Effects of NOS inhibitors on the benzodiazepines-induced memory impairment of mice in the modified elevated plus-maze task.

The aim of the present study was to examine the effects of nitric oxide synthase (NOS) inhibitors on responses, elicited by benzodiazepines (BZs) in a modified elevated plus-maze task in mice. It was shown that acute doses of diazepam (DZ; 1 and 2 mg/kg) and flunitrazepam (FNZ; 0.05, 0.1 and 0.2 mg/kg) significantly increased the time of transfer latency (TL2) in a retention trial, thus confirming memory impairing effects of BZs. l-NAME (N(G)-nitro-l-arginine methyl ester; 200 mg/kg), a non-selective inhibitor of NOS, and 7-NI (7-nitroindazole; 40 mg/kg), a selective inhibitor of NOS, further intensified DZ-induced memory impairment. On the other hand, L-NAME (50, 100 and 200 mg/kg) and 7-NI (10, 20 and 40 mg/kg) prevented FNZ-induced memory compromising process. The results of this study indicated that suppressed NO synthesis enhanced DZ-induced but prevented FNZ-induced memory impairment. Taken together, these findings could suggest NO involvement in BZs-induced impairment of memory processes. The precise mechanism of these controversial effects, however, remains elusive.

Quantitative analyses of antagonism: combinations of midazolam and either flunitrazepam or pregnanolone in rhesus monkeys discriminating midazolam.

Adverse effects of benzodiazepines limit their clinical use; these effects might be reduced without altering therapeutic effects by administering other positive GABA(A) modulators (i.e., neuroactive steroids) with benzodiazepines. One concern with this strategy involves reversing these combined effects in case of overdose. The current study examined whether flumazenil can attenuate the combined effects of two benzodiazepines, midazolam and flunitrazepam, and the combined effects of midazolam and the neuroactive steroid pregnanolone, in four monkeys discriminating midazolam. Each positive modulator produced ≥80% midazolam-lever responding. Interactions between midazolam and either flunitrazepam or pregnanolone were additive. Flumazenil antagonized the benzodiazepines when they were administered alone or in combination. Schild analyses yielded slopes that did not deviate from unity, regardless of whether benzodiazepines were administered alone or together; the pA(2) value for flumazenil was 7.58. In contrast, flumazenil enhanced the effects of pregnanolone with 0.32 mg/kg flumazenil shifting the pregnanolone dose-effect curve 2-fold leftward. Flumazenil attenuated the combined effects of midazolam and pregnanolone, although antagonism was not dose-dependent. Thus, the interaction between two benzodiazepines was similar to that of a benzodiazepine and a neuroactive steroid; however, flumazenil more efficiently attenuated a combination of two benzodiazepines compared with a combination of a benzodiazepine and a neuroactive steroid. Although the magnitude of antagonism of a benzodiazepine combined with a neuroactive steroid was reduced, these results support continued exploration of the use of combinations of positive modulators to enhance therapeutic effects while reducing adverse effects.

Mercury interaction with the GABA(A) receptor modulates the benzodiazepine binding site in primary cultures of mouse cerebellar granule cells.

Mercury compounds are neurotoxic compounds with a great specificity for cerebellar granule cells. The interaction of mercury compounds with proteins in the central nervous system may underlie some of their effects on neurotransmission. In this work we study the interaction of mercuric chloride (HgCl2) and methylmercury (MeHg) with the GABA(A) receptor in primary cultures of cerebellar granule cells. Both compounds increased, dose dependently, the binding of [3H]flunitrazepam to the benzodiazepine recognition site. EC50 values for this effect were 3.56 and 15.24 microM for HgCl2 and MeHg, respectively, after 30 min exposure of intact cultured cerebellar granule cells. The increase of [3H]flunitrazepam binding by mercury compounds was completely inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxinin, and by the organochlorine pesticide alpha-endosulfan. It was also partially inhibited by the anion transporter blocker DIDS, however this effect could be due to a possible chelation of mercury by DIDS. Intracellular events, like intracellular calcium, kinase activation/inactivation or antioxidant conditions did not affect [3H]flunitrazepam binding or its increase induced by mercury compounds. The sulfhydryl alkylating agent N-ethylmaleimide mimicked the effect of mercury compounds on [3H]flunitrazepam binding suggesting a common mechanism. We conclude that mercury compounds interact with the GABA(A) receptor by the way of alkylation of SH groups of cysteinyl residues found in GABA(A) receptor subunit sequences.

Benzodiazepine receptor ligands. III. Synthesis and biological evaluation of 2- and/or 3-substituted pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides.

A new series of 2- and/or 3-substituted pyrazolo [5,1-c][benzotriazine 5-oxides and their 8-chloro derivatives were synthesized, and their benzodiazepine receptor (BZR) affinities were evaluated in vitro in comparison to lead compound 3-ethoxycarbonyl-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (29) [1,2]. None of the new compounds showed significant affinity for BZR. On the basis of a pharmacophore/receptor model suggested for lead compound 29, some hypotheses to explain the inactivity of new derivatives are discussed.

The effect of chronic benzodiazepines exposure on body weight in rats.

Changes in body weight (BW) in female rats treated for 5 weeks (wk) with weekly subcutaneous implantation of silastic capsules containing different benzodiazepines (BZs): diazepam (DZ) 90, 180, 360 and 540 mg wk-1; nordiazepam (ND) 600 mg wk-1; oxazepam (OX) 600 mg wk-1 and flunitrazepam (FN) 540 mg wk-1 and in male rats exposed to DZ (540 mg wk-1) were evaluated herein. Rats (female and male) implanted with empty capsules served as controls. The BW gain was significantly higher in male than in female rats (both DZ-treated and controls). The BW gain increased with increasing doses of DZ but slowed with time of exposure. In comparison to control rats, the BW gain was significantly higher in DZ-(540 mg wk-1) and OX- but not in ND- and FN-treated female rats. However, the differences between BZs were not of statistical significance. In rats exposed to empty capsules (male, female); DZ (male); ND and OX (female) the BW gain increased with time (1-4 wk) while in rats exposed to DZ and FN (female) the BW stabilised within 2 wk. Acute injection of the central BZ receptor antagonist, flumazenil (40 mg kg-1, i.v., 5th wk of chronic exposure), tended to inhibit the time-related BW gain in rats exposed to empty capsules (male, female), DZ (male), ND and OX (female) but did not affect the BW in DZ- (540 mg wk-1) and FN-exposed rats (female) where BW stabilised prior to FLU injection. Repeated administration of flumazenil (30 mg kg-1 wk-1, i.p.) did not affect the BW gain in DZ- and ND-treated female rats. The present data indicate that different BZs have different effects on BW gain in the rat suggesting that different subtypes of BZ receptors are involved.

Voltage-dependent deactivation and desensitization of GABA responses in cultured murine cerebellar granule cells.

1. Electrophysiological recordings of GABAergic IPSCs and responses to applications of exogenous GABA were made from cultured murine cerebellar granule cells. In both the presence and absence of tetrodotoxin, depolarization of the postsynaptic cell consistently produced a broadening of the IPSC. This voltage-dependent change in kinetics arose entirely from a slowing of the rate of current decay. The duration of miniature IPSCs was increased by a significant but lesser amount by the GABA uptake inhibitor nipecotic acid (300 microM). 2. Five millisecond applications of 1 mM GABA elicited rapidly activating, biexponentially deactivating currents in patches derived from granule cell bodies. Deactivation of these responses was slowed by membrane depolarization. This effect arose from an increased fractional participation of the slow component of deactivation. The benzodiazepine flunitrazepam (1 microM) slowed deactivation at a holding potential of -70 mV but not at +50 mV. 3. Longer-lasting applications of GABA produced substantial biexponential macroscopic desensitization. The rate of desensitization was faster at a holding potential of +50 mV than at -70 mV. The speeding of desensitization at depolarized membrane potentials arose from an increase in the fractional contribution of the fast component of desensitization. 4. When two 5 ms, 1 mM GABA applications were made at an interstimulus latency of 150 ms, the second response was consistently smaller than the first. The depression of the second response was significantly heightened when the membrane potential was depolarized from -70 to +50 mV. 5. The degree of desensitization produced was closely linked to receptor occupancy. The rate of current deactivation was also voltage dependent when non-saturating, and therefore less desensitizing, applications of GABA were analysed. In contrast, both the GABA EC50 (approximately 30 microM) and the current activation kinetics at near EC50 agonist concentrations appeared to be voltage independent.

N3-phenacyluridine, a novel hypnotic compound, interacts with the benzodiazepine receptor.

N3-Phenacyluridine (3-phenacyl-1-beta-D-ribofuranosyluracil) has potent sedative and hypnotic activities following intracerebroventricular injection in mice. To study the mechanism of action of N3-phenacyluridine, the interaction of this compound with the benzodiazepine receptor has been investigated. Results obtained showed that this compound inhibited specific binding of [3H]flunitrazepam to synaptic membranes of bovine cortex in a concentration-dependent fashion (IC50 = 129 microM). Scatchard analysis of [3H]flunitrazepam binding revealed that N3-phenacyluridine interacted with the ligand at the benzodiazepine receptor binding site in a competitive manner. Ro15-1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a ]1, 4-benzodiazepine), a benzodiazepine receptor antagonist, also inhibited the specific binding of [3H]flunitrazepam in the presence of the compound. The results suggest that the pharmacological activity of N3-phenacyluridine may be partially mediated through the benzodiazepine receptor.

Molecular mechanisms of benzodiazepine-induced down-regulation of GABAA receptor alpha 1 subunit protein in rat cerebellar granule cells.

1. Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit protein, that was dose-dependent (1 nM-1 microM) and prevented by the benzodiazepine antagonist flumazenil (1 microM). After 2 days of treatment with 1 microM flunitrazepam the alpha 1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treatment. Chronic flunitrazepam treatment did not significantly alter the GABAA receptor alpha 6 subunit protein over the 2-12 day period. 2. GABA treatment for 2 days down-regulates the alpha 1 subunit protein in a dose-dependent (10 microM-1 mM) manner that was prevented by the selective GABAA receptor antagonist bicuculline (10 microM). At 10 microM and 1 mM GABA the reduction in alpha 1 subunit expression compared to controls was 31% and 66%, respectively. 3. The flunitrazepam-induced decrease in alpha 1 subunit protein is independent of GABA, which suggests that it involves a mechanism distinct from the GABA-dependent action of benzodiazepines on GABAA receptor channel activity. 4. Simultaneous treatment with flunitrazepam and GABA did not produce an additive down-regulation of alpha 1 subunit protein, but produced an effect of the same magnitude as that of flunitrazepam alone. This down-regulation induced by the combination of flunitrazepam and GABA was inhibited by flumazenil (78%), but unaffected by bicuculline. 5. The flunitrazepam-induced down-regulation of alpha 1 subunit protein at 2 days was completely reversed by the protein kinase inhibitor staurosporine (0.3 microM). 6. This study has shown that both flunitrazepam and GABA treatment, via their respective binding sites, caused a reduction in the expression of the GABAA receptor alpha 1 subunit protein; an effect mediated through the same neurochemical mechanism. The results also imply that the benzodiazepine effect is independent of GABA, and that the benzodiazepine and GABA sites may not be equally coupled to the down-regulation process, with the benzodiazepine site being the more dominant. The biochemical mechanism underlying the benzodiazepine-mediated down-regulation of the alpha 1 subunit protein seems to involve the activity of staurosporine-sensitive protein kinases.

Synthesis and pharmacological properties of novel 8-substituted imidazobenzodiazepines: high-affinity, selective probes for alpha 5-containing GABAA receptors.

The synthesis and pharmacological properties of imidazobenzodiazepines with both high affinity and selectivity for alpha 5-containing GABAA receptors are described. Four of these compounds (5, 6, 8, and 9) inhibited [3H]flunitrazepam binding to recombinant alpha 5 beta 2 gamma 2 GABAA receptors with IC50 values between approximately 0.4 and 5 nM. These compounds were > or = 24-75-fold more selective for recombinant receptors containing alpha 5 subunits compared to other, "diazepam-sensitive" GABAA receptors containing either alpha 1, alpha 2, or alpha 3 subunits. Imidazobenzodiazepine 9 (used as the prototypical alpha 5 selective ligand) inhibited [3H]flunitrazepam binding to hippocampal membranes with high- and low-affinity components (IC50 0.6 +/- 0.2 and 85.6 +/- 13.1 nM, respectively), representing approximately 16% and approximately 84% of the receptor pool. Inhibition of [3H]flunitrazepam binding to cerebellar membranes with imidazobenzodiazepine 9 was best fitted to a single population of sites with an IC50 of 79.8 +/- 18.3 nM. These imidazobenzodiazepines behaved as GABA negative ligands in recombinant GABAA receptors expressed in Xenopus oocytes and were convulsant in mice after parenteral administration. The relative potencies of flumazenil and zolpidem in blocking convulsions induced by 9 and DMCM, respectively, indicated that occupation of alpha 5-containing GABAA receptors substantially contributed to the convulsant properties of acetylene analog 9. These 8-substituted imidazobenzodiazepines (5, 6, 8 and 9) should prove useful in examining the physiological roles of GABAA receptors bearing an alpha 5 subunit and may also lead to the development of novel, subtype selective therapeutic agents.

High-affinity partial agonist imidazo[1,5-a]quinoxaline amides, carbamates, and ureas at the gamma-aminobutyric acid A/benzodiazepine receptor complex.

A series of imidazo[1,5-a]quinoxaline amides, carbamates, and ureas which have high affinity for the gamma-aminobutyric acid A/benzodiazepine receptor complex was developed. Compounds within this class have varying efficacies ranging from antagonists to full agonists. However, most analogs were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Many of these compounds were also effective in antagonizing metrazole-induced seizures in accordance with anticonvulsant and possible anxiolytic activity. Selected quinoxalines displayed limited benzodiazepine-type side effects such as ethanol potentiation and physical dependence in animal models. Dimethylamino urea 41 emerged as the most interesting analog, having a partial agonist profile in vitro while possessing useful activity in animal models of anxiety such as the Vogel and Geller assays. In accordance with its partial agonist profile, 41 was devoid of typical benzodiazepine side effects.

Comparison of benzodiazepine receptor ligands with partial agonistic, antagonistic or partial inverse agonistic properties in precipitating withdrawal in squirrel monkeys.

Benzodiazepine receptor (BZR) ligands previously characterized as differing in intrinsic efficacy were evaluated first for potency in antagonizing flunitrazepam-induced sleep in monkeys. Data from these experiments were used to define approximately equieffective doses for subsequent use in precipitating withdrawal in diazepam-treated monkeys. It was shown that partial agonists with intermediate intrinsic efficacy (bretazenil, Ro 41-7812) were relatively ineffective in precipitating withdrawal reactions in diazepam-treated squirrel monkeys. The potent and specific BZR antagonist flumazenil, which possesses weak intrinsic efficacy, was more effective in precipitating a withdrawal reaction in diazepam-treated monkeys. In contrast, the highest dose of the BZR antagonist ZK 93426 that could be administered failed to precipitate withdrawal under the same experimental conditions. Finally, the BZR partial inverse agonist sarmazenil was the most effective of these BZR ligands in eliciting a precipitated withdrawal reaction. Thus, the results of the present investigation strongly suggest that BZR ligands differing in intrinsic efficacy differentially precipitate withdrawal in squirrel monkeys treated chronically with diazepam.

[The effect of flumazenil in reversing midazolam, flunitrazepam or diazepam].

In 31 adult patients who had undergone spinal or epidural anesthesia, we evaluated the effect of flumazenil in reversing midazolam, flunitrazepam or diazepam. The patients received midazolam 5 mg, flunitrazepam 1 mg or diazepam 5 mg 15 min after the spinal or epidural anesthesia. After the completion of operation, flumazenil (0.2 mg-1.0 mg) was administered until the patient became awake. Blood pressure, pulse rate and respiratory rate before and after administration of flumazenil showed no statistically significant changes in these groups. There were no significant differences in necessary amount of flumazenil among these groups. The time necessary for the patient to be awake in midazolam group was significantly shorter than that in flunitrazepam or diazepam group. Half of the patients in flunitrazepam and diazepam groups slept again after leaving the operating room, but they presented no clinical problems. In conclusion, we consider that flumazenil does not affect circulation and respiration, so it seems to be safe and effective for reversing benzodiazepins in clinical situation.

Direct effects of the anabolic/androgenic steroids, stanozolol and 17 alpha-methyltestosterone, on benzodiazepine binding to the. gamma-aminobutyric acid(a) receptor.

Various exogenous and endogenous steroids have been demonstrated to have both enhancing and inhibiting effects on ligand binding to the gamma-aminobutyric acid(A) receptor (GABAA receptor) in previous studies. In the present study we have explored the possibility that an additional class of synthetic steroidal compounds, anabolic/androgenic steroids (AAS), mediate some of their CNS effects through direct interaction with the GABAa receptor. At micromolar concentrations, two AAS, stanozolol and 17 alpha-methyltestosterone (17 alpha-MT), significantly inhibited 1 nM [3H]flunitrazepam ([3H]Fln) binding to rat brain cerebrocortical membranes. Inhibition of 1 nM [3H]Fln binding by stanozolol was similar for both males and females (approximately 50% inhibition at 50 microM stanozolol). 17 alpha-MT was much less efficacious, but did significantly inhibit 1 nM [3H]Fln binding at concentrations > 10 microM. In equilibrium binding assays, stanozolol (50 microM) raised the apparent KD for [3H]Fln binding. The observed changes in the [3H]Fln binding curve, when analyzed by Rosenthal analysis, reveal complex equilibrium binding behavior. In females, the Rosenthal plot was best fit by a two site binding model. Stanozolol (50 microM) inhibited binding to the higher affinity site in a manner consistent with competitive inhibition, increasing the KD without changing the BMAX. However, the effect of stanozolol on the binding to the low affinity site was more complex, with an increase in the the KD and the BMAX. In males the data were best fit by a single binding site model. This single site exhibited a slight increase in the KD and a decrease in the BMAX in the presence of 50 microM stanozolol.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of thyroxine and its related compounds on cerebral GABA receptors: inhibitory action on benzodiazepine recognition site in GABAA receptor complex.

The effects of thyroxine and its related derivatives on gamma-aminobutyric acid (GABA) receptors in the rat brain were examined. D-Thyroxine strongly inhibited [3H]flunitrazepam binding to benzodiazepine receptor in crude synaptic membrane from the rat brain. The Scatchard analysis of the [3H]flunitrazepam binding in the presence of D-thyroxine indicated the decreases in the affinity and maximum number of binding site. Furthermore, D-thyroxine inhibited the enhancing effect of flunitrazepam on GABA-stimulated 36Cl- influx into membrane vesicles, although GABA-stimulated 36Cl- influx alone was not affected by D-thyroxine. On the other hand, the effects of thyroxine and its related derivatives on cerebral GABAB receptor binding were not noted. These results suggest that D-thyroxine may be a drug which is able to modulate the function of GABAA receptor complex via the inhibitory action on benzodiazepine recognition site.

GABAA receptor subtypes: ligand binding heterogeneity demonstrated by photoaffinity labeling and autoradiography.

Heterogeneity of binding affinities for a variety of ligands was observed for gamma-aminobutyric acid type A (GABAA) receptors in the rat CNS, at both GABA and benzodiazepine recognition sites. Photoaffinity labeling by [3H]flunitrazepam and [3H]muscimol to affinity column-purified receptor proteins was examined by gel electrophoresis in sodium dodecyl sulfate. Anesthetic barbiturates (pentobarbital) and steroids (alphaxalone) both differentially stimulated the incorporation of [3H]flunitrazepam more so into the 51-kDa alpha 1 subunit than into the 53-kDa alpha 2 polypeptide, and incorporation of [3H]muscimol into the 55-kDa beta 2 subunit more so than the 58-kDa beta 3 polypeptide. Binding to these polypeptides was also affected differentially by other allosteric modulators and competitive inhibitors, including the benzodiazepine "type 1" selective ligand CL218,872. Heterogeneity in affinity of this drug for the single 51-kDa alpha 1 polypeptide strongly suggests that type I receptors, like type II, are heterogeneous. In brain sections, the extent of enhancement of [3H]muscimol binding showed significant regional variation, similar for both steroids and barbiturates, and the GABA analogues THIP and taurine inhibited muscimol binding with regional variations in affinity that were almost opposites of each other. Modulation of [3H]flunitrazepam binding by steroids, barbiturates, and THIP significantly varied with regions. Taken together, ligand binding heterogeneity exhibited by photoaffinity labeling and autoradiography demonstrate the existence of multiple pharmacological-binding subtypes resulting from the combination of multiple polypeptide gene products into several oligomeric isoreceptors. Comparison of the regional distribution of binding subtypes with that of different subunit gene products allows the following conclusions about possible subunit compositions of native pharmacological receptor subtypes present in the brain: Benzodiazepine pharmacology of the oligomeric receptor isoforms is dependent on the nature of alpha and subunits other than alpha, GABA-benzodiazepine coupling is dependent on the nature of the alpha subunits, GABA site pharmacology is dependent on the nature of the beta subunits, and several subunits including alpha and beta contribute to the degree of sensitivity to steroids and barbiturates. Finally, the presence of discrete subunits may be necessary but is not sufficient to postulate a defined pharmacological property.

Antagonismo do flumazenil ao flunitrazepam / Flunitrazepam with flumazenil

Flumazenil is a competitive inhibitor of the specific biding of benzodiazepines to central nervous system receptor sites and it is valuable in reversing their sedative effect. Twenty-four healthy patients (ASA I and II), both male and female, with age between 19 and 58 years and weight between 46 and 79 kg, scheduled for elective surgery under flunitrazepam oral premedication and intravenous sedation after spinal anesthesia, were submitted to a trial with flumazenil for reversal of sedation. Judged by the degree of sedaation, comprehension, cooperation and time and space orientation, flumazenil showed good results. Only 12 por cent of the patients presented resedation after 120 minutes. No adverse effect was observed.

Effects of midazolam and flunitrazepam on the release of dopamine from rat striatum measured by in vivo microdialysis.

We have studied the effects of midazolam and flunitrazepam on extracellular concentrations of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum in freely moving animals using in vivo microdialysis. I.v. injections of midazolam 0.075 and 0.15 mg kg-1 decreased striatal dopamine concentrations in a dose-dependent manner without affecting the concentrations of DOPAC and HVA. Flunitrazepam 0.015 and 0.03 mg kg-1 also decreased striatal dopamine concentrations in a dose-related manner, but the reductions in DOPAC and HVA were not significant. Flumazenil 6 micrograms kg-1 alone did not affect striatal concentrations of dopamine, DOPAC and HVA, but it prevented the effects of midazolam and flunitrazepam. Flunitrazepam 10 mumol litre-1 also decreased striatal dopamine release when infused through a dialysis probe placed into the striatum, but it failed to affect striatal dopamine release when infused into the ipsilateral substantia nigra. Central administrations of midazolam were effective only when the drug was infused into both sites simultaneously (10 and 100 mumol litre-1) or given by intraventricular injection (0.5 and 1 micrograms). These results suggest that midazolam and flunitrazepam affect striatal dopamine release in a different manner.

[Sedation with flunitrazepam and its antagonism with flumazenil in endoscopic urological procedures: our clinical experience]. / Sedazione con flunitrazepam e suo antagonismo con flumazenil nelle procedure urologiche endoscopiche: nostra esperienza clinica.

Patients, especially males, do not easily stand endoscopic urological diagnostic examinations. So flunitrazepam (0.01 mg/kg IV) has been used to insure a suitable sedation. At the end of such examination flumazenil, a selective benzodiazepine antagonist has been injected. The levels of sedation and orientation so obtained and the cardiorespiratory parameters have been collected during a one hour follow-up from the intravenous injection of the antagonist. This method presents minimal side effects (a marginal, but statistically significative, systolic arterial pressure reduction), a completely normal coordination of movements and awareness of space and time relationship at the end of the observation period. Therefore it is a very good solution for short time examinations giving the chance to safely and shortly dismiss the patient.

Differential ontogenesis of type I and II benzodiazepine receptors in mouse cerebellum.

The ontogeny of type I and type II benzodiazepine binding sites was studied in mouse cerebellum by displacement of [3H]flunitrazepam binding by zolpidem, a ligand specific for the type I sites. Type I binding sites predominate throughout development and in the adult while type II sites account for 25% of total cerebellar benzodiazepine binding sites at birth and, during development, decrease to 10% or less in the adult. On a per cerebellum basis type II sites increase during the first postnatal week and then remain at a steady level while type I sites increase until adulthood. These results may indicate a specific localization of the type II sites (and of the corresponding alpha-protein subunits in the GABA/benzodiazepine receptor complex) in structures already present at birth and developing during a short early postnatal period. The affinity of zolpidem for its high affinity (type I) binding sites increases during cerebellar ontogeny, this increase possibly indicates an epigenetic (post-translational) 'maturation' process of the corresponding receptor molecule. Hill numbers indicate the existence of an additional binding site heterogeneity greater during development but still present in the adult; probably this is to be related to the simultaneous presence of different 'maturation' stages during development and with a certain variety of the final products.

Hypnotic action of flunitrazepam is reversed by proglumide in rats.

1. Caerulein, an analogue of cholecystokinin (CCK-8), like CCK-8, has been shown to produce hypnotic effects similar to those of benzodiazepine (flunitrazepam). 2. Proglumide antagonizes the action of CCK-8 and of its analogue. 3. The aim of the present study was to demonstrate whether proglumide would affect the potent hypnotic action of flunitrazepam in rats. 4. The association of proglumide with flunitrazepam suppress the increase of total sleep time and slow wave sleep seen after flunitrazepam alone. Proglumide alone has no effect on sleep stages. The authors report here for the first time that the hypnotic action of flunitrazepam is antagonized by proglumide in rat.