Status epilepticus alters neurogenesis and decreases the number of GABAergic neurons in the septal dentate gyrus of 9-day-old rats at the early phase of epileptogenesis.

The effects of a prolonged seizure, i.e. status epilepticus (SE), on neurogenesis of dentate granule cells (DGCs) in the immature dentate gyrus (DG) and possible changes in the phenotypes of the newborn neurons have remained incompletely characterized. We have now studied neurogenesis of DGCs in 9-day-old (postnatal, P9) rats 1 week after kainate (KA)-induced SE using 5-bromo-2-deoxyuridine (BrdU) immunostaining. The phenotype characterization of the newborn cells was carried out by immunofluorescence double labeling using doublecortin (DCX) and nestin as markers for immature cells, and glial fibrillary acid protein (GFAP) as a marker for glial cells. Newborn GABAergic neurons were further identified with antibodies for parvalbumin, glutamate decarboxylase 67 (GAD67), and the GABAA receptor α1 subunit, and mRNA expression of GABAergic and immature neurons was measured with quantitative real-time PCR (qPCR) in the DG. Our results show that the number of newborn as well as GABAergic neurons was significantly decreased after SE in the superior blade of the septal DG. The majority of the newborn BrdU-stained neurons co-expressed DCX, but neither nestin nor GFAP. In both experimental groups, newborn neurons were frequently localized in close contact, but not co-localized, with the cells positively stained for the GABAergic cell markers. Nestin and calretinin mRNA expression were significantly increased after SE. Our results suggest that SE-induced disruption of DGC neurogenesis and decreased number of GABAergic neurons could modify the connectivity between these cells and disturb the maturation of the GABAergic neurotransmission in the immature DG at the early epileptogenic phase.

Status epilepticus alters zolpidem sensitivity of [3H]flunitrazepam binding in the developing rat brain.

GABA, the main inhibitory neurotransmitter in the adult brain, exerts its effects through multiple GABA(A) receptor subtypes with different pharmacological profiles, the alpha subunit variant mainly determining the binding properties of benzodiazepine site on the receptor protein. In adult experimental epileptic animals and in humans with epilepsy, increased excitation, i.e. seizures, alters GABA(A) receptor subunit expression leading to changes in the receptor structure, function, and pharmacology. Whether this also occurs in the developing brain, in which GABA has a trophic, excitatory effect, is not known. We have now applied autoradiography to study properties of GABA(A)/benzodiazepine receptors in 9-day-old rats acutely (6 h) and sub-acutely (7 days) after kainic acid-induced status epilepticus by analyzing displacement of [(3)H]flunitrazepam binding by zolpidem, a ligand selective for the alpha1beta2gamma2 receptor subtype. Regional changes in the binding properties were further corroborated at the cellular level by immunocytochemistry. The results revealed that status epilepticus significantly decreased displacement of [(3)H]flunitrazepam binding by zolpidem 6 h after the kainic acid-treatment in the dentate gyrus of the hippocampus, parietal cortex, and thalamus, and in the hippocampal CA3 and CA1 cell layers 1 week after the treatment. Our results suggest that status epilepticus modifies region-specifically the pharmacological properties of GABA(A) receptors, and may thus disturb the normal, strictly developmentally-regulated maturation of zolpidem-sensitive GABA(A) receptors in the immature rat brain. A part of these changes could be due to alterations in the cell surface expression of receptor subtypes.

Kainic acid-induced status epilepticus alters GABA receptor subunit mRNA and protein expression in the developing rat hippocampus.

Kainic acid-induced status epilepticus leads to structural and functional changes in inhibitory GABAA receptors in the adult rat hippocampus, but whether similar changes occur in the developing rat is not known. We have used in situ hybridization to study status epilepticus-induced changes in the GABAAalpha1-alpha5, beta1-beta3, gamma1 and gamma2 subunit mRNA expression in the hippocampus of 9-day-old rats during 1 week after the treatment. Immunocytochemistry was applied to detect the alpha1, alpha2 and beta3 subunit proteins in the control and treated rats. In the saline-injected control rats, the alpha1 and alpha4 subunit mRNA expression significantly increased between the postnatal days 9-16, whereas those of alpha2, beta3 and gamma2 subunits decreased. The normal developmental changes in the expression of alpha1, alpha2, beta3 and gamma2 subunit mRNAs were altered after the treatment. The immunostainings with antibodies to alpha1, alpha2 and beta3 subunits confirmed the in situ hybridization findings. No neuronal death was detected in any hippocampal subregion in the treated rats. Our results show that status epilepticus disturbs the normal developmental expression pattern of GABAA receptor subunit in the rat hippocampus during the sensitive postnatal period of brain development. These perturbations could result in altered functional and pharmacological properties of GABAA receptors.

Mechanisms of kainate-induced region-specific neuronal death in immature organotypic hippocampal slice cultures.

Excessive activation of excitatory amino acid receptors has been implicated in neuronal death in a number of central nervous system insults. We have here investigated, the time course and mechanisms of kainate (KA)- induced neuronal death in immature organotypic hippocampal slice cultures (OHCs) using Fluoro-Jade B (FJB) staining as a marker of cell death, and immunoblotting, immunocytochemistry, and electron microscopy as methods to clarify the mechanisms. After 6 KA treatment (5 microM), no significant neuronal death was detected in any hippocampal subregion, whereas the treatment of 12, 24, and 48 h resulted in neuronal death in the CA3 regions, but not in CA1. The 48 h resting period in normal medium after KA-treatment did not rescue the cells but further increased the number of dead neurons in CA3 as compared to the corresponding acute phase. In Western blotting, the expression levels of the active, 17 kDa form of caspase-3, and the 84-85 kDa cleaved fragment of poly(ADP ribose)polymerase (PARP) were not altered from the control levels. Moreover, no active caspase-3 labelled cells were detected in immunocytochemical study 24 h after KA treatment either in the acute or resting groups. Electron microscopy showed non-apoptotic injury in the CA3a/b pyramidal neurons in KA-treated slices. Our results suggest that KA-induced neuronal death in immature OHCs is a strictly region-specific, irreversible, necrotic process.

Differential expression and localization of the phosphorylated and nonphosphorylated neurofilaments during the early postnatal development of rat hippocampus.

Neurofilament (NF) proteins are expressed in most mature neurons in the central nervous system. Although they play a crucial role in neuronal growth, organization, shape, and plasticity, their expression pattern and cellular distribution in the developing hippocampus remain unknown. In the present study, we have used Western blotting and immunocytochemistry to study the low- (NF-L), medium- (NF-M), and high- (NF-H) molecular-weight NF proteins; phosphorylated epitopes of NF-M and NF-H; and a nonphosphorylated epitope of NF-H in the early postnatal (through P1-P21) development of the rat hippocampus. During the first postnatal week, NF-M was the most abundantly expressed NF, followed by NF-L, whereas the expression of NF-H was very low. Through P7-P14, the expression of NF-H increased dramatically and later began to plateau, as also occurred in the expression of NF-M and NF-L. At P1, no NF-M immunopositive cell bodies were detected, but cell processes in the CA1-CA3 fields were faintly immunopositive for NF-M and for the phosphorylated epitopes of NF-M and NF-H. At P7, CA3 pyramidal neurons were strongly immunopositive for NF-L and NF-H, but not for NF-M. The axons of granule cells, the mossy fibers (MFs), were NF-L and NF-M positive through P7-P21 but were NF-H immunonegative at all ages. Although they stained strongly for the phosphorylated NF-M and NF-H at P7, the staining intensity sharply decreased at P14 and remained so at P21. The cell bodies of CA1 pyramidal neurons and granule cells remained immunonegative against all five antibodies in all age groups. Our results show a different time course in the expression and differential cell type and cellular localization of the NF proteins in the developing hippocampus. These developmental changes could be of importance in determining the reactivity of hippocampal neurons in pathological conditions in the immature hippocampus.

Neuronal activity regulates GABAA receptor subunit expression in organotypic hippocampal slice cultures.

The postnatal expression of GABA(A) receptor subunit mRNAs in the rat brain, including the hippocampus, exhibits a unique temporal and regional developmental profile in vivo, which may be altered by external stimuli. Using the in situ hybridization technique we have now studied the in vitro expression of alpha1,alpha2, alpha 4, alpha 5, beta 1, beta 3, gamma 2, and gamma 3 subunit mRNAs of GABA(A) receptors in organotypic hippocampal slices cultured for 7 days. To find out whether neuronal activity regulates the subunit expression, a subset of cultures was chronically treated either with a GABA(A) receptor antagonist picrotoxin, or by a non-N-methyl-D-aspartate (non-NMDA)-receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). In untreated control cultures, the expression pattern of the subunits varied regionally, the most abundantly expressed subunits being alpha 2 and alpha 5 in all subregions. All studied subunits were expressed in CA3a/b and CA1, whereas in CA3c and in granule cells of the dentate gyrus (DG) no signal of alpha 4 and gamma 3 was detected. The drug treatment differently affected the regional subunit expression. In picrotoxin-treated cultures, the expression of alpha1, alpha 5 and gamma 2 mRNAs was significantly increased in pyramidal cell layers, and in DNQX-treated cultures the expression of alpha2 mRNA in CA3c and DG, and that of beta1 in DG. Changes in the expression of GABA(A) receptor subunit mRNAs in treated cultures suggest that neuronal activity can regulate their regional expression in vitro. Since the expression profile in untreated control cultures closely resembled that observed earlier in vivo, organotypic hippocampal slice cultures could serve as a good model system to study the regulatory mechanisms of receptor expression under well-controlled experimental conditions in the developing hippocampus.

Event-related desynchronization and synchronization during a memory task in children.

OBJECTIVE: To examine the event-related desynchronization (ERD) and synchronization (ERS) responses of several narrow electroencephalographic (EEG) frequency bands in children during an auditory memory task. METHODS: ERD/ERS responses of the 4-6, 6-8, 8-10 and 10-12 Hz EEG frequency bands were studied in 12 children (mean age 12 years) while they performed an auditory memory task. Twelve adult subjects served as a control group. RESULTS: The children's ERD/ERS responses differed from those of the adult's in the 4-6, 6-8 and 8-10 Hz EEG frequency bands, especially during retrieval from memory. The children's 4-6 Hz initial ERS responses were of lesser amplitude and of delayed latency as compared to those of the adults. In the 6-8 and 8-10 Hz frequency bands, especially during retrieval from memory, the children's ERD responses were of lesser magnitude than those of the adults. In the 10-12 Hz frequency band, no differences were observed between the ERD/ERS responses between the children and adults. CONCLUSIONS: Our results suggest that theta and alpha response systems might participate in auditory information processing already at this age, although not being fully developed. Memory systems involving retrieval may be the last to mature.

Changes in neurofilament protein-immunoreactivity after kainic acid treatment of organotypic hippocampal slice cultures.

Neurofilament (NF) proteins are expressed in the majority of neurons in the central nervous system, and play a crucial role in the organization of neuronal shape and function. In the present study, we have used immunoblotting and immunocytochemical methods to study the light (NF-L), medium (NF-M ), and heavy (NF-H) molecular weight NF proteins in cultured organotypic hippocampal slices during the in vitro maturation and the changes after kainic acid (KA) treatment. In control cultures at 11 DIV throughout 25 DIV, CA3 pyramidal neurons and their proximal dendrites were heavily labeled with the antibodies against all three NF proteins. In CA1 pyramidal neurons, no staining was detected in any age group. A few weakly NF-L positive granule cells with fibers were detected in each age group, whereas NF-M and NF-H positive granule cells first appeared in the older cultures. The application of KA (5 microM) to the cultures for 48 hr, induced a pronounced cell death in the CA3 cell layers, and also moderately damaged granule cells. After the treatment, the immunoblot signal of NF-L and NF-M markedly decreased, whereas that of NF-H almost completely disappeared. The amount of NF-L positive fibers, however, dramatically increased in the molecular and hilar regions of the dentate gyrus in both age groups. Our results show the cellular heterogeneity in the distribution of NF protein triplet in cultured organotypic hippocampal slices. Kainic acid treatment induced changes, which mimicked those observed in the hippocampal region of epileptic animals.

Do antiepileptics phenytoin, carbamazepine, and loreclezole show GABA(A) receptor subtype selectivity in rat brain sections?

[35S]t-Butylbicyclophosphorothionate ([35S]TBPS), a convulsant site ligand of GABA(A) receptors, was used in autoradiography with rat brain sections to test suggested receptor subtype-selective actions of antiepileptics phenytoin, carbamazepine and loreclezole on native GABA(A) receptors. At maximal 100 microM concentration, both phenytoin and carbamazepine decreased [35S]TBPS binding only by 20%, indicating that their low potency and efficacy prevents their use as alpha1 subunit-identifying compounds. Ten microM loreclezole did not affect the binding, but a further increase in loreclezole concentration strongly decreased it. The action of loreclezole, assumed to reflect beta2/3 subunit-containing receptors, varied from brain region to region, but the effects were unrelated to the regional expression profiles of beta subunit variants. We conclude that in autoradiographic [35S]TBPS binding assay neither carbamazepine, phenytoin nor loreclezole are useful tools in characterizing brain regional heterogeneity of GABA(A) receptors in rats and that only loreclezole exhibits high, pharmacologically relevant efficacy.

Decreased binding of [11C]flumazenil in Angelman syndrome patients with GABA(A) receptor beta3 subunit deletions.

We used positron emission tomography (PET) to study brain [11C]flumazenil (FMZ) binding in four Angelman syndrome (AS) patients. Patients 1 to 3 had a maternal deletion of 15q11-q13 leading to the loss of beta3 subunit of gamma-aminobutyric acidA/benzodiazepine (GABA(A)/BZ) receptor, whereas Patient 4 had a mutation in the ubiquitin protein ligase (UBE3A) saving the beta3 subunit gene. [11C]FMZ binding potential in the frontal, parietal, hippocampal, and cerebellar regions was significantly lower in Patients 1 to 3 than in Patient 4. We propose that the 15q11-q13 deletion leads to a reduced number of GABA(A)/BZ receptors, which could partly explain the neurological deficits of the AS patients.

Proton spectroscopy in children with epilepsy and febrile convulsions.

An association between complex febrile convulsions and the development of hippocampal atrophy, which is characterized by neuron loss and gliosis, has been suggested but is still controversial. In proton magnetic resonance spectroscopy (1H-MRS) a reduction in N-acetylaspartate (NAA), a neuron marker, or in its ratio to other metabolites, that is, creatine and phospocreatine (Cr) and choline-containing compounds (Cho), is considered a sensitive method for detecting neuron loss. We performed 1H-MRS of mesial temporal regions, including hippocampi, in two different groups of children with epilepsy: in children with a history of complex febrile convulsions (CFCs) (n = 7; mean age 7.1 years) and in children without any history of CFCs, referred to herein as the non-CFC group (n = 6; mean age 7.6 years). Changes in the metabolite ratios were detected in 57% of children in the CFC group and in 67% of children in the non-CFC group. In both groups, NAA/(Cho + Cr), NAA/Cho, and NAA/Cr were significantly decreased ipsilaterally to the seizure focus when compared with the control group, but no significant differences were detected between the CFC and non-CFC groups. Also on the contralateral side, NAA/(Cho + Cr) and NAA/Cr were significantly decreased in both patient groups, but the differences were not significant between the CFC and non-CFC groups. Metabolite abnormalities in the mesial temporal region were detected in children with intractable epilepsy and in children whose epilepsy is well controlled by antiepileptic medication. The noninvasive 1H-MRS can be considered an additional diagnostic method to promote early detection of mesial temporal abnormalities that, in the light of this study, seem to be underdiagnosed in children with either temporal lobe epilepsy or other seizure types.

Abnormal frequency mismatch negativity in mentally retarded children and in children with developmental dysphasia.

The auditory event-related potential waveform termed "mismatch negativity" was examined in 12 mentally retarded children with delayed development of speech and language (aged 5-8 years) and in 13 children with developmental dysphasia (aged 5-9 years). The mismatch negativity waves were elicited with pure sine-wave tone stimuli using the oddball paradigm. We measured the peak latency and peak amplitude of mismatch negativity responses to frequency (500/553 Hz) difference. The mismatch negativity patterns were compared with those of 10 children with normal development of linguistic skills (aged 5-9 years). In both the mentally retarded and dysphasic groups, the peak amplitude of the frequency mismatch negativity was significantly attenuated when compared with the control group, but no significant difference was observed between the mentally retarded and dysphasic groups. Attenuated frequency mismatch negativity was related to impairment of linguistic skills irrespective of the child's cognitive skills. Because the mismatch negativity response reflects central auditory processing and is modal specific for auditory stimuli, this change-specific response can serve as an objective tool to elucidate central auditory deficits in children.

Attenuated auditory event-related potential (mismatch negativity) in children with developmental dysphasia.

An attention-independent negative wave-form termed 'mismatch negativity' (MMN) of the auditory event-related potentials (ERPs) was studied in ten children (3-6 years) with developmental dysphasia and in fourteen control children (3-7 years) with normal speech and language development. The MMNs were elicited with pure sine tone stimuli using the oddball paradigm. The peak latency and peak amplitude of MMN response to frequency (500/553 H2) difference was measured. The grand average amplitude of frequency MMN was significantly attenuated in dysphasic children as compared to controls, but no significant difference was observed in the latency of peak frequency MMN. The results indicate that dysphasic children have a defect in automatic auditory processing of frequency differences. Because the MMN response reflects central auditory processing and is modal specific for auditory stimuli we argue that the MMN method can serve as an objective tool to assess central auditory deficits in children.

Temporal lobe pathology in epilepsy: proton magnetic resonance spectroscopy and positron emission tomography study.

Hippocampal atrophy characterized by neuronal loss is a common feature in intractable temporal lobe epilepsy in adults. In proton magnetic resonance spectroscopy, a reduction in N-acetylaspartate or in its ratio to other metabolites is considered a sensitive method for detecting neuronal loss. This noninvasive approach was used to study the temporal lobes, especially the hippocampal regions of children with partial epilepsy. In all cases, 2-[18F]fluoro-2-deoxy-D-glucose scans were studied to verify the extension of the hypometabolic area. Five children manifested temporal lobe epilepsy; in 2 of them, epilepsy was intractable. Both manifested temporal hypometabolism on positron emission tomography, hippocampal atrophy on magnetic resonance imaging, and significant reductions in the metabolite ratios on spectroscopy ipsilateral to the seizure focus, as verified by EEG. Three children with temporal lobe epilepsy had infrequent seizures. One of them had a reduction in metabolite ratios in the hippocampus and hippocampal atrophy on magnetic resonance imaging ipsilateral to the seizure focus. The results show that the value of proton spectroscopy lies not only in lateralizing hippocampal atrophy but also in detecting bilaterality and the extent of neuronal loss outside hippocampi. This noninvasive preoperative diagnostic method can be considered an additional technique to be used in conjunction with other imaging techniques for clinical assessment of children with intractable temporal lobe epilepsy.