Petar Polc

A three-state model of the benzodiazepine receptor explains the interactions between the benzodiazepine antagonist Ro 15-1788, benzodiazepine tranquilizers, β-carbolines, and phenobarbitone

SummaryThe potent benzodiazepine receptor ligands β-carboline-3-carboxylic acid ethyl ester (β-CCM) and the corresponding methylester (β-CCM) administered i.v. depressed segmental dorsal root potentials in spinal cats, reversed the prolongation of dorsal root potentials by phenobarbitone, and abolished the depression of a motor performance task induced by phenobarbitone in mice; β-CCE enhanced the low-frequency facilitation of pyramidal population spikes in the hippocampus of anaesthetized rats. These effects of β-carbolines reflect a depression of GABAergic synaptic transmission and, thus, are diametrically opposed to the enhancing action of benzodiazepine tranquilizers. The specific benzodiazepine antagonist, Ro 15-1788, while not affecting dorsal root potentials, hippocampal population spikes or phenobarbitone-induced motor performance depression, abolished the effects of β-CCE on the three parameters and similar effects of β-CCM on the spinal cord and motor performance.A three-state model of the benzodiazepine receptor is proposed in which benzodiazepine tranquilizers act as agonists enhancing the function of the benzodiazepine receptor as a coupling unit between GABA receptor and chloride channel, β-carbolines act as “inverse agonists” reducing this coupling function, and Ro 15-1788 represents a competitive antagonist blocking both the enhancing effect of agonists and the depressant effect of “inverse agonists” on GABAergic synaptic transmission.

The effect of diazepam on spinal cord activities: Possible sites and mechanisms of action

SummaryThe effect of diazepam on several activities of the spinal cord was investigated in decerebrate and high-spinal cats by recording neurograms from lumbosacral ventral and dorsal roots and by measuring the levels of γ-aminobutyric acid (GABA) in the lumbosacral spinal cord. Chlorpromazine and benzoctamine were included for comparison with diazepam in part of the investigation.Diazepam depressed but did not abolish monosynaptic and polysynaptic ventral root reflex (VRR) responses; it was 3 to 5 times more potent in the decerebrate than in the spinal cat. Spontaneous gamma fibre activity was markedly and almost equally reduced by diazepam in both preparations. Dorsal root potentials (DRPs) and the presynaptic inhibition of monosynaptic VRRs elicited by stimulation of peripheral afferents were enhanced and prolonged by diazepam to the same extent in spinal and decerebrate animals; however, the enhancement of DRPs elicited by stimulation of the medullary reticular formation required approximately 3 time higher doses of diazepam. The effect of diazepam on presynaptic inhibition and DRPs was antagonized by bicuculline in a surmountalbe manner. Following amino-oxy-acetic acid (AOAA), which more than doubled the levels of endogenous GABA in the spinal cord, presynaptic inhibition and DRPs were enhanced but the amplitude of monosynaptic VRR responses was unaffected; diazepam further enhanced presynaptic inhibition and DRPs but no longer depressed monosynaptic VRR responses. Thiosemicarbazide, which decreased the level of GABA in the spinal cord by about 60%, reduced presynaptic inhibition and DRPs and prevented the augmenting effect of diazepam on these parameters. Doses of the organic solvent of diazepam in the 10 times higher amounts than used in the experiments with diazepam had only negligible and short-lasting effects; it seems very unlikely that the solvent contributed appreciably to the effect of diazepam solutions.It is concluded that 1) diazepam affects various activities of the spinal cord predominantly by a spinal site of action, 2) normal levels of GABA in the spinal cord seem to be a prequisite for the augmenting effect of diazepam on presynaptic inhibition in the spinal cord, 3) diazepam may act by altering the metabolism or disposition of GABA. Whether the enhancement of presynaptic inhibition fully accounts for the depressant effect of diazepam on monosynaptic and polysynaptic VRRs and on the gamma activity cannot be decided yet.In contrast to diazepam, chlorpromazine and benzoctamine did not enhance DRPs. Chlorpromazine depressed monosynaptic and polysynaptic VRRs and gamma activity only in the decerebrate cat. Benzoctamine was approximately as potent as diazepam in depressing monosynaptic and polysynaptic VRR responses in both preparations and in reducing gamma fibre activity in decerebrate cats, but was less potent than diazepam on the gamma fibre activity in spinal animals.

Electrophysiological studies on the specific benzodiazepine antagonist Ro 15-1788.

SummaryThis is an electrophysiological study in cats and rats of the imidazobenzodiazepinone derivative, Ro 15-1788, the first representative of specific benzodiazepine antagonists.(1)In unanaesthetized spinal cats, 1–10 mg kg−1 Ro 15-1788 i.v. did not affect segmental dorsal root potentials (DRPs), polysynaptic ventral root reflexes (VRRs), Renshaw cell responses to antidromic ventral root volleys and spontaneous γ-motoneurone activity. However, at 1 mg kg−1 i.v., it antagonized the enhancement of DRPs as well as the depression of polysynaptic VRRs, Renshaw cell discharges and γ-motoneurone activity induced by meclonazepam (0.1 mg kg−1 i.v.), diazepam (0.3 mg kg−1 i.v.) or zopiclone (1 mg kg−1 i.v.). The same dose of Ro 15-1788 failed to reduce similar effects of phenobarbital (10 mg kg−1 i.v.) on spinal cord activities.(2)In unanaesthetized “encéphale isolé” rats, 3 mg kg−1 Ro 15-1788 i.v. abolished the decrease induced by 5 mg kg−1 midazolam i.v. of spontaneous multiunit activity (MUA) in the substantia nigra pars compacta, nucleus raphé dorsalis, nucleus locus coeruleus and the CAl area of the hippocampus dorsalis, but not the decrease produced by 10 mg kg−1 pentobarbital i.v. Ro 15-1788 (12 mg kg−1 i.v.) by itself did not affect MUA in the substantia nigra, but slightly depressed MUA in the other 3 areas.(3)In intact immobilized rats, the increase of power induced by 1 mg kg−1 flunitrazepam i.v. in the 0.5–48 Hz range of the electrocorticogram as well as in the 0.5–8 Hz, 8–32 Hz and 32–48 Hz frequency bands was transiently abolished by 5 mg kg−1 Ro 15-1788 i.v.(4)In unrestrained cats, 5 mg kg−1 Ro 15-1788 i.p. had no effect on the electrical threshold for eliciting a rage reaction evoked by electric hypothalamic stimulation, but abolished the threshold increase caused by 1 mg kg−1 diazepam i.p. These results are in line with biochemical and behavioural findings and support the selective antagonism by Ro 15-1788 of central effects of benzodiazepines through an interaction at benzodiazepine receptors.

Effects of two benzodiazepines, phenobarbitone, and baclofen on synaptic transmission in the cat cuneate nucleus

SummaryThe effects of diazepam, flunitrazepam, phenobarbitone and baclofen on excitatory as well as on pre- and postsynaptic inhibitory processes in the cuneate nucleus were studied in decerebrate cats.Afferent presynaptic inhibition in the cuneate nucleus, evoked by volleys in the median nerve, and assessed by the size of the positive cuneate surface potential (P wave), the dorsal column reflex (DCR), and the increased excitability of primary afferent terminals of the ulnar nerve, was markedly enhanced by diazepam (0.1–3.0 mg/kg i.v.) and flunitrazepam (0.01–0.3 mg/kg i.v.), slightly enhanced by lower doses of phenobarbitone (3–20 mg/kg i.v.), but depressed by baclofen (1–10 mg/kg i.v.). Diazepam, flunitrazepam and phenobarbitone also increased postsynaptic inhibition in the cuneate nucleus which was measured by the decrease after conditioning volleys in the median nerve of the short-latency lemniscal response to cuneate stimulation. The GABA receptor blocking agent, picrotoxin, antagonized the effects of diazepam on pre- and postsynaptic inhibition in a surmountable way. After thiosemicarbazide (TSC), an inhibitor of GABA synthesis, both pre-and postsynaptic inhibition were greatly reduced and the augmenting effect of diazepam on both types of inhibition was nearly abolished. Aminooxyacetic acid (AOAA), an inhibitor of GABA degradation, slightly enhanced pre- and postsynaptic inhibition; the effects of diazepam were unaffected by AOAA. Diazepam, flunitrazepam and phenobarbitone did not alter the resting excitability of primary afferent endings or of cuneo-thalamic relay (CTR) cells in the cuneate nucleus.After higher doses (30 mg/kg i.v.) of phenobarbitone pre- and postsynaptic inhibition, which were enhanced by 10 mg/kg of this drug, tended to return to pre-drug values or below. Phenobarbitone, in contrast to benzodiazepines, also depressed in a dose-dependent way the N wave, which is an index of the orthodromic excitation of the CTR cells. Baclofen strongly depressed the cuneate N wave, decreased the excitability of CTR cells, reduced pre- and postsynaptic inhibition, but had no effect on the resting excitability of primary afferent endings.Our findings suggest the following modes of action of the above mentioned drugs: 1. benzodiazepines enhance selectively the GABA-mediated pre- and postsynaptic inhibition in the cuneate nucleus; 2. phenobarbitone slightly enhances pre- and postsynaptic inhibition only in a narrow dose range, and in addition reduces the excitatory processes in the cuneate nucleus; 3. baclofen seems to depress the excitation of cuneate relay cells and interneurones postsynaptically; the depression of relay cells is probably non-specific.

Caffeine antagonizes several central effects of diazepam

Abstract In spinal cats, caffeine (3–30 mg·kg −1 i.v.) reduced the increase of dorsal root potentials (DRPs) caused by diazepam (0.1–1 mg·kg −1 i.v.) without affecting the prolongation of DRPs evoked by phenobarbitone (10–20 mg·kg −1 i.v.). Caffeine antagonized the depression by diazepam, but not that by phenobarbitone, of the ventral root-evoked Renshaw cell discharge. In unrestrained cats, 50 mg·kg −1 caffeine i.p. abolished the elevation induced by 1 mg·kg −1 diazepam i.p. of the threshold for eliciting a rage reaction by stimulation of the lateral hypothalamus, but was ineffective against the threshold increase caused by 20 mg·kg −1 phenobarbitine i.p. In the horizontal wire test in mice, caffeine was more potent in reversing the depression of performance induced by diazepam that that by phenobarbitone (ED50 1.8 mg·kg −1 and 139 mg·kg −1 p.o., respectively). The reduction of skeletal muscle tone in mice produced by diazepam was antagonized by low doses of caffeine (ED50 0.53 mg·kg −1 p.o.). While caffeine at low doses (0.3-3 mg·kg −1 p.o.) abolished the anticonflict effect of diazepam in rats, high doses (ED50 160 mg·kg −1 p.o.) were necessary to antagonize the anticonvulsant effect of diazepam on pentylene-tetrazole-induced seizures in mice. The interaction between caffeine and diazepam is not due to a competition at the benzodiazepine receptors but may involve purinergic mechanisms.

Ro 15-4513: Partial inverse agonism at the BZR and interaction with ethanol☆

The imidazobenzodiazepinone derivative Ro 15-4513 has the activity profile of a partial inverse (low efficacy) agonist at the benzodiazepine receptor (BZR). It reverses central nervous depressant effects of diazepam, and, in part, of phenobarbitone and ethanol in mice, rats and cats in behavioural, electrophysiological, and neurochemical paradigms. The interaction of Ro 15-4513 with barbiturates and ethanol is due to its inverse agonistic (negative allosteric modulatory) property at the BZR, as it was reversed by the selective BZR blocker flumazenil (Ro 15-1788). In the present experiment situations, other BZR partial inverse agonists in subconvulsant or overt convulsant doses were less effective against ethanol effects than Ro 15-4513. Possible mechanisms for this differential activity of BZR inverse agonists are discussed.

Effects of several centrally active drugs on the sleep-wakefulness cycle of cats.

Abstract Eighteen drugs were injected intraperitoneally at different doses in cats and their effects on the sleep-wakefulness cycle studied in 6-hr sessions of telemetric EEG and EMG recording. At the lowest doses that affected the sleep-wakefulness cycle, clinically used and potential antidepressants (imipramine, clomipramine, desipramine, amitriptyline, nortriptyline, maprotiline, Ro 03-5939, mianserin, iprindole, viloxazine, pargyline, Ro 11-1163) as well as 5-hydroxy- l -tryptophan selectively depressed rapid eye movement (REM) sleep. Other psychotropic drugs also reduced REM sleep but, in contrast to the above antidepressants, simultaneously affected non-rapid eye movement (NREM) sleep at minimum effective doses: LSD, methylphenidate and, to a lesser degree, haioperidol, depressed, whereas cyproheptadine augmented NREM sleep. Chlorpromazine non-significantly reduced REM sleep at a dose that induced ataxia. The results reveal a selective suppressant effect of antidepressant drugs on REM sleep in cats which seems to reflect an increased activation of 5-hydroxytryptamine and/or noradrenaline receptors in the brain as the consequence of either inhibition ofmonoamine uptake or monoamine oxidase, increased release of monoamines or some yet unknown mechanisms.

Effect of the Delta Sleep-Inducing Peptide (DSIP) on the sleep-wakefulness cycle of cats

Abstract The effect of intravenously injected delta sleep-inducing peptide (DSIP) on the sleep-wakefulness cycle of the cat was studied using telemetric EEG and EMG recording in 6-h sessions. A small dose of DSIP (30 nmol kg −1 ) shortened latency of onset of sleep, reduced waking time, enhanced non-rapid eye movement (NREM) sleep and, even more markedly, rapid eye movement (REM) sleep. In contrast, a higher dose of DSIP (300 nmol kg −1 ) produced no significant effects. The results suggest that, in the cat, appropriate intravenous doses of DSIP have a facilitatory action on both NREM and REM sleep, an effect which is absent after higher doses.

2-amino-7-phosphonoheptanoic acid depresses γ-motoneurons and polysynaptic reflexes in the cat spinal cord

The effects of 2-amino-7-phosphonoheptanoic acid (APH), a selective antagonist of N-methyl-D-aspartate (NMDA) receptors, were studied on spinal cord functions in unanaesthetized spinal cats. APH (10 mg/kg i.v.) depressed spontaneous activity of gamma-motoneurons and segmental polysynaptic ventral root reflexes (VRRs) without affecting monosynaptic VRRs. The NMDA-induced enhancement of polysynaptic VRRs and activation of gamma-motoneurons were antagonized by APH. The results support the hypothesis that NMDA receptors are involved in the polysynaptic excitation of motoneurons, including gamma-motoneurons, and thus participate in motor functions of the spinal cord.

Benzodiazepines enhance the bicuculline-sensitive part of recurrent Renshaw inhibition in the cat spinal cord.

Abstract The effects of intravenous diazepam and midazolam on recurrent Renshaw inhibition of lumbar motoneurones were studied in unanaesthetized spinal cats. Both benzodiazepines enhanced the bicuculline-sensitive, late part of recurrent inhibition without affecting its strychnine-sensitive, early component. The results support the view that some Renshaw cells are GABAergic and extend the potentiating effect of benzodiazepines to a spinal GABAergic synapse that mediates postsynaptic inhibition.