Flight induced by infusion of bicuculline methiodide into periventricular structures

Microinjections of different doses of bicuculline methiodide (BM) were performed into the mesencephalic central gray (CG), the medial hypothalamus (MH) and lateral hypothalamus (LH). Flight reactions could be induced by microinjections of BM into either the CG or the MH. However, the type of flight behavior was different whether the injection was made in the CG or the MH. Furthermore, microinjections of 35 ng BM in either structure produced an increase in locomotor activity whose time course differed according to the injected structure, and an increase in rearings was induced at MH but not at CG sites. At lateral hypothalamic sites, BM produced an increase in locomotor activity and rearings but no jump. These effects were antagonized in a dose-dependent manner by a local pretreatment with 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP), a GABA agonist. These results suggest that (1) at the level of both the MH and the CG, a GABAergic link is involved in the inhibition of a substrate whose activation produces aversive effects, and (2) the aversive effect induced by CG BM microinjection seems to be different from that induced by MH BM microinjection.     

Convulsive seizures in rats induced by N-methyl-D-aspartate injection into the massa intermedia.

The behavioral and electroencephalographic effects of N-methyl-D-aspartate (NMDA, 25 nmol/1 microliter) injection into the massa intermedia (MI) was examined in rats. The injection caused violent running/jumping and shrill vocalization without evidence of EEG seizure in the hippocampus (HP) and amygdala (AM). Animals with the injection site located in the reuniens nucleus subsequently developed generalized tonic and then clonic seizure, leading to fatal status epilepticus in some animals. Intermittent or continuous EEG discharge in the limbic system was found during clonic seizures. These findings suggest that the NMDA receptor in the reuniens nucleus in the MI participates in the generation and expression of convulsive seizure in rats.     

Convulsive seizures induced by N-methyl-D-aspartate microinjection into the mesencephalic reticular formation in rats

Effects of microinjections of a single 2 or 10 nmol dose of N-methyl-D-aspartate (NMDA) into the unilateral mesencephalic reticular formation (MRF) on behavior and electroencephalogram were examined in rats (n=18) during a 15 min period (Exp. 1), and subsequent effects of sound stimulation with key jingling applied at 15, 30, and 45 min after the injections were observed (Exp. 2). The microinjections of 2 nmol dose of NMDA (n=10) induced hyperactivity (9 of 10 rats) and running/circling (8 of 10 rats) in Exp. 1, and hyperactivity (3 of 10 rats) in Exp. 2. Moreover, the microinjections of 10 nmol dose of NMDA (n=8) induced not only hyperactivity (8 of 8 rats) and running/circling (7 of 8 rats) but also generalized tonic-clonic seizures (GTCS) (5 of 8 rats) in Exp. 1; these seizure patterns were also elicited by sound stimulation in Exp. 2. The seizure patterns were accompanied by electroencephalographic seizure discharges in the MRF and the motor cortex. In contrast, the control group rats (n=10) which received a single dose of saline microinjection into the unilateral MRF showed no behavioral or electroencephalographic changes in both Exp. 1 and 2. These findings suggest that the MRF has an important role in the development of GTCS, which follows hyperactivity and running/circling, and that potentiation of excitatory neurotransmission in the MRF participates in the development of audiogenic seizures as well as GTCS.     

Amygdaloid kindling with bicuculline methiodide in rats

The effect of repeated intraamygdaloid injection of bicuculline methiodide (BM) was studied in rats. Chemitrodes for both microinjection and electrographic recording were implanted into the left basolateral amygdala. Two weeks after the surgery, a subconvulsive dose of BM (0.2 or 0.4 nmol) was administered through the chemitrodes every fourth day. Repeated injections caused a progressive seizure development which was comparable to that seen with electrical kindling. The kindling effect persisted after a 6-month interruption of the stimulation. When a mixture of BM and GABA agonist (GABA 20 nmol, muscimol 2 nmol, or baclofen 5 nmol) was injected into the amygdala of kindled rats, seizure activity was markedly suppressed to stage 0 or 1. On the other hand, an intraamygdaloid injection of picrotoxin (0.8 nmol) brought about the same seizure as induced by BM, whereas no seizure was observed with strychnine (4 nmol). No histological change specific to this kindling was detected. The present results indicate that chemical kindling can be induced by repeated local injection of BM into the amygdala, and that the mechanism underlying this kindling is closely associated with local postsynaptic GABA receptors in the amygdala. This GABAergic system may also be important in other types of kindling.     

Convulsive seizures induced by N-methyl- -aspartate microinjection into the mesencephalic reticular formation in rats

Effects of microinjections of a single 2 or 10 nmol dose of N-methyl- -aspartate (NMDA) into the unilateral mesencephalic reticular formation (MRF) on behavior and electroencephalogram were examined in rats (n=18) during a 15 min period (Exp. 1), and subsequent effects of sound stimulation with key jingling applied at 15, 30, and 45 min after the injections were observed (Exp. 2). The microinjections of 2 nmol dose of NMDA (n=10) induced hyperactivity (9 of 10 rats) and running/circling (8 of 10 rats) in Exp. 1, and hyperactivity (3 of 10 rats) in Exp. 2. Moreover, the microinjections of 10 nmol dose of NMDA (n=8) induced not only hyperactivity (8 of 8 rats) and running/circling (7 of 8 rats) but also generalized tonic–clonic seizures (GTCS) (5 of 8 rats) in Exp. 1; these seizure patterns were also elicited by sound stimulation in Exp. 2. The seizure patterns were accompanied by electroencephalographic seizure discharges in the MRF and the motor cortex. In contrast, the control group rats (n=10) which received a single dose of saline microinjection into the unilateral MRF showed no behavioral or electroencephalographic changes in both Exp. 1 and 2. These findings suggest that the MRF has an important role in the development of GTCS, which follows hyperactivity and running/circling, and that potentiation of excitatory neurotransmission in the MRF participates in the development of audiogenic seizures as well as GTCS.     

Comparison of seizures and brain lesions produced by intracerebroventricular kainic acid and bicuculline methiodide

Intracerebroventricular kainic acid produces in rats brain lesions similar to Ammon's horn sclerosis in humans. To test the hypothesis that these lesions result indirectly from prolonged seizure activity and not from a direct action of kainic acid on the neurons that are destroyed, the effects of intracerebroventricular kainic acid and bicuculline methiodide were compared. Although bicuculline methiodide seizures differed dramatically from kainic acid seizures, both electrographically and behaviorally, the resulting brain lesions were similar for a given total limbic seizure duration. These results, in combination with other data, support the view that lesions made by intracerebroventricular administration of convulsants are indeed caused by prolonged limbic seizures. The total duration of seizure activity appears to be one important variable.     

Convulsive seizures induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid microinjection into the mesencephalic reticular formation in rats

Effects of microinjections of a single 2 or 10 nmol dose of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) into the unilateral mesencephalic reticular formation (MRF) on behavior and on the electroencephalogram were examined in rats (n=30) over a 15-min period (Exp. 1); subsequent effects of sound stimulation with key jingling applied at 15, 30, and 45 min after the injection were observed (Exp. 2). The microinjections of a 2 nmol dose of AMPA (n=15) induced hyperactivity (15 of 15 rats) and running/circling (10 of 15 rats) in Exp. 1, and hyperactivity (5 of 15 rats) in Exp. 2. Moreover, the microinjections of a 10 nmol dose of AMPA (n=15) induced hyperactivity (15 of 15 rats), running/circling (13 of 15 rats), generalized tonic-clonic seizures (GTCS) (4 of 15 rats), and amygdala kindling-like seizures (AMKS) (8 of 15 rats) in Exp. 1; electroencephalographic seizure discharges were predominantly observed in the MRF during hyperactivity, running/circling and GTCS, while those predominantly observed in the amygdala were during AMKS. In Exp. 2, hyperactivity (15 of 15 rats), running/circling (14 of 15 rats) and GTCS (6 of 15 rats) were elicited by sound stimulation, although AMKS were not. The control group of rats (n=15) which received a single dose of saline microinjection into the unilateral MRF showed no behavioral or electroencephalographic changes in both Exp. 1 and 2. These findings suggest that potentiation of excitatory amino acid neurotransmission induced by AMPA injection into the MRF plays an important role not only in the development of hyperactivity, running/circling, GTCS and AMKS, but also in the development of audiogenic seizures.     

Antagonism between intracerebroventricularly administered N-methyl-D-aspartate and bicuculline methiodide in induction of clonic seizures in mice

N-Methyl-D-aspartate and bicuculline were administered alone or as a combination by intracerebroventricular injection to mice, and their convulsant activity was monitored. Both of these compounds elicited clonic seizures, though by different mechanisms. However, their simultaneous administration resulted in less than additive induction of clonic activity.     

Convulsant action of systemically administered glutamate and bicuculline methiodide in immature rats

Developmental changes of transport of drugs into the brain play an important role in ontogenetic neuropharmacology. Two convulsant drugs with different mechanisms of action (glutamate and bicuculline methiodide) were chosen to demonstrate these changes in developing rats. High dose of glutamate (4 g/kg i.p.) induced both minimal (predominantly clonic) and generalized tonic-clonic seizures in rat pups 7, 12, and 18 days old. In contrast, seizures were only exceptionally observed in 25 and 90 days old animals. Bicuculline methiodide was administered in a dose of 2 or 20 mg/kg i.p. The first sign of bicuculline methiodide action in all age groups was represented by automatisms, a symptomatology never seen after bicuculline hydrochloride administration. Minimal seizures were induced in 12-day-old and in a few 18-day-old and adult rats. Generalized seizures were common after the higher dose of bicuculline methiodide in 7- and 12-day-old rat pups, seldom in 18-day-old ones and never seen in 25-day-old and adult animals. Both glutamate and bicuculline methiodide enter the brain in immature rats but the mechanisms are probably different - glutamate is transported actively through the blood-brain barrier whereas no similar system is known for bicuculline methiodide.     

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

Summary 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 GABA_A antagonistic effect of bicuculline in BCCA animals thus preventing the normal seizure inducing effect of this substance.Supported by Deutsche Forschungsgemeinschaft, SFB 330, Organprotektion     

The development of cholecystokinin in the interpeduncular nucleus of rats.

This study describes the development and late disappearance of cholecystokinin-like immunoreactivity (CCK-LI) in the interpeduncular nucleus (IPN) of rats. Between one and 7 days of age, CCK-positive fiber labeling was sparse and restricted to the lateral subnuclei of the caudal IPN. By 28 days of age the density of CCK-positive fibers increased and labeling was found in the lateral, rostral and apical subnuclei, and medial to the dorsolateral subnuclei. At 35 days of age CCK-LI fiber labeling was absent in the lateral subnuclei and medial to the dorsolateral subnuclei. No additional changes in fiber labeling were observed after 35 days of age. These results suggest that significant anatomical or biochemical reorganization may occur in projections to the IPN between 28 and 35 days of age.     

Geniculate spikes during epileptic seizures induced in dogs by pentylenetetrazol and bicuculline

Previous studies have indicated that in cats generalized seizures induced by pentylenetetrazol (PTZ) or bicuculline (BIC) may be preceded by high frequency multi-unit activity in brain-stem structures. This activity is not readily demonstrable using conventional EEG recording bandwidths and precedes the onset of spikes and spikes-wave activity in thalamo-cortical systems. Recording with chronic subdural and depth electrodes during PTZ and BIC-induced seizures in 18 dogs essentially replicated these results. However, a new and consistent finding was that the high frequency discharges accompanying seizure onset were most often seen, and most often appeared first, in the lateral geniculate body, neocortex and hippocampus being involved later. The findings are considered to suggest a significant role for subcortical structures in this particular model of generalized cortico-reticular epilepsy.     

Cardiovascular effects of intracerebroventricular bicuculline in rats with absence seizures

Gamma-aminobutyric acid (GABA) plays an important role in both central cardiovascular homeostasis and pathogenesis of epileptic seizures. Previous studies have indicated a critical role of the amygdala in the spread of seizures from brainstem to forebrain and in the regulation of autonomic responses such as blood pressure and heart rate. The purpose of the present study was to examine blood pressure and heart rate effects of bicuculline, a GABA(A) antagonist, and the effect of lesions of the central or the basolateral nuclei of the amygdala on bicuculline-induced cardiovascular responses in conscious WAG/Rij rats with absence epilepsy. Intracerebroventricular administration of 0.3 and 0.5 nmol of bicuculline produces an increase in blood pressure and a slight bradycardia in non-epileptic Wistar rats. The blood pressure response to intracerebroventricular bicuculline is significantly potentiated in epileptic WAG/Rij rats. Bilateral lesions of the basolateral nucleus of the amygdala of WAG/Rij rats completely prevent the pressor response to 0.5 nmol of bicuculline, whereas unilateral lesion of the basolateral nucleus does not affect blood pressure changes in epileptic WAG/Rij rats. Additionally, the pressor effect of 0.5 nmol of bicuculline is not attenuated by bilateral electrolytic ablation of the central nucleus of the amygdala in WAG/Rij rats. Heart rate response to bicuculline is not significantly changed in the lesioned groups. These findings indicate (a) altered GABAergic function in blood pressure regulation; and (b) a critical role of the basolateral nucleus in GABA(A)-mediated blood pressure control in epileptic WAG/Rij rats.     

Cerebral blood flow and metabolic rate early and late in prolonged epileptic seizures induced in rats by bicuculline.

Cerebral blood flow (CBF) and cerebral metablic rate for oxygen (CMRO2) have been studied during sustained epileptic seizures induced by bicuculline (1-2 mg/kg, i.v.) in paralysed Wistar rats, artificially ventilated with nitrous oxide/oxygen. CBF was determined by venous outflow collection, and by 133Xe desaturation, using sagittal sinus blood (for cerebral cortical flow) or retroglenoid venous blood (for 'whole brain' flow). The procedure employed ensured that arterial oxygenation remained normal and blood glucose concentration was normal or high throughout the seizure. Arterial hypotension was prevented by the infusion of donor blood. CBF increased concurrently with seizure onset, reaching a maximum nine times higher than control value after 15-60 s. This was due to a marked rise in mean arterial pressure (to greater than 180 torr) and a dramatic fall in cerebrovascular resistance to less than 15 per cent of control). Subsequently, with decreasing blood pressure, CBF slowly diminished, being more than four times higher than control at 20 min, and slightly less than three times higher than control at two hours. The different procedures for measuring CBF gave closely similar results. A threefold increase relative to control CMRO2 (7-6 ml/100 g-1/min-1 for 'whole brain,' and 10-2 ml/100 g-1/min-1 for cerebral cortex) was measured after 1-20 min of seizure activity (utilizing either the venous outflow or the 133Xe desaturation procedure for CBF determination). After two hours of seizure activity CMR02 was still more than twice as high as the control. This high metabolic rate during sustained seizure activity will increase the susceptibility of the brain to 'ischaemic' damage during prolonged seizures in man in which an additional metabolic stress may be imposed by cerebral hypoxia, arterial hypotension, hyperpyrexia or hypoglycaemia.     

Infusion of bicuculline methiodide into the tectum: model specificity of pro- and anticonvulsant actions.

Microinjection of drugs, such as muscimol, into the substantia nigra pars reticulata (SNpr) can inhibit several types of experimental seizures. Some findings suggested that this was a result of disinhibition of neurons receiving input from GABAergic nigrotectal cells. Indeed, it was reported that bicuculline methiodide (BMI), infused into the tectal region that was reported to receive nigral input, produced an anticonvulsant effect against maximal electroshock (MES) convulsion. Since previous work had suggested that the anticonvulsant effect of intranigral muscimol depended on the particular experimental seizure used, three different experimental seizures were used in the present study to evaluate the effects of BMI infusion into the tectum. Guide cannulas aimed at the tectal region receiving nigral innervation were stereotaxically implanted in rats a week before testing. Bilateral intratectal infusions of BMI (25 ng/side) had an anticonvulsant effect against MES convulsions, confirming a previous report. In contrast, the same BMI pretreatment worsened convulsions produced by either systemic pentylenetetrazol (40 mg/kg, i.p.) or bicuculline (0.5 mg/kg, i.v.). The effects of intratectal BMI were seizure model-dependent, suggesting different functional interconnections between tectum and those pathways responsible for generalization of MES as compared to PTZ or bicuculline convulsions.     

The subnuclear organization of acetylcholinesterase-containing neurons in the interpeduncular nucleus of rats

The interpeduncular nucleus (IPN) is a heterogeneous structure comprised of seven subnuclei which differ with regard to their cytoarchitecture, synaptology, connectivity, and content of neuropeptides and biogenic amines. In the present study, we used Butcher's pharmacohistochemical regimen to assess the subnuclear distribution and staining intensity of acetylcholinesterase (AChE)-containing neurons in the IPN. Although AChE-positive somata were present in every subnucleus, their staining intensity differed within and between the subnuclei. The most intensely stained somata were found in the apical and central subnuclei; however, they comprised only 10–25% of the total population of AChE-positive somata in these subnuclei. Heavily stained somata were observed in the apical, central, and lateral subnuclei; moderately stained somata in the central, lateral, intermediate, and rostral subnuclei; and lightly stained somata in the lateral, intermediate, rostral, dorsal lateral, and rostral lateral subnuclei. The present findings indicate that AChE-containing neurons are differentially distributed between subnuclei of the IPN.     

Nigral infusions of muscimol or bicuculline facilitate seizures in developing rats

The substantia nigra (SN) appears to be a crucial site involved in the modification of seizures. The aim of this study was to elucidate the role of the GABA nigral system in the expression of seizures by comparing the effects of multiple doses of a GABA agonist (muscimol) and a GABA antagonist (bicuculline methobromide) on the development of flurothyl seizures in 16-day-old rat pups. The drugs were infused bilaterally either in the SN or dorsal to the SN. An additional group of pups were infused with bicuculline in the corpus striatum. Results indicate that both drugs facilitated the development of seizures in a dose-related manner when infused into the SN. Infusions of muscimol dorsal to the SN had no effect on seizure latencies while infusions of bicuculline dorsal to SN or corpus striatum still increased the susceptibility of rat pups to seizures. The data suggest that only the effects of muscimol on seizures are specific for the SN and that early in life muscimol may exert its proconvulsant effects via a different receptor site or mechanism than bicuculline.     

Cerebral phosphoinositide, triacylglycerol and energy metabolism during sustained seizures induced by bicuculline

In ventilated rats, levels of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), diacylglycerol (DAG), triacylglycerol (TAG), free fatty acids (FFA) and phosphatidic acid, as well as their fatty acid contents, were measured in forebrain tissue after 1, 20 and 60 min of seizures induced by bicuculline. Cerebral energy state was also measured. PI decreased progressively throughout 60 min of seizures, whereas the levels of PIP and PIP2 did not change. DAG increased modestly and persistently. FFA increased markedly during the early seizure period, but decreased later. Following an initial drop, TAG rose above control. Phosphatidic acid did not change. The levels of ATP and energy charge potential decreased slightly and lactate accumulated. Stearic acid (18:0) and arachidonic acid (20:4) primarily accounted for the changes in the levels of the lipids. At the onset of seizures, the decrease of 18.0 and 20:4 in PI occurred in parallel with an enrichment of these fatty acids in FFA and DAG. Despite the fact that the losses of 18:0 and 20:4 from PI were quantitatively similar to each other at all times examined, the increase in free 18:0 was much larger than the increase in free 20:4 at 20 min of seizures. Concurrently there was a rise of 20:4 in TAG. As the FFA levels declined thereafter, 20:4 and docosahexaenoate (22:6) in TAG continued to increase. The results are consistent with the view that seizure activity stimulates the hydrolytic breakdown of brain phosphoinositides--the pathway catalyzed by phosphodiesterase of the phospholipase C type followed by lipases, and probably the pathway catabolized by phospholipases A as well. Preferential incorporation of polyunsaturated fatty acids into TAG-acyl residues may represent a mechanism to reduce the level of their free forms when the latter are produced in large amounts.     

Neuroethological evaluation of audiogenic seizures and audiogenic-like seizures induced by microinjection of bicuculline into the inferior colliculus. I. Effects of midcollicular knife cuts.

Audiogenic seizures (AS) are a model of generalized tonic-clonic seizures. The inferior colliculus (IC) and the GABAergic neurotransmission seems to be the most critical site and neurotransmitter system, respectively, of the auditory midbrain involved in AS origin and development. Thus, audiogenic-like seizures are evoked by GABAA antagonists such as bicuculline (BIC). Wistar audiogenic AS resistant (R) rats were sham-transected through the midcollicular line and microinjected with IC bicuculline (BIC; 80 ng/0.2 microliters) (n = 8); transected through the midcollicular line and microinjected with IC saline 0.9% (n = 8); transected through the cortex above the midcollicular line and microinjected with IC BIC (n = 3); transected through the midcollicular line up to 6.0 mm depth and microinjected with IC BIC (80 ng/0.2 microliters or 120 ng/0.3 microliters (n = 8). Wistar AS susceptible (S) rats were submitted to cortical transections (n = 8) and midcollicular transections (n = 7). Animals were studied by means of an ethological method before and after microinjections and/or transections in order to evaluate possible pathways in the AS-like evoked seizures. Bicuculline-evoked seizures were very similar to those evoked by acoustic stimulation, but lacked the tonic-clonic component. No modification in animal behavior was observed in the presence of sound, once the AS-like behavior was initiated. A small percentage of the animals, however, presented procursive behavior which was increased by sound. The IC BIC-evoked patterns were almost totally blocked by midcollicular but not cortical transections. Furthermore, midcollicular but not cortical transections blocked the tonic-clonic component of AS in genetically S animals without modifying the wild running component. These data suggest that the inferior colliculus-superior colliculus connection may be involved in the sensorimotor transduction necessary for AS-like behaviors.     

Paradoxical anticonvulsant activity of the gamma-aminobutyrate antagonist bicuculline methiodide in the rat striatum

Bicuculline methiodide (BMI), a gamma-aminobutyrate (GABA) antagonist, is a powerful convulsant agent when injected into the cerebral ventricles, amygdala, hippocampus, thalamus, neocortex, and deep prepiriform cortex in rats. In contrast, bilateral microinjection of BMI into the rat striatum confers protection against seizures induced by the cholinergic agonist pilocarpine (380 mg/kg, i.p.), with an ED50 of 94 fmol (range 45-195 fmol). No topographical variation in the anticonvulsant action of BMI was detected throughout rostrocaudal and dorsoventral aspects of the striatum. The anticonvulsant action of BMI in the striatum was reversed by coadministration of the GABA agonist muscimol or by blocking GABA-mediated inhibition in either the substantia nigra pars reticulata or in the entopeduncular nucleus. The results show that blockade of GABA-mediated inhibition in the striatum has a powerful anticonvulsant effect in the pilocarpine model, suggesting that GABAergic transmission in the striatum modulates the seizure propagation in the forebrain.