W. Haefely

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.

Benzodiazepine antagonist Ro 15-1788: binding characteristics and interaction with drug-induced changes in dopamine turnover and cerebellar cGMP levels.

Abstract: The recently discovered benzodiazepine antagonist Ro 15‐1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug‐induced changes in dopamine turnover and cerebellar cGMP level. Ro 15‐1788 reduced [3H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [3H]diazepam binding in vitro (IC50= 2.3 ± 0.6 nmol/liter). [3H]Ro 15‐1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation constant (KD) of 1.0 ± 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [3H]Ro 15‐1788 from its binding site was of the same rank order as found previously in [3H]diazepam binding. Autoradiograms of [3H]Ro 15‐1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [3H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine‐ or stress‐induced elevation of homovanillic acid in rat brain was antagonized by Ro 15‐1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15‐1788. Thus, Ro 15‐1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15‐1788.

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.nThese 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.

Interaction of benzodiazepines with neuroleptics at central dopamine neurons

SummarySeveral benzodiazepines (chlordiazepoxide, clonazepam, diazepam and flunitrazepam) markedly counteracted the elevation of the homovanillic acid (HVA) content of the rat brain induced by neuroleptics (haloperidol, pimozide, chlorpromazine, and clozapine). A similar effect was obtained with the inhibitor of GABA transaminase, aminooxyacetic acid (AOAA). The interaction of benzodiazepines with the neuroleptic-induced HVA increase was similar in the striatum and in the limbic forebrain, and was antagonized by the GABA receptor-blocking agent, picrotoxin. Both the benzodiazepines used and AOAA potentiated the cataleptic effect of the four neuroleptics.It is concluded that benzodiazepines, by intensifying GABA-ergic transmission, enhance the ongoing inhibition of mesencephalic dopamine neurons exerted by the striatonigral GABA system. As a consequence, the feedback activation of dopamine neurons induced by the neuroleptic blockade of dopamine receptors in the striatum and the limbic system is attenuated. This results in a reduction of the neuroleptic-induced increase of HVA and in the potentiation of the cataleptic effect of neuroleptics.

Benzodiazepine receptors resolved.

To date, attempts to map the distribution and density of benzodiazepine receptors in the CNS have been dominated by radiohistochemical techniques with conventional receptor binding. Their limited resolution, however, prompted us to try an immunohistochemical approach. Purified GABA/benzodiazepine receptors, prepared from bovine cerebral cortex, have been used to raise monoclonal antibodies for this purpose. Immunoreactive sites in rat brain, spinal cord and retina as well as in bovine and post-mortem human brain were found to be concentrated on neuronal cell bodies and processes in those regions known to be innervated by GABAergic neurons. Electron microscopic analysis revealed a selective staining of axosomatic and axodendritic pre- and postsynaptic contacts.

The effects of 5-hydroxytryptamine and some related compounds on the cat superior cervical ganglion in situ

SummaryThe effects of 5-hydroxytryptamine (5-HT) and related indole derivatives injected as i.a. bolus injections in a large dose range were studied in the cat superior cervical ganglion (SCG) by recording ganglionic and postganglionic electrical activity. 5-HT had two independent and opposite actions: a long-lasting inhibition of ganglionic transmission occurred already with extremely low doses and increased up to highest ones: both pre-and postsynaptic sites of action seem to be involved. In a medium and high dose range, the inhibitory effect was temporarily masked by a short-lasting stimulation and a depolarisation which was small when compared with that produced by nicotinic agents. The depolarization triggered a secondary hyperpolarization. The stimulant, but not the inhibitory action was very prone to tachyphylaxis.LSD, methysergide, psilocybin and N,N-dimethyltryptamine (DMT) possess only the inhibitory property of 5-HT. The two N,N-dimethylated derivatives bufotenine and DMT, as well as the α-methylated 5-HT stimulated nicotinic receptors, bufotenine in addition to, and 5-hydroxy-α-methyltryptamine in the absence of typical 5-HT-like inhibitory and excitatory activity. Tryptamine had neither inhibitory nor excitatory 5-HT-like actions, it depolarized and blocked transmission by an unknown mechanism. 5-Hydroxy-α-methyltryptamine and α-methyltryptamine seem to have a marked affinity for, but no intrinsic activity at excitatory 5-HT receptors; they blocked the excitatory effect of 5-HT.

The effect of diphenylhydantoin, diazepam and clonazepam on the activity of Purkinje cells in the rat cerebellum

SummarySpike discharges of single cerebellar Purkinje cells were recorded continuously with extracellular microelectrodes in unanaesthetized curarized rats. The intravenous injection of diphenylhydantoin in doses between 10 and 100 mg kg−1 did not substantially alter the activity of Purkinje cells within 2–3 h. The two benzodiazepines, diazepam and clonazepam, already in low i.v. doses (0.03–0.1 mg kg−1) consistently and reversibly depressed the firing rate. Our results do not support the previously advanced hypothesis that these drugs reduce epilepti-form activities by increasing the output from the cerebellar cortex. They rather point to the possibility that a reduced firing rate of cerebellar Purkinje cells mediates at least in part ataxia and muscular hypotonia observed after these drugs.

The effects of 1,1-dimethyl-4-phenyl-piperazinium (DMPP) in the cat superior cervical ganglion in situ

SummaryThe response of the cat superior cervical ganglion (SCG) in situ to injections of DMPP into its arterial blood supply was investigated by recording continuously both the ganglionic surface (demarcation) potential and the activity in a postganglionic nerve.Almost all ganglionic effects of DMPP can be explained by two basic mechanisms, namely a) the shunt of the ganglion cell membrane resistance caused by the interaction of DMPP with the nicotinic receptors on the ganglion cell membrane, and b) the activity of an electrogenic Na+-pump triggered by the disturbance of the ionic distribution resulting from the primary action.When medium doses of DMPP are injected by bolus-injections, the ganglionic response can easily be separated into two phases representing in almost pure form the consequences of the two mechanisms. An early phase of depolarization with ganglionic excitation, facilitation and depression of synaptic transmission is due directly to nicotinic receptor stimulation. A longer lasting late phase of hyperpolarization with non-specific depression of ganglionic excitability is caused by an electrogenic Na+-pumping which lasts until the pre-injection resting conditions of ionic gradients are restored.An additional presynaptic site of action of DMPP is postulated to explain the transient recovery of ganglionic transmission separating the initial depolarization block from the late phase of inhibition.Certain circulatory conditions and high doses of DMPP render the responses more complex, but the interaction of the two basic processes are revealed by appropriate pharmacological procedures.

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.