Central-type Benzodiazepine Binding Sites Research Articles

Habituation and inhibitory avoidance training alter brain regional levels of benzodiazepine-like molecules and are affected by intracerebral flumazenil microinjection.

The effects of habituation and inhibitory avoidance training on the rat brain regional levels of benzodiazepine (BZD)-like molecules and on central type BZD binding sites were examined. BZD-like immunoreactivity was decreased by 26-50% in the amygdala, cerebral cortex and septum of rats sacrificed immediately after stepping-down from the platform of an inhibitory avoidance apparatus (non-trained group) as compared to naive controls. Rats submitted to a second step-down session 20 h later (habituated group) have significantly lower BZD-like immunoreactivity in the septum (-60%) as compared to non-trained animals. Rats exposed to an inhibitory avoidance training, i.e. stepping-down and receiving a footshock (trained group), showed a significant reduction in the content of BZD-like molecules in cerebral cortex (-44%), amygdala (-68%), septum (-80%) and hippocampus (-82%) as compared to non-trained rats. In addition, the density of central type BZD binding sites was slightly increased in the hippocampus and septum of trained rats. No changes were observed in the apparent dissociation constant. No changes were observed in parallel measurements of [3H]-L-quinuclidinyl benzylate binding constants at cholinergic muscarinic binding sites. The immediate posttraining intrahippocampal bilateral injection of the central type BZD receptor antagonist flumazenil (10 nmol/hippocampus), enhanced the retention of habituation but not when injected in the amygdala or septum. In contrast, retention of the inhibitory avoidance task was significantly increased by flumazenil administered bilaterally into any of the 3 brain structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative in vivo analysis of benzodiazepine binding sites in the human brain using positron emission tomography

Central-type benzodiazepine binding sites were characterized in a single normal human subject, using positron emission tomography (PET) and the radiolabelled benzodiazepine antagonist, carbon-11 labelled flumazenil ([ 11C]Ro 15-1788). The subject was scanned using tracer alone and tracer plus 4 different concentrations of unlabelled Ro 15-1788, including one concentration of unlabelled Ro 15-1788, chosen to produce maximum displacement of [ 11C]Ro 15-1788 from specific binding sites. Concentrations of free, unmetabolized [ 11C]Ro 15-1788 in plasma were estimated using a simple extraction and ultrafiltration method. Radioactivity in the regional exchangeable pool in brain was estimated under non-saturation conditions from the ratio of radioactivity in brain to plasma, under saturation conditions and the kinetics of free ligand in plasma. The specific binding was, then, estimated by the difference between the total radioactivity in brain and exchangeable pool radioactivity. Scatchard analyses were performed to yield f max and K d values under pseudo-equilibrium conditions, which was observed as an increase of specific binding/free with reduction in specific binding. In cerebral cortex and cerebellum, the B max values were about 62–73 nmol 1 and the K d values were 3.6–6 nM in the estimation of free ligand in plasma and 12–15 nM in the estimation of exchangeable pool in brain, as free in brain.

Central-type benzodiazepines inhibit release of α-melanocyte-stimulating hormone from the rat hypothalamus

In a previous work, we have shown that GABA inhibits the release of α-melanocyte-stimulating hormone (α-melanotropin) from hypothalamic neurons through activation of GABA A receptors [Delbende et al. (1989) Brain Res. 497, 86–93]. Since GABA-gated channel activity can be allosterically modulated by a variety of compounds including benzodiazepines, we have investigated the effect of benzodiazepines in the control of α-melanotropin release by the rat basal hypothalamus. This study was conducted in vitro using perifused rat hypothalamic slices and the amount of α-melanotropin release was monitored with a sensitive and highly specific radioimmunoassay. Infusion of clonazepam (50 μM), a selective agonist for central-type benzodiazepine binding sites, induced an inhibition of KCl (50 mM)evoked α-melanotropin release. The inhibitory effect of clonazepam was rapid and reversible. Administration of Ro 15-1788 (100 μM), a specific antagonist for central-type benzodiazepine receptors or SR 95531, a GABA A receptor antagonist, completely reversed the inhibitory effect of clonazepam. In addition, Ro 15-1788 and SR 95531 both enhanced the amplitude of the response observed during prolonged KCl infusion on α-melanotropin neurons, suggesting the existence of a tonic inhibitory effect of endogenous GABA and/or benzodiazepines in the release of α-melanotropin by hypothalamic neurons. To investigate further the effect of benzodiazepines in the regulation of α-melanotropin neurons, rats were treated in vivo with clonazepam (5 mg/kg) or the non-selective benzodiazepine receptor agonist diazepam (3 mg/kg). Both compounds caused a significant increase in the content of α-melanotropin and β-endorphin in the rat hypothalamus within 3 h. At the pituitary level, diazepam induced a significant increase of α-melanotropin and β-endorphin concentrations in the neurointermediate lobe and corticotropin concentration in the anterior lobe. In contrast, clonazepam had no effect on peptide concentrations in the neurointermediate lobe and slightly increased corticotropin level in the anterior pituitary. Altogether, these results indicate that benzodiazepines exert an inhibitory control on the release of α-melanotropin from hypothalamic neurons via modulation of the GABA A receptor complex. In contrast, the action of benzodiazepines on α-melanotropin-related hormones at the pituitary level appears to be mainly mediated through peripheral-type benzodiazepine receptors.

Stress‐protective Action of β‐Phenyl(GABA): Involvement of Central and Peripheral Type Benzodiazepine Binding Sites

Forced swimming stress caused a significant increase in the density of central type benzodiazepine binding sites in rat cerebral cortex and hippocampus. The number of peripheral type benzodiazepine binding sites was also enhanced on blood platelets. The affinity of neither central nor peripheral type benzodiazepine binding sites was changed considerably after swimming stress. Pretreatment of rats with beta-(phenyl)GABA (100 mg/kg), a GABAB agonist, almost completely eliminated the described changes of the both types of benzodiazepine binding sites caused by swimming stress. In an elevated plus-maze model of anxiety beta-(phenyl)GABA itself was inactive but like diazepam effectively counteracted the behavioural effects of DMCM, a beta-carboline derivative with anxiogenic properties. The possible involvement of benzodiazepine receptors in the mechanism of action of beta-(phenyl)GABA is discussed.

Stereoselective inhibition of the binding of [ 3H]PK 11195 to peripheral-type benzodiazepine binding sites by a quinolinepropanamide derivative

The specific binding of [ 3H]PK 11195 to the peripheral-type benzodiazepine binding site is inhibited by the 1-enantiomer of N,N-diethyl-α-methyl-2-phenyl-4-quinolinepropanamide ((−)Q 1) but not by its d-enantiomer ((+)Q 1). (−)Q 1 inhibited [ 3H]PK 11195 binding to several rat tissues with an IC 50 of less than 10nM whereas (+)Q 1 was at least 500 times less potent. This stereoselectivity was observed in all the tissues tested (brain, heart, kidney and adrenals). The same stereoselectivity was found for the displacement of the binding of [ 3H]PK 11195 in vivo, where (−)Q 1 had an ID 50 between 4–15 mg/kg and (+)Q 1 was completely inactive at all doses tested (i.e. up to 40 mg/kg). Neither isomer had appreciable affinity for central-type benzodiazepine binding sites ([ 3H]diazepam) nor for voltage-sensitive calcium channels ([ 3H]PN 200210 and [ 3H]verapamil).

Clonazepam-induced hyperphagia in nondeprived rats: Tests of pharmacological specificity with R05-4864, Ro5-3663, Ro15-1788 and CGS 9896

Nondeprived male rats were familiarised with daily 60 min access to a highly palatable diet, consisting of powdered rat diet, sweetened condensed milk and water. Clonazepam (0.625–5.0 mg/kg, IP) produced a substantial increase in food consumption within the first 30 min of access. The increase was similar across all dose conditions, suggesting that a maximal effect may have been achieved with a dose as small as 0.625 mg/kg. The hyperphagia induced by clonazepam was reversed by the benzodiazepine receptor antagonist, Ro15-1788 (5.0–20.0 mg/kg), indicating that the effect was benzodiazepine receptor-mediated. Treatments with the peripheral-type benzodiazepine agonist, Ro5-4864, did not induce a hyperphagic response. Instead, food consumption was significantly depressed following the administration of Ro5-4864 at 20 and 40 mg/kg, IP. A comparison of the clonazepam and Ro5-4864 data suggests that benzodiazepine-induced hyperphagia is mediated by central-type benzodiazepine binding sites. The pyrazoloquinoline, CGS 9896, binds with high affinity to benzodiazepine sites and has recently been described as a nonsedating anxiolytic. CGS 9896 (2.5–20.0 mg/kg, administered either IP or PO) did not affect consumption of the highly palatable diet. In consequence, anxiolytic and hyperphagic effects of drug actions at benzodiazepine receptors may be dissociated in the case of this compound. The atypical 1,4-benzodiazepine, Ro5-3663, a GABA antagonist which may act at the picrotoxinin site, produced a dose-related reduction in food consumption. Comparison with the results for Ro5-4864 rules out an interpretation for the anorexia in terms of anxiogenic effects. Ro5-3663 (1.25–5.0 mg/kg, IP) did not diminish the hyperphagia induced by clonazepam (1.25 mg/kg), indicating that it was ineffective as an anorexic agent in the presence of clonazepam. Taken together, these results define more clearly the nature of drug-receptor interactions which may underlie benzodiazepine-induced hyperphagia.

Central-type benzodiazepine binding sites in rat pituitary gland are of the BZ 1 subtype

Experiments were performed to investigate the subtype of central-type benzodiazepine binding site present in rat pituitary gland. B max values for [ 3H] n-propyl β-carboline-3-carboxylate and [ 34H] flunitrazepam (for central-type binding only) were similar. Displacement of [ 3H] flunitrazepam by n-propyl β-carboline-3-carboxylate gave a linear Hofstee plot and a Hill coefficient close to unity. These results suggest that the central benzodiazepine binding sites in the pituitary are predominantly of the BZ 1 subtype, as found in the cerebellum.

Central type benzodiazepine binding sites: A positron emission tomography study in the baboon's brain

An in vivo characterization of specific central type benzodiazepine (BZD) binding sites, labelled with [ 11C]Ro 15-1788 was performed, using positron emission tomography. After i.v. injection of 10 mCi [ 11C]Ro 15-1788 (corresponding to 1 nmol/kg), sequential quantitative tomographic slices of the brain were obtained during 80 min. In some experiments various doses of different cold drugs (BZD agonist or antagonist) were injected i.v. subsequently in order to explore the specificity of the binding of the radioligand in brain structures. The main criteria usually utilized in vitro to demonstrate a specific binding to receptors, such as regional distribution, stereospecificity and saturability of the binding and pharmacological effect linked to the receptor's occupancy, were demonstrated in the brain of a living baboon.

Differential effects of GABA on peripheral and central type benzodiazepine binding sites in brain

The binding of the clinically inactive benzodiazepine [ 3H]RO-5-4864 to brain membranes was investigated. The peripheral type benzodiazepine binding site was demonstrated in brain with an apparent K d of 1.6 nM and a B max of 290 fmol/mg protein. Monophasic Scatchard plots indicate a homogenous population of high affinity sites. The major inhibitory transmitter GABA, has no effect on [ 3H]RO-5-4864 binding to extensively washed brain membranes. The peripheral type benzodiazepine binding site found in brain is therefore not modulated by GABA.