Bicuculline-induced Seizures Research Articles

Transient reduction of cerebral blood flow leads to longlasting increase in GABA content in vulnerable structures and decreased susceptibility to bicuculline induced seizures

Rats were exposed for 24 min to bilateral clamping of the common carotid arteries (BCCA) in pentobarbital anaesthesia. The GABA content was measured 24 hours, 48 hours, 4 days, 14 days and 3 months after BCCA. In other groups of rats seizures were elicited by i.p. injection of (+)-bicuculline (3 mg/kg) 24 hours, 48 hours, 4 days, 14 days and 3 months after BCCA. Analysis of the GABA content revealed significant increase compared with controls in the hippocampus, frontal cortex and substantia nigra from 24 hours up to 3 months. Bicuculline treatment induced tonic/clonic seizures and status epilepticus in sham operated animals; these effects were drastically diminished at various time points after BCCA. The present results suggest that BCCA produces a longlasting increase in GABA content and as a consequence protection from bicuculline-induced seizures.

Effects of pancuronium bromide on cerebral blood flow changes during seizures in newborn pigs.

We investigated the effects of pancuronium bromide pretreatment on cerebral blood flow (CBF) during bicuculline-induced seizures in anesthetized piglets. Arterial blood pressure, gases, pH, cerebral electrocortical activity, and CBF (radioactive microsphere) were monitored at baseline, 10 min after administration of pancuronium (0.3 mg/kg i.v.; n = 9) or vehicle (normal saline; n = 8), and again at 5, 15, and 60 min after bicuculline (3 mg/kg i.v.). No change in CBF from baseline was observed at 10 min after either saline or pancuronium treatment, before induction of seizures. In the saline group, CBF was 36 +/- 3 mL.min-1.100 g-1 before bicuculline and increased to 166 +/- 24 and 205 +/- 35 mL.min-1.100 g-1 at 5 and 15 min, respectively, after bicuculline, returning toward baseline by 60 min. In the pancuronium group at 5 min after bicuculline, CBF increased from 45 +/- 7 to 169 +/- 26 mL.min-1.100 g-1, but fell to 88 +/- 17 mL.min-1.100 g-1 at 15 min in contrast to saline-treated piglets. Also, at 15 min of seizures, differences between groups were observed in arterial blood pressure, gases, and pH. Although these variables were in the normal range with pancuronium treatment, the saline-treated animals had increased arterial blood pressure (81 +/- 6 mm Hg) and PCO2 (6 +/- 0.4 kPa) and decreased PO2 (7 +/- 0.5 kPa) and pH (6.91 +/- 0.06). Electrocortical activity was abnormal during seizures in both groups. At 60 min, reversal to normal activity was observed in six of nine pancuronium-treated animals versus two of eight saline-treated animals. These data suggest that pancuronium limits cerebral hyperemia during prolonged seizures by attenuating increases in blood pressure as a result of elimination of skeletal muscle activity. This leads to minimal alteration of arterial PCO2, PO2, and pH during seizures.

Local blood flow and glucose consumption in the rat brain during sustained bicuculline-induced seizures

ABSTRACT The present study addresses the problem of whether brain structures which have been shown to develop neuronal cell damage in recurrent or prolonged epileptic seizures have higher metabolic rates and/or less pronounced increases in blood flow rates than others during sustained seizures. To that end, local cerebral blood flow (CBF) and glucose utilization (CMRgl) were measured autoradiographically in ventilated rats, in which seizures of 20, 60, or 120 min duration were induced by i.v. bicuculline. After 20 and 60 min of seizure activity, local CBF increased 2- to 4-fold in most of the 21 structures analysed. However, there was a marked heterogeneity with CBF values varying between 150% (caudoputamen) and 500% (globus pallidus) of control. After 120 min, CBF in several structures, notably cortical and limbic regions, fell in spite of unchanged blood pressure and continued seizure activity. Changes in local CMRgl were equally heterogenous, and correlated poorly with blood flow rates. Some structures (the cerebral cortices and 3 limbic areas) showed a sustained 2-4 fold increase in CMRgl. In these, metabolic rate and blood flow were inititially matched but CBF subsequently fell to yield a pattern of relative hypoperfusion. Other structures showed no, or only modrate, increases in CMRgl. In spite of this, CBF increased markedly to yield a pattern of relative hyperemia. It is concluded that bicucullineinduced seizures represent a condition in which structures, observed to be prone to develop cell damage, show grossly enhanced metabolic rate and develop relative underperfusion. Furthermore, the results suggest that structures with a large increase of the metabolic rate during seizures, develop a striking mismatch between local metabolic rate and blood flow.

Age-related anticonvulsant activity of NMDA in the rat caudate-putamen

Striatal pathways are important for modulating the threshold for seizures in the rat forebrain. N- Methyl- d-aspartate (NMDA), an excitatory amino acid derivative and powerful anticonvulsant agent, when injected into the brain, has been shown to protect adult rats against kindling and pilocarpine-induced seizures when injected into the caudate-putamen. The present study examines whether the anticovulsant action of NMDA in the caudate-putamen varies with age. Bilateral striatal administration of NMDA was effective in suppressing bicuculline-induced seizures in rats older than 23 days of age. The results suggest that striatal pathways involved in the anticonvulsant activity of NMDA in the caudate-putamen are not functionally active in developing rats before the 4th week of life.

Induction of immediate early gene encoded proteins in the rat hippocampus after bicuculline-induced seizures: Differential expression of KROX-24, FOS and JUN proteins

Immunocytochemistry with specific antisera was used to assess regional levels of six immediate early gene encoded proteins (KROX-24, c-FOS, FOS B, c-JUN, JUN B and JUN D) in the rat hippocampus after 15 min of bicuculline-induced seizures. Serial sections of the dorsal hippocampus were examined at various postictal recovery periods up to 24 h. The results demonstrate a complex temporal and spatial pattern of immediate early gene synthesis and accumulation. Three major categories of immediate early gene products could best be distinguished in the dentate gyrus: KROX-24 and c-FOS showed a concurrent rapid rise with peak levels at 2 h and a return to baseline levels within 8 h after seizure termination. FOS B, c-JUN and JUN B levels increased more gradually with peak intensities in the dentate gyrus reached at 4 h. These immediate early gene products showed above normal levels in various hippocampal subpopulations up to 24 h. JUN D exhibited the most delayed onset combined with a prolonged increase of seizure-induced immunoreactivity. Irrespective of this differential temporal expression profile of individual transcription factors, the sequence of induction in the hippocampal subpopulations was identical for all immediate early gene-encoded proteins examined: first in the dentate gyrus granule cells followed by CA1 and CA3 neurons, respectively. Our data indicate an asynchronous synthesis of several immediate early gene-encoded proteins in the brain after status epilepticus. FOS and JUN proteins act via homo- or heterodimer complexes at the AP-1 and other DNA binding sites. The different time-courses for individual immediate early gene products strongly suggest, that at different time-points after status epilepticus, different AP-1 complexes are effective. In vitro studies have shown that different AP-1 complexes possess different DNA binding affinities as well as different transcriptional regulatory effects. Our results suggest that these molecular mechanisms are also effective in vivo.

Influence of sex on the blood brain barrier permeability during bicuculline-induced seizures.

Sex related differences of the blood brain barrier permeability was investigated during bicuculline-induced seizures in Wistar rats. The rats were anesthetized with diethyl-ether. Evans-blue, which was used as a blood brain barrier tracer, was injected into femoral vein 5 minutes before administering bicuculline to induced grand mal seizures. Evans-blue albumin extravasation was determined as a macroscopical finding; and a quantitative estimation with spectrophotometer using homogenized brain to release the dye was also performed to evaluate the macroscopic findings. During convulsions the mean arterial blood pressure increased in both female and male rats, but the difference was in the extravasation of Evans-blue being more pronounced in the females. Blood brain barrier lesions were present in 85% of female rats and 61% of male rats. Mean value for Evans-blue dye in the whole brain was found to be 1.197 +/- .402 mg % in the group consisting of all the female rats, and .755 +/- .247 mg % in the group of all male rats during bicuculline-induced seizure. This difference between female and male rats was found to be statistically significant (p < .001). Severe protein leakage was seen in the thalamus, hypothalamus, hippocampus, globus pallidus, nucleus caudatus, periaqueductal gray and mesencephalon bilaterally in female rats. However, in male rats, Evans-blue leakage was similar to that of female rats except that the frequency and intensity of blood brain barrier breakdown was less after convulsions. Our results showed that the extravasation of Evans-blue albumin was most pronounced in the brains of female rats compared to male rats after bicuculline induced seizure.

Effect of GABA antagonist-induced seizures on3H-muscimol and3H-diazepam binding in the rat striatum

The binding of 3H-muscimol and 3H-diazepam to rat striatum membranes after picrotoxin- and bicuculline-induced seizures was characterized. No alteration in the maximal binding capacity (Bmax) of 3H-muscimol was observed. However, bicuculline produced a 27% decrease in Kd. Both picrotoxin and bicuculline increased the binding capacity of 3H-diazepam. Bicuculline produced a 86% increase in Kd. These results suggest that the GABA antagonists-induced seizures may modulate 3H-muscimol and 3H-diazepam binding in rat striatum.

Interaction of felbamate and diazepam against maximal electroshock seizures and chemoconvulsants in mice

The anticonvulsant effects of felbamate alone or in combination with diazepam were investigated against maximal electroshock-, pentylenetetrazol-, isoniazid- and bicuculline-induced seizures in mice. A single subprotective dose of felbamate, a dose which offers no protection to animals when combined with diazepam, enhanced the protective effects of diazepam against seizures induced by electroshock, pentylenetetrazol and isoniazid, as measured by significant reduction of ED 50 values. However, felbamate failed to significantly affect the protective action of diazepam against bicuculline. Felbamate does not interact directly with the GABA-benzodiazepine-ionophore complex. Thus the enhancement of anticonvulsant activity of diazepam by felbamate against maximal electroshock and pentylenetetrazol may involve an indirect effect at benzodiazepine receptors. The anticonvulsant action felbamate against isoniazid does not seem to involve benzodiazepine receptors and may be due to reversing the inhibitory effect of isoniazid on glutamate decarboxylase (GAD) activity. The interaction between felbamate and diazepam may also involve other mechanisms.

Information processing in short-term memory and event-related brain potentials

Rats were either given 80 escapable shocks, yoked inescapable shocks, restraint or given no treatment. Two hours later all subjects received i.p. injection of bicuculline (4, 6 or 8 mg/kg) and were immediately tested for latency to initial myoclonic jerk and clonus. The latency to clonic convulsion was dramatically affected by prior shock treatment, and the direction of this change depended upon the escapability/inescapability of the shock. Subjects that were given escapable shock a delay of onset to seizure, while subjects inescapably shocked demonstrated a decreased latency to clonus in comparison to restrained and naive controls. It was also demonstrated that if the subjects were tested immediately following a stress experience, both the 80 escapable and inescapable shock condition protected against bicuculline-induced seizures in comparison to the control condition. Finally Experiment 2 confirmed a previous finding that less stress, i.e., 20 inescapable shocks, protects against seizures when the animals are challenged with bicuculline either immediately or 2 h later. Our suggestion is that control over stress may facilitate GABAergic transmission, and this may be the mechanism whereby coping protects against the behavioral and physiological disruption produced by exposure to a stressor.

Autoradiographic changes in central benzodiazepine binding sites and their coupling to gamma-aminobutyric acid receptors after seizures in the developing rat.

Benzodiazepines are psychoactive substances classically used for their anticonvulsant properties in neonates as well as in adults. In a previous work, we have shown that seizures lead to an age-dependent upregulation of central benzodiazepine binding sites measured in isolated rat cerebral membranes. However, information concerning regional changes in the receptor density was lacking. In our present study, the effects of bicuculline-induced seizures were investigated by quantitative autoradiography of central benzodiazepine receptors in developing rats and in adults. Animals were killed 30 min after an intraperitoneal injection of either saline or a convulsive dose of bicuculline. Benzodiazepine binding sites in brain sections were labeled by [3H]flunitrazepam. Generalized seizures induced a widespread increase in benzodiazepine receptors, with a marked enhancement in structures that mediate seizure activity, such as substantia nigra, amygdala, septum, and hippocampus. The addition of exogenous gamma-aminobutyric acid to the incubation medium increased benzodiazepine binding by the same order of magnitude whether rats were given saline or bicuculline, suggesting that additional benzodiazepine sites are also functionally linked to gamma-aminobutyric acid receptors. The age-related postictal increase in benzodiazepine receptors might reflect a compensatory response for protection against recurrent seizures, especially in newborns.

Autoradiographic changes in brain adenosine A 1 receptors and their coupling to G proteins following seizures in the developing rat

In the central nervous system, adenosine has been shown to be a major regulator of neuronal activity in convulsive disorders, mainly via the A 1 receptor subtype. In a previous work, we have shown that seizures lead to an age-dependent upregulation of cerebral adenosine A 1 sites measured in isolated rat cerebral membranes. However, information concerning regional changes in the receptor density was so far lacking. In the present study, the effects of bicuculline-induced seizures were investigated by quantitative autoradiography of central adenosine A 1 receptors in developing rats and in adults. Animals were sacrificed 30 min after an intraperitoneal injection of either saline or a convulsive dose of bicuculline. Adenosine A 1 receptors in brain section, were labeled by [ 3H]N 6-cyclohexyladenosine (CHA), a potent receptor agonist. Generalized seizures induced a widespread increase in CHA-specific binding, with a marked enhancement in structures that mediate seizure activity, such as substantia nigra, amygdala, septum and hippocampus. Moreover, the addition of guanylyl-5′-imidodiphosphate, a GTP analogue, to the incubation medium reduced CHA binding by the same order of magnitude whether rats were given saline or bicuculline, suggesting that additional adenosine A 1 receptors are also functionally linked to G proteins. The age-related postictal increase in adenosine receptors might contribute to facilitate adenosine anticonvulsant effect, especially in newborns.

Selective protection by adenosine receptor agonists against DMCM-induced seizures

The anticonvulsant actions of the adenosine receptor agonists, 1-phenylisopropyladenosine, 2-chloroadenosine and cyclohexyladenosine, against DMCM (methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate)-induced seizures in mice were studied with an infusion technique. 2-Chloroadenosine and cyclohexyladenosine were active at 1 mg/kg whereas l-phenyl-isopropyladenosine was active at 0.03 mg/kg given i.p. At 10 mg/kg, 1-phenylisopropyladenosine was only weakly active against pentylenetetrazol-induced seizures and not active against bicuculline-induced seizures. The selective effect of l-phenylisopropyladenosine against DMCM-induced seizures suggests that adenosine receptor agonists may allosterically counteract the negative modulating effect of DMCM on GABA coupling to the chloride channel. This indicates that adenosine receptors may have a physiological function within the GABA/benzodiazepine receptor complex in the brain.

Adrenalectomy increases bicuculline-induced seizure sensitivity in long-sleep and short-sleep mice

Susceptibility to bicuculline-induced seizure onset and tonus was increased in LS and SS mice after adrenalectomy (ADX). Replacement with 10% corticosterone (CCS) in ADX animals resulted in a return to seizure latencies equal to those of sham-operated (SHAM) mice. In SS mice, dexamethasone (DEX) and cholesterol-control replacement was as effective as 10% CCS in returning seizure thresholds to SHAM values. In LS mice, DEX was only effective at a low bicuculline dose. Within the sham-operated group SS mice were more susceptible to bicuculline-induced seizure onset than LS mice; however, after ADX latencies did not differ between the two lines. These results suggest that seizure thresholds are regulated to some extent by the hypothalamic-pituitary-adrenal (HPA) axis. The effects of ADX on GABA-related seizure activity may also be influenced by genotype, such that genetic differences in GABA A receptor function and adrenocortical responses in LS and SS mice may be responsible for the differential seizure latencies observed in sham-operated mice.

Chronic administration of β-carboline-3-carboxylic acid methylamide by continuous intraventricular infusion increases gabaergic function

The repeated, intraperitoneal administration of the benzodiazepine receptor inverse agonist, FG 7142 (β-carboline-3-carboxylic acid methylamide), leads to pharmacological kindling and an associated decrease in GABA-stimulataed influx of 36Cl − into cortical membrane preparations. The chronic administration of benzodiazepine agonists results in the development of tolerance and also results in a decrease in GABA-stimulated uptake of 36Cl −. The present study was designed to evaluate further the paradoxical reports that both chronic treatment with benzodiazepine receptor agonists and inverse agonists results in a decreased ability of GABA to stimulate uptake of 36 − into cortical membrane preparations. The effects of continuous administration of FG 7142 on GABA-stimulated uptake of 36Cl − the threshold for bicuculline-induced seizures and the proconvulsant actions of acute administration FG 7142 were evaluated. The continuous administration of FG 7142 resulted in an increased capacity of GABA to stimulate the uptake of 36Cl − into cortical membrane preparations and a significant increase in the seizure threshold for bicuculline following the acute administration of FG 7142. These data, therefore, indicate that changes in GABAergic function following chronic administration of FG 7142 are dependent on the regimen of administration of drug. The results also suggest that the GABA receptor homeostatically responds to continuous occupation by inverse agonists by an upregulation of its functional response to GABA.

Decreased susceptibility to seizures induced by bicuculline after transient bilateral clamping of the carotid arteries in rats

Rats were exposed for 24 min to bilateral clamping of the common carotid arteries (BCCA) in pentobarbital anaesthesia. 14 days later the animals were subjected to subcutaneous injection of (+)-bicuculline (3 or 4 mg/kg). A significantly decreased susceptibility to bicuculline-induced seizures could be observed in BCCA treated rats compared with sham operated controls. It is suggested that BCCA treatment protects animals against status epilepticus and lethal toxicity produced by bicuculline. Electrographic recordings of the BCCA animals revealed no ictal activity within 1 h after bicuculline injection. An analysis of the GABA content showed a significant increase in the hippocampus (HPC), frontal cortex (FCX), parietal cortex and substantia nigra in BCCA animals compared with controls. It is therefore possible that an increase in GABA content postsynaptically counteracts the GABAA antagonistic effect of bicuculline in BCCA animals thus preventing the normal seizure inducing effect of this substance.

Effect of postnatal anoxia on seizure susceptibility in rats: Bicuculline-induced seizures and pretreatment with iproniazid

The effects of anoxia at 10 days of age on seizures induced by bicuculline (BIC), a specific antagonist of γ-aminobutyric acid (GABA), and BIC-induced seizures after pretreatment with iproniacid (IPR), a monoamine oxidase inhibitor, were investigated in male adult rats. The rats with postnatal anoxia (Group A) were significantly more susceptible to BIC than the control rats (Group C). On the other hand, BIC-induced seizures in both groups were suppressed by IPR pretreatment. Especially, the suppression of BIC-induced seizures which was made by pretreatment with IPR at small doses was more apparent in Group A as compared with Group C. The present study suggests that the enhanced seizure susceptibility of adult rats with postnatal anoxia involves brain monoaminergic system.

ChemInform Abstract: Synthesis and Pharmacological Evaluation of 6,8‐Diaryl‐1,2,4‐triazolo‐(4,3‐b)‐ and ‐1,2,3,4‐tetrazolo(1,5‐b)pyridazines.

A series of 6,8-diaryl-1,2,4-triazolo[4,3-b] and 1,2,3,4-tetrazolo[1,5-b]pyridazines was synthesized from suitable chloropyridazines. The compounds were screened in mice for their ability to antagonize maximal electroshock-, pentylenetetrazole- and bicuculline-induced seizures; sedative effects were evaluated by a study of the spontaneous motor activity. Some of pyridazine derivatives exhibited appreciable anticonvulsant activity. Substituting the phenyl ring in the 6-position with an halogen atom led to a substantial increase of activity. Furthermore, none of the compounds was notably active in tests predictive of anxiolytic activity.

Cyclic alteration in the anticonvulsant effect of nitrous oxide in rats.

The anticonvulsant action of nitrous oxide and its time course were studied in rats. Bicuculline, a GABA-receptor antagonist, was administered intravenously at a rate of 0.2 mg.kg(-1).min(-1) during exposure to air (n = 60) or 75% nitrous oxide in oxygen (n = 80). The convulsant dose of bicuculline was determined. The rats were divided into subgroups according to the duration of exposure to air or nitrous oxide, from 0 to 120 min at 15 min intervals. Although the convulsant dose of bicuculline was consistent in the air group (1.03 +/- 0.06 mg.kg(-1), mean +/- SEM), it showed two peaks at 30- and 90 min exposures to nitrous oxide. The threshold dose in the nitrous oxide group was significantly higher than in the air group at only 15- and 30 min exposures (1.50 +/- 0.16, 2.15 +/- 0.25 mg.kg(-1), respectively, P < 0.05). We conclude that nitrous oxide has an anticonvulsant action against bicuculline-induced seizure, and that a cyclic nature exists in its action.

Alterations in cytosolic free calcium in the cat cortex during bicuculline-induced epilepsy

Influx of calcium ion (Ca ++) into the neurons has recently been implicated in the generation of seizure activity. Utilizing indo-1, a fluorescent Ca ++ indicator, changes in cytosolic free calcium ([Ca ++]i), NAD/NADH redox state and hemodynamics were simultaneously measured in vivo from the cat cortex during bicuculline-induced seizure activity. A ratio of indo-1-Ca ++ fluorescence at 400 and 506 nm during ultraviolet excitation (340 nm) was utilized as a measure of changes in [Ca ++]i. Alterations in the NAD/NADH redox state and local cortical blood volume (ICBV) were assessed at 464 nm and 340 nm, respectively. Local cortical blood flow (1CBF) was calculated from 1CBV and mean transit time determined from cortical hemodilution curves. Electroencephalogram (EEG) was monitored from the same cortical region as the optical measurements. The [Ca ++]i signal ratio started to increase 19 ± 2 sec prior to the onset of seizure activity on the EEG and remained elevated until the activity was suppressed by an intravenous administration of diazepam (2 mg/kg). The early increase in [Ca ++]i is presumably due to a synaptic Ca ++ entry associated with facilitated excitatory neurotransmission. The NAD/NADH redox state became oxidized during the seizure activity and started to recover as the EEG activity was suppressed. The 1CBV and lCBF increased by 17 ± 8% and 68 ± 16%, respectively, 10 min into the seizure activity. This study provides direct in vivo evidence suggesting a possible role of calcium entry into the neurons in the epileptogenesis.

35S]TBPS binding sites are decreased in the colliculi of mice with a genetic predisposition to bicuculline-induced seizures

Several differences have been found in GABAergic function between the long sleep (LS) and short sleep (SS) mice which were genetically selected for different ethanol-induced sleeptimes, and it has been suggested that these differences may explain their differential ethanol sensitivity. However, these lines also differ in seizure susceptibility, a behavior which may also be mediated by GABAergic pathways. Thus, it is difficult to associate differences in GABA neurochemistry with either of these behaviors, particularly when only two selected lines are used. We measured differences in the density and affinity of the [ 35S]TBPS binding site on the GABA A receptor/Cl − ionophore complex in discrete brain areas; and in order to determine the relationship between receptor binding and behavioral differences, we included mice from 5 of the LS and SS recombinant inbred strains (LS × SS RI) in addition to mice from the LS and SS lines. [ 35S]TBPS binding in sagittal brain sections was analyzed by quantitative autoradiography, and the amount of binding differed depending on whether bicuculline was added to inhibit endogenous GABA binding. In the presence of bicuculline, the number of [ 35S]TBPS sites in SS mice was highest in the colliculi (4.5 ± 0.5 pmol/mg protein), cerebellum (4.8 ± 0.6 pmol/mg), hippocampus (3.2 ± 0.7 pmol/mg) and cortex (2.9 ± 0.3 pmol/mg). The B max was two-fold lower in both superior and inferior colliculi (IC) of LS mice. There were no differences between lines in B max in any other area and in K d values in any area (58 ± 4.0 nM). When we compared [ 35S]TBPS binding in the LS × SS RI strains, B max values in IC were negatively correlated with latency to bicuculline-induced seizures but were not correlated with the duration of ethanol-induced sleeptime. These data support the hypothesis that differences in GABAergic neurochemistry in these mouse lines may not always be related to differences in ethanol sensitivity and point to GABAergic receptors in IC as possibly being involved in determining seizure sensitivity to bicuculline.