NMDA and Not Non-NMDA Receptor Antagonists Are Protective against Seizures Induced by Homocysteine in Neonatal Rats

Homocysteine induces seizures in adult, as well as in immature, experimental animals, but the mechanism of its action is still unknown. The aim of the present study was to examine whether homocysteine in immature animals may act via excitatory amino acids receptors. Seizures were induced in 7-day-old rats by ip administration of homocysteine (16.5 mmol/kg) and the effects of selected antagonists at NMDA and non-NMDA receptor sites were investigated. The anticonvulsant effect was evaluated not only in terms of behavioral changes, but also in terms of some indicators of brain energy metabolism. Rat pups were sacrificed during generalized clonic-tonic seizures, corresponding approximately to 15-30 min after homocysteine administration. Comparable time intervals were used for sacrificing pups in the groups with protective drugs. Non-NMDA antagonists, -glutamic acid diethylester (GDEE) (4 mmol/kg, ip) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline (NBQX) (two doses, 30 mg/kg each, ip), failed to protect neonatal rats against homocysteine-induced seizures. Although NBQX prevented the tonic phase, the severity of clonic movements was even more pronounced. Metabolic changes accompanying the seizures (decreases of glucose and glycogen and a rise of lactate) were also not influenced by GDEE or NBQX pretreatment. On the contrary, NMDA antagonists, both competitive (AP7, 0.33 mmol/kg, ip) and noncompetitive (MK-801, 0.5 mg/kg, ip), had a clear-cut anticonvulsant effect. They not only suppressed the behavioral signs of seizures, but also prevented most of the metabolic changes accompanying seizures. Whether incomplete normalization of lactate levels reflects subclinical seizure activity or whether it is due to the effect of anatagonists (AP7, MK-801) themselves is not clear. All drugs, besides GDEE, when given alone caused a rise in brain glucose. This is the first demonstration that seizures induced by homocysteine can be blocked by NMDA antagonists. The findings thus suggest that, at least in neonatal rats, enhanced activation of NMDA receptors is implicated in homocysteine-induced seizures.     

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.     

Convulsant Action of D,L-Homocysteic Acid and Its Stereoisomers in Immature Rats

Summary: Purpose: We wished to characterize the convulsant effect of homocysteic acid (HCA) in developing rats.
Methods: Seizures were induced in 7-, 12-, 18-, and 25-day-old rats by intraperitoneal (i.p.) administration of D,L-HCA and in 12-day-old rats by i.p. injection of L- and D-stereoisomers of HCA. The animals were observed for 30 min after injection. The incidence, latencies, pattern of motor seizures, and all behavioral phenomena were noted. Fifty percent convulsant dose (CD₅₀) values were calculated by probit analysis. Electrocor-ticograms (ECoG) were recorded after injection.
Results: HCA did not elicit minimal clonic seizures whereas generalized tonic-clonic seizures (GTCS) occurred in all the age groups studied. Flexion (emprosthotonic) convulsions occurred to postnatal day 18. ECoG recordings exhibited delta activity in younger pups and sharp graphoelements in older pups, but electroclinical correlation was poor. Young animals were more sensitive to the convulsant effect of D,L-HCA. In addition, D-HCA was significantly more effective than L-HCA in inducing both flexion and generalized seizures.
Conclusions: Our data clearly indicate that seizures induced by HCA differ from those evoked by homocysteine. There are no qualitative differences in the motor pattern of seizures induced by the two stereoisomers of HCA, but marked differences were apparent in the very first signs of their action. These differences might be due to interaction with different glutamate receptor subtypes.     

Anticonvulsant Action of both NMDA and Non-NMDA Receptor Antagonists against Seizures Induced by Homocysteine in Immature Rats

Seizures were induced in immature 18-day-old rats by i.p. administration of homocysteine (11 mmol/kg) and the effects of selected antagonists of NMDA receptors [MK-801 (0.5 mg/kg), AP7 (0.33 mmol/kg), CGP 40116 (10 mg/kg)] and non-NMDA receptors [GDEE (4 mmol/kg), NBQX (two doses, 30 mg/kg each)] were studied. The effect of MgSO₄(two doses, 2 mmol/kg each) was also tested. The anticonvulsant effect was evaluated not only from the behavioral manifestations of seizures, but also in terms of some indicators of brain energy metabolism. Rat pups were sacrificed during generalized clonic-tonic seizures, corresponding to 16–45 min after homocysteine administration. Comparable time intervals were used for sacrificing the pups which had received the protective drugs. In contrast to neonatal rats, in which only NMDA antagonists could prevent homocysteine-induced seizures, both NMDA and non-NMDA receptor antagonists exerted an anticonvulsant effect in 18-day-old rats. In addition, the pronounced anticonvulsant effect could be achieved by the combined treatment with low subthreshold doses of NMDA (MK-801) and non-NMDA (NBQX) receptor antagonists. The protection was evident not only in suppressing behavioral symptoms of seizures, but also in preventing most of the metabolic changes accompanying seizures, mainly glycogen degradation. More than a sevenfold accumulation of lactate occurring during seizures was markedly reduced by all the tested drugs, but was not completely eliminated. All antagonists, when given alone in the same doses as those used for seizure protection, remained without any effect on lactate levels. Comparison of the present data with previous findings concerning neonatal rats suggests that there may be a developmental change in anticonvulsant efficacy of non-NMDA receptor antagonists against homocysteine-induced seizures in rats.     

Wednesday July 5, 200612:00–13:30Hall 3DPlatform SessionBasic Science III: Pharmacology and Treatment

¹ P. Mareš and ¹ A. Mikulecká (   ¹ Institute of Physiology, Academy of Sciences of The Czech Republic, Czech Republic )
Purpose: To study possible anticonvulsant action of ifenprodil and its side effects on motor performance in immature rats.
Method: Motor seizures were elicited by pentylenetetrazol (100 mg/kg s.c.) in 7-, 12-, 18- and 25-day-old rats. The incidence and latency of minimal clonic and generalised tonic–clonic seizures were evaluated. Motor performance was tested in 12-, 18- and 25-day-old rats using age specific tests; spontaneouos locomotion was measured in open field. Ifenprodil (10, 20 or 40 mg/kg i.p.) was administered 20 min before pentylenetetrazol injection or behavioural testing. Open field was tested once more one hour after ifenprodil administration.
Results: Minimal clonic seizures could be elicited only in 18- and 25-day-old rats. Ifenprodil did not influence their incidence and latency. Specific anticonvulsant action (suppression of the tonic phase of generalised seizures) was found in 7-, 12- and 18-day-old rats. This effect decreased with age and it failed to appear in 25-day-old animals. Latency of generalised seizures was prolonged in 7-day-old but paradoxically shortened in older animals. Locomotor activity was transiently increased by ifenprodil only in 12-day-old rats; it remained uninfluenced in older animals. Motor performace was moderately compromised in the youngest group only. Motor activity was normalised one hour after the administration.
Conclusion: Ifenprodil exhibits specific action against the tonic phase of generalised seizures in the first three postnatal weeks. Unwanted side effects on motor activitites were observed only in 12-day-old rats but not in older animals. Ifenprodil thus differs from other NMDA receptor antagonists. Supported by research projects LC554 and AV0Z5011009.     

Age-Dependent Differences in the Anticonvulsant Effects of 2–Amino-7-Phosphono-Heptanoic Acid or Ketamine Infusions Into the Substantia Nigra of Rats

Infusions of 2-amino-7-phosphonoheptanoic acid (AP7) or ketamine into the substantia nigra pars reticulata (SNPR) of adult rats increase the latency of onset to seizures induced by the convulsant ether flurothyl. Nigral infusions of AP7 or ketamine in concentrations up to 10 times greater than the adult dose are ineffective in 16-day-old rats. These results suggest that differences in seizure susceptibility between adult and immature rats may be related to differences in excitatory amino acid neurotransmission in the SN.     

Different effects of two N-methyl-D-aspartate receptor antagonists on seizures, spontaneous behavior, and motor performance in immature rats

Typical N-methyl-D-aspartate (NMDA) receptor antagonists exhibit anticonvulsant action and unwanted effects, even in developing rats. Therefore, we studied the actions of the low-affinity, noncompetitive antagonist memantine and the NR2B-specific antagonist ifenprodil. Seizures (minimal clonic and generalized tonic-clonic) were elicited with pentylenetetrazol (100mg/kg subcutaneously) in rats 7, 12, 18, and 25 days old pretreated with memantine (2.5-40 mg/kg intraperitoneally) or ifenprodil (10-60 mg/kg intraperitoneally). The effects of both drugs were studied in open field and motor performance tests in 12-, 18-, and 25-day-old rats. Memantine suppressed generalized tonic-clonic seizures in all age groups; minimal seizures were potentiated. Ifenprodil abolished the tonic phase of generalized tonic-clonic seizures in 7-, 12-, and 18-day-old rats only; minimal seizures remained untouched. Memantine induced locomotor hyperactivity and compromised motor performance in all age groups. Ifenprodil exerted these effects only in 12-day-old rats; older animals were less active in open field tests. Memantine exhibits both anti- and pro-convulsant and behavioral effects typical of NMDA antagonists. Ifenprodil exerted the same effects in 12-day-old rats, but its anticonvulsant action in 18-day-old rats was accompanied by a decrease in locomotion.     

Anticonvulsant effects of phenobarbital and primidone during ontogenesis in rats

The influence of phenobarbital (PHB, 10, 20, 40 or 80 mg/kg i.p.) and primidone (PRI, 40 or 80 mg/kg i.p.) on metrazol-induced motor seizures was studied in rats 7, 12, 15, 18, 25 and 90 days old. PHB blocked both types of seizures induced by metrazol — minimal (mMS) as well as generalized tonic-clonic (MMS) — in all age groups where they appeared under control conditions. The effect against major seizures was always better expressed than against mMS. Pretreatment with PHB led to the appearance of mMS in 7- and 12-day-old rat pups, where control animals did not exhibit this type of seizure. Combined administration of the 2 high doses of PHB and metrazol resulted in the appearance of behavioral automatisms in young rats. PRI abolished MMS in adult rats only, no changes were seen in 25-day-old animals and specific suppression of the tonic phase of MMS was observed in younger rats. mMS were influenced only in 7-and 12-day-old rats, where an increase in their incidence was recorded. Pretreatment with PRI never induced automatisms. The different actions of PHB and PRI speak in favor of an anticonvulsant action of PRI itself.     

NMDA-induced seizures in developing rats cause long-term learning impairment and increased seizure susceptibility

N-methyl-D-aspartate (NMDA) receptors play a prominent role in the pathogenesis of epilepsy, yet few studies have used NMDA as a convulsant in whole animals. In developing rats, systemic NMDA induces seizures with a unique seizure phenotype ("emprosthotonic" or hyperflexion seizures) and electrographic pattern (electrodecrement). These features are not seen in kainic acid-induced seizures, suggesting that seizures activated by NMDA might cause different long-term consequences. Therefore, we investigated the effects of NMDA seizures during development on cognitive function and susceptibility to seizures in adulthood. Rat pups (P12-20) were injected with saline (n=36) or NMDA (n=64) at convulsant doses (15-30mg/kg, i.p.). After NMDA injection, a characteristic sequence of seizure activity was seen: initial behavioral arrest, followed by hyperactivity, agitation, and then emprosthotonus and generalized tonic-clonic seizures. Seizures were terminated 30min later by ketamine (50mg/kg, i.p.). On P85, rats underwent behavioral testing in the water maze. Rats that had experienced NMDA seizures as pups took significantly longer to learn the platform location over 5 days of testing, compared to controls. On P90, rats were injected with pentylenetetrazol (PTZ, 50mg/kg, i.p.) to assess their susceptibility to generalized seizures. NMDA-treated rats had decreased latency and increased duration of class V PTZ seizures. Cresyl violet-stained sections of cortex and hippocampus had no obvious cell loss or gliosis. In summary, NMDA causes a unique seizure phenotype in the developing brain, with subsequent deficits in spatial learning and an increased susceptibility to PTZ seizures in adulthood. This study provides additional evidence for long-term alterations of neuronal excitability and cognitive capacity associated with seizures during development.     

MPEP, an antagonist of metabotropic glutamate receptors, exhibits anticonvulsant action in immature rats without a serious impairment of motor performance

An antagonist of type I metabotropic glutamate receptors MPEP was found to exhibit anticonvulsant action in adult rodents. Present experiments were focused on action of this drug against pentetrazol-induced motor seizures in immature rats 12-, 18- and 25-days old. Dose of pentetrazol (100 mg/kg s.c.) was chosen to elicit minimal clonic seizures and (after a longer latency) generalized tonic-clonic seizures. Pretreatment with MPEP (doses from 10 to 80 mg/kg i.p.) resulted in a dose-dependent suppression of the tonic phase of generalized tonic-clonic seizures in all age groups studied. Efficacy of MPEP was higher and the effect lasted longer in 12- than in 25-day-old rats. In addition, minimal clonic seizures were suppressed in 18-day-old rats. Motor abilities of immature animals were not compromised by MPEP in doses of 20 and/or 40 mg/kg i.p., only righting reflex was a little slowed down in 12- and 18-day-old rats. In contrast to antagonists of ionotropic glutamate receptors anticonvulsant doses of MPEP do not induce unwanted side effects in motor performance of developing rats.