Nootropic nefiracetam inhibits proconvulsant action of peripheral-type benzodiazepines in epileptic mutant EL mice.

Piracetam and structurally related nootropics are known to potentiate the anticonvulsant effects of antiepileptic drugs. It remains to be seen, however, whether these nootropics inhibit proconvulsant actions of many toxic agents including Ro 5-4864, a specific agonist for peripheral-type benzodiazepine receptors (PBR). The present study was designed to address this issue using EL mice, an animal model of epilepsy. In behavioral pharmacological experiments, EL mice were highly susceptible to convulsions induced by Ro 5-4864 (i.p.) in comparison with nonepileptic DDY mice. Nefiracetam administered orally to EL mice inhibited spontaneous seizures. In DDY mice, convulsions induced by Ro 5-4864 were prevented by nefiracetam when administered by i.v. injection. Aniracetam (i.v.) was partially effective, but piracetam and oxiracetam were ineffective as anticonvulsants. Binding assay for brain tissues revealed a higher density of mitochondrial PBR in EL mice compared with DDY mice. Binding of the PBR ligands Ro 5-4864 to either EL or DDY mouse brain was inhibited by micromolar concentrations of these nootropic agents in the sequence of nefiracetam > aniracetam >> oxiracetam, piracetam. This rank order is identical to potency as anticonvulsants. These data suggest that nefiracetam may prevent toxic effects of PBR agonists through interacting with PBR.

Involvement of steroids in anti-inflammatory effects of peripheral benzodiazepine receptor ligands.

Mouse paw oedema induced by carrageenan is used to determine if glucocorticoids are involved in the anti-inflammatory effects of peripheral benzodiazepine receptor ligands. The anti-inflammatory responses elicited by i.p. treatment with 1-(2-chlorophenyl)-N-methyl-N (1-methyl-propyl)-3-isoquinoline carboxamide (PK11195) and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2-H-1, 4-benzodiazepin-2 (Ro5-4864) were reversed by aminoglutethimide, an inhibitor of steroidal synthesis. Intraplantar injection into the ipsilateral paw of Ro5-4864, but not PK11195, inhibited the formation of paw oedema and this effect was reversed by aminoglutethimide. These results suggest that glucocorticoids are involved in the systemic and local anti-inflammatory effects of Ro5-4864 and only in the systemic response to PK11195.

Identification of a peptide antagonist to the peripheral-type benzodiazepine receptor that inhibits hormone-stimulated leydig cell steroid formation.

Peripheral-type benzodiazepine receptor (PBR) is an 18-kDa high-affinity cholesterol and drug ligand-binding protein involved in various cell functions, including cholesterol transport and steroid biosynthesis. To aid our investigation of the biological function of PBR, we have set out to identify functional antagonists. By screening phage display libraries, we have identified peptides that displace the high-affinity PBR benzodiazepine drug ligand, Ro5-4864 (4'-chlorodiazepam). Among these peptides, STPHSTP was the most potent (IC(50) = 10 microM). All of the isolated peptides showed a conserved motif STXXXXP. The role of these peptides in Leydig cell steroidogenesis was examined using a transducible peptide composed of the TAT domain of human immunodeficiency virus and the peptides under investigation. Synthesized peptides efficiently transduced into MA-10 Leydig cells, and the peptide TAT-STPHSTP inhibited Ro5-4864- and human chorionic gonadotropin-stimulated steroid production in a dose-dependent manner (ED(50) = 5 microM). TAT-STPHSTP behaved as a competitive PBR antagonist, which did not affect 22R-hydroxycholesterol-supported steroidogenesis. These results yield leads for the development of potent PBR antagonists and indicate that endogenous PBR agonist-receptor interaction is critical for hormone-induced steroidogenesis.

Blockade of behavioral effects of bretazenil by flumazenil and ZK 93,426 in pigeons.

Benzodiazepine receptor partial agonists manifest full efficacy in preclinical tests of anxiolytic drug action but do not fully reproduce the discriminative stimulus effects of benzodiazepine receptor full agonists in pigeons. The partial agonist, bretazenil, binds to both diazepam-sensitive and diazepam-insensitive GABAA receptors. Previous studies have suggested a role for each of these receptor populations in some behavioral effects of bretazenil in pigeons. A possible role for these receptor subtypes in the behavioral effects of bretazenil was further investigated through drug interaction studies with the benzodiazepine receptor antagonists, flumazenil and ZK 93,426. Whereas flumazenil binds with high affinity to both receptor isoforms, ZK 93,426 binds preferentially to diazepam-sensitive binding sites. Bretazenil markedly increased punished responding of pigeons without significantly affecting nonpunished responding. In pigeons discriminating the full benzodiazepine receptor agonist, midazolam, from saline, bretazenil produced only 60-75% maximal effect. Flumazenil and ZK 93,426 neither increased punished responding nor substituted for midazolam, but dose-dependently blocked the effects of bretazenil on punished responding. Flumazenil also dose-dependently blocked the effects of bretazenil in midazolam-discriminating pigeons, whereas ZK 93,426 only attenuated this effect. These results indicate that bretazenil's actions as a partial agonist at diazepam-sensitive benzodiazepine receptors mediate increases in punished responding and substitution for the discriminative stimulus effects of midazolam in pigeons. The differences in the effects of flumazenil and ZK 93,426 on the discriminative stimulus effects of bretazenil suggest a potential contribution of diazepam-insensitive sites to this behavioral effect.

Impairment of stress adaptive behaviours in rats by the CCKA receptor antagonist, devazepide.

1. Cholecystokinin (CCK) is released during stress both in limbic and hypothalamic areas suggesting that CCK could participate in modulating neuroendocrine as well as behavioural responses to stress. 2. In this study we have examined the effect of CCK receptor antagonists on the retention of the immobility response to a forced-swim stress in rats. In this test, rats are forced to swim during 15 min (conditioning period) and 24 h later, the duration of immobility is measured during a period of 5 min (re-test period). During the conditioning period rats display a period of vigorous activity, followed by progressive inactivity. During the re-test period rats remain 70-80% of the time in an immobile posture. 3. The CCKA receptor antagonist, devazepide (MK-329) but not the CCKB receptor antagonist, L-365,260, administered s.c. immediately before the conditioning period, decreased the duration of acquired immobility during the re-test period. The effect of devazepide was prevented by cholecystokinin octapeptide (CCK-8; 40 micrograms kg-1, s.c) as well as by the selective glucocorticosteroid GII receptor agonist, dexamethasone (30 micrograms kg-1, s.c.) 4. Neither corticosterone nor ACTH plasma levels measured both after the re-test period and after the conditioning period were modified by devazepide treatment. 5. The results suggest a role for CCK in the behavioural adaptation to stress and indicate a relationship between CCK systems and glucocorticoids in the neuronal mechanisms involved in the acquisition of adaptive behaviours to stress.

Modulatory mechanisms of cyclic AMP-stimulated steroid content in rat brain cortex.

The modulation of cyclic AMP dependent neurosteroidogenesis was studied in minces prepared from the cerebral cortex of adult rat. Forskolin or dibutyryl-cyclic AMP enhanced pregnenolone and progesterone production in a time and dose-dependent manner. The forskolin effect was mimicked by the cyclic AMP phosphodiesterase inhibitor isobutyl-methyl-xanthine, but not by the adenylate cyclase inactive forskolin analogue 1,9,dideoxy-forskolin. 4'-Chloro-diazepam, a high affinity ligand for the mitochondrial diazepam binding inhibitor (DBI) receptor, also elicited a time dependent increase in steroidogenesis. The forskolin and the 4'-chloro-diazepam stimulated pregnenolone increase was prevented by preexposing the rat brain cortical minces to 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline carboxamide (PK 11195), a high affinity ligand for the mitochondrial DBI receptor endowed with antagonistic properties. The protein synthesis inhibitor cycloheximide prevented the forskolin and 4'-chloro-diazepam stimulation of pregnenolone formation. In brain cortical minces of adrenalectomised/orchiectomised rats dibutyryl-cyclic AMP increased both pregnenolone and progesterone formation, while forskolin only increased progesterone. These data show that cyclic AMP enhances brain steroidogenesis by acting on a labile protein substrate which interacts with the mitochondrial DBI receptor.

Prevention of ethanol-induced gastric damage by the imidazobenzodiazepines Ro 15-4513 and Ro 15-3505 in rats.

The protective effects of the imidazobenzodiazepines Ro 15-4513 and Ro 15-3505 against ethanol-induced gastric damage were investigated. Gastric lesions were induced in rats by the oral administration of 1 ml of absolute ethanol. Ro 15-4513 (2.5-10 mg/kg, IP) or Ro 15-3505 (5-20 mg/kg, IP), administered 30 min before ethanol, protected against ethanol-induced gastric damage. The protective effects of these compounds were blocked by the benzodiazepine antagonist, flumazenil (10 mg/kg, IP). These results present evidence for the involvement of the GABA-benzodiazepine receptor complex in the pathogenesis of ethanol-induced gastric damage.

Cibacron blue-induced enhancement of agonist binding to cholecystokinin (CCK) receptors in solubilized pancreatic membranes.

The pancreatic receptor for cholecystokinin (CCK) typifies many G protein-coupled receptors in that its ability to bind agonist can be reduced by GTP or the solubilization of membranes. We found, however, that a dye, cibacron blue, caused up to a 6-fold increase in binding of the CCK receptor agonist, 125I-CCK-8, to rat pancreatic membranes solubilized with digitonin. Binding optimally enhanced in this manner was comparable to binding of 125I-CCK-8 to native membranes with respect to time-course, maximal amount bound, reversibility, and sensitivity to inhibition by various CCK receptor ligands. Increases in affinity of the CCK receptor for CCK-8 accounted fully for the enhancement of binding of 125I-CCK-8. Cibacron blue did not enhance binding of 125I-CCK-8 to native membranes, and also failed to enhance binding of the CCK receptor antagonist, [3H]L-364,718, to solubilized or native membranes. The ability of cibacron blue to enhance binding of agonist but not that of antagonist suggests that this dye may mimic or perhaps stimulate the effects of G protein on CCK receptors. Such a phenomenon may provide new insights into the mechanisms by which receptors distinguish agonists from antagonists.

Benzodiazepines inhibit neutrophil chemotaxis and superoxide production in a stimulus dependent manner; PK-11195 antagonizes these effects.

Diazepam, which binds both central (neuronal) and peripheral (non-neuronal) benzodiazepine binding sites, and Ro5-4864, a ligand selective for benzodiazepine peripheral binding sites (PBS), both inhibited the FMLP induced chemotaxis in human neutrophils at concentrations as low as 10(-8) M. A selective peripheral benzodiazepine antagonist, PK-11195 (10(-5) M), partially reversed the benzodiazepine inhibition of chemotaxis. Diazepam also inhibited the superoxide production induced by FMLP, NaF, and A23187, but not that induced by PMA whose stimulant action was insensitive even to 10(-4) M diazepam. The FMLP-induced superoxide production was most sensitive to diazepam inhibition (ID50 = 2.25 x 10(-6) M diazepam); the effect of NaF was slightly less sensitive (ID50 = 1.34 x 10(-5) M diazepam); and the effect of A23187 was least sensitive as it was suppressed only at 10(-4) M diazepam concentrations. Like diazepam, Ro5-4864 inhibited the FMLP-induced superoxide production, and PK-11195 (10(-5) M) significantly antagonized both diazepam and Ro5-4864 inhibition. Binding studies showed the presence of a saturable benzodiazepine 'peripheral' type binding site (PBS) on human neutrophils with a Kd of 1.2 +/- 0.06 x 10(-8) M (+/- SEM), and a Bmax of 1028 +/- 86.2 fmol/10(6) cells (+/- SEM) for [3H]Ro5-4864; the binding was displaceable by PK-11195, Ro5-4864 and diazepam but not by clonazepam.

Effect of benzodiazepine and beta-carboline antagonists and partial agonists on loprazolam- and ZK 93423-induced hypothermia.

The pharmacology of the hypothermia induced by benzodiazepine and beta-carboline full agonists in mice has been investigated using partial agonists and antagonists from both chemical series. The benzodiazepine antagonist flumazenil (10 mg/kg i.p.) blocked the hypothermia induced by loprazolam (3 mg/kg i.p.) but not that induced by the beta-carboline ZK 93423 (3 mg/kg i.p.). Both hypothermic responses were reduced by the beta-carboline antagonist ZK 93426 (3 mg/kg i.p.) and the benzodiazepine partial agonist Ro 17-1812 (10 mg/kg i.p.). On the other hand, the beta-carboline partial agonist ZK 91296 (30 mg/kg i.p.) blocked ZK 93423-hypothermia but not that induced by loprazolam. Thus, the hypothermic actions of benzodiazepine and beta-carboline agonists appear to be differentially antagonised by antagonists and partial agonists of the two chemical classes suggesting receptor subtype interactions which are non-uniformly related to the chemical class. These results cannot be explained simply in terms of pharmacokinetics or thermoregulatory effects of the compounds themselves. They suggest that, at least in the hypothalamus, different subtypes of benzodiazepine receptor may exist.

Modulatory role for CCK-B antagonists in Parkinson's disease.

We examined the ability of selective CCK-A and CCK-B receptor antagonists to induce or modulate the locomotor stimulant effects of dopamine agonists in MPTP-treated squirrel monkeys. Administration of 1-100 micrograms/kg i.p. of either the selective CCK-A receptor antagonist devazepide (MK-329) or the CCK-B receptor antagonist L-365,260 alone failed to stimulate a locomotor response in parkinsonian monkeys. In contrast, treatment with L-365,260 caused a 50-60% potentiation of the locomotor stimulatory effects of L-DOPA or (+)-PHNO. No such modulatory effects were observed following pretreatment with devazepide. We suggest that CCK-B receptor antagonists may be useful adjuncts to existing dopamine replacement therapy for improved management of Parkinson's disease.

Effects of peripheral benzodiazepine receptor ligands on hypothalamic-pituitary-adrenal axis function in the rat.

High concentrations of the "peripheral" benzodiazepine (pBZD) binding site ("receptor") have been described in the hypothalamus, the pituitary and the adrenal glands. This study was undertaken to examine the effects of ligands of this binding site on the hypothalamic-pituitary-adrenal axis (HPA). To accomplish this we administered graded doses of the pBZD receptor agonist 4-chloro-diazepam (Ro5-4864) i.v. to catheterized, freely moving adult male Sprague-Dawley rats. Serial blood samples for plasma adrenocorticotropin hormone (ACTH) and corticosterone determinations were drawn from the catheter before and after the injection of the drug. Ro5-4864 significantly stimulated ACTH and corticosterone secretion in a dose-dependent fashion. To examine whether this effect could be antagonized by the pBZD binding site antagonist PK 11195, we treated rats with PK 11195 at doses 10- and 50-times higher than Ro5-4864 before administration of a maximally effective dose of Ro5-4864. Neither dose of PK 11195 antagonized Ro5-4864-induced plasma ACTH or corticosterone elevations. However, this agent, given alone, stimulated ACTH and corticosterone release. Similarly, carbamazepine (CBZ), which binds to the pBZD binding site with low affinity, stimulated weakly the HPA in vivo, reaching statistical significance only at the highest dose tested. To examine the site(s) of action of these compounds on the HPA, we evaluated their effects on hypothalamic corticotropin-releasing hormone (CRH) and pituitary ACTH secretion in vitro. Ro5-4864 stimulated hypothalamic CRH, but not pituitary ACTH secretion. Neither PK 11195 nor CBZ had any agonist effect on hypothalamic CRH secretion in vitro, whereas both antagonized Ro5-4864-induced CRH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory effects of cholecystokinin on isolated perifused islets inhibited by potent and specific antagonist L 364718 [corrected].

The influence of L 364718 on islet responsiveness to sulfated cholecystokinin (CCK-8S) was investigated. In islets whose inositol-containing phospholipids were prelabeled during a 2-h incubation period, subsequent exposure to L 364718 (1 nM) significantly impaired the secretion of insulin usually noted in response to 200 nM CCK-8S in the simultaneous presence of 7 mM glucose. A higher level of the antagonist (10 nM) completely abolished insulin secretion. L 364718 (1-10 nM) reduced the efflux of 3H from myo-[2-3H]-inositol prelabeled islets in parallel with the reduction in secretion. L 364718 (10 nM) significantly reduced the accumulation of 3H-containing inositol phosphates usually noted with CCK-8S addition. L 364718, at levels 10- to 100-fold greater than those necessary to attenuate CCK-8S-induced insulin secretion, had no adverse effect on the insulin secretory response of freshly isolated islets to 10 mM glucose alone, 5 mM D-glyceraldehyde, 15 mM alpha-ketoisocaproate, or 50 ng/ml gastric inhibitory polypeptide. L 364718 (1000 nM) had no adverse influence on carbamylcholine (1 mM)-induced phosphoinositide hydrolysis. These results establish L 364718 as a potent and highly selective antagonist of cholecystokinin's stimulatory actions on beta-cells. Because of its potency, selectivity, and oral effectiveness, in vivo studies with L 364718, aimed at unraveling the pleiotropic effects of CCK-8S on glucose and insulin homeostasis, seem feasible.

Tetrazepam: a benzodiazepine which dissociates sedation from other benzodiazepine activities. I. Psychopharmacological profile in rodents.

In the 1,4-benzodiazepine (BZD) series the nature of the C(5) substituent is critical for activity. In tetrazepam this substituent is a cyclohexenyl ring, in all other clinically effective 1,4-BZD derivatives it is a phenyl ring. The activities of tetrazepam and diazepam, whose chemical structures differ only by the nature of the C(5) substituent, were compared in rodent models which are predictive of anticonvulsant, anxiolytic, muscle relaxant and sedative effects. In mice, tetrazepam and diazepam antagonized pentylenetetrazol (PTZ)-induced seizures, increased novel food consumption, decreased rearing behavior in the Staircase test and impaired performance in the Traction test. The effects of both drugs were abolished by Ro 15-1788. In rats, both drugs antagonized PTZ-induced seizures, released punished responding in an approach-avoidance conflict procedure and blocked the PTZ discriminative cue. In mice and rats, and with both BZDs, maximal activity occurred 15 to 30 min after drug administration. In mice and rats, the overall anxiolytic and muscle relaxant potencies of tetrazepam were one-fourth and one-sixth those of diazepam, respectively. The anticonvulsant potency of tetrazepam varied from one-half (rats) to one-twentieth (mice) that of diazepam. In rats and mice, the sedative potency of tetrazepam (Rotorod test; locomotor activity) was approximately one-sixteenth that of diazepam. Finally, tetrazepam induced a loss of righting reflex in mice, with an ED50 value which was approximately 70-times greater than that of diazepam. It is concluded that replacing the 5-phenyl ring by a 5-cyclohexenyl ring leads to a relative dissociation of the pharmacological actions of the 1,4-BZDs.

Interactions of the imidazodiazepine Ro 15-4513 with chemical convulsants.

1. The proconvulsant effects of the imidazodiazepine Ro 15-4513, were investigated in mice by use of intravenous infusion of a variety of convulsant drugs. 2. Dose-response and time course studies of Ro 15-4513 against gamma-aminobutyric acid (GABA) antagonists were performed. On the basis of these studies a maximally effective dose of 5 mg kg-1 was administered 5 min before the determination of seizure thresholds in subsequent experiments. 3. Ro 15-4513 (5 mg kg-1) significantly lowered seizure thresholds to pentylenetetrazole, bicuculline and the convulsant benzodiazepine Ro 5-3663, but failed to alter seizure thresholds to picrotoxin, strychnine, caffeine and quipazine. 4. Ro 15-4513 significantly raised seizure threshold to the benzodiazepine receptor inverse agonist methyl 6,7-dimethoxy-4 ethyl-beta-carboline-3-carboxylate (DMCM). 5. These results are discussed in relation to other studies investigating the proconvulsant and alcohol-antagonizing effects of Ro 15-4513.

Cueing effects of anxiolytic benzodiazepines (DZP and Ro 11-3128): stereospecificity and antagonism by the convulsant benzodiazepine Ro 5-3663.

Two groups of gerbils were trained in a T-maze to discriminate between the vehicle condition (4 ml/kg) and either of the benzodiazepine (BDZ) agonists diazepam (DZP) and Ro 11-3128; administration (5.6 mg/kg) was IP 5 min prior to training onset. Once trained, novel doses and drugs were assessed in test sessions interposed between the regular training days. A dose-related generalization effect occurred with both compounds (range 0.1-5.6 mg/kg), the effect being similar at both the 5 and 15 min test intervals; the two intervals were evaluated after a single injection. The lack of generalization of Ro 11-3624 (range 5.6-56 mg/kg) indicates a stereoisomeric separation of BDZ agonist activity. Ro 5-4864 (range 17.5-56 mg/kg), an agent chemically/structurally related to DZP, did not produce DZP responding at either of the two test inverals; clear-cut convulsant activity occurred at the 15 min interval. The convulsant BDZ compound Ro 5-3663 (3 and 10 mg/kg) antagonized the DZP stimulus irrespective of whether Ro 5-3663 was given either prior to, simultaneously with, or shortly after the DZP injection.

Gallamine binding to muscarinic M1 and M2 receptors, studied by inhibition of [3H]pirenzepine and [3H]quinuclidinylbenzilate binding to rat brain membranes.

Whereas classic muscarinic antagonist ligands appear to recognize only a single class of muscarinic receptor sites, the recently discovered antagonist pirenzepine appears to distinguish at least two classes of sites. Its unique binding properties, demonstrated in both indirect and direct binding studies, have led to an emerging concept of high affinity (M1) and low affinity (M2) sites. This concept has been supported by pharmacologic studies of functional muscarinic responses, as well as by data suggesting different effector relationships for the two sites. Gallamine possesses muscarinic antagonist properties, and it also recognizes heterogeneity among muscarinic receptors. The purpose of this study was to define gallamine-recognized heterogeneity in terms of the pirenzepine-defined M1, M2 concept. This has been done by studying the ability of gallamine to inhibit [3H]pirenzepine binding to the M1 site, and to inhibit [3H]quinuclidinylbenzilate ([3H]QNB) binding in cerebellar membrane preparations, which contain almost exclusively the M2 site. The results show that gallamine binds with high affinity to the M2 site, with Ki = 2.4 nM, and lower affinity to the M1 site with Ki = 24 nM. Within these classes gallamine does not recognize heterogeneity. The ability of gallamine to inhibit [3H]QNB binding to cortex is best described by a two-site model comprised of 77% low affinity gallamine sites (M1) and 23% high affinity gallamine sites (M2). Thus, the heterogeneity among muscarinic receptors which is recognized by gallamine within the receptor binding paradigms of this study can be attributed to the M1, M2 subtypes as defined by pirenzepine binding. In addition, gallamine at low concentrations appears to bind as a pure competitive antagonist at these two sites, indicated by linear Schild plots with slopes of 1.0, the lack of an effect on dissociation of radioligands, and the ability to protect [3H]pirenzepine and [3H]QNB-binding sites from alkylation by propylbenzylcholine mustard. These studies do not exclude the possibility of a non-competitive interaction of gallamine with the muscarinic receptor observed by other investigators at high gallamine concentrations, and postulated to occur at a site adjacent to the primary muscarinic site. It is proposed that gallamine is capable of interacting with both the primary muscarinic site and an allosteric site. These results support the emerging concept of M1 and M2 muscarinic subclasses and suggest that gallamine and related compounds may be useful in defining muscarinic receptor subclasses, given their higher affinity for the M2 site.

Convulsant effect of Ro 5-4864, a peripheral type benzodiazepine, on the baboon (Papio papio).

The effects of Ro 5-4864, a 1,4-benzodiazepine with a high affinity for the peripheral-type benzodiazepine (Bz) binding site, were investigated in the baboon (Papio papio), which is genetically predisposed to epilepsy. A proconvulsant effect of low doses (1-3 mg/kg, i.v.) of Ro 5-4864 was observed by studying its effect on the photic responses induced by intermittent light stimulation in non-photosensitive baboons. Higher doses of Ro 5-4864 (10 mg/kg, i.v.) were overtly convulsant. The Bzs clonazepam and diazepam blocked these convulsant actions of Ro 5-4864 whereas neither Ro 15-1788, an antagonist of central Bz binding sites, nor PK 11 195, an antagonist of peripheral Bz binding sites, had any effect. It thus appeared that the convulsant effect of Ro 5-4864 was not mediated by Bz binding sites of either the central or the peripheral type. It is possible that Ro 5-4864 exerts its convulsant action at the picrotoxin site of the central Bz receptor - gamma-aminobutyric acid receptor-chloride ionophore complex.

Antagonism of the effects of the atypical benzodiazepine, Ro 5-4864 on intracranial self-stimulation in the rat.

The effects of Ro 5-4864 (chlordiazepam) were examined on responding for self-stimulation of the mid-lateral hypothalamus. Rewarding stimuli were delivered according to a 10-sec variable interval schedule of reinforcement. Ro 5-4864 (10-30 mg/kg, subconsulsive doses in these rats) decreased responding. This effect was antagonized by chlordiazepoxide (5-10 mg/kg) and phenobarbitone (35 mg/kg) but not by the benzodiazepine receptor antagonist Ro 15-1788 (10-20 mg/kg), the ligand for peripheral benzodiazepine receptors PK 11195 (60 mg/kg) or phenytoin (60 mg/kg). The pattern of interactions of Ro 5-4864 with these compounds differs from the pattern obtained with other procedures, and suggests that Ro 5-4864 has effects on systems unrelated to anxiety, convulsive activity or sedation.

The peripheral benzodiazepine receptor ligand Ro 5-4864 induces supraspinal convulsions in rabbits. Reversal by the central benzodiazepine antagonist Ro 15-1788.

The peripheral BDZ receptor ligand Ro 5-4864 was administered to rabbits in doses ranging from 0.2 to 7 mg/kg IV. Changes in electrocortical activity appeared within 1 min after administration, characterized by trains of slow waves in the posterior sensorimotor and optic cortices (0.6-2 mg/kg) and by grand mal seizures (2-10 mg/kg). The low doses also induced alterations in the basic rhythms both of the hippocampus (reduced amplitude and spike-like waves) and of the nucleus ventralis of thalamus (trains of slow waves), not associated with observable behavioural changes. The paroxysmal EEG activity observed at higher doses of the drug was first recorded in the cortical areas and then spread to the subcortical structures. No change in electrical activity could be observed in the spinal cord. The paroxysmal activity was associated with tonic-clonic convulsions and scialorrea. The EEG and behavioural manifestations were inhibited by administration of Ro 15-1788. This drug at doses of 0.6 and 6 mg/kg antagonized the effects of Ro 5-4864 at doses of 0.6-5 mg/kg and 6-7 mg/kg, respectively. This effect began 1-3 min after administration of the antagonist, and led to EEG synchronization. These data suggest that in rabbits the convulsant effect of Ro 5-4864 is due to interference of the drug at the GABA-BDZ-picrotoxin receptor oligomeric complex. Such an effect seems to be mediated at least in part by central BDZ receptors.