Benzodiazepine antagonists reduce epileptiform discharges in rat hippocampal slices.

PURPOSE: The antiepileptic effects of benzodiazepine-receptor (BZR) agonists have been well documented. Surprisingly, an antiepileptic effect for the BZR antagonist, flumazenil, has also been described, the mechanism of which is unknown. We investigated the effects of nanomolar concentrations of flumazenil and a structurally dissimilar BZR antagonist, propyl-beta-carboline-3-carboxylate (beta-CCP), on normal synaptic responses and epileptiform discharges induced by a variety of methods in the CA1 region of rat hippocampal slices. METHODS: Extracellular field potentials were recorded from stratum pyramidale of the CA1 region. Orthodromic stimulation was delivered by a bipolar electrode placed in the stratum radiatum at the border of the CA2/CA3 regions. Drugs were bath applied, and epileptiform discharges were quantified by using the Coastline Bursting Index, which calculates the total length of the discharge waveform of evoked multiple population spikes. For statistical comparisons, we calculated the Coastline Bursting Index for the average of five traces at the end of the control period (20 min), drug application (20 min), and washout (20-40 min). RESULTS: Flumazenil was without effect on normal synaptic responses; however, flumazenil reduced epileptiform discharges evoked in the presence of high [K+]o, leu-enkephalin, the BZR inverse agonist, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), or after a cold-shock procedure. beta-CCP exhibited an action similar to that observed for flumazenil, suggesting that the antiepileptic effect is due to properties common to BZR antagonists. CONCLUSIONS: We suggest that the antiepileptic effect we observed for flumazenil and beta-CCP is mediated at the BZR and might be due to competition with endogenous BZR inverse agonists released preferentially during epileptiform activity.

Benzodiazepine antagonist flumazenil reduces hippocampal epileptiform activity.

We examined the effects of the benzodiazepine antagonist, flumazenil, on epileptiform discharges evoked in the hippocampal CA1 region in vitro. Application of 100 nM flumazenil did not affect normal synaptic responses; however, flumazenil did depress epileptiform discharges induced by 8 mM [K+]o. Epileptiform discharges induced by the GABAA channel antagonist picrotoxin or by the K+ channel blocker 4-aminopyridine were unaffected. Application of the high-affinity, low-efficacy benzodiazepine partial inverse agonist, Ro 19-4603, blocked the anticonvulsant effect of flumazenil, indicating that this action of flumazenil is mediated at a benzodiazepine binding site located on the GABAA receptor. A likely explanation of the present results is that flumazenil antagonizes the action of an endogenous benzodiazepine inverse agonist, which is released during epileptiform discharges evoked in high K+ ACSF.

Involvement of endogenous benzodiazepine receptor ligands in brain disorders: therapeutic potential for benzodiazepine antagonists?

Many therapeutic effects of benzodiazepines are mediated by neuronal high-affinity binding sites, i.e. benzodiazepine receptors (BR), located on GABAA receptors. Recently, endogenous BR ligands have partially been identified which, as agonists, either increase or, as inverse agonists, decrease GABAergic inhibition in the brain. BR antagonists, previously described as intrinsically inactive, induce effects in animals and humans under particular circumstances emphasizing a functional relevance of endogenous BR ligands. Several brain disorders, e.g. anxiety, insomnia, epilepsy, spasticity, alcoholism, coma, dementia, may be associated with a disequilibrium of opposing endogenous BR ligands changing the excitability of neurons implicated in aforementioned diseases. It is proposed that, depending on the relative role endogenous BR ligands play in the pathophysiology of these disorders, BR antagonists might demonstrate a variable efficacy in improving their symptomatology. In fact, such therapy would restore the homeostatic balance among various endogenous BR ligands being disturbed during an illness.

Benzodiazepine antagonist flumazenil reduces bicuculline-induced enhancement of neuronal activity in the spinal cord.

The interaction between the GABAA receptor antagonist bicuculline and the benzodiazepine receptor (BR) antagonist flumazenil was studied in the lumbosacral cord of spinal cats. Bicuculline (0.8 mg/kg i.v.) evoked epileptiform bursting of motoneurons in parallel with a depression of peripherally elicited dorsal root potentials (DRP) and dorsal root reflexes (DDR). A low dose of flumazenil (0.03 mg/kg i.v.) reduced the bursting activity of motoneurons as well as partially reversed the depression of DRP and DRR induced by bicuculline. It is suggested that an endogenous BR ligand down-regulating GABAA receptors is released during the bicuculline-evoked enhancement of neuronal activity and may contribute to the epileptiform discharge of motoneurons in the spinal cord.

Afferent stimulation frequency modulates GABAergic phenomena in the spinal cord: reversal by benzodiazepine antagonists.

Switching from a low (0.5 Hz) to a higher (5 Hz) frequency repetitive stimulation of hindlimb muscle nerve afferents reduced GABA-mediated dorsal root potentials and dorsal root reflexes in the lumbosacral cord of spinal cats. Benzodiazepine receptor antagonists flumazenil and Ro 15-3505 in a very low dose (0.03 mg/kg i.v.) reversed this effect suggesting a stimulation-coupled release of an endogenous benzodiazepine receptor ligand down-regulating GABAA-receptors in the spinal cord.

Pharmacological profile of moclobemide, a short-acting and reversible inhibitor of monoamine oxidase type A.

The novel antidepressant moclobemide is a reversible inhibitor of monoamine oxidase (MAO), preferentially of type A. Moclomide was active in three animal models considered predictive for antidepressant activity: 1) it prevented dose-dependently akinesia and blepharospasm induced in mice and rats by Ro 4-1284, a short-acting amine releasing agent. Prevention of akinesia by moclobemide also depended upon the dose of Ro 4-1284. For comparison also, effects of cimoxatone, harmaline, tranylcypromine and clorgyline are presented: 2) in cats, it selectively and dose-dependently suppressed rapid eye movement sleep without disturbing the sleep-wakefulness cycle; and 3) in the behavioral despair test in mice, it decreased the immobility score to a similar degree as amitriptyline or imipramine. In addition, moclobemide potentiated 5-hydroxytryptophan-induced stereotypies in rats with a potency similar to cimoxatone and with a duration of action of less than 24 hr. Moclobemide had almost no effect on the spontaneous behavior in mice, rats, cats and monkeys. Only in higher doses, marginal sedation and slight impairment in motor performance were seen. Moclobemide did not prevent pilcarpine-induced salivation in mice, demonstrating the absence of anticholinergic activity. Blood pressure and heart rate of freely moving, spontaneously hypertensive rats were only slightly decreased for less than 3 hr. Moclobemide moderately potentiated the pressor effect of p.o. tyramine in rats. In conclusion, the reversible MAO inhibitor moclobemide is active in animal models sensitive to all major drugs used in the treatment of depression. In contrast to imipramine-like antidepressants, it lacks anticholinergic activity and it differs from classic MAO inhibitors by potentiating only weakly the pressor effect of p.o. tyramine.

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.

Electrophysiology of benzodiazepine receptor ligands: multiple mechanisms and sites of action.

Electrophysiology of BZR ligands has been reviewed from different points of view. A great effort was made to critically discuss the arguments for and against the temporarily leading hypothesis of the mechanism of action of BZR ligands, the GABA hypothesis. As has been discussed at length in the present article, an impressive body of electrophysiological and biochemical evidence suggests an enhancement of GABAergic inhibition in CNS as a mechanism of action of BZR agonists. Biochemical data even indicate a physical coupling between GABA recognition sites and BZR which, together with the effector site build-up by Cl- channels, form a supramolecular GABAA/BZR complex. By binding to a specific site on this complex, BZR agonists allosterically increase and BZR inverse agonists decrease the gating of GABA-linked Cl- channels, whereas BZR antagonists bind to the same site without an appreciable intrinsic activity and block the binding and action of both agonists as well as inverse agonists. While this model is supported by many electrophysiological experiments performed with BZR ligands in higher nanomolar and lower micromolar concentrations, it does not explain much controversial data from animal behavior and, more importantly, is not in line with electrophysiological effects obtained with low nanomolar BZ concentrations. The latter actions of BZR ligands in brain slices occur within a concentration range compatible with concentrations of BZ observed in CSF fluid, which would be expected to be found in the biophase (receptor level) during anxiolytic therapy in man. Enhanced K+ conductance seems to be a suitable candidate for this effect of BZR ligands. This direct action on neuronal membrane properties may underlie the many electrophysiological observations with extremely low systemic doses of BZR ligands in vivo which demonstrated a depressant effect on spontaneous neuronal firing in various CNS regions. Skeletomuscular spasticity and epilepsy are two neurological disorders, where both the enhanced GABAergic inhibition and increased K+ conductance may contribute to the therapeutic effect of BZR agonists, since electrophysiological and behavioral studies strongly support GABA-dependent as well as GABA-independent action of BZR ligands elicited by low to intermediate doses of BZ necessary to evoke anticonvulsant and muscle relaxant effects. Somewhat higher doses of BZR ligands, inducing sedation and sleep, lead perhaps to the only pharmacologically relevant CNS concentrations (ca. 1 microM) which might be due entirely to increased GABAergic inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)

NMDA receptors mediate background and excessive activity of gamma-motoneurones in the spinal cord.

The effects of 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), a selective and potent antagonist of N-methyl-D-aspartic acid (NMDA) receptors, were studied on gamma-motoneurone activity in the spinal cord of unanaesthetized spinal cats. CPP (1-10 mg/kg i.v.) dose dependently reduced the spontaneous activity of gamma-motoneurones. The kainic acid (1 mg/kg i.v.)-induced bursting firing pattern of gamma-motoneurones was unaffected or enhanced by CPP. In contrast, CPP suppressed the increase of gamma-motoneuronal excitability caused by NMDA (3 mg/kg i.v.), suggesting that both the background and the exaggerated activity of gamma-motoneurones are mediated by an amino acid that activates NMDA receptors.

L-cycloserine: behavioural and biochemical effects after single and repeated administration to mice, rats and cats.

L-Cycloserine dose-dependently inhibited the activity of gamma-aminobutyric acid (GABA)-transaminase (GABA-T) and elevated the level of GABA in whole mouse brain with a peak effect 3-4 hr after a single intraperitoneal injection. At a dose (30 mg/kg) which elevated the level of GABA almost 4-fold, L-cycloserine moderately increased the content of alanine and slightly reduced that of aspartate, glutamate and glycine in the brain. L-Cycloserine (10-30 mg/kg, p.o. or i.p.) prevented tonic seizures induced by 3-mercaptopropionic acid (3-MPA) and audiogenic seizures in DBA/2 mice, without affecting those evoked by pentylenetetrazol, bicuculline and electroshock. Similarly small doses of L-cycloserine reduced the level of cGMP in the cerebellum of rats, prevented its elevation by 3-MPA and attenuated the hypothalamically-elicited rage reaction in cats. Larger doses of L-cycloserine (greater than 30-100 mg/kg) impaired the performance of mice in the rotarod, chimney and horizontal wire tests, and reduced spontaneous locomotor activity of rats. Upon repeated administration the inhibitory effect of L-cycloserine on the activity of GABA-T and on seizures elicited by 3-MPA in mice increased. In contrast, the depressant action of L-cycloserine on motor performance and locomotion declined in subchronically-treated mice and rats. The levels of amino acids in brain after repeated administration did not differ markedly from those in acutely-treated mice. It is suggested that small doses of L-cycloserine, probably by increasing GABAergic inhibition, reduce hyperexcitability in the brain in acute- and subchronically-treated animals. Larger doses of L-cycloserine, possibly by inducing multiple neurochemical changes, evoke central depressant effects which diminish during subchronic treatment.

2-Amino-7-phosphonoheptanoic acid depresses gamma-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.

Some pharmacological effects of delta-sleep-inducing peptide (DSIP).

The synthetic nonapeptide DSIP was studied in rabbits and cats under normal conditions and under conditions of disturbed sleep. In other experiments, the effect of the oligopeptide on withdrawal jumping provoked by naloxone in morphine-dependent mice was studied. In rabbits, DSIP at 25 micrograms X kg-1 i.v. and 1 mg X kg-1 s.c. augmented spindle-dominated, light nonREM sleep and prevented hyposomnia after a stressful situation. In cats, 25 micrograms X kg-1 i.v. and 100 micrograms X kg-1 s.c. preferentially augmented REM sleep and abolished the sleep suppressant effect of morphine. In morphine-dependent mice, 25.5 micrograms X kg-1 i.v. as well as doses beyond 85 micrograms X kg-1 s.c. attenuated naloxone-induced withdrawal jumping. In most experimental situations, indications for bell-shaped dose-response curves of DSIP were found.

Low-dose benzodiazepine neuronal inhibition: enhanced Ca2+-mediated K+-conductance.

The water-soluble inhibitory benzodiazepine, midazolam, was applied in low nanomolar concentrations to CA1 hippocampal neurons in vitro, recorded intracellularly. The drug caused a long-lasting hyperpolarization and moderate conductance increase, which persisted with TTX-induced synaptic blockade or with intracellular injection of Cl- ions, but not in zero Ca2+ perfusate. Calcium spikes elicited in the presence of TTX were enhanced by midazolam. It was concluded that these low nanomolar concentrations, which did not enhance GABA actions, inhibited by augmenting Ca2+ mediated K+-conductance.

The excitatory effects of the specific benzodiazepine antagonist Ro14-7437, measured intracellularly in hippocampal CA1 cells.

The specific benzodiazepine antagonist, Ro14-7437, in nanomolar concentrations, caused depolarization, increased spontaneous spiking, and conductance decrease when applied to CA1 cells in vitro. These effects were resistant to intracellularly injected Cl- ions or synaptic blockade by TTX, were prevented in Ca2+-free medium, and occurred with or without prior application of midazolam, an inhibitory benzodiazepine. Ca2+-mediated AHPs and Ca2+ spikes in TTX medium were diminished by the blocker, suggesting that Ro14-7437 acted by inhibiting Ca2+-mediated K+ conductance.

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

The potent benzodiazepine receptor ligands beta-carboline-3-carboxylic acid ethyl ester (beta-CCE) and the corresponding methylester (beta-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; beta-CCE enhanced the low-frequency facilitation of pyramidal population spikes in the hippocampus of anaesthetized rats. These effects of beta-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 beta-CCE on the three parameters and similar effects of beta-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, beta-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.

Enhancement of GABAergic inhibition: a mechanism of action of benzodiazepines, phenobarbital, valproate and L-cycloserine in the cat spinal cord.

Unanaesthetized spinal cats were used to assess the effects of several central depressant drugs on GABA-mediated presynaptic inhibition. Benzodiazepines enhance GABAergic inhibition by interaction with specific receptors in the spinal cord. Phenobarbital facilitates GABAergic inhibition by a mechanism unrelated to that of benzodiazepines, perhaps by an effect on the Cl- ionophore within the GABA-benzodiazepine-barbiturate receptor complex. A similar mechanism or the inhibition of GABA re-uptake might be responsible for the facilitating effect of valproate on GABA transmission. By selectively inhibiting GABA transaminase, L-cycloserine presumably increases the amount of GABA available for release.

Ethyl beta-carboline-3-carboxylate antagonizes the action of GABA and benzodiazepines in the hippocampus.

In urethane-anaesthetized rats, the beta-carboline derivative beta CCE (0.3-1.0 mg/kg i.v.) excited hippocampal pyramidal cells which were inhibited by GABA (applied iontophoretically) and benzodiazepines (applied iontophoretically or intravenously). While benzodiazepines facilitated the action of GABA, the effects of GABA and benzodiazepines were antagonized by beta CCE. This electrophysiological study supports the behavioural observations that beta CCE is a benzodiazepine receptor antagonist.