Microinjections of methyl-beta-carboline-3-carboxylate into the dorsal raphe nucleus: behavioural consequences

Very small quantities (0.01-10 ng) of the inverse benzodiazepine receptor agonist, methyl-beta-carboline-3-carboxylate (beta-CCM) microinjected into the dorsal raphe nucleus (DRN) of the rat selectively reduced social interaction, an effect consistent with an increase in anxiety. Similarly, intraperitoneally injected beta-CCM, within a limited dose range, reduced social interaction without affecting locomotor activity. The benzodiazepine receptor antagonist, RO15-1788 (1 ng) microinjected into the DRN, reversed the suppression of social interaction induced by either intra-raphe or intraperitoneal beta-CCM. Histological examination of the beta-CCM microinjection sites showed that locations within the DRN were almost invariably associated with decreases in social interaction; microinjections failing to decrease social interaction were located primarily outside the DRN. We conclude that the DRN has a major role in expressing the anxiogenic effect of beta-CCM and it may therefore be an important area in the neuronal system controlling anxiety.     

RO 15-1788 produces naloxone-reversible analgesia in the rat

Intraperitoneal and intracerebroventricular administration of the benzodiazepine antagonist RO 15-1788 produced analgesia to both thermal and mechanical pain. This effect was reversed by pretreatment with the opioid antagonist naloxone but was unaffected by pretreatment with the benzodiazepine agonist midazolam. Furthermore, administration of the benzodiazepine antagonist RO 15-3505 was without analgesic effect. It is, therefore, proposed that the intrinsic action induced by RO 15-1788 is exerted via the indirect activation of endogenous opioid systems and that the observed effect is not due to the action of the antagonist on the benzodiazepine receptor.     

Analgesia and motor activity following administration of THIP into the periaqueductal gray and lateral ventricle of rats

Intracerebral microinjections of THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), a GABAergic agonist that produces analgesia when administered systemically, were made to investigate the central sites of action of this agent. Comparisons were also made with intracerebral microinjections of morphine and with systemic administration of each agent. Injections into the ventral lateral periqueductal gray (PAG) produced analgesia on the 55^°C hot-plate test following a 2.0 μg dose, but were without effect at the other doses examined (0.5–20.0 μg). In the tail-flick test, hyperalgesia was seen following the 5.0- and 20.0-μg doses. Motor activity was increased following the 1.0-, 2.0-, and 10.0-μg doses and was accompanied by ipsilateral turning. Injections into the lateral cerebral ventricle (ICV) were without effect at doses of less than 50 μg. No significant effects were observed in the hot-plate test following 50 μg produced severe ataxia, precluding testing. Following 50 μg, hyperalgesia was seen in the tail-flick test. THIP at both doses decreased motor activity. The present findings further demonstrate some novel aspects of the pharmacology of THIP and suggest that much of the drug's analgesic activity is produced by interaction with GABAergic receptors outside the PAG and structures easily accessed by ICV administration.     

The anxiogenic action of RO 5-4864 in the social interaction test: effect of chlordiazepoxide,RO 15-1788 and CGS 8216

Summary RO 5-4864 (20 mg/kg), a benzodiazepine with high affinity for peripheral-type benzodiazepine binding sites in rat kidney and brain, but not for the classical CNS sites, reduced the time spent by pairs of rats in active social interaction, without reducing locomotor activity, possibly reflecting an anxiogenic action. This anxiogenic effect was not reversed by chlordiazepoxide (5 or 10 mg/kg) given acutely, but was reversed by chlordiazepoxide (5 mg/kg) given for 5 days prior to testing. RO 15-1788 (10 mg/kg), a drug that antagonises several effects of benzodiazepines but has little affinity for peripheral-type sites, had no action on the reduction in social interaction induced by RO 5-4864. However, CGS 8216 (10 mg/kg) which also antagonises the effects of benzodiazepines and has little affinity for RO 5-4864 recognition sites, significantly enhanced the reduction in social interaction caused by RO 5-4864, and the combination produced a significant decrease in locomotor activity. These results are discussed in terms of possible sites of action of RO 5-4864 on the GABA-benzodiazepine receptor complex.     

Specific benzodiazepine antagonist RO15-1788 and thirst-induced drinking in the rat

Midazolam, a new water-soluble benzodiazepine, increased the water intake of water deprived rats. The effect was abolished by concurrent treatment with the benzodiazepine antagonist, RO15-1788. The lack of effect of RO15-1788 on phenobarbitone-induced hyperdipsia confirmed the specificity of the antagonist action. Alone RO15-1788 (0.3-30 mg . kg-1) had no effect on thirst-induced drinking. Thus, it was not necessary to posit the action of an endogenous benzodiazepine ligand in thirst-induced drinking.     

Proconvulsant and 'anxiogenic' effects of n-butyl beta carboline-3-carboxylate, an endogenous benzodiazepine binding inhibitor from brain

The discovery of n-butyl beta carboline-3-carboxylate (beta CCB) as an endogenous substance of brain capable of interacting with the central benzodiazepine receptor, and the fact that this beta carboline increases in the cerebral cortex of rats undergoing acute stress, led us to study the pharmacological properties of beta CCB in mice. Using 3-mercaptopropionic acid in subconvulsant doses, it was found that this beta carboline, although not being a convulsant, has a proconvulsant action, as indicated by the number of mice undergoing convulsions and the reduction in latency. This proconvulsant effect was observed both with IP or ICV injections and was blocked by the benzodiazepine receptor antagonist RO 15-1788. In an open-field test the injection of 0.3 mg/kg of diazepam increased the number of squares crossed, while beta CCB had the opposite effect, reducing the squares crossed in a dose dependent manner between 1 and 30 mg/kg. This drug also increased the time of freezing and decreased the number of rearings. These changes were partially counteracted by the injection of 3.6 mg/kg of RO 15-1788. In the plus-maze test, 10 mg/kg chlordiazepoxide increased the number of entries and the time spent in the open arms, while the beta carboline produced the opposite effect. The conclusion reached is that beta CCB has both proconvulsant and anxiogenic actions, behaving as an inverse agonist for the central benzodiazepine receptor.     

Central effects of nicotinamide and inosine which are not mediated through benzodiazepine receptors.

The actions of nicotinamide and inosine were investigated on rat cerebellar Purkinje cells using ionophoretic and extracellular recording techniques. Ionophoretic application of nicotinamide or inosine showed that they were potent inhibitors of Purkinje cell firing. This inhibition differed from that induced by benzodiazepines in that it was not reversed by the GABA antagonists bicuculline methiodide and picrotoxin. RO 15-1788, the specific benzodiazepine antagonist, did not reverse the effects of nicotinamide. Chlordiazepoxide has been shown to increase significantly social interaction between pairs of male rats and this increase can be reversed by RO 15-1788, 20 mg kg-1 i.p. Nicotinamide also caused a small increase in social interaction but this effect was not reversed by the benzodiazepine antagonist. Inosine did not increase social interaction. [3H]-flunitrazepam binding studies showed that nicotinamide and inosine have only low affinities for the benzodiazepine binding site. These results suggest that while nicotinamide may exert some neuronal depressant and anxiolytic activity, its site of action appears not to be associated with the benzodiazepine receptor site. Similarly, inosine exerts a neuronal depressant effect dissimilar from that of benzodiazepines.     

Chronic treatment with Ro 15-1788 distinguishes between its benzodiazepine antagonist,agonist and inverse agonist properties

Ro 15-1788 (flumazepil) is an imidazodiazepine that is able to antagonise most of the behavioural actions of the benzodiazepines, as well as having some intrinsic effects. Acute administration of Ro 15-1788 (10 mg/kg) decreases social interaction between male rats and elevates exploratory head-dipping. After 5 days of pretreatment there was tolerance to the former effect, although Ro 15-1788 retained its ability to antagonise the effects on social interaction of the -carboline, FG 7142. Ro 15-1788 also retained its ability to elevate head-dipping: additionally, the chronically-treated rats had elevated motor activity and rearing scores. The acute effects of lorazepam in the holeboard were unchanged by chronic pretreatment with Ro-15-1788. The plasma and brain concentrations after acute administration of lorazepam were unchanged following chronic administration of Ro 15-1788. After chronic treatment the brain concentrations of Ro 15-1788 were unchanged. It is unlikely that pharmacokinetic factors could underlie the different behavioural changes following chronic treatment.     

Involvement of local orphanin FQ in the tolerance induced by repeated microinjections of morphine into ventrolateral periaqueductal gray in rats

The present study evaluated the role of ventrolateral periaqueductal gray (vlPAG)-located orphanin-FQ (OFQ) in the opioid tolerance induced by repeated microinjections of morphine (MOR) into vlPAG. Microinjection of MOR (5 microg/0.5 microl) into vlPAG caused antinociception as quantified with the tail flick and the hot plate tests. When MOR microinjection was repeated twice daily, the antinociceptive effect disappeared within 2 days (tolerance). However, if MOR microinjection was preceded by the OFQ receptor antagonist nocistatin (NST; 1 ng/0.5 microl), the microinjections of MOR did not induce tolerance. If NST microinjections were suspended, subsequent MOR microinjections induced tolerance. In MOR-tolerant rats, a single NST microinjection into vlPAG was enough to restore the antinociceptive effect of MOR. Furthermore, if OFQ (1 ng/0.5 microl) was microinjected into vlPAG, then a MOR microinjection administered 15 min later into vlPAG did not elicit antinociception. Finally, opioid tolerance induced by repeated systemic MOR injections (5 mg/kg, i.p.) was reversed by a single microinjection of NST into vlPAG. This emphasizes the central importance of vlPAG-located OFQ in the MOR tolerance.     

Beneficial actions of RO 15-1788, a benzodiazepine receptor antagonist, in hemorrhagic shock

We studied RO 15-1788, a new benzodiazepine receptor antagonist, [ethyl 8-fluoro-5, 6-dihydro-5-methyl-6-oxo-4H-imidazo (1, 5a) (1, 4) benzodiazepine-3-carboxylate] to determine its effects in a murine model of hemorrhagic shock. Hemorrhaged rats treated with RO 15-1788 maintained post-reinfusion mean arterial blood pressure (MABP) at significantly higher values compared to rats receiving only the vehicle (final MABP 114 +/- 4 vs 82 +/- 4 mmHg, p less than 0.001). Moreover, RO 15-1788 decreased the release of the lysosomal hydrolase, cathepsin D (p less than 0.02) into the circulation and blunted the plasma accumulation of free amino-nitrogen groups (p less than 0.01). Furthermore, the plasma activity of a myocardial depressant factor (MDF) was significantly lower in RO 15-1788 treated rats subjected to hemorrhagic shock than in those given the vehicle (18 +/- 2 vs 42 +/- 4 U/ml, p less than 0.01). Additionally, in vitro analysis indicated that RO 15-1788 antagonizes PAF induced coronary vasoconstriction and cardiac depression observed in perfused rat hearts, as well as inhibiting PAF induced platelet aggregation in cat platelet rich plasma. Our results suggest that antagonism of PAF actions can contribute significantly to the beneficial effects of RO 15-1788 in hemorrhagic shock.     

The anxiogenic action of benzodiazepine antagonists

Two benzodiazepine antagonists were tested in an animal model of anxiety, the social interaction test. Ethyl beta-carboline-3-carboxylate (1 and 2 mg/kg) had a potent anxiogenic action; the imidazodiazepine RO 15-1788 (4-10 mg/kg) had a weak anxiogenic effect that with a larger dose (20 mg/kg) disappeared and RO 15-1788 (10 mg/kg) significantly counteracted the anxiogenic effect of the beta-carboline (1 mg/kg). The implications of these results for the understanding of the pharmacological basis of anxiety and for the existence and nature of an endogenous ligand for the benzodiazepine binding site are discussed.     

The effects of GABA and benzodiazepine receptor antagonists on the anti-conflict actions of diazepam or ethanol

The effects of picrotoxin, bicuculline or RO 15-1788 on the anti-conflict action(s) of diazepam or ethanol were studied in rats using a modified Vogel's conflict test procedure. RO 15-1788 antagonized the anti-punishment effects of diazepam (2.5 mg/kg, IP), whereas various doses of bicuculline or picrotoxin did not interfere with diazepam's anti-conflict effect in this test situation. The anti-conflict action of ethanol (2 g/kg, IP) was antagonized by picrotoxin (1.0 mg/kg, IP), whereas both bicuculline and RO 15-1788 were without effect on the increased punishment response produced by ethanol. These data suggest that the anti-conflict properties of ethanol are at least partially mediated through an enhancement of central GABAergic activity.     

Specific antagonism by RO 15-1788 of benzodiazepine-induced increases in food intake in rhesus monkeys

Food intake of rhesus monkeys was limited to a single daily 2 hr session of banana flavored pellet availability, seven days a week. Following stabilization of intake, the effects of intragastric bromazepam, diazepam and pentobarbital, when given alone, were determined by delivering a dose twice weekly, 60 min before the session. Dose-dependent increases in food intake were observed with the following descending order of potencies: bromazepam, diazepam and pentobarbital. RO 15-1788 (0.5-1.0 mg/kg, IM), when given alone, five min before the session had no effect on food intake. When given in combination with bromazepam and diazepam, RO 15-1788 completely blocked the increases in food intake observed when the benzodiazepines were given alone. The specificity of this antagonism was shown by the failure of RO 15-1788 to alter the food intake increases induced by pentobarbital. These results confirm and extend previous reports of the specific antagonism of benzodiazepine behavioral effects by RO 15-1788 to an additional species and another behavioral effect of benzodiazepines.     

Benzodiazepine antagonist RO 15-1788 partly reverses some anxiolytic effects of ethanol in the mouse

The effects of the benzodiazepine (BZD) receptor antagonist RO 15-1788 (3 mg/kg) on the anxiolytic properties of ethanol in mice confronted with a light/dark choice procedure and with the staircase test were investigated. RO 15-1788 reversed the effects of ethanol on some of the behavioural parameters without eliciting intrinsic effects when given alone. These data closely resemble those we previously obtained with several BZD receptor inverse agonists such as RO 15-3505, RO 15-4513 or -CCM. Since anxiogenic-like properties of low doses of RO 15-1788 have been identified by other authors, it is suggested that the antagonistic action of this drug against some of the behavioural effects of ethanol could be due to its being a partial BZD inverse agonist.     

Contrasting effects of ethyl beta-carboline-3-carboxylate (beta CCE) and diazepam on cerebellar cyclic GMP content and antagonism of both effects by Ro 15-1788, a specific benzodiazepine receptor blocker

Ethyl beta-carboline-3-carboxylate (beta CCE), a benzodiazepine antagonist, was found to increase basal levels of cyclic GMP in rat cerebellum. beta CCE also augmented the elevation of cyclic GMP concentrations induced by isoniazid, in contrast to diazepam which blocked this effect of isoniazid. Administration of beta CCE and diazepam together cancelled each other's effect on the elevation of cyclic GMP levels after isoniazid. Ro 15-1788, another potent benzodiazepine antagonist, was found to have virtually no effect on cyclic GMP levels in naive or isoniazid-treated rats. Ro 15-1788 antagonized diazepam's lowering of the elevation of cyclic GMP content of cerebellum after isoniazid. Ro 15-1788 also blocked the increase in cyclic GMP levels elicited by beta CCE, indicating that this effect of beta CCE involves its interaction at benzodiazepine receptors. Some pharmacological actions of beta CCE might be based on hindering GABA transmission.     

Interaction of suriclone with central type benzodiazepine receptors in living baboons

The interaction of suriclone and two of its main metabolites with central type benzodiazepine receptors, which had been labeled in vivo with the radioligand [11C]RO 15-1788, was investigated in living baboons. The concentration of radioligand bound to the receptors, as measured in brain transverse sections by positron emission tomography, decreased rapidly after the i.v. administration of suriclone at doses known to induce pharmacological effects. The rate and extent to which [11C]RO 15-1788 binding was displaced increased with increasing doses of suriclone. The half-inhibitory dose (ID50) was determined to be 0.08 mg/kg in vivo. The rapid inhibitory effect of suriclone on the in vivo binding of [11C]RO 15-1788 in the brain seems to reflect its ability to act at the GABA-benzodiazepine receptor complex, at or near to the benzodiazepine binding site, to induce its pharmacological activity. The i.v. injection of the demethylated metabolite of suriclone, RP 35,489, only caused a slight displacement of [11C]RO 15-1788 binding even at a dose of 2 mg/kg. Thus, suriclone appears to be more potent than RP 35,489 to displace the benzodiazepine 11C antagonist in vivo. The sulfoxide metabolite, RP 46,166, did not significantly change the kinetics of [11C]RO 15-1788 binding in the brain. The slight effects produced by high doses of RP 35,489 and RP 46,166 on [11C]RO 15-1788 binding in the brain suggest that these metabolites are probably not responsible for the expression of biological activity of suriclone mediated by benzodiazepine receptors.     

Recovery from lorazepam tolerance and the effects of a benzodiazepine antagonist (RO 15-1788) on the development of tolerance

After 3 days of dosing rats with lorazepam (0.25 mg/kg), tolerance developed to its sedative effects. Recovery from this tolerance was rapid. No differences could be detected in undrugged behaviour 24 h after the last dose and no differences in response to a probe injection could be found when 2 drug-free days intervened between the chronic treatment and test dose. RO 15-1788 (1–4 mg/kg) antagonised the sedative effects of acute lorazepam (0.5 and 0.25 mg/kg), but chronic treatment with these doses concomitantly with lorazepam did not prevent the development of tolerance. However, 4 mg/kg RO 15-1788 administered for 5 days at the same time as lorazepam (0.5 mg/kg) and again 45 min later attenuated the development of tolerance. Plasma concentrations after acute and chronic treatment did not differ for 0.25 mg/kg lorazepam, but they were lower following chronic treatment with 0.5 mg/kg. Therefore the development of behavioural tolerance in rats to the sedative effects of benzodiazepines probably involves changes in benzodiazepine receptors, in addition to a pharmacokinetic contribution after treatment with high doses.Wellcome Trust Senior Lecturer     

Pentylenetetrazol-induced seizure is not mediated by benzodiazepine receptors in vivo

The selective benzodiazepine antagonist RO 15-1788, labelled with carbon 11 [11C] RO 15-1788, as a specific marker, together with positron emission tomography, allows the in vivo study of benzodiazepine receptors in primates. In addition, when coupled with recordings of electroencephalographic activity, this method offers the feasibility of studying the correlation between occupancy of benzodiazepine receptors and the convulsant action of drugs acting at the benzodiazepine-GABA receptor complex in vivo. The present study showed that convulsant doses of pentylenetetrazol (PTZ) could affect the binding of [11C] RO 15-1788 in vivo in two ways, depending on the doses tested: at concentrations of 20 and 30 mg/kg, pentylenetetrazol increased the binding of [11C] RO 15-1788 whereas larger concentrations displaced the binding of [11C] RO 15-1788. The direct correlation between the occupancy of respective benzodiazepine receptors, afforded by increasing convulsant doses of pentylenetetrazol, revealed that competitive interaction with benzodiazepine receptors was not necessary for pentylenetetrazol to induce the appearance of seizures in vivo.     

Partial anxiolytic action of morphine sulphate following microinjection into the central nucleus of the amygdala in rats.

In the social interaction test of anxiety, bilateral microinjections of morphine sulphate (10 microgram) into the central nucleus of the amygdala counteracted the reduction in social interaction normally seen when the test arena is unfamiliar to rats. However, these injections did not counteract the decrease in social interaction that is observed as illuminance of the arena is increased. Morphine injections into the medial site depressed social interaction below the levels shown by control animals. In the open field test, morphine produced a facilitation of peripheral activity when injected into the central nucleus whilst a decrease in rearing was observed following similar injections into the medial nucleus. Overall, these data indicate a partial anxiolytic action of morphine in the central amygdaloid nucleus. Results are discussed in relation to possible differences in opioid peptide innervation of these two amygdaloid nuclei.     

3-((+-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and phencyclidine produce a deficit of passive avoidance retention in rats

3-((+-)-2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), phencyclidine (PCP) and diazepam were evaluated for their ability to produce a deficit for a single trial step-through passive avoidance response in rats. Pretraining administration with CPP at doses ranging from 2.0 to 10.0 mg/kg s.c. significantly decreased retention latencies 24 h after passive avoidance training. Similar effects were found with PCP at doses ranging from 0.5 to 1.7 mg/kg s.c. and diazepam at doses between 5.0-18.0 mg/kg s.c. Pretraining administration with the benzodiazepine antagonist, RO15-1788 at doses between 0.1-15 mg/kg s.c., did not alter retention latencies. Co-administration of RO15-1788 (0.01-15.0 mg/kg s.c.) with CPP (6.0 mg/kg s.c.) or PCP (1.0 mg/kg s.c.) failed to block decreases in latencies. However, when RO15-1788 was co-administered with diazepam (9.0 mg/kg s.c.) a dose-related antagonism of diazepam's effects were found. These results suggest that the behavioral actions of CPP and PCP on passive avoidance retention are not mediated via the benzodiazepine receptor complex.