Antiepileptic potential and behavioral profile of L-pGlu-(2-propyl)-L-His-L-ProNH2, a newer thyrotropin-releasing hormone analog.

Thyrotropin-releasing hormone (TRH) and its analogs have a number of neurobiological functions and therapeutic uses in disorders of the central nervous system. In this study, the newly synthesized TRH analogs were evaluated for central nervous system activity in pentobarbital-induced sleeping in mice. The most potent TRH analog (L-pGlu-(2-propyl)-L-His-L-ProNH(2) coded as NP-647) was evaluated for its antiepileptic potential in various seizure models in mice in comparison with TRH. Intravenous pretreatment with NP-647 (10 and 20 micromol/kg body wt) significantly delayed the onset and reduced the frequency of convulsions in the pentylenetetrazole model, but not in the maximum electroshock seizure model. Also, it was found to be protective against picrotoxin- and kainic acid-induced seizures. However, NP-647 did not significantly affect theophylline-induced seizures. Further study of the effect of NP-647 on locomotor activity and a functional observational battery revealed that it did not significantly exhibit any undesirable effects as compared with vehicle and TRH. NP-647 did not significantly affect cerebral blood flow, whereas the native peptide TRH markedly increased cerebral blood flow. Furthermore, NP-647 exerted antiepileptic activity without significantly altering plasma thyroid-stimulating hormone levels and mean arterial blood pressure. This suggests that NP-647 is more selective for central nervous system activity and devoid of hormonal and cerebrovascular system effects. In contrast, TRH exhibited cardiac and endocrine effects as marked by significant elevation in mean arterial blood pressure and plasma thyroid-stimulating hormone levels. This study demonstrates that NP-647 has potential antiepileptic activity devoid of undesirable effects and, thus, can be exploited for the prevention and treatment of epilepsy.

Stimulation of 5-HT 1A receptors increases the seizure threshold for picrotoxin in mice.

To evaluate the possible role of 5-HT 1A and 5-HT 2A receptors in the anticonvulsant effect of swim stress, mice were pre-treated with agonists and antagonists of these receptors prior to exposure to stress and the intravenous infusion of picrotoxin. 8-OH-DPAT ((+/-)-8-hydroxy-2-(di-n-propylamino) tetralin) and WAY-100635 (a selective agonist and antagonist of 5-HT 1A receptors), DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) and ketanserin (a 5-HT 2A/2C receptor agonist and antagonist) were used. Results demonstrated that 1 and 3 mg/kg of 8-OH-DPAT increased the doses of picrotoxin producing running/bouncing clonus, tonic hindlimb extension and death in stressed and unstressed mice, respectively. Pre-treatment with WAY (0.3 mg/kg) prevented the effect of 8-OH-DPAT (3 mg/kg). DOI (2.5 mg/kg) and ketanserin (1 mg/kg) failed to affect the seizure threshold for picrotoxin. The results show that stimulation of 5-HT 1A receptors exerts anticonvulsant actions in stressed and unstressed mice, while stimulation of 5-HT 2A/2C receptors does not interfere with the effect of stress on picrotoxin-induced convulsions.

Effects of ethosuximide and tetramethylsuccinimide on cultured cortical neurons.

In cultured cortical neurons, ethosuximide (ESM) had unexpected actions for an antiepileptic drug; it slightly diminished the effects of inhibitory neurotransmitters, GABA, and glycine. ESM had no direct membrane effects, did not change action potential characteristics or alter spontaneous activity, and did not reverse the effects of convulsants that are GABA antagonists. The structurally related convulsant tetramethylsuccinimide (TMSM) reduced the amplitude of GABA-mediated inhibitory postsynaptic potentials and antagonized responses to applied GABA, effects not reversed by ESM. The convulsant effects of TMSM include a blockade of postsynaptic GABA actions, but the antiepileptic effects of ESM are not due to an enhancement of GABA-mediated inhibition.

Inhibitory mechanisms of the hyper-irritability caused by picrotoxin in the rat.

Topically applied norepinephrine, dopamine, serotonin, GABA and glycine, and systemically administered clonidine, L-DOPA (plus carbidopa) and 5-hydroxytryptophan completely suppressed the cutaneous hyper-irritability produced in the trigeminal sensory distribution by picrotoxin overlying the caudal medulla. Cholinergic agents and apomorphine were ineffective. Of the positive compounds, norepinephrine, serotonin and GABA showed the shortest latencies and norepinephrine and serotonin required the lowest concentrations in order to inhibit the hyper-irritability. If L-DOPA (plus carbidopa) was injected after pre-treatment with FLA-63, the effects of L-DOPA did not appear. Similar depression of the hyper-irritability was caused by electrical stimulation of the central gray. The inhibitory effects of stimulation of the central gray was suppressed after administration of tetrabenazine, but again it produced markedly by injection of L-DOPA. From these observations it was concluded that the hyper-irritability could be suppressed by serotonergic as well as noradrenergic fibers terminating at the spinal trigeminal nucleus caudalis. The potential clinical use of L-DOPA in patients with hyperesthesia is discussed.

Anticonvulsant and convulsant properties of flurazepam.

The anticonvulsant efficacy of the benzodiazepine flurazepam was tested in rats treated with a convulsant dose of either picrotoxin or 3-mercaptopropionic acid. Against picrotoxin, a significant anticonvulsant effect was observed in the range of 30-60 mg/kg, i.p. Epileptogenicity increased as doses of flurazepam were increased from 60 to 200 mg/kg, and at a dose of 400 mg/kg all subjects died. In similar tests, the benzodiazepines clonazepam and triazolam produced only dose-dependent increases in anticonvulsant efficacy. Flurazepam also proved to be an effective anticonvulsant in tests against 3-mercaptopropionic acid. The results of this study provide further evidence that benzodiazepines have varying degrees of epileptogenicity.

Structure-activity and dose-effect relationships of the antagonism of picrotoxin-induced seizures by cholecystokinin, fragments and analogues of cholecystokinin in mice.

Intraperitoneal administration of cholecystokinin octapeptide sulphate ester (CCK-8-SE) and nonsulphated cholecystokinin octapeptide (CCK-8-NS) enhanced the latency of seizures induced by picrotoxin in mice. Experiments with N- and C-terminal fragments revealed that the C-terminal tetrapeptide (CCK-5-8) was the active centre of the CCK octapeptide molecule. The analogues CCK-8-SE and CCK-8-NS (dose range 0.2-6.4 mumol/kg) and caerulein dose range 0.1-0.8 mumol/kg) showed bell-shaped dose-effect curves, with the greatest maximum inhibition for CCK-8-NS. The peptide CCK-5-8 had weak anticonvulsant activity in comparison to the octapeptides, 3.2 mumol/kg and larger doses of the reference drug, diazepam, totally prevented picrotoxin-induced seizures and mortality. The maximum effect of the peptides tested was less than that of diazepam. Experiments with analogues and derivatives of CCK-5-8 demonstrated that the effectiveness of the beta-alanyl derivatives of CCK-5-8 were enhanced and that they were equipotent with CCK-8-SE. Of the CCK-2-8 analogues, Ser(SO3H)7-Ac-CCK-2-8-SE and Thr(SO3H)7-Ac-CCK-2-8-SE and Hyp(SO3H)-Ac-CCK-2-8-SE were slightly more active than CCK-8-SE.

Do the reductions in social interaction produced by picrotoxin and pentylenetetrazole indicate anxiogenic actions?

The effects of picrotoxin (2 and 4 mg/kg) and of pentylenetetrazole (PTZ 5, 10 and 20 mg/kg) were examined in a social-interaction test of anxiety. Picrotoxin (2 mg/kg) caused a significant reduction in active social interaction, without a concomitant reduction in motor activity, indicating an anxiogenic action. Picrotoxin (4 mg/kg) and pentylenetrazole (20 mg/kg) reduced social interaction by more than 75% and motor activity by 40%. Although it is likely that the reduction in motor activity is secondary to the very low levels of social interaction, an unambiguous interpretation of an anxiogenic effect is not possible. Increased concentrations of corticosterone in plasma following administration of picrotoxin were consistent with the drug being anxiogenic. Chlordiazepoxide (5 mg/kg) significantly reversed the reductions in social interaction and locomotor activity following injection of picrotoxin (4 mg/kg) and PTZ (20 mg/kg).

Desensitization of GABAB receptors and antagonism by CGP 35348, prevent bicuculline- and picrotoxin-induced antinociception.

The effect of the GABAA antagonists, bicuculline and picrotoxin, in the hot plate and writhing tests in mice and the paw-pressure test in rats was assessed. Subconvulsant doses of bicuculline (1.3-4 mumol kg-1, s.c.) or picrotoxin (0.8-2.5 mumol kg-1, s.c.) induced a dose-related increase in latency of licking in the hot plate test in mice, whereas subconvulsant doses of strychnine and thiosemicarbazide (0.9 and 6 mg kg-1, s.c. respectively), did not modify the threshold to thermal stimuli in mice. The effects of bicuculline and picrotoxin were not modified by naloxone (3 mg kg-1, i.p., a dose which inhibited the antinociceptive effect of morphine) or by atropine (5 mg kg-1, i.p., a dose which prevented oxotremorine-induced antinociception) but were antagonized by the GABAB antagonist CGP 35348 (2.5 micrograms, i.c.v., a dose which prevented (+/-)baclofen-induced antinociception). Mice, rendered tolerant to baclofen-induced antinociception by twice daily injection of increasing doses of baclofen (5-18 mg kg-1, s.c.), were unresponsive to the antinociceptive effects of bicuculline and picrotoxin but still responded to morphine. Bicuculline and picrotoxin, in the same range of doses which affected the three models of antinociception used, inhibited pentobarbital-induced hypnosis. Large doses of bicuculline and picrotoxin (4 and 2.5 mumol kg-1, s.c. respectively), reduced locomotor activity and impaired rota-rod performance in mice. The changes in response to noxious stimuli, induced by bicuculline and picrotoxin, are interpreted as an antinociceptive effect. It is then suggested that this effect might depend on an indirect activation of GABAB receptors through release of GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Augmentation by chlordiazepoxide of the inhibitory effects of taurine, beta-alanine and gamma-aminobutyric acid on spike discharges in guinea-pig cerebellar slices.

1. Chlordiazepoxide (Cdp, 1 to 100 micrometer) enhanced the inhibitory action of externally applied gamma-aminobutyric acid (GABA) upon spontaneous spike discharges in guinea-pig cerebellar slices; the actions of externally applied beta-alanine and taurine, but not externally applied glycine, were also enhanced by Cdp. 2. It was suggested the Cdp might exert its action by enhancing the increase of membrane permeability to K+ induced by the amino acid, but not to Cl-. 3. Cdp (5 to 100 micrometer) reversed the antagonism of picrotoxin to the inhibitory action of externally applied GABA and also the antagonism of strychnine to the actions of externally applied beta-alanine and taurine. 4. The inhibition of the spontaneous spike discharges of Purkinje cells, evoked by electrical stimulation of the slice, was also enhanced by Cdp (10 to 100 micrometer). 5. The blocking action of picrotoxin (10 to 20 micrometer) on the stimulus-evoked inhibition of spike discharges was reversed by Cdp (10 micrometer). 6. In a similar manner, strychnine (10 or 20 micrometer) was also found to block the stimulus-evoked inhibition of spike discharges. It is suggested that in the cerebellum strychnine-sensitive amino acid(s) may be involved in synaptic transmission. Strychnine blockade was also reversed by Cdp (10 micrometer).

Modulation of the GABAA receptor complex by steroids in slices of rat cuneate nucleus.

1. Several derivatives of pregnane and androstane that have hypnotic properties have been investigated for their ability to potentiate responses to the GABAA receptor agonist muscimol and to reduce the effect of the non-competitive GABAA antagonist picrotoxin. 2. Depolarizing responses to muscimol in slices of rat cuneate nucleus were potentiated most potently by 3 alpha-hydroxy,5 alpha-pregnane-11,20-dione (alphaxalone), which gave half-maximal potentiation at 0.15 microM. The 5 beta isomer of alphaxalone had little effect up to 3 microM but in analogues lacking an 11-keto substituent (pregnanolones), both the 5 alpha- and 5 beta-isomers potentiated with potencies 20 and 10 times lower, respectively, than that of alphaxalone. The alpha configuration of the 3-hydroxy was essential in alphaxalone, the 3 beta-hydroxy isomer being inactive. However, there was little difference between the potencies of the 3 alpha- and 3 beta-hydroxy configurations in the pregnanolones, although the maximal effects of the 3 beta-hydroxy isomers were rather lower than those of the 3 alpha-hydroxy isomers. 3. Reductions in the effect of picrotoxin as an antagonist of muscimol were caused most potently by the 3 alpha-hydroxy pregnanolones, with a ten fold reduction in picrotoxin potency at 1 microM concentrations of these steroids. Alphaxalone and its 5 beta-isomer were about half as potent. Androsterone was about 10 times less potent and the 3 beta-hydroxy pregnanolones were ineffective. 4. This difference in the structure-activity relationships for steroidal potentiation of muscimol and reduction in picrotoxin antagonism of muscimol is reminiscent of an analogous distinction found with the barbiturates.

Interactions of the imidazodiazepine Ro 15-4513 with chemical convulsants.

1. The proconvulsant effects of the imidazodiazepine Ro 15-4513, were investigated in mice by use of intravenous infusion of a variety of convulsant drugs. 2. Dose-response and time course studies of Ro 15-4513 against gamma-aminobutyric acid (GABA) antagonists were performed. On the basis of these studies a maximally effective dose of 5 mg kg-1 was administered 5 min before the determination of seizure thresholds in subsequent experiments. 3. Ro 15-4513 (5 mg kg-1) significantly lowered seizure thresholds to pentylenetetrazole, bicuculline and the convulsant benzodiazepine Ro 5-3663, but failed to alter seizure thresholds to picrotoxin, strychnine, caffeine and quipazine. 4. Ro 15-4513 significantly raised seizure threshold to the benzodiazepine receptor inverse agonist methyl 6,7-dimethoxy-4 ethyl-beta-carboline-3-carboxylate (DMCM). 5. These results are discussed in relation to other studies investigating the proconvulsant and alcohol-antagonizing effects of Ro 15-4513.

Effects of gamma-hydroxybutyrate on chick behaviour, electrocortical activity and crossed extensor reflexes.

1. Convulsant and anticonvulsant effects of gamma-hydroxybutyrate (GHB) have been studied in chicks.2. When administered alone, GHB produced weak myoclonic seizures accompanied by electrocortical synchrony and spikes.3. GHB was found to protect chicks against leptazol- and picrotoxin-induced seizures. A slight potentiation of strychnine-induced seizures was evident.

Effect of elevated brain GABA concentrations on the actions of bicuculline and picrotoxin in mice.

gamma-Acetylenic GABA and gamma-vinyl GABA, two catalytic irreversible inhibitors of mammalian brain GABA transaminase that produce several-fold increases in brain GABA concentrations were tested for their effects on bicuculline and picrotoxin-induced seizures and mortality in mice. Neither inhibitor influenced the frequency of seizures or death produced by either bicuculline or picrotoxin. Both inhibitors, however, produced a dose-dependent prolongation of the time to onset of seizures and death induced by picrotoxin but by bicuculline. These results suggest differences in the antagonism by bicuculline and picrotoxin of GABA-mediated neural functions.

Facilitation of diazepam action by anticonvulsant agents against picrotoxin induced convulsions.

A subeffective dose (2 mg/kg) of diazepam produced only 50% protection against picrotoxin-induced (PTX) convulsions in rats. Simultaneous administration of GABA and other GABA-ergic substances such as piracetam and sidium valproate, which did not have any effect by themselves, potentiated diazepam action. The onset of convulsions and mortality due to PTX were significantly delayed. The other conventional anticonvulsants phenobarbitone, phenytoin and ethosuximide also enhanced the protective effect of diazepam. Inosine, a putative benzodiazepine ligand, also enhanced diazepam action. These observations are explained on the basis of data from in vitro studies indicating that GABA-ergic agents and barbiturates enhance both the number of benzodiazepine binding sites and benzodiazepine binding. The protective effect of clonidine, however, may be mediated by a different mechanism unrelated to the GABA-ergic system.

Picrotoxin in the medial prefrontal cortex impairs sensorimotor gating in rats: reversal by haloperidol.

RATIONALE: Neuropathological data indicate a GABAergic dysfunction in the prefrontal cortex and hippocampus of schizophrenics. On this basis, the construct validity of an animal model of schizophrenia was tested. OBJECTIVE: This study assessed prepulse inhibition (PPI) of startle in rats after injections of the GABA antagonist picrotoxin into the prefrontal cortex and the ventral hippocampus. It was also tested if reductions in PPI are reversed by the dopamine antagonist haloperidol. PPI is a measure of sensorimotor gating and is impaired in schizophrenia patients. The hypothesis underlying this study was that blockade of prefrontocortical and hippocampal GABA receptors disrupts PPI in a dopamine-dependent way. This hypothesis was based on neuropathological data from schizophrenics indicating a loss of GABAergic neurons in the prefrontal cortex and hippocampus and on the observation that PPI is reduced in schizophrenics. METHODS: Picrotoxin (0, 5, 10 ng/0.5 microl) was infused through chronically indwelling cannulae into the medial prefrontal cortex (mPFC), into the lateral prefrontal cortex and into the ventral hippocampus. The effect on PPI was measured directly after picrotoxin infusion. The neuroleptic compound haloperidol (0.1 mg/kg) was administered intraperitoneally 30 min before testing. RESULTS: Picrotoxin in the mPFC dose-dependently reduced PPI and this effect was antagonized by systemic pretreatment with the dopamine antagonist haloperidol. No significant effects on PPI were observed after picrotoxin infusions into the lateral prefrontal cortex or into the ventral hippocampus. CONCLUSIONS: These findings indicate that acute blockade of GABA receptors in the mPFC impairs sensorimotor gating in a dopamine-dependent manner. Since PPI in rats has been shown to possess face, predictive, and construct validity as an animal model for some psychotic symptoms, we discuss the potential relevance of our findings for the pathophysiology of schizophrenia.

Role of GABA-A and mitochondrial diazepam binding inhibitor receptors in the anti-stress activity of neurosteroids in mice.

Neuroactive steroidal modulation of immobilization-stress and possible involvement of GABA-A and mitochondrial diazepam binding inhibitor (DBI) receptors (MDR) has been investigated in mice. Immobilization of mice for 2 h induced intense antinociception, anxiety state, and associated with a fall in adrenal ascorbic acid levels. Pretreatment with high dose of progesterone (10 mg/kg), a precursor of neurosteroids, significantly decreased the stress-induced antinociception, anxiety and fall in adrenal ascorbic acid, while low doses (1 and 5 mg/kg) or hydrocortisone (10 and 100 mg/kg) were ineffective. In contrast, progesterone (1 mg/kg, for 9 days) produced a significant antistress effect, which was blocked by GABA-A antagonists picrotoxin (1 mg/kg) and bicuculline (1 mg/kg), but not by flumazenil (2 mg/kg), a specific benzodiazepine (BZD) antagonist. 4'-chlordiazepam (0.1 and 0.25 mg/kg), a specific high affinity MDR agonist, produced significant anti-stress effect in a flumazenil-insensitive manner, but was blocked by pretreatment with PK11195 (1.5 mg/kg), a selective partial agonist of MDR, and with bicuculline (1 mg/kg), a potent GABA-A receptor antagonist. At higher doses, progesterone and 4'-chlordiazepam which are effective in immobilization stress also reduced locomotion. However, lower doses of progesterone (6.5 mg/kg) neither affected locomotion, nor produced any motor toxicity on rota-rod test. At the lower doses, the MDR ligand 4'-chlordiazepam (50 micrograms/kg) decreased locomotor activity, without altering motor toxicity on rota-rod test. Further, the per se effects of these treatments on unstressed mice were not significantly different from those of untreated controls, except for plus-maze test. The antistress profile of progesterone may be attributed to the in vivo production of neurosteroid allopregnanolone, thus resembled that of BZDs. Furthermore, the antistress actions are flumazenil-resistant, reaffirming that there may be an increase in the levels of pregnane neurosteroids in vivo, which may act on a specific allosteric site on GABA-A receptors distinct from BZD site. Because 4'-chlordiazepam binds to MDRs and stimulate mitochondrial neurosteroidogenesis, the anti-stress effects of 4'-chlordiazepam may be imputed to its MDR-induced neurosteroids, which then act on GABA-A receptors. These data suggest a pivotal role for GABA-A and mitochondrial DBI receptors in the antistress actions of neurosteroids and reinforces their ameliorative effect in physiological stress.

Neuroactive steroids modulate GABA inhibition of hypothalamic somatostatin release.

The reduced steroids 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and 3 alpha,21-di-hydroxy-5 alpha-pregnan-20-one (allotetrahydroDOC) are potent ligands of GABAA receptors. This study examined the possible modulatory effect of these metabolites on GABA inhibition of somatostatin release in cultured hypothalamic neurones. Allopregnanolone potentiates GABA inhibition, and reversed picrotoxin and bicuculline-induced augmentation of somatostatin release in a dose-dependent manner. AllotetrahydroDOC also inhibits the stimulated release induced by the antagonists, but did not modify that induced by depolarizing concentrations of K+. Pregnenolone sulphate had no effect on picrotoxin-induced somatostatin release. These findings clearly establish that 3 alpha-hydroxysteroids modulate GABA inhibition of hypothalamic somatostatin release.

Inhibitory effect of NPY on the picrotoxin-induced activity in the hippocampus: a behavioural and electrophysiological study.

The effect of neuropeptide Y (NPY) on the picrotoxin-induced activity was studied in rat brain hippocampal slices in vitro and after intrahippocampal injection in vivo. In the hippocampal slices, NPY (0.1-0.5 microM) inhibited the picrotoxin-induced epileptiform activity recorded extracellularly in CA1 and CA3 hippocampal pyramidal cells. Similar inhibition was induced by the Y2 receptor agonist NPY13-36, which indicates that the effect of NPY was due to activation of Y2 receptors. In behavioural studies, rats with chronically implanted cannulae were injected unilaterally into the CA1 hippocampal region with a 1 ml volume of the studied substances. Picrotoxin in a dose of 1 mg (1.6 nmol) induced behavioural excitation, shakes and weak signs of epileptic behaviour. NPY in a dose of 2 mg (470 pmol), but not 1 mg, inhibited some excitatory effects of picrotoxin, but did not change the epileptic symptoms. The obtained results suggest that NPY has an inhibitory action in the hippocampus, which can be observed in vitro and also in a behavioural study.

Anticonvulsant action of an arylamine-containing fraction from Agelenopsis spider venom.

A low molecular weight fraction (AG2) containing arylamine compounds has been isolated from venom of the spider Agelenopsis aperta. When administered intravenously or intracerebroventricularly, AG2 produces dose-dependent suppression of behavioral convulsions induced in rats by kainic acid, picrotoxin, or bicuculline. The low molecular weight compounds in spider venoms may provide novel tools for anticonvulsant research and therapy.

Benzodiazepine receptors modulate circulating plasma vasopressin concentration.

Chlordiazepoxide pretreatment decreased basal levels of plasma arginine-vasopressin (AVP) and attenuated picrotoxin-induced increases in plasma AVP and blood pressure compared to saline-pretreated spinal animals. Prior administration of RO 15-1788 blocked the effects of chlordiazepoxide on basal plasma AVP as well as picrotoxin-evoked changes in plasma AVP and blood pressure. Thus, interactions at the benzodiazepine receptor may influence basal and evoked changes in plasma AVP concentration.