Pavel Mareš

Seizures elicited by corneal 6-Hz stimulation are widely acknowledged as a model of temporal lobe seizures. Despite the intensive research in rodents, no studies hint at this model in developing animals. We focused on seven age groups of both male and female rats. Biphasic pulses with 0.3 ms duration and current intensities from 20 to 80 mA were applied transcorneally for 3 s to calculate threshold intensities for individual age groups. Threshold stimulation intensity necessary for elicitation of clonic seizures was highly age- and sex-dependent. The highest threshold was observed in the youngest (15-day-old) group then it decreased to the age of 25 days and increased again up to adulthood. The threshold current tended to be lower in females of all age groups. The incidence of convulsive seizures increased with stimulation intensity up to postnatal day 25 in either sex. In rats of 31 days old and older convulsions occurred irregularly regardless of the stimulation current and sex. For subsequent analysis, the animals were categorized into two groups: juveniles, aged 15 to 25 days, and adolescents/adults, aged 31 days and older. Our statistical analyses revealed an increased risk of convulsions after the stimulation with higher intensities in juvenile but not adolescent/adult rats. Females tended to be more sensitive to the stimulation with lower currents than males. Seizure severity was higher in females 18- to 25-day old compared to males of the same age and the seizure duration increased with stimulation intensities in juvenile but not adolescent/adult animals. The data extend the use of the rat 6 Hz model to immature animals and may be useful as a model of pediatric temporal lobe seizures.

The aim of the present study was to analyze the location of degenerating neurons in the dorsal (insular) claustrum (DCL, VCL) and the dorsal, intermediate and ventral endopiriform nucleus (DEn, IEn, VEn) in rat pups following lithium–pilocarpine status epilepticus (SE) induced at postnatal days [P]12, 15, 18, 21 and 25. The presence of Fluoro-Jade B-positive neurons was evaluated at 4, 12, 24, 48 h and 1 week later. A small number of degenerated neurons was observed in the CL, as well as in the DEn at P12 and P15. The number of degenerated neurons was increased in the CL as well as in the DEn at P18 and above and was highest at longer survival intervals. The CL at P15 and 18 contained a small or moderate number of degenerated neurons mainly close to the medial and dorsal margins also designated as DCl (“shell”) while isolated degenerated neurons were distributed in the VCl (“core”). In P21 and 25, a larger number of degenerated neurons occurred in both subdivisions of the dorsal claustrum. The majority of degenerated neurons in the endopiriform nucleus were found in the intermediate and caudal third of the DEn. A small number of degenerated neurons was dispersed in the whole extent of the DEn with prevalence to its medial margin. Our results indicate that degenerated neurons in the claustrum CL and endopiriform nucleus are distributed mainly in subdivisions originating from the ventral pallium; their distribution correlates with chemoarchitectonics of both nuclei and with their intrinsic and extrinsic connections.

Status epilepticus (SE) is the most common neurologic emergency in children. Both clinical and laboratory studies have demonstrated that SE in early life can cause brain damage and permanent behavioral abnormalities, trigger epileptogenesis, and interfere with normal brain development. In experimental rodent models, the consequences of seizures are dependent upon age, the model used, and seizure duration. In studies involving neonatal and infantile animals, the model used, experimental design, conditions during the experiment, and manipulation of animals can significantly affect the course of the experiments as well as the results obtained. Standardization of laboratory approaches, harmonization of scientific methodology, and improvement in data collection can improve the comparability of data among laboratories.

The GluN2A subunit of N-methyl-D-aspartate (NMDA) receptors becomes dominant during postnatal development, overgrowing the originally dominant GluN2B subunit. The aim of our study was to show changes of anticonvulsant action of the GluN2A subunit-preferring antagonist during postnatal development of rats. Possible anticonvulsant action of GluN2A-preferring antagonist of NMDA receptors P = [[[(1S)-1-(4-bromophenyl)ethyl]amino](1,2,3,4-tetrahydro-2,3-dioxo-5-quinoxalinyl)methyl]phosphonic acid tetrasodium salt (PEAQX) (5, 10, 20 mg/kg s.c.) was tested in 12-, 18-, and 25-day-old rats in three models of convulsive seizures. Pentylenetetrazol-induced generalized seizures with a loss of righting reflexes generated in the brainstem were suppressed in all three age groups in a dose-dependent manner. Minimal clonic seizures with preserved righting ability exhibited only moderately prolonged latency after the highest dose of PEAQX. Anticonvulsant action of all three doses of PEAQX against cortical epileptic afterdischarges (generated in the forebrain) was found in the 25-day-old animals. The highest dose (20 mg/kg) was efficient also in the two younger groups, which might be due to lower specificity of PEAQX and its partial affinity to the GluN2B subunit. Our results are in agreement with the postero-anterior maturation gradient of subunit composition of NMDA receptors (i.e., an increase of GluN2A representation). In spite of the lower selectivity of PEAQX, our data demonstrate, for the first time, developmental differences in comparison with an antagonist of NMDA receptors with a dominant GluN2B subunit.

Background Postictal potentiation presented immediately after cortical seizures in immature rats might be due to imbalance between excitation and inhibition. The aim of the present study was to determine whether augmentation of inhibition mediated by GABAA receptors could also suppress the postictal potentiation. Methods Twelve-day old rats with implanted electrodes were used in our study. Five drugs were tested: the agonist muscimol, the positive modulator midazolam and three neurosteroids affecting GABAA receptors—allopregnanolone, pregnanolone sulphate and pregnanolone glutamate. Results None of the five drugs was able to suppress potentiation appearing immediately after cortical epileptic afterdischarges, but all of them exhibited delayed anticonvulsant action 10 (in the case of midazolam and muscimol) or 20 min (all three steroids) after cortical seizures. Conclusion Our results support a role of GABA in augmentation of cortical after discharges after longer intervals, whereas immediate postictal potentiation is not affected by GABAergic drugs. Due to similar effect with GABAergic drugs, the main mechanism of action of the three steroids tested is potentiation of GABAergic inhibition.

Benzodiazepines (BZDs) are widely used in patients of all ages. Unlike adults, neonatal animals treated with BZDs exhibit a variety of behavioral deficits later in life; however, the mechanisms underlying these deficits are poorly understood. This study aims to examine whether administration of clonazepam (CZP; 1 mg/kg/day) in 7–11-day-old rats affects Gama aminobutyric acid (GABA)ergic receptors in both the short and long terms. Using RT-PCR and quantitative autoradiography, we examined the expression of the selected GABA_A receptor subunits (α1, α2, α4, γ2, and δ) and the GABA_B B2 subunit, and GABA_A, benzodiazepine, and GABA_B receptor binding 48 h, 1 week, and 2 months after treatment discontinuation. Within one week after CZP cessation, the expression of the α2 subunit was upregulated, whereas that of the δ subunit was downregulated in both the hippocampus and cortex. In the hippocampus, the α4 subunit was downregulated after the 2-month interval. Changes in receptor binding were highly dependent on the receptor type, the interval after treatment cessation, and the brain structure. GABA_A receptor binding was increased in almost all of the brain structures after the 48-h interval. BZD-binding was decreased in many brain structures involved in the neuronal networks associated with emotional behavior, anxiety, and cognitive functions after the 2-month interval. Binding of the GABA_B receptors changed depending on the interval and brain structure. Overall, the described changes may affect both synaptic development and functioning and may potentially cause behavioral impairment.

Mechanism of ictogenesis of D- and L-stereroisomers of homocysteic acid was studied in 12-day-old rats by means of antagonists of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. There was no qualitative difference between the two stereoisomers in generation of emprosthotonic (flexion) as well as generalized tonic-clonic seizures. Moderate differences were observed in the first, nonconvulsive effects of the two isomers. As generation of the two types of seizures is concerned, NMDA and AMPA participate in generalized tonic-clonic seizures whereas NMDA receptors play a dominant role in generation of flexion seizures.

Ifenprodil as a specific antagonist of NMDA receptors containing a dominant NR2B subunit exhibits age-dependent anticonvulsant action. Possible changes of this action due to status epilepticus (SE) elicited at early stage of development were studied using cortical epileptic afterdischarges (ADs) as a model. Lithium-pilocarpine SE was induced at postnatal day 12 and effects of ifenprodil were studied 3, 6, 9, and 13 days after SE in rat pups with implanted epidural electrodes. Controls (LiPAR) received saline instead of pilocarpine. ADs were elicited by low frequency stimulation of sensorimotor cortex. Intensity of stimulation current increased in 18 steps from 0.2 to 15 mA. Ifenprodil (20 mg/kg) was administered intraperitoneally (ip) after the stimulation with 3.5-mA current. Threshold for four different phenomena as well as duration of ADs were evaluated. The threshold for the transition into the limbic type of ADs was higher in 15-day-old SE rats than in LiPAR controls. Opposite difference was found in 18-day-old animals, older rats did not exhibit any difference. Isolated significant changes in total duration of ADs were found after high stimulation intensities. These changes appeared in 18-day-old rats where ADs were shorter in SE than in control LiPAR rats. Changes in ifenprodil action were found only in the first week after SE but not in the second week. Interpretation of the results is complicated by failure of significant differences between SE and LiPAR rats probably due to a high dose of paraldehyde.