max and K d of central benzodiazepine receptors in multiple brain regions for clinical research.

Multiple neurochemical action of clozapine: A quantitative autoradiographic study of DA 2, opiate and benzodiazepine receptors in the rat brain after long-term treatment

The atypical neuroleptic clozapine has clinical and behavioral properties that differ not only from the typical compounds, but also from atypical ones. It interacts with the dopaminergic systems, but also produces effects on the serotoninergic, GABA-ergic, cholinergic systems. In spite of the amount of papers devoted to its study, the profile of the neurochemical action of this drug is still confuse. In this paper we investigated the DA2-, opiate- and benzodiazepine-receptor modifications induced by the long term (21 days) treatment with clozapine 20 mg/kg/day in the rat brain. We found a decrease of DA2 receptor density in the target areas of the mesolimbocortical system (ventral n. caudate-putamen, cerebral cortex except for the anterior cingulate at the most anterior level and the n. accumbens) and a decrease of opiate and benzodiazepine receptors in the cerebral cortex and in the olfactory tubercle. Opiate receptors increase in the patches of the striatum. We also compared these effects with those produced by long-term (21 days), low-dosage (0.5 mg/kg day) haloperidol.

Ontogeny of the benzodiazepine receptor in human brain: fluorographic, immunochemical, and reversible binding studies.

The prenatal and postnatal human ontogeny of the central benzodiazepine receptor was investigated in six different brain regions between week 24 postconception and age 14 years. Binding studies, which were performed with [3H]flunitrazepam [( 3H]FNZ), revealed a steep increase in receptor density postnatally in frontal cortex and cerebellum. Bmax values were higher in medulla oblongata, pons, and thalamus than in cortex and cerebellum up to week 26. After that, receptor densities declined significantly in medulla and olive. The same tendency was apparent in pons, whereas receptor density remained unchanged in thalamus. The early ontogeny of the benzodiazepine receptor was also evaluated in fluorographs [( 3H]FNZ) and immunoblots using the alpha 1-subunit-specific monoclonal antibody (mAb) bd-24. Specific radiolabeled proteins with molecular weights of 53K and 59K were visible in cortical membranes from gestational week 8, the earliest time investigated. During further development, the intensity of the 53K band increased without changes in the 59K band. As in other species, postmortem proteolysis in human brain led to a specifically labeled peptide of 47K. The mAb bd-24 immunolabeled only the 53K protein and the 47K peptide.

Compartmental analysis of [11C]flumazenil kinetics for the estimation of ligand transport rate and receptor distribution using positron emission tomography.

The in vivo kinetic behavior of [11C]flumazenil ([11C]FMZ), a non-subtype-specific central benzodiazepine antagonist, is characterized using compartmental analysis with the aim of producing an optimized data acquisition protocol and tracer kinetic model configuration for the assessment of [11C]FMZ binding to benzodiazepine receptors (BZRs) in human brain. The approach presented is simple, requiring only a single radioligand injection. Dynamic positron emission tomography data were acquired on 18 normal volunteers using a 60- to 90-min sequence of scans and were analyzed with model configurations that included a three-compartment, four-parameter model, a three-compartment, three-parameter model, with a fixed value for free plus nonspecific binding; and a two-compartment, two-parameter model. Statistical analysis indicated that a four-parameter model did not yield significantly better fits than a three-parameter model. Goodness of fit was improved for three- versus two-parameter configurations in regions with low receptor density, but not in regions with moderate to high receptor density. Thus, a two-compartment, two-parameter configuration was found to adequately describe the kinetic behavior of [11C]FMZ in human brain, with stable estimates of the model parameters obtainable from as little as 20-30 min of data. Pixel-by-pixel analysis yields functional images of transport rate (K1) and ligand distribution volume (DV"), and thus provides independent estimates of ligand delivery and BZR binding.

Genetic difference in sensitivity to β-carboline: Evidence for the involvement of brain benzodiazepine receptors

The convulsive effects of methyl β-carboline-3-car☐ylate (β-CCM), a benzodiazepine receptor ligand, are different in two inbred strains of mice: BALB/cBy mice are more sensitive to β-CCM than C57BL/6J mice. In the present article, we report the effects of [3H]flunitrazepam binding in these two strains, which suggest a possible explanation of the differences in their sensitivity to β-CCM by the involvement of brain benzodiazepine receptors.

SPECT imaging of the benzodiazepine receptor in non-human primate brain with [ 123I]Ro 16-0154

We have used SPECT (single photon emission computed tomography) imaging in non-human primates to examine the time course and pharmacological specificity of 123I-labeled Ro 16-0154 as an in vivo probe of the benzodiazepine receptor. Maximal brain uptake was reached approximately 70 min post i.v. administration of the radioligand and represented approximately 10% of the injected dose. The regional distribution of radioactive densities was consistent with the known distribution of benzodiazepine receptor in primate brain, with highest uptake localized over the occipital area. Washout of radioactivity was relatively slow with a rate of 3% per hour after the time of peak radioactivity. Injection of the benzodiazepine antagonist Ro 15-1788 (0.2–0.3 mg/kg i.v.) caused a rapid decrease of more than 90% of radioactivity from brain. In summary, [ 123I]Ro 16-0154 is a promising in vivo SPECT radioligand for the benzodiazepine receptor, with high brain uptake, a stable period of peak radioactivity, appropriate regional distribution, and ability to be displaced by other benzodiazepine receptor agents.

Changes in benzodiazepine receptors of rat brain after long-term treatment with the Ca 2+-antagonists nifedipine, verapamil, flunarizine and with the calmodulin antagonist trifluoperazine

1. The binding of [ 3H]flunitrazepam to benzodiazepine receptors in the cerebral cortex and hippocampus (membrane fraction P 2) was studied after 13-day oral treatment of male Wistar rats with the Ca 2+-antagonists nifedipine (20 mg/kg), verapamil (50 mg/kg), flunarizine (10 mg/kg) and with the calmodulin (CaM)-antagonist trifluoperazine (TFP) (3 mg/kg). 2. The changes in the binding characteristics of the benzodiazepine receptors in the frontal cortex were studied in vitro after the addition of nifedipine (10 -6 and 10 -5M) and verapamil (10 -6 and 10 -5M). 3. A significant decrease of the binding capacity ( B max) of [ 3H]flunitrazepam was established after in vivo treatment with the three Ca 2+-antagonists as well as after TFP, the decrease being much more pronounced in the hippocampus. 4. Changes in the affinity values ( K d) of [ 3H]flunitrazepam binding were found in neither of the groups. 5. No data for a direct interaction of nifedipine and verapamil with the brain benzodiazepine receptors were obtained in in vitro experiments.

Endogenous Benzodiazepine Receptor Ligands in Human and Animal Hepatic Encephalopathy

The role of endogenous benzodiazepine receptor ligands in the pathogenesis of hepatic encephalopathy was studied in humans and in rat models of hepatic encephalopathy. Endogenous benzodiazepine ligands were extracted from rat brain and human CSF by acid treatment and purification by HPLC. Detection and partial characterization of these endogenous benzodiazepine ligands were carried out using both radioreceptor binding assays and radioimmunoassays with anti-benzodiazepine antibodies. Four different benzodiazepine receptor ligands were identified in human and rat tissue, two of which may be diazepam and desmethyldiazepam, based on elution profiles and anti-benzo-diazepine antibody reactivity. Human CSF and serum from patients with hepatic encephalopathy contained approximately 10 times more endogenous benzodiazepine receptor ligand than CSF from controls or nonencephalopathic patients with liver disease. The levels of brain benzodiazepine receptor ligand compounds were also increased approximately 10-fold in rats suffering from fulminant hepatic failure, but not in rats with portacaval shunts, a model of chronic hepatic disease. The increased concentrations of these substances could be behaviorally significant and may contribute to the pathogenesis of hepatic encephalopathy.