Linda K. Friedman

Suppression of hippocampal neurogenesis is associated with developmental stage, number of perinatal seizure episodes, and glucocorticosteroid level☆

Seizures increase dentate granule cell proliferation in adult rats but decrease proliferation in young pups. The particular period and number of perinatal seizures required to cause newborn granule cell suppression in development are unknown. Therefore, we examined cell proliferation with bromodeoxyuridine (BrdU) immunohistochemistry during the peak of neurogenesis (e.g., P6 and P9) and at later postnatal ages (e.g., P13, P20, or P30) following single and multiple episodes of perinatal status epilepticus induced by kainate (KA). Because an inverse relationship exists between glucocorticosteroids (CORT) levels and granule cell proliferation, plasma CORT levels and electroencephalographic (EEG) activity were simultaneously monitored to elucidate underlying mechanisms that inhibit cell proliferation. In control animals, the number of BrdU-labeled cells increased then declined with maturation. After 1x KA or 2x KA administered on P6 and P9, the numbers of BrdU-labeled cells were not different from age-matched controls. However, rat pups with 3x KA (on P6, P9, and P13) had marked suppression of BrdU-labeled cells 48-72 h after the last seizure (43 +/- 6.5% of control). Cell proliferation was also significantly inhibited on P20 after 2x KA (to 56 +/- 6.9%) or 3x KA (to 54 +/- 7.9%) and on P30 with 3x KA (to 74.5 +/- 8.2% of age-matched controls). Cell death was not apparent as chromatin stains showed increased basophilia of only inner cells lining the granule cell layers, in the absence of eosinophilia, argyrophilia, or terminal deoxynucleotidyl dUTP nick endlabeling (TUNEL) labeling at times examined. In P13 pups with 3x KA, electron microscopy revealed an increased number of immature granule cells and putative stem cells with irregular shape, condensed cytoplasm, and electron dense nuclei, and they were also BrdU positive. The EEG showed no relationship between neurogenesis and duration of high-synchronous ictal activity. However, endocrine studies showed a correlation with BrdU number and age, sustained increases in circulating CORT levels following 1x KA on P6 (0.7 +/- 0.1 to 2.40 +/- 0.86 microg/dl), and cumulative increases that exceeded 10 microg/dl at 4-8 h after 3x KA on P13 or P20. In conclusion, a history of only one or two perinatal seizure(s) can suppress neurogenesis if a second or third seizure recurs after a critical developmental period associated with a marked surge in CORT. During the first 2 weeks of postnatal life sustained increases in postictal circulating CORT levels but not duration or intensity of ictal activity has long-term consequences on neurogenesis. The occurrence of an increased proportion of immature granule cells and putative stem cells with irregular morphology in the absence of neurodegeneration suggests that progenitors may not differentiate properly and remain in an immature state.

Intraischemic but not postischemic hypothermia prevents non-selective hippocampal downregulation of AMPA and NMDA receptor gene expression after global ischemia

Hypothermia may afford histological neuroprotection induced by ischemia by preventing aberrant Ca2+ influx through NMDA (N-methyl-D-aspartic acid) or Ca2+-permeable AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors. Expression of hippocampal GluR1A, GluR2B, GluR3C and NMDAR1 (NR1) subunits was investigated by in situ hybridization at 1 and 7 days after 10-min transient global ischemia in the presence and absence of intraischemic or postischemic brain hypothermia (30 degrees C). At 1 day, normothermic ischemia markedly suppressed the expression of GluR1A, GluR2B, and GluR3C receptor mRNAs to a similar degree in the vulnerable CA1. Less vulnerable CA3a-c subregions were also acutely downregulated. NR1 mRNA expression was reduced in CA1 but to a lesser extent than AMPA mRNAs. At 7 days after normothermic ischemia, a time of marked CA1 cell loss, all three AMPA transcripts were nearly absent in CA1 while a percentage (33.9+/-7.2%) of NR1 mRNA remained. Intraischemic hypothermia fully blocked the damage and non-selective mRNA downregulations at 1 and 7 days. By contrast, postischemic hypothermia postponed neurodegeneration but only partially rescued the expression of AMPA and NR1 mRNAs at 7 days and not at 1 day after the insult. Therefore, hippocampal AMPA receptor mRNAs decline at a relatively similar rate after normothermic global ischemia and cellular neuroprotection by intraischemic hypothermia occurred independently of altered subunit composition of AMPA receptors. Since decreases persist within resistant neurons under the postischemic condition, AMPA receptor-mediated Ca2+ currents probably do not contribute to selective vulnerability.

Sex differences in GABAAergic system in rat substantia nigra pars reticulata

The substantia nigra pars reticulata (SNR) is involved in the control of movement disorders including seizures through its GABAergic neurons. Microinfusions of muscimol (a GABAA receptor agonist) produce specific effects on seizures depending on sex, infusion site (SNRanterior or SNRposterior) and age. To assess whether these effects are due to sex differences in GABAergic indices within the SNR we analyzed the expression of α1 subunit mRNA of the GABAA receptor and the levels of GABA immunoreactivity (IR) of male and female rats at postnatal day 15 (PN15) and PN30. In each age, within the same SNR region, expression of α1 subunit mRNA and intensity of GABA IR per neuron was higher in females compared to males. At PN15, in both sexes, there were no regional differences in expression of α1 subunit mRNA and intensity of GABA IR. However, at PN30 in both sexes, expression of α1 subunit mRNA and intensity of GABA IR per cell was higher in SNRanterior than in SNRposterior. These results demonstrate that expression of α1 subunit mRNA for GABAA receptor and levels of GABA IR in the SNR are sex‐ and site‐specific, which may contribute to sex‐, regional‐ and age‐related differences in the expression of movement disorders and seizures.

GluR2 hippocampal knockdown reveals developmental regulation of epileptogenicity and neurodegeneration

In adult rats, kainic acid-induced status epilepticus reduces GluR2 subunit expression prior to neurodegeneration of hippocampal CA3 neurons. Increased formation of Ca2+ permeable AMPA receptors may contribute to the delayed neurodegenerative process. In rat pups, highly prone to seizures but resistant to seizure-induced hippocampal damage, GluR2 mRNA and protein expression remain constant in CA3 neurons possibly contributing to their survival. To investigate whether reduced GluR2 expression in hippocampus may lead to enhanced hippocampal vulnerability in an age-dependent manner and whether changes correspond to altered electroencephalography (EEG) patterns, unilateral microinfusion of GluR2 antisense oligodeoxynucleotides (AS-ODNs) into hippocampus was performed at three ages (postnatal (P8), P13, and adult). At P13, GluR2 knockdown resulted in spontaneous seizure-like behavioral manifestations and neurodegeneration of CA3 neurons lateral and distal from the cannula infusion site. EEG recordings revealed high rhythmic activity associated with seizure-like behavior. In P8 pups and adult rats, there were no behavioral manifestations; distant hippocampal damage of the CA3 was not observed. Results indicate that unilateral knockdown of hippocampal GluR2 subunit expression induces age-dependent seizure-like behavioral manifestations, altered EEG recording patterns, and reduces the survival of CA3 neurons in the hippocampus of young rats during a specific postnatal period (3rd week), when GluR2 expression peaks in development and glutamatergic inputs are maturing.

GluR2 knockdown reveals a dissociation between [Ca2+]i surge and neurotoxicity

Reduction in GluR2 subunit expression and subsequent increases in AMPA receptor mediated Ca(2+) currents were postulated to exacerbate glutamate neurotoxicity following seizures or global ischemia. To directly test the effects of shifting the GluR1/GluR2 subunit ratio on excitotoxicity, GluR2 antisense deoxyoligonucleotides (AS-ODNs) were applied to dissociated hippocampal cultures for 1-8 days. The GluR1/GluR2 protein ratio was examined immunohistochemically and by Western blotting. [Ca(2+)](i) concentrations were determined by ratiometric imaging of Fura 2-loaded cells. The cultures were exposed to glutamate, AMPA, NMDA or kainic acid (KA) 3 days after GluR2 knockdown and cell viability was determined 1 day later by MTT reduction assay or Trypan blue exclusion. Although GluR2 AS-ODNs increased the GluR1/GluR2 protein ratio in a time dependent manner, neurons and glia appeared healthy and MTT reduction values were similar to untreated and sense controls. Basal [Ca(2+)](i) levels were unchanged but [Ca(2+)](i) was selectively increased by agonist stimulation of AMPA receptors. Unexpectedly, delayed neurotoxicity was attenuated at saturating doses of glutamate while little difference in cell viability was observed at lower doses or with the other excitotoxins at any concentration. Therefore, there was a dissociation between rises in AMPA receptor-mediated Ca(2+) influx and neurotoxicity despite marked decreases in GluR2 but not GluR1 immunoreactivity. It is proposed that a modification of AMPA receptor stochiometry that raises agonist-stimulated Ca(2+) influx during an excitotoxic insult may have eventual neuroprotective effects.

Distinct regulation of metabotropic glutamate receptor (mGluR1 alpha) in the developing limbic system following multiple early-life seizures

The effects of repeated neonatal seizures on metabotropic glutamate receptors (mGluRs) during critical periods of brain development are unknown. Therefore, we characterized the expression of Group I (mGluR1 and mGluR5) and Group II (mGluR2/3) metabotropic glutamate receptor proteins in the developing limbic system in response to a varied neonatal seizure history. Status epilepticus was induced with kainic acid (KA) either once (1x KA) on postnatal (P) day (P13), twice (2x KA) on P6 and P9 or P13, or three times (3x KA) on P6, P9, and P13. In control hippocampus, mGluR1alpha protein expression differed at all stages of development examined, whereas mGluR2/3 and mGluR5 protein expression patterns were mature by P15. After KA-induced status epilepticus, there was a significant elevation in mGluR1alpha protein expression within a select group of inhibitory interneurons of the CA1 stratum oriens-alveus that was enhanced with increasing number of neonatal seizures. mGluR2/3 and mGluR5 subtypes were unchanged. Increases were also observed within neurons of the amygdala and piriform cortex. Selective increases of mGluR1alpha subtypes within limbic structures may contribute to the resistance and tolerance of the immature hippocampus from damage. This may occur by excessive stimulation of excitatory synapses to collectively enhance the inhibitory drive of the immature brain by increasing GABA release. Data suggest that the mGluR1alpha subtype plays an important role in regulating hippocampal network activity after early-life seizures.

Maturational Effects of Single and Multiple Early-Life Seizures on AMPA Receptors in Prepubescent Hippocampus

The effects of single versus multiple episodes of status epilepticus on the expression of AMPA receptors during a critical growth spurt are unknown. To determine whether the pattern of hippocampal AMPA receptor subunit expression depends upon the age of the animal, timing and number of perinatal seizures, we characterized maturational changes in AMPA receptor protein levels of the hippocampus with immunohistochemistry and Western blotting in rats of juvenile ages with and without a history of neonatal seizures. Kainic acid (KA) was used to induce a single episode of status epilepticus (1 × KA) in rats on P20 or P30. Animals with a history of multiple seizures (3 × KA) were given KA on P6, P9, and then on P20 or P30. After 1 × KA, in P20 and P30 rats that are preferentially sensitive to CA1 damage, GluR1 immunoreactivity was depleted remarkably in CA1 stratum pyramidale and stratum lucidum and only morphologically healthy cells were faintly labeled. At P30, GluR2 subunit expression was nearly absent in the healthy cells and increased within the injured CA1 neuronal population. Western blot analysis confirmed that the GluR1/GluR2 ratio was decreased at P20 and further decreased at P30. A history of perinatal seizures (3 × KA) prevented the age-dependent alterations in the CA1. Except for areas of cell loss, NR1 and NR2A/B antibody labeling was relatively stable throughout the hippocampus at both ages and conditions examined. Data suggest that (i) Ca2+ permeable AMPA receptors may not be responsible for neuronal injury or irreversible cell loss and that (ii) the expression of AMPA receptors after status epilepticus depends upon the age of the animal, the timing of the first insult and subsequent formation of AMPA receptor subunit compositions within specific populations of hippocampal neurons.

Perinatal seizures preferentially protect CA1 neurons from seizure-induced damage in prepubescent rats

Neonatal seizures may increase neuronal vulnerability later in life. Therefore, status epilepticus was induced with kainate (KA) during the first and second postnatal (P) weeks to determine whether early seizures shift the window of neuronal vulnerability to a younger age. KA was injected (i.p.) once (1x KA) on P13, P20 or P30 or three times (3 x KA), once on P6 and P9, and then either on P13, P20 or P30. After 1x KA, onset to behavioral seizures increased with age. Electroencephalography (EEG) showed interictal events appeared with maturation. After 3 x KA, spike number, frequency, spike amplitude, and high-frequency synchronous events and duration were increased at P13 when compared to age-matched controls. In contrast, P20 and P30 rats had decreases in EEG parameters relative to P20 and P30 rats with 1x KA despite that these animals had the same history of perinatal seizures on P6 and P9. In P13 rats with 1x KA, silver impregnation, hematoxylin/eosin and TUNEL methods showed no significant hippocampal injury and damage was minimal with 3 x KA. In contrast, P20 and P30 rats with 1x KA had robust eosinophilic or TUNEL positive labeling and preferential accumulation of silver ions within inner layer CA1 neurons. After 3 x KA, the CA1 but not CA3 of P20 and P30 rats was preferentially protected following 3 or 6 days. Although paradoxical changes occur in the EEG with maturation, the results indicate that early perinatal seizures do not significantly shift the window of hippocampal vulnerability to an earlier age but induce a tolerance that leads to long-term neuroprotection that differentially affects endogenous properties of CA1 versus CA3 neurons.

Age- and region-dependent patterns of Ca2+ accumulations following status epilepticus.

Elevated Ca2+ concentrations have been implicated in cell death mechanisms following seizures, however, the age and brain region of intracellular Ca2+ accumulations [Ca2+]i, may influence whether or not they are toxic. Therefore, we examined regional accumulations of 45Ca2+ by autoradiography from rats of several developmental stages (P14, P21, P30 and P60) at 5, 14, and 24 h after status epilepticus. To determine whether the uptake was intracellular, Ca2+ was also assessed in hippocampal slices with the dye indicator, Fura 2AM at P14. Control animals accumulated low homogeneous levels of 45Ca2+; however, highly specific and age‐dependent patterns of 45Ca2+ uptake were observed at 5 h. 45Ca2+ accumulations were predominant in dorsal hippocampal regions, CA1/CA2/CA3a, in P14 and P21 rats and in CA3a and CA3c neurons of P30 and P60 rats. Selective midline and amygdala nuclei were marked at P14 but not at P21 and limbic accumulations recurred with maturation that were extensive at P30 and even more so at P60. At 14 h, P14 and P21 rats had no persistent accumulations whereas P30 and P60 rats showed persistent uptake patterns within selective amygdala, thalamic and hypothalamic nuclei, and other limbic cortical regions that continued to differ at these ages. For example, piriform cortex accumulation was highest at P60. Fura 2AM imaging at P14 confirmed that Ca2+ rises were intracellular and occurred in both vulnerable and invulnerable regions of the hippocampus, such as CA2 pyramidal and dentate granule cells. Silver impregnation showed predominant CA1 injury at P20 and P30 but CA3 injury at P60 whereas little or no injury was found in extrahippocampal structures at P14 and P20 but was modest at P30 and maximal at P60. Thus, at young ages there was an apparent dissociation between high 45Ca2+ accumulations and neurotoxicity whereas in adults a closer relationship was observed, particularly in the extrahippocampal structures.

Certain secondary antiepileptic drugs can rescue hippocampal injury following a critical growth period despite poor anticonvulsant activity and cognitive deficits

Clinical and experimental studies have shown that many common secondary antiepileptic drugs (AEDs) are ineffective at blocking seizures in adulthood; however, some afford neuroprotection. In early development, certain AEDs cause apoptosis; however, it is unknown whether these drugs are neurotoxic to the juvenile brain following a developmentally regulated proapoptotic period and whether they alter the seizure threshold, seizure-induced neuronal vulnerability, and/or cognitive function. Lamotrigine (LTG), carbamazepine (CBZ), phenytoin (PHT), valproate (VPA), and topiramate (TPM) were systemically administered to rat pups for 7days beginning on postnatal (P) day 14 (P14), then half the animals were injected with kainate (KA) to trigger seizures, an age when the CA1 subregion becomes preferentially sensitive to status epilepticus. Histological outcome, seizure severity, and learning and memory were determined with an electroencephalograph (EEG), silver impregnation, and a water-maze swim task. None of the AEDs tested significantly attenuated behavioral or electrographic seizures. Phenytoin increased mortality, identifying a detrimental side effect of this drug. The other drugs (LTG, VPA, TPM, and CBZ) afforded different amounts of protection to the CA1 subregion but not to the CA3 subregion or extrahippocampal structures. With the exception of VPA, AED-treated animals lagged behind during swim task acquisition. All groups improved in the water-maze swim task over time, particularly on the last trials; however, the average escape latency was still impaired for TPM-treated animals and all AED+KA-treated groups. Thus, while certain AEDs demonstrated some neuroprotective effects, poor antiepileptic activity, memory impairment, and other deleterious side effects were observed with these drugs suggesting that the search for potentially more effective and tolerated agents is essential for improving clinical outcome in children and adolescents with epilepsy.