Chronic GABAergic blockade in the spinal cord in vivo induces motor alterations and neurodegeneration.

Inhibitory GABAergic and glycinergic neurotransmission in the spinal cord play a central role in the regulation of neuronal excitability, by maintaining a balance with the glutamate-mediated excitatory transmission. Glutamatergic agonists infusion in the spinal cord induce motor neuron death by excitotoxicity, leading to motor deficits and paralysis, but little is known on the effect of the blockade of inhibitory transmission. In this work we studied the effects of GABAergic and glycinergic blockade, by means of microdialysis perfusion (acute administration) and osmotic minipumps infusion (chronic administration) of GABA and glycine receptors antagonists directly in the lumbar spinal cord. We show that acute glycinergic blockade with strychnine or GABAergic blockade with bicuculline had no significant effects on motor activity and on motor neuron survival. However, chronic bicuculline infusion, but not strychnine, induced ipsilateral gait alterations, phalange flaccidity and significant motor neuron loss, and these effects were prevented by AMPA receptor blockade with CNQX but not by NMDA receptor blockade with MK801. In addition, we demonstrate that the chronic infusion of bicuculline enhanced the excitotoxic effect of AMPA, causing faster bilateral paralysis and increasing motor neuron loss. These findings indicate a relevant role of GABAergic inhibitory circuits in the regulation of motor neuron excitability and suggest that their alterations may be involved in the neurodegeneration processes characteristic of motor neuron diseases such as amyotrophic lateral sclerosis.

Psychopharmacological effects of tianeptine analogous hetero[2,1] benzothiazepine derivatives.

The psychopharmacological effects of a number of thieno and pyrazolo[2,1] benzothiazepine derivatives as well as several synthetic intermediate compounds were investigated in mice. Previously published studies in mice have shown that some of these compounds were effective in the tetrabenazine and Porsolt tests. In the present study, 7 of the 15 compounds under study clearly antagonized the apomorphine (16 mg/kg s.c.)-induced hypothermia, but no significant potentiation of the 5-hydroxytryptophan (5-HTP) and amphetamine actions was found. Five of them inhibited the syndrome induced by 5-HTP (250 mg/kg i.p.). Moreover, some of them were effective in the plus-maze test and antagonized the apomorphine (3 mg/kg s.c.)-induced effects. On the other hand, these compounds produced a moderate inhibition of exploratory behaviour in the hole-board test, but they had no significant muscle relaxant and anticonvulsant activities. The results indicate that some of the compounds under study combine a spectrum of antidepressant, anxiolytic and neuroleptic properties in mice with a lack of muscle relaxant and anticonvulsant activities.

Anticonvulsive activity of Butea monosperma flowers in laboratory animals.

The bioassay-guided fractionation of dried flowers of Butea monosperma (BM) was carried out to isolate the active principle responsible for its anticonvulsant activity. The petroleum ether extract was fractionated by column chromatography using solvents of varying polarity such as n-hexane, n-hexane:ethyl acetate, ethyl acetate, and methanol. The anticonvulsive principle of B. monosperma was found to be a triterpene (TBM) present in the n-hexane:ethyl acetate (1:1) fraction of the petroleum ether extract. TBM exhibited anticonvulsant activity against seizures induced by maximum electroshock (MES) and its PD(50) was found to be 34.2+/-18.1 mg/kg. TBM also inhibited seizures induced by pentylenetetrazol (PTZ), electrical kindling, and the combination of lithium sulfate and pilocarpine nitrate (Li-Pilo). However, TBM was not effective against seizures induced by strychnine and picrotoxin. TBM exhibited depressant effect on the central nervous system. After repeated use for 7 days, the PD(50) (MES) of TBM increased to 51.5+/-12.1 mg/kg. Similarly, after repeated use of TBM, the duration of sleep induced by pentobarbital was not reduced significantly. Further studies are required to investigate its usefulness in the treatment of epilepsy.

The central nervous effects of Mitragyna africanus (Willd) stembark extract in rats.

The acute toxicity and the central effects of Mitragyna africanus (M. africanus) stembark methanol extract were studied in rats. The extract did not produce any death in the treated rats even at the highest dose (6400 mg kg(-1)) used. It produced depressant effects on the central nervous system. The stembark extract potentiated amylobarbitone sleeping time in rats dose-dependently, induced sleep in rats and also produced significant local anaesthetic effect on rabbits, the effects being comparable to that of xylocaine. The extract protected rats treated with a convulsive dose of strychnine (2 mg kg(-1)) and increased the period of onset of convulsions and decreased the number of spasms.

Design of semicarbazones and their bio-isosteric analogues as potential anticonvulsants.

A series of semicarbazones and hydrazones were prepared and evaluated for anticonvulsant activity. Some compounds provided significant protection against maximal electroshock (MES) and subcutaneous strychnine induced seizures (ScSty). Compound 2a emerged as the most active compound at a dose of 30 mg/kg in ScSty test. The compounds 1a, 1g and 2a-e showed significant potentiation of sedative and hypnotic activity of pentobarbitone sodium. Thus compound 2a could serve as a prototype for future developments.

Glycine receptor mediated responses in rat histaminergic neurons.

Patch-clamp whole-cell recordings were made in the hypothalamic tuberomammillary (TM) nucleus from isolated histaminergic neurons, identified by their expression of histidine decarboxylase. We compared strychnine-sensitive glycine-mediated currents with maximal currents activated by gamma-Aminobutyric acid (GABA, 0.5 mM) which were blocked by gabazine. The maximal glycine response (1 mM) in histaminergic cells with larger somata (25 microm) was about half of the maximal GABA response whereas in the cells with a smaller soma size (19.5 microm) the glycine response was absent or very small. We conclude that histaminergic cells are heterogeneous with respect to their sensitivity to glycine and this correlates with their size.

Progesterone and testosterone modulate the convulsant actions of pentylenetetrazol and strychnine in mice.

The influence of progesterone and testosterone on the incidence of seizures after administration of intraperitoneal pentylenetetrazol and subcutaneous strychnine was evaluated in mice. Pentylenetetrazol and strychnine were administered in doses that induced seizures in 40-50% of control mice in dioestrus (48 and 0.9 mg/kg, respectively). The percentage of seizures induced by pentylenetetrazol and strychnine was significantly lower in female mice in prooestrus/oestrus, when progesterone levels are high, than in dioestrus, when progesterone levels are low. Pretreatment of pentylenetetrazol-challenged mice with progesterone (250 microg/kg) increased the incidence of seizures in prooestrus/oestrus, without affecting seizures in dioestrus. The same pretreatment in strychnine-challenged mice also increased the incidence of seizures in prooestrus-dioestrus, but significantly reduced the incidence of seizures in dioestrus. In addition, progesterone pretreatment significantly increased the percentage of deaths induced by strychnine in prooestrus-oestrus, reducing deaths in dioestrus. Orchidectomized male mice had a significantly higher incidence of seizures after administration of pentylenetetrazol and strychnine than control mice. Administration of 11 daily doses of 250 microg/kg of testosterone to castrated mice significantly reduced the incidence of seizures induced by pentylenetetrazol. These results confirm the modulatory influence of reproductive steroids on the excitability of the central nervous system and the possible clinical importance of progesterone and testosterone in the management of partial epilepsy.

Evaluation of semicarbazones for anticonvulsant and sedative-hypnotic properties.

A series of semicarbazones and thiosemicarbazones were synthesized and evaluated for anti-convulsant activity. Some compounds provided significant protection against Maximal Electroshock (MES) and subcutaneous strychnine induced seizures. Compound 1 was the most active in the series with activity in a dose of 30 mg/kg in the strychnine seizure pattern test and an ED50 of 10 mg/kg in the MES test. Hence it could serve as a prototype molecule for future development. Also compounds with a p-nitrophenyl substitution in place of the amino hydrogen of semicarbazone moiety showed activity in a dose of 30 mg/kg and an ED50 of 83 mg/kg in the MES test.

Milacemide, a glycine pro-drug, inhibits strychnine-allodynia without affecting normal nociception in the rat.

The blockade of spinal glycine receptors with intrathecal (i.t.) strychnine (STR) produces reversible, segmentally localized allodynia in the rat. The purpose of this study was: (1) to investigate the effect of the anticonvulsant agent, milacemide, a glycine pro-drug on STR-allodynia; (2) to compare this effect with that of milacemide on normal nociception (without STR); and (3) to determine the sensitivity of the anti-allodynic effect of milacemide to pretreatment with selective monoamine oxidase (MAO)-A (clorgyline) and MAO-B (L-deprenyl) inhibitors. Male Sprague-Dawley rats, fitted with chronic i.t. catheters, were lightly anesthetized with urethane. Hair deflection (HD) evoked maximum changes in blood pressure and heart rate were recorded from left carotid artery, and cortical electroencephalographic (EEG) activity was continuously monitored using subdermal needle electrodes before and after i.t. STR (40 microg). Rats were pretreated with a single intravenous (i.v.) injection of milacemide (100-600 mg/kg), 1 h before i.t. STR. To sustain the allodynic state, STR was injected every hour for up to 4 h. HD was applied to the affected dermatomes (2 min duration) using a cotton-tipped applicator at 5-min intervals for the duration of the STR effect. Normally innocuous HD elicited a marked increase in mean arterial blood pressure and heart rate, an immediate motor responses, and desynchronisation of EEG when applied to the cutaneous dermatomes affected by i.t. STR. Milacemide (100-600 mg/kg, i.v.) dose-dependently inhibited the heart rate and pressor responses (ED50 = 398 mg/kg; 95%CI = 196-873) and the motor responses (ED50 = 404 mg/kg; 95%CI = 275-727). Maximum inhibition was observed approximately 2 h after i.v. injection. The duration of action ranged from 3 h (400 mg/kg) to 4 h (600 mg/kg). Milacemide had no effect on the percent synchrony in the EEG. At the time of maximum inhibition of STR-allodynia (2 h post-infusion), responses evoked by noxious pinch were unaffected by milacemide. Pretreatment with L-deprenyl (3 mg/kg, i.p.), but not clorgyline (10 mg/kg, i.p.) significantly blocked the anti-allodynic effect of milacemide (600 mg/kg i.v). These data indicate that i.v. milacemide significantly attenuates the allodynia arising from spinal glycine receptor blockade, and are consistent with: (1) the selective modulation of low threshold afferent input by STR-sensitive, glycine interneurons in the rat spinal cord; and (2) the pharmacological actions of milacemide as a glycine pro-drug.

L-NAME inhibits pentylenetetrazole and strychnine-induced seizures in mice.

Nitric oxide (NO) formation has been shown in many neuronal tissues subserves a variety of functions. N-Methyl-D-aspartate (NMDA) receptor stimulation which releases nitric oxide and raises cGMP levels, mediates epileptiform activity induced by various agents. Disinhibition of inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and/or activation of NMDA receptor appears to be factors involved in the initiation and generalization of the pentylenetetrazole (PTZ) induced seizures. In the present study, we examined the effects of N(omega)-nitro-L-arginine methylester (L-NAME) which inhibits nitric oxide synthase, on PTZ and strychnine induced seizures in mice. L-NAME (100 mg/kg) significantly prolonged the onset time of tonic generalized extension without affecting myoclonic jerks and tonic-clonic convulsions. L-NAME (200 mg/kg) significantly delayed three characteristic behavioral changes including first myoclonic jerk (FMJ), generalized clonic seizure (GCS) and tonic generalized extension (TGE). The effects of L-NAME were reversed by L-arginine (1000 mg/kg). L-NAME (100 and 200 mg/kg) significantly delayed the onset time of strychnine induced TGE. The effects of both doses of L-NAME were reversed by L-arginine. In conclusion, our results demonstrate that NO synthase inhibition suppresses the onset time of PTZ and strychnine induced seizures. Under the light of our current knowledge NO synthase inhibitors seem far away to be considered as a group of antiepileptic drugs. On the other hand there are some strong evidences about the role of NO in central pathophysiological mechanisms.

Neuronal model of tactile allodynia produced by spinal strychnine: effects of excitatory amino acid receptor antagonists and a mu-opiate receptor agonist.

Touch evoked agitation (allodynia) can be induced by spinal delivery of strychnine and this effect is antagonized by intrathecal NMDA and non-NMDA receptor antagonists, but not by mu-opiate receptor agonists. In this study, we sought to characterize the effect of focal glycine-receptor inhibition on spontaneous and evoked activity in dorsal horn neurons of the chloralose-anesthetized cat. Strychnine (1 mM) applied near the neurons through a dialysis fiber caused an enhanced response to hair deflection, enlargement of the low threshold receptive fields and in some cells, an increase in afterdischarge. These changes were observed only in cells that were activated by both hair deflection and high intensity mechanical stimulation. Subsequent co-administration of an NMDA receptor antagonist (AP-7, 2.0 mM) preferentially blocked strychnine-associated effects without changing the original receptive field characteristics. Co-administration of a non-NMDA excitatory amino acid receptor antagonist (CNQX, 1 mM) with the strychnine served to block low (brush) and high intensity (pinch) afferent input. In contrast, addition of a mu-opiate receptor agonist (alfentanil 2.4 mM) to the strychnine perfusate selectively reduced responsiveness to high intensity stimulation, while having no effect on the exaggerated response to hair deflection. Given the functional and pharmacological similarity of the effects of spinal strychnine to post-nerve injury states in man, disinhibition due to a loss of glycinergic input may be associated with large myelinated fiber-mediated nociceptive states. Consistent with these data is the contention that under normal circumstances, afferent hair follicle input onto convergent neurons is regulated by a tonic glycinergic circuit. Removal of this regulatory influence leads to a magnification of low threshold tactile throughput in dorsal horn. This model may help to provide pharmacological insights into more efficacious treatments for such pain states that are relatively refractory to opioid therapies.

Alphaxalone, a steroid anesthetic, inhibits the startle-enhancing effects of corticotropin releasing factor, but not strychnine.

Corticotropin releasing factor (CRF) is a 41 amino acid peptide implicated in the expression of stress- and fear-enhanced behaviors. CRF potentiates the amplitude of the startle reflex, and this effect is reversed by benzodiazepines (BDZ), suggesting that the startle-enhancing effects of CRF are modulated by changes in the GABA/BDZ receptor complex. In the present study, CRF-potentiated startle is inhibited by alphaxalone, a pregnane steroid anesthetic that is thought to act via the GABA/BDZ receptor complex. Alphaxalone (ALX) does not reduce CRF-potentiated startle by producing a generalized reduction in reactivity, since blockade of CRF-stimulated startle was not accompanied by an ALX-induced reduction in baseline startle amplitude and ALX does not reduce strychnine-potentiated startle. The effects of alphaxalone on CRF-potentiated startle may not be generalized to all CRF-stimulated behaviours, since alphaxalone failed to disrupt CRF-stimulated locomotor activity. CRF-potentiated startle is a useful assay for studying the effects of novel anxiolytic agents, and alphaxalone appears to be a steroid anesthetic with anxiolytic properties in this assay.

Effects of intrathecal strychnine and bicuculline on nerve compression-induced thermal hyperalgesia and selective antagonism by MK-801.

We studied the effects of intrathecally administered strychnine (STR; glycine antagonist; 10 or 30 micrograms) and bicuculline (BIC; GABAA antagonist 1 or 3 micrograms) on the thermal hyperalgesia which occurs following sciatic nerve constriction injury in rats. Following unilateral application of loose ligatures around the sciatic nerve, all rats typically displayed an ipsilateral thermal hyperalgesia on day 7. Intrathecal STR or BIC administered just after the nerve lesion and on days 1 and 2 after the nerve lesion significantly enhanced in a dose-dependent fashion the magnitude of the thermal hyperalgesia normally observed on day 7, as compared to intrathecal saline (for STR: 30 micrograms > 10 micrograms > or = saline; for BIC: 30 micrograms > 10 micrograms > or = saline, p < 0.05). Intrathecal MK-801, an N-methyl-D-aspartate antagonist, was without effect upon the response latency of the normal or sham operated paw, but selectively reversed the hyperalgesia. These results suggest that the loss of a spinal STR- and BIC-sensitive inhibition augments development of the hyperalgesia induced by chronic nerve compression.

Antagonism of non-NMDA receptors inhibits handling-induced, strychnine-potentiated convulsions.

The effects of blockade of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-proprionate (AMPA) and kainate subtypes of NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) and compared to the effects of N-methyl-D-aspartate (NMDA) receptor blockade with D-CPPene (D-(E)-4-(3-phosphonoprop-2-enyl)-piperazine-2-carboxylic acid), a competitive NMDA antagonist. Both compounds exerted peak blocking activity 30 min after intraperitoneal administration. Using this pretreatment interval, a dose-response relationship for blocking handling-induced, strychnine-potentiated convulsions was generated for each compound. D-CPPene blocked seizures with an ED50 of 0.72 (0.59-0.87) mg/kg and NBQX blocked seizures with an ED50 of 68.0 (36.72-125.94) mg/kg. These results indicate that both NMDA and non-NMDA subtypes of excitatory amino acid receptors are activated in handling-induced, strychnine-potentiated convulsions.

Anticonvulsant and sodium channel blocking effects of ralitoline in different screening models.

Ralitoline, a thiazolidinone derivative chemically distinct from known antiepileptic drugs, possesses remarkable anticonvulsant properties as demonstrated in various animal models of epilepsy. The efficacy of this compound seems to be comparable or even better than that of conventional antiepileptics. In the present study, the activity of ralitoline was investigated in four seizure models in rodents in order to characterize the anticonvulsant profile of action further. In the maximal electroshock seizure test (mice), this compound showed marked anticonvulsant effects (ED50 2.8 mg/kg i.p.). The efficacy of clinically established anti-epileptics was significantly increased when ralitoline was given as co-medication. In the strychnine seizure test (mice), ralitoline (5 and 10 mg/kg) prolonged the latency of tonic seizures as well as the survival time. On the other hand, in the subcutaneous pentylenetetrazol seizure threshold test (mice), this drug revealed limited protective actions at higher doses and increased the effectiveness of ethosuximide. In unrestrained rats with chronically implanted electrodes, ralitoline (5 mg/kg) significantly reduced the duration of electrically-evoked hippocampal discharges and raised the focal stimulation threshold (10 mg/kg). In the rotorod ataxia test (mice), a TD50 value of 14.5 mg/kg i.p. was determined for ralitoline (protective index TD50/MES-ED50 5.2). With regard to the possible mode of action, whole-cell voltage-clamp experiments on cultured neonatal rat cardiomyocytes showed that ralitoline may act specifically on voltage-sensitive sodium channels. The compound inhibited the fast sodium inward current in a frequency- and voltage-dependent manner. In conclusion, the findings confirm the potent anticonvulsant effects of ralitoline, especially against generalized tonic-clonic and complex partial seizures. Moreover, in combination with antiepileptics, an additive synergism can be found at lower concentrations. Regarding the mode of action, this drug was capable of depressing the fast sodium inward current in cultured heart ventricular cells, suggesting that the local anesthetic properties may be important for the anticonvulsant activity of ralitoline.

Cyclopentenyl ethylamines active on CNS.

Two new cyclopentenylethylamines were prepared and were submitted to a pharmacological screening together with some others previously described and now reprepared. All compounds exhibited different degrees of depressive activity on CNS and good analgesic activity. Compound 5, bearing a phenyl group on the carbon atom to which the amino group is connected, appears rather interesting being the most active as analgesic and the least toxic. Compounds 2 and 3 are able to antagonize in a certain degree lethal doses of physostigmine and also, respectively, of pentylenetetrazole and strychnine.

MDL 27,531 selectively reverses strychnine-induced seizures in mice.

1. Strychnine-sensitive glycine receptors are primarily localized in the brainstem and spinal cord where they are the major mediators of postsynaptic inhibition. A compound which acts functionally like a glycine receptor agonist would be potentially useful as a pharmacological tool and as a therapeutic agent for treating disorders of glycinergic transmission. 2. MDL 27,531 (4-methyl-3-methylsulphonyl-5-phenyl-4H-1,2,4-triazole) blocked strychnine-induced tonic extensor seizures in mice following either intraperitoneal (ED50 = 12.8 mg kg-1; 30 min) or oral (ED50 = 7.3 mg kg-1; 30 min) administration. Time course studies revealed a maximal effect at 30-60 min, though significant activity was still seen after 24 h. 3. MDL 27,531 was selective in antagonizing strychnine seizures and little or no activity was seen against seizures produced by other chemical convulsants (bicuculline; quinolinic acid; mercaptopropionic acid); by electrical stimuli (maximal electroshock); or by sensory stimuli (audiogenic seizure susceptible mice). MDL 27,531 blocked pentylenetetrazol-induced seizures with an ED50 = 55 mg kg-1. This profile differed from those of the anticonvulsants diazepam, valproic acid, and diphenylhydantoin. 4. The antagonism of strychnine seizures by MDL 27,531 occurred at doses that did not produce signs of sedation (suppression of spontaneous motor activity), motor ataxia (disruption of rotarod performance), muscle relaxation (inhibition of morphine-induced Straub tail), or CNS depression (potentiation of hexobarbitone sleep time). MDL 27,531 had less side effect potential (as derived from ratios obtained from the above measures) relative to those of the known muscle relaxants diazepam and baclofen. 5. Although MDL 27,531 behaved functionally like a selective agonist at the strychnine-sensitive glycine receptor, the compound did not alter the in vitro binding of [3H]-strychnine to mice brainstem/spinal cord membranes at concentrations of up to 100 microM. In further in vitro binding assays, MDL 27,531 at concentrations of up to 100 microM, did not displace the binding of [3H]-muscimol, [3H]-flunitrazepam, or["S]-t-butylbicyclophosphorthionate to rat cortical membranes. These ligands bind to the 7y-aminobutyric acid (GABA), benzodiazepine, and picrotoxin-convulsant binding sites, respectively.6. MDL 27,531 (10-100mgkg-', i.p.) enhanced binding of the benzodiazepine antagonist [3H]-Ro15-1788 to mouse cerebral cortex in vivo without directly affecting GABA levels.7. Ro 15-1788 (16, 32 mg kg-') significantly blocked the MDL 27,531 antagonism of strychnineinduced seizures, though this antagonism was not competitive. The same doses of Ro 15-1788 produced parallel rightward shifts in the dose-response curves for diazepam inhibition of pentylenetetrazol-induced seizures, consistent with a competitive antagonism.8. Thus, MDL 27,531 acts functionally like an agonist at the strychnine-sensitive glycine receptor in its capacity to reverse selectively strychnine-induced seizures. Though the precise mechanism of action of MDL 27,531 is unknown, MDL 27,531 may act at an allosteric site on the strychnine-sensitive receptor which produces agonist-like activity.

Pretreatment with aldosterone or corticosterone blocks the memory-enhancing effects of nimodipine, captopril, CGP 37,849, and strychnine in mice.

Oral pretreatment with aldosterone or corticosterone blocked the memory-enhancing effects of the calcium antagonist nimodipine, the ACE inhibitor captopril, the NMDA blocker CGP 37,849, and the glycine antagonist strychnine in a passive-avoidance test in mice. The memory-disturbing effects of phenobarbitone, diazepam, CGP 37,849 and scopolamine were not influenced by the hormonal pretreatment. These findings could indicate the involvement of a steroid-sensitive mechanism in drug-induced improvement of memory. In the light of clinical observations showing elevated cortisol levels in Alzheimer patients, the results might also explain why only a limited number of these patients respond to therapy with memory enhancers.