Effects of some centrally acting muscle relaxants on spinal root potentials: a comparative study.

The effects of intravenously administered mephenesin, tolperisone, baclofen, diazepam and midazolam on reflex activity were studied in unanesthetized spinal cats. Monosynaptic, as well as polysynaptic ventral root reflexes, the dorsal root potential and the dorsal root reflex were recorded simultaneously from L6-S1 segments. An analogue integrating method was developed for quantitative monitoring and recording ipsilateral spinal root potentials evoked by stimulation of the tibial nerve. Mephenesin (12.5-50 mg/kg) caused a significant and dose-dependent reduction in the polysynaptic and the dorsal root reflexes, slightly decreased the dorsal root potential but minimally affected the monosynaptic ventral root reflex. Tolperisone (2.5-10 mg/kg) dose-dependently inhibited both ventral root reflexes and the dorsal root reflex. It slightly prolonged the dorsal root potential without affecting the amplitude. Baclofen (0.5 mg/kg) abolished the monosynaptic reflex, partially inhibited the polysynaptic reflex, while dorsal root responses were less attenuated. Both benzodiazepines exerted similar actions, both qualitatively as well as quantitatively: the polysynaptic reflex was partially reduced while the monosynaptic reflex was not modified by diazepam or midazolam. Dorsal root responses were enhanced and the half-time of decay of the dorsal root potential was prolonged. Different patterns of action of muscle relaxants studied here are discussed in terms of their possible mechanisms of action. Profound depressant effects of mephenesin and tolperisone on the dorsal root reflex are in contrast to the small effect of both drugs on the dorsal root potential and might reflect their inhibition of spike-generating mechanisms. For a yet unknown reason, various spinal pathways are affected differentially by baclofen. In spinal cats, the reduction by benzodiazepines of the polysynaptic reflex may be related to the potentiation of some unidentified GABA-ergic inhibitory processes. The use of water-soluble midazolam, as a model compound instead of diazepam, is suggested because the usual organic solvents for diazepam may affect its action.

Activity of 2,3-benzodiazepines at native rat and recombinant human glutamate receptors in vitro: stereospecificity and selectivity profiles.

The activity and selectivity of the glutamate receptor antagonists belonging to the 2,3-benzodiazepine class of compounds have been examined at recombinant human non-NMDA glutamate receptors expressed in HEK293 cells and on native rat NMDA and non-NMDA receptors in vitro. The racemic 2,3-benzodiazepines GYKI52466, LY293606 (GYKI53405) and LY300168 (GYKI53655) inhibited AMPA (10 microM)-mediated responses in recombinant human GluR1 receptors expressed in HEK293 cells with approximate IC50 values of 18 microM, 24 microM and 6 microM, respectively and AMPA (10 microM) responses in recombinant human GluR4 expressing HEK293 cells with approximate IC50 values of 22 microM, 28 microM and 5 microM, respectively. GYKI 52466, LY293606 and LY300168 were non-competitive antagonists of AMPA receptor-mediated responses in acutely isolated rat cerebellar Purkinje neurons with approximate IC50 values of 10 microM, 8 microM and 1.5 microM, respectively. The activity of racemic compounds LY293606 and LY300168 was established to reside in the (-) isomer of each compound. At a concentration of 100 microM, GYKI52466, LY293606 and LY300168 produced < 30% inhibition of kainate-activated currents evoked in HEK293 cells expressing either human homomeric GluR5 or GluR6 receptors or heteromeric GluR6+KA2 kainate receptors. The activity of the 2,3-benzodiazepines at 100 microM was weak at kainate receptors, but was stereoselective. Similar levels of inhibition were observed for kainate-induced currents in dorsal root ganglion neurons. Intact tissue preparations were also used to examine the stereoselective actions of the 2,3-benzodiazepines. In the cortical wedge preparation, the active isomer of LY300168, LY303070, produced a non-competitive antagonism of AMPA-evoked depolarizations with smaller changes in depolarizations induced by kainate and no effect on NMDA-dependent depolarizations. LY303070 was also effective in preventing 30 microM AMPA-induced depolarizations in isolated spinal cord dorsal roots with an approximate IC50 value of 1 microM. Synaptic transmission in the hemisected spinal cord preparation was stereoselectively antagonized by the active isomers of LY300168 and LY293606. In summary, these results indicate that 2,3-benzodiazepines are potent, selective and stereospecific antagonists of the AMPA subtype of the non-NMDA glutamate receptor.

Low extracellular magnesium unmasks N-methyl-D-aspartate-mediated graft-host connections in rat neocortex slice preparation.

The main purpose of this study was to investigate the role of N-methyl-D-aspartate receptors in host-graft synaptic transmission in the neocortex. The effects of low extracellular magnesium, the glutamate agonist N-methyl-D-aspartate and N-methyl-D-aspartate antagonists on the synaptic activation of connections between embryonic neocortical graft tissue and the surrounding host tissue were studied in 17 perfused slices of rat neocortex. In standard artificial cerebrospinal fluid, stimulation of the host white matter evoked field potentials in four of 17 grafts. However, in Mg(2+)-free medium, the same stimulation evoked field potentials in an additional six grafts, with significant increases in the mean duration of the evoked responses in the 10 responsive grafts. In five of these slices stimulation of the graft also evoked field potentials in the host tissue, suggesting reciprocal interaction between graft and host. Simultaneous extracellular recordings from graft and host tissues in Mg(2+)-free medium showed that spontaneous epileptiform discharges developed in the graft and host tissue synchronously. In Mg(2+)-free medium, application of N-methyl-D-aspartate induced a shift of the baseline with superimposed epileptiform discharges in both graft and host. Application of the non-competitive N-methyl-D-aspartate antagonist ketamine and the competitive antagonist D,L-2-amino-5-phosphonovaleric acid attenuated or reversibly blocked both the spontaneous epileptiform discharges and the evoked field potentials. Our data provides evidence that N-methyl-D-aspartate receptors are present at synapses created between fetal graft and host neocortex, and that the N-methyl-D-aspartate-activated receptor-channel complex plays an active role in mediating excitatory synaptic transmission in host-graft circuitry.

2,3-benzodiazepine AMPA antagonists.

The 2,3-benzodiazepine GYKI 52466 (1-(4-aminophenyl)-4 methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine) and its analogues represent a family of selective AMPA antagonists. They modulate AMPA channel functions through an allosteric site on the receptor, which is probably different from the ones involved in the actions of cyclothiazide and aniracetam. These compounds are frequently used as research tools to elucidate glutamate receptor-mediated functions. The most effective members of the family inhibit AMPA-induced currents in the submicromolar range. In addition, they are active at low systemic doses in various in vivo experimental models and also possess a good oral bioavailability. In vitro and in vivo pharmacological results with 2,3-benzodiazepine AMPA antagonists indicate their potential therapeutical value in treating a great variety of central nervous system diseases, of which epilepsy and neurodegenerative disorders are regarded as the most important.

Comparison of spontaneous and evoked epileptiform activity in three in vitro epilepsy models.

Rat neocortical slices express spontaneous epileptiform activity after incubation with GABA(A) receptor blocker bicuculline (BIC, 20 microM), with potassium channel blocker 4-aminopyridine (4-AP, 50 microM) or in Mg(2+)-free medium (LMG). Various parameters of spontaneous and evoked epileptiform discharges and their pharmacological sensitivity were analysed using extracellular field potential recordings in this comparative in vitro study. All types of convulsant solution induced spontaneous epileptiform activity, however, the analysed parameters showed that characteristics of epileptiform discharges are rather different in the three models. The longest duration of discharges was recorded in LMG, while the highest frequency of spontaneous events was detected in 4-AP. The epileptiform field responses elicited by electrical stimulation appeared in an all-or-none manner in BIC. On the contrary, in 4-AP and in LMG the amplitude of the responses increased gradually with increasing stimulation intensities. The NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (APV, 25 microM) abolished the LMG induced spontaneous epileptiform activity and significantly reduced the frequency of the epileptiform discharges in BIC and 4-AP. Blocking the AMPA type of glutamate transmission with 1-(aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466, 40 microM) rapidly abolished BIC-induced spontaneous epileptiform activity and caused a significant decrease in the frequency of 4-AP induced spontaneous epileptiform discharges. However, it had only a weak effect on the LMG-induced epileptiform activity. We conclude that the contribution of NMDA and AMPA types of glutamate receptors to the development and maintenance of epileptiform activity in cortical cell assemblies is different in the three models. There are significant alterations in contribution of NMDA and AMPA types of glutamate receptors to the above-mentioned processes in the different convulsants. In BIC the synchronisation is mainly due to the altered network properties, namely inhibition is reduced in the local circuits. Although inhibition is reduced in the local circuits, the AMPA receptor antagonist relatively easily blocked the synchronised excitation. In 4-AP, and especially in LMG, changes in the membrane characteristics of neurones play a crucial role in the increased excitability. In this case the AMPA antagonist was less effective.

Apparent antinociceptive and anti-inflammatory effects of GYKI 52466.

GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine) was examined in a battery of analgesia and anti-inflammatory tests in rats and mice, respectively. Its 3-N-acetyl (GYKI 53773) and 3-N-methylcarbamoyl (GYKI 53784) derivatives were also examined in some assays. These 2,3-benzodiazepines, known as prototypic non-competitive antagonists of AMPA receptors, showed a peculiar profile in some routinely used antinociceptive tests. They were found fairly potent in rat tail flick and mouse phenylquinone writhing assays but the dose-response curves were rather shallow as compared to that of morphine. Their action is stereoselective, i.e., the (+) isomers were found inactive, in agreement with the previous in vitro studies. Their antinociceptive effect could not be reversed by naloxone and the GYKI compounds did not potentiate significantly the morphine-induced analgesia. In the mouse hot plate assay the 2,3-benzodiazepines were active only in doses inducing visible motor incapacitation. In rats, GYKI 52466 weakly reduced the hypersensitivity accompanying acute carrageenan edema. However, it potently inhibited the hyperalgesia during Freund adjuvant-induced chronic arthritis. In the latter assay GYKI 52466 also attenuated the body weight loss without altering the paw edema. The present findings confirm reports in the literature which indicate AMPA receptors may contribute to certain forms of pathological hyperalgesia, e.g., to that detectable in inflamed tissues.

Electrophysiological studies with a 2,3-benzodiazepine muscle relaxant: GYKI 52466.

The effects of GYKI 52466, a new 2,3-benzodiazepine with muscle relaxant and anticonvulsant properties, were investigated and compared to those of midazolam in electrophysiological experiments. The effects of the drugs on the reflex potentials evoked by afferent nerve stimulation and recorded from the spinal roots in unanesthetized spinal cats were studied. GYKI 52466 exerted a strong inhibitory effect on the monosynaptic as well as the polysynaptic ventral root reflexes, while the dorsal root responses decreased slightly. In contrast, midazolam markedly enhanced the dorsal root responses, did not modify the monosynaptic reflex and partially inhibited the polysynaptic reflex. The spontaneous firing of cerebellar Purkinje cells was depressed by midazolam, but not by GYKI 52466. These results suggest strongly that, contrary to the classical 1,4-benzodiazepines, potentiation of the GABA-A receptor-mediated inhibition does not play a significant role in the pharmacological actions of GYKI 52466.

Differential modulation of the GYKI 53784-induced inhibition of AMPA currents by various AMPA-positive modulators in cerebellar Purkinje cells.

The effects of various (S)-alpha-amino-3-hydroxy-5-methyl-4-izoxazole-propionate (AMPA) receptor modulators on AMPA-induced whole-cell currents were compared in isolated rat cerebellar Purkinje cells. The positive modulators, aniracetam, cyclothiazide, 1-(1, 3-benzodioxol-5-ylcarbonyl)-piperidine (1-BCP), and 1-(quinoxaline-6-ylcarbonyl)-piperidine (BDP-12), dose-dependently potentiated the steady-state component of AMPA currents. The negative modulator, (-)1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-4,5-dihydro-3-methylcarbamoyl-2,3-benzodiazepine (GYKI 53784), dose-dependently suppressed AMPA responses. Its concentration-response curve was shifted to the right in a parallel fashion by all positive modulators, indicating a competitive type of interaction. However, the relative potencies of the positive modulators were different with regard to the enhancement of AMPA responses and the reversal of GYKI 53784-induced inhibition, respectively. It is supposed that positive modulators act at multiple allosteric sites and that they interact with GYKI 53784 at only one of these sites.

Morphine and [D-Met2,Pro5]enkephalinamide do not show specific neuroleptic activity.

Several conventionally used in vivo pharmacological assays were applied to examine whether morphine (M) and a potent enkephalin analogue, [D-Met2,Pro5]enkephalinamide (DMPEA) have haloperidol (H)-like neuroleptic activity. The apomorphine (A)-induced stereotypy and the conditioned reflex activity were inhibited by extremely low doses of H, while somewhat higher doses were needed to induce catalepsy or to suppress the A-elicited turning behaviour in rats with unilateral nigral lesion. M produced these effects only in doses higher than needed for analgesia. DMPEA, however, attenuated the A-elicited stereotypy already at a subanalgesic dose level but it was very weak in the other tests. Furthermore, neither M nor DMPEA inhibited the A-elicited stereotypy completely. Consequently, these drugs exhibit strikingly dissimilar relative potencies in the in vivo assays considered specific for neuroleptics. Our findings, in accordance with much of the data available, suggest that neither M nor DMPEA has a specific neuroleptic activity.

Development of long-term potentiation in the somatosensory cortex of rats of different ages.

The age dependence of possible long-term potentiation (LTP) induction in rat somatosensory cortex was studied in in vitro slice experiments. Coronal slices were prepared from the somatosensory cortex of rats of different ages, and excitatory postsynaptic potentials evoked by stimulation of the white matter (0.1 Hz, subthreshold for spike) were recorded intracellularly. In 70% of the slices taken from 2-week-old rats, a moderate potentiation (20-30%) could be induced by either 5 or 100 Hz stimulation. No LTP was observed in younger (1 week) or older (3 weeks) cortex. On the basis of our experiments an important ontogenetic role of increased synaptic efficacy is suggested in a critical developmental period of rats after birth.

Bicarbonate and thiocyanate ions affect the gating of gamma-aminobutyric acid(A) receptors in cultured rat cortical cells.

The ionophore function of gamma-aminobutyric acid(A) (GABA(A)) receptors was studied by whole-cell patch clamp electrophysiology in primary cultures of rat cerebral cortex. Chloride ions were replaced for SCN(-) (thiocyanate) and HCO(-3) (bicarbonate) ions. The EC(50) values of the GABA(A) agonist muscimol (HCO(-3)>Cl(-)>SCN(-)) varied parallel with the free energies of dehydration of the anions, while the inhibition constants of the GABA(A) antagonist bicuculline methiodide were not affected. These findings might be relevant in the interpretation of the contribution of HCO(-3) versus Cl(-) currents to the pharmacological differences between depolarizing and hyperpolarizing GABA responses.

An increased intensity of N-methyl-D-aspartate (NMDA) but not non-NMDA receptor activation may be responsible for the enhancement of excitatory processes in the neocortex of two-week-old rats: a brain slices study.

During the brain maturation a critical period is detectable when the sensitivity of the neocortex is high. Enhanced excitatory activity is characteristic at that time while the inhibitory processes are underdeveloped. The goal of this study was to determine the effectiveness of different types of excitatory amino acid antagonists reducing the electrically evoked excitatory synaptic responses of the somatosensory cortex. Effects of the specific competitive N-methyl-D-aspartate (NMDA) antagonist 4-amino-phosphono-valerate (APV), and the specific non-competitive, non-NMDA antagonist 1-(4-aminophenyl)-3-methylcarbamoyl-7,8-methylenedioxy-5H-2,3-benz odiazepine (GYKI 53655) were analysed on neocortex slices prepared from 2-week-old and adult rats. APV caused a partial inhibition of the electrically evoked response more effectively in young animals than in adults, while the effective IC50 values were similar. In contrast, the non-NMDA antagonist had a similar effect on the slices of both age-groups.

AMPA receptor antagonists, GYKI 52466 and NBQX, do not block the induction of long-term potentiation at therapeutically relevant concentrations.

The involvement of alpha-amino-3-hydroxy-5-methylizoxazole-4-propionic acid (AMPA) receptors in induction of long-term potentiation (LTP) was examined in rat hippocampal slice preparation. Using conventional extracellular recording, excitatory postsynaptic potentials (EPSPs) and population action potentials (PSs), evoked by low-frequency stimulation of the Schaffer collateral-commissural fibres, were recorded in the CA1 region. The effects of a competitive AMPA receptor antagonist, 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2, 3-dione (NBQX), and that of a non-competitive blocker, 1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (GYKI 52466) have been examined. 0.25-0.5 microM of NBQX and 20-40 microM of GYKI 52466 did not suppress the induction of LTP. LTP was attenuated only at the highest concentrations tested (1 microM NBQX or 80 microM GYKI 52466). These in vitro concentrations, however, exceed the brain levels needed for in vivo anticonvulsant action. Furthermore, even at the highest concentrations both compounds suppressed only the expression but not the induction of LTP. Namely after their washout LTP reappeared. Thus, at pharmacologically relevant concentrations these AMPA receptor antagonists apparently do not suppress LTP, a cellular mechanism underlying memory formation. These experiments suggest that in clinical practice AMPA receptor blockade may have some advantage over N-methyl-D-aspartate receptor antagonism, which is accompanied by severe memory impairment.

Effect of glutamate receptor antagonists on excitatory postsynaptic potentials in striatum.

Glutamate, as the main transmitter of corticostriatal pathway, has a crucial role in the regulation of the activity of striatal cells as well as in pathogenesis of some diseases characterized by striatal malfunction caused by overexcitation of neurons. In the present study, the role of ionotropic excitatory amino acid receptors was investigated in the striatal synaptic transmission. Using conventional intracellular electrophysiological methods in brain slices, we have investigated the effects of the N-methyl-D-aspartate (NMDA) antagonist (+/-) 2-amino-5-phosphono-valerate (APV) and the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) antagonist (+/-) 1-(4-aminophenyl)-3-methyl-carbamoyl-7,8-methylenedioxy-5H-2,3-benzodiaz epine (GYKI 53655) on the excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of corpus callosalpham. The AMPA antagonist significantly decreased electrically evoked responses and a weak inhibition was also observed after APV application. The results were compared to similar data obtained in a cortical slice study.

Changes in seizure activity of the neocortex during the early postnatal development of the rat: an electrophysiological study on slices in Mg(2+)-free medium.

NMDA receptor-mediated process of rat neocortex slices prepared from 2-24-day-old rats were studied in Mg(2+)-free solution. The response to NMDA application as well as the susceptibility to epileptiform discharges showed age-dependent changes during the first 4 weeks. Slices from the youngest age group seemed to be the most sensitive to NMDA, whereas epileptic activity developed most readily at around the third week.

Age-dependent modification of aspecific cellular effects of the benzodiazepine flunitrazepam.

The experiments presented here demonstrate an aspecific effect of the benzodiazepine derivative flunitrazepam (FNZ). It differs in sites and mechanisms of action, both from benzodiazepine (BZ) specific effects on gamma-aminobutyric acid (GABA) blocking transmission via central BZ receptors and from BZ effects mediated by peripheral BZ receptors. The aspecific effect of FNZ can suitably be examined on isolated and identified neurons of the mollusc Lymnaea stagnalis (pond snail). The physiological sites of action are outside the synaptic zone, on the neuron somatic membrane and affect 'intrinsic' properties of membrane, including calcium, calcium-activated potassium and chloride channels. The aspecific FNZ effect exerts an influence on the metabolism of the cell by decreasing the permeability of the calcium channel, diminishing the excitability of the neuron membrane, and hyperpolarizing the cell, thus potentiating the specific effect of FNZ. The senile alterations of the neuron function intensify the aspecific effects of FNZ to such a degree that it must be taken seriously in consideration in anesthesia of elderly patients.

Effect of morphine in combination with chlorpromazine and haloperiodol on operant behavior.

Using a discrete-trial avoidance-escape schedule in rats the interactions of morphine (M), chlorpromazine (C) and Haloperidol (H) were studied. All three drugs given alone or in combination disrupted operant behavior. At low doses C and H potentiated each other's effect, while at high doses antagonism could be observed. When combinations of M with C or H were tested, no antagonism or synergism was detected. Based on the statistical considerations used, it seems to be confirmed that the reflex inhibitory action of neuroleptics and M are mediated by independent receptor populations.