Immature Rat Hippocampus Research Articles

Silencing miR-155–5p alleviates hippocampal damage in kainic acid-induced epileptic rats via the Dusp14/MAPK pathway

Epilepsy with recurrent seizures is characterized by neuronal damage and glial proliferation induced by brain inflammation. Recurrent seizures can lead to changes in the microRNA (miRNA) spectrum, significantly influencing the inflammatory response of microglia. MiR-155–5p, as a pro-inflammatory miRNA, is increased in the epileptic brain. However, its specific role in acute seizures remains unknown. The study aimed to develop a new strategy for treating epilepsy by investigating how silencing of miR-155–5p initiated its anticonvulsive mechanism. The level of miR-155–5p was up-regulated in the hippocampus of epileptic immature rats induced by kainic acid (KA). The use of antago-miR-155–5p exerted significant beneficial effects on the seizure scores, brain discharges and cognition in immature rats following KA-induced epilepsy. Antago-miR-155–5p also inhibited neuron damage and microglial activation. Moreover, the silencing of miR-155–5p significantly inhibited the Dual-specificity phosphatase 14 (Dusp14)/ mitogen-activated protein kinase (MAPK) axis in vivo. MiR-155–5p interacted with dusp14 to regulate MAPK signaling way expression, verified by a dual-luciferase reporter assay. The results suggested that the silencing of miR-155–5p might reduce hippocampal damage in epileptic immature rats induced by KA via Dusp14/MAPK signaling way. This implied that miR-155–5p could serve as a therapeutic tool to prevent the development of epilepsy.

Adenosine Kinase Isoforms in the Developing Rat Hippocampus after LiCl/Pilocarpine Status Epilepticus.

LiCl/pilocarpine status epilepticus (SE) induced in immature rats leads, after a latent period, to hippocampal hyperexcitability. The excitability may be influenced by adenosine, which exhibits anticonvulsant activity. The concentration of adenosine is regulated by adenosine kinase (ADK) present in two isoforms—ADK-L and ADK-S. The main goal of the study is to elucidate the changes in ADK isoform expression after LiCl/pilocarpine SE and whether potential changes, as well as inhibition of ADK by 5-iodotubercidin (5-ITU), may contribute to changes in hippocampal excitability during brain development. LiCl/pilocarpine SE was elicited in 12-day-old rats. Hippocampal excitability in immature rats was studied by the model of hippocampal afterdischarges (ADs), in which we demonstrated the potential inhibitory effect of 5-ITU. ADs demonstrated significantly decreased hippocampal excitability 3 days after SE induction, whereas significant hyperexcitability after 20 days compared to controls was shown. 5-ITU administration showed its inhibitory effect on the ADs in 32-day-old SE rats compared to SE rats without 5-ITU. Moreover, both ADK isoforms were examined in the immature rat hippocampus. The ADK-L isoform demonstrated significantly decreased expression in 12-day-old SE rats compared to the appropriate naïve rats, whereas increased ADK-S isoform expression was revealed. A decreasing ADK-L/-S ratio showed the declining dominance of ADK-L isoform during early brain development. LiCl/pilocarpine SE increased the excitability of the hippocampus 20 days after SE induction. The ADK inhibitor 5-ITU exhibited anticonvulsant activity at the same age. Age-related differences in hippocampal excitability after SE might correspond to the development of ADK isoform levels in the hippocampus.

Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus

Long non‐coding RNAs (lncRNAs) have been implicated in the regulation of gene expression at various levels. However, to date, the expression profile of lncRNAs in status epilepticus (SE) was unclear. In our study, the expression profile of lncRNAs was investigated by high‐throughput sequencing based on a lithium/pilocarpine‐induced SE model in immature rats. Furthermore, weighted correlation network analysis (WGCNA), gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to construct co‐expression networks and establish functions of the identified hub lncRNAs in SE. The functional role of a hub lncRNA (NONRATT010788.2) in SE was investigated in an in vitro model. Our results indicated that 7082 lncRNAs (3522 up‐regulated and 3560 down‐regulated), which are involved in cell proliferation, inflammatory responses, angiogenesis and autophagy, were dysregulated in the hippocampus of immature rats with SE. Additionally, WGCNA identified 667 up‐regulated hub lncRNAs in turquoise module that were involved in apoptosis, inflammatory responses and angiogenesis via regulation of HIF‐1, p53 and chemokine signalling pathways and via inflammatory mediator regulation of TRP channels. Knockdown of an identified hub lncRNA (NONRATT010788.2) inhibited neuronal apoptosis in vitro. Taken together, our study is the first to demonstrate the expression profile and potential function of lncRNAs in the hippocampus of immature rats with SE. The defined hub lncRNAs may participate in the pathogenesis of SE via lncRNA‐miRNA‐mRNA network.

MiR-96 attenuates status epilepticus-induced brain injury by directly targeting Atg7 and Atg16L1

Status epilepticus (SE) can cause brain damage and lead to neural dysfunction. Developing novel targets for SE therapy and diagnosis is important and necessary. Previously, we found several differentially expressed microRNAs (miRNAs) in the developing hippocampus following SE, including the autophagy-related miR-96. In the present study, we employed immunofluorescence staining and Western blot analysis to assess the expression of autophagy-related 7 (Atg7) and Atg16L1 and the status of autophagosome formation in the hippocampus of immature rats with SE. Additional in vivo intervention was also performed to investigate the potential therapeutic function of miR-96 in developing rats with SE. We found that Atg7 and Atg16L1 were up-regulated in the neurons after SE, together with an increase in autophagosome formation. Meanwhile, overexpression of miR-96 significantly prevented brain damage in SE rats by inhibiting Atg7 and Atg16L1 expression and autophagosome formation in the hippocampus. Furthermore, Rapamycin negated miR-96 mediated brain injury attenuation through inducing autophagosome formation. Our study indicates that miR-96 might be a potential target for therapy of pediatric SE.

General Anesthesia Causes Epigenetic Histone Modulation of c-Fos and Brain-derived Neurotrophic: Factor, Target Genes Important for Neuronal Development in the Immature Rat Hippocampus

(Anesthesiology. 2016;124(6):1311–1327) Research studies have shown that general anesthesia (GA) exposure among young animals causes cognitive impairments in not only first generation offspring but also nonexposed second generation offspring born to dams. Studies on rodents have revealed that GA exposure in utero has proven this to be true, thus confirming that transient GA exposure during a critical period of neuronal remodeling does result in changes that become embedded in the genetic information thus causing cognitive impairments. This study hypothesized that during critical stages of synaptogenesis, transient exposure to GA results in epigenetic changes in chromatin with adverse effects on transcription of target genes which are important for proper synapse formation and cognitive development.

General Anesthesia Causes Epigenetic Histone Modulation of c-Fos and Brain-derived Neurotrophic Factor, Target Genes Important for Neuronal Development in the Immature Rat Hippocampus.

Early postnatal exposure to general anesthesia (GA) may be detrimental to brain development, resulting in long-term cognitive impairments. Older literature suggests that in utero exposure of rodents to GA causes cognitive impairments in the first-generation as well as in the second-generation offspring never exposed to GA. Thus, the authors hypothesize that transient exposure to GA during critical stages of synaptogenesis causes epigenetic changes in chromatin with deleterious effects on transcription of target genes crucial for proper synapse formation and cognitive development. They focus on the effects of GA on histone acetyltransferase activity of cAMP-responsive element-binding protein and the histone-3 acetylation status in the promoters of the target genes brain-derived neurotrophic factor and cellular Finkel-Biskis-Jinkins murine sarcoma virus osteosarcoma oncogene (c-Fos) known to regulate the development of neuronal morphology and function. Seven-day-old rat pups were exposed to a sedative dose of midazolam followed by combined nitrous oxide and isoflurane anesthesia for 6 h. Hippocampal neurons and organotypic hippocampal slices were cultured in vitro and exposed to GA for 24 h. GA caused epigenetic modulations manifested as histone-3 hypoacetylation (decrease of 25 to 30%, n = 7 to 9) and fragmentation of cAMP-responsive element-binding protein (two-fold increase, n = 6) with 25% decrease in its histone acetyltransferase activity, which resulted in down-regulated transcription of brain-derived neurotrophic factor (0.2- to 0.4-fold, n = 7 to 8) and cellular Finkel-Biskis-Jinkins murine sarcoma virus osteosarcoma oncogene (about 0.2-fold, n = 10 to 12). Reversal of histone hypoacetylation with sodium butyrate blocked GA-induced morphological and functional impairments of neuronal development and synaptic communication. Long-term impairments of neuronal development and synaptic communication could be caused by GA-induced epigenetic phenomena.

Status Epilepticus in Immature Rats Is Associated with Oxidative Stress and Mitochondrial Dysfunction.

Epilepsy is a neurologic disorder, particularly frequent in infants and children where it can lead to serious consequences later in life. Oxidative stress and mitochondrial dysfunction are implicated in the pathogenesis of many neurological disorders including epilepsy in adults. However, their role in immature epileptic brain is unclear since there have been two contrary opinions: oxidative stress is age-dependent and does not occur in immature brain during status epilepticus (SE) and, on the other hand, evidence of oxidative stress in immature brain during a specific model of SE. To solve this dilemma, we have decided to investigate oxidative stress following SE induced in immature 12-day-old rats by three substances with a different mechanism of action, namely 4-aminopyridine, LiCl-pilocarpine or kainic acid. Fluoro-Jade-B staining revealed mild brain damage especially in hippocampus and thalamus in each of the tested models. Decrease of glucose and glycogen with parallel rises of lactate clearly indicate high rate of glycolysis, which was apparently not sufficient in 4-AP and Li-Pilo status, as evident from the decreases of PCr levels. Hydroethidium method revealed significantly higher levels of superoxide anion (by ∼60%) in the hippocampus, cerebral cortex and thalamus of immature rats during status. SE lead to mitochondrial dysfunction with a specific pronounced decrease of complex I activity that persisted for a long period of survival. Complexes II and IV activities remained in the control range. Antioxidant treatment with SOD mimetic MnTMPYP or peroxynitrite scavenger FeTPPS significantly attenuated oxidative stress and inhibition of complex I activity. These findings bring evidence that oxidative stress and mitochondrial dysfunction are age and model independent, and may thus be considered a general phenomenon. They can have a clinical relevance for a novel approach to the treatment of epilepsy, allowing to target the mechanisms which play a crucial or additive role in the pathogenesis of epilepsies in infants and children.

Maternal Exposure to Ethanol During Pregnancy and Lactation Affects Glutamatergic System and Induces Oxidative Stress in Offspring Hippocampus.

Alcohol abuse during pregnancy leads to intellectual disability and morphological defects in the offspring. The aim of this study was to determine the effect of chronic maternal ethanol (EtOH) consumption during pregnancy and lactation on glutamatergic transmission regulation, energy deficit, and oxidative stress in the hippocampus of the offspring. EtOH was administered to dams in drinking water at increasing doses (2 to 20%) from the gestation day 5 to lactation day 21. EtOH and tap water intake by treated and control groups, respectively, were measured daily. Results showed that EtOH exposure does not affect fluid intake over the course of pregnancy and lactation. The toxicity of maternal exposure to EtOH was demonstrated by decreased offspring body weight at experimental age, on postnatal day 21. Moreover, maternal EtOH exposure decreased (45) Ca(2+) influx in the offspring's hippocampus. Corroborating this finding, EtOH increased both Na(+) -dependent and Na(+) -independent glial [(14) C]-glutamate uptakein hippocampus of immature rats. Also, maternal EtOH exposure decreased glutamine synthetase activity and induced aspartate aminotransferase enzymatic activity, suggesting that in EtOH-exposed offspring hippocampus, glutamate is preferentially used as a fuel in tricarboxylic acid cycle instead of being converted into glutamine. In addition, EtOH exposure decreased [U-14C]-2-deoxy-D-glucose uptake in offspring hippocampus. The decline in glucose transport coincided with increased lactate dehydrogenase activity, suggesting an adaptative response in EtOH-exposed offspring hippocampus, using lactate as an alternative fuel. These events were associated with oxidative damage, as demonstrated by changes in the enzymatic antioxidant defense system and lipid peroxidation. Taken together, the results demonstrate that maternal exposure to EtOH during pregnancy and lactation impairs glutamatergic transmission, as well as inducing oxidative stress and energy deficit in immature rat hippocampus.

PO-0849 Expression Pattern Of Brain Specific Mir-124, Mir-134, And Mir-9 In An Immature Rat Model And Children With Mesial Temporal Lobe Epilepsy

Background and aims Mesial temporal lobe epilepsy (MTLE) is a particularly devastating form of human epilepsy with significant incidence of medical intractability. MicroRNAs (miRs) are small, noncoding RNAs that regulate post-transcriptional expression of protein-coding mRNAs, which may have key roles in the pathogenesis of MTLE development. We aimed to detect the dynamic expression pattern of brain specific miR-124, miR-134 and miR-9 in the hippocampi of immature rats and children with MTLE. Methods To study the dynamic expression pattern of brain specific miR-124, miR-134 and miR-9, we performed real-time quantitative PCR on the hippocampi of immature rats at 25 days of age. Expression was monitored in the acute, latent, and chronic stages of disease (2 h and 3 and 8 weeks after induction of lithium-pilocarpine status epilepticus, respectively), and in control hippocampal tissues corresponding to the same timeframes. Similar expression method was applied to hippocampi obtained from children with MTLE and normal controls. Results The expression pattern for the miR-124, miR-134 and miR-9 nearly showed the same dynamics in the three stages of MTLE development to be upregulated in the acute and chronic stages and nearly equal to the control in the latent stage, they upregulated also in the children with MTLE. Conclusions The significant upregualtion for the brain specific miR-124, miR-134 and miR-9 in the seizures related stages and children suggested that both can be a potential targets for anticonvulsant drugs in the epileptic developing brains.

O-177 Dynamic Expression Pattern Of Two Pro-inflammatory Cytokines And Two Inflammation Related Mocrornas In Mesial Temporal Lobe Epilepsy Devlopment In The Devloping Brains

Background and aims This study investigated the dynamic expression pattern of interleukin-1β and tumour necrosis factor alpha (TNF-α) as a proinflammatory cytokines and microRNA (miR)-146a and miR-155 as a posttranscriptional inflammation- related miRs in the hippocampi of an immature rat model andchildren with MTLE. Methods To study the expression of IL-1β, TNF-α, miR-146a and miR-155, we performed a reverse transcription polymerase chain reaction, Western blot, andreal-time quantitative PCR on the hippocampi of immature rats. Expressions were monitored in the acute, latent, and chronic stages of disease (2 h and 3 and 8 weeks after induction of lithium-pilocarpine status epilepticus, respectively), and in control hippocampal tissues corresponding to the same timeframes. Similar expression methods were applied to hippocampi obtained from children with MTLE and normal controls. Results The expression of IL-1β, TNF-α, miR-146a and miR-155 were up-regulated in children with MTLE. In the immature rat model, IL-1β and miR-146a are significantly up-regulated, but in opposite ways: in the acute stage IL-1β expression is highest, when miR-146a expression is at its lowest level; thereverse of this expression occurs in the latent stage, while both are up-regulated in the chronic stage. TNF-α and miR-155 dynamic expressions showed the same fluctuating upregulation in the three stages of the MTLE development. Conclusion The dynamic expressions of (IL-1β and miR-146a) and (TNF-α and miR-155) in the different stages of MTLE suggesting an interactive relationship. Therefore, modulation of IL-1β–miR-146a and TNF-α–miR-155 axesmay be novel therapeutic targets in the treatment of MTLE.

Activation of glycine receptors modulates spontaneous epileptiform activity in the immature rat hippocampus

While the expression of glycine receptors in the immature hippocampus has been shown, no information about the role of glycine receptors in controlling the excitability in the immature CNS is available. Therefore, we examined the effect of glycinergic agonists and antagonists in the CA3 region of an intact corticohippocampal preparation of the immature (postnatal days 4-7) rat using field potential recordings. Bath application of 100 μM taurine or 10 μM glycine enhanced the occurrence of recurrent epileptiform activity induced by 20 μM 4-aminopyridine in low Mg(2+) solution. This proconvulsive effect was prevented by 3 μM strychnine or after incubation with the loop diuretic bumetanide (10 μM), suggesting that it required glycine receptors and an active NKCC1-dependent Cl(-) accumulation. Application of higher doses of taurine (≥ 1 mM) or glycine (100 μM) attenuated recurrent epileptiform discharges. The anticonvulsive effect of taurine was also observed in the presence of the GABAA receptor antagonist gabazine and was attenuated by strychnine, suggesting that it was partially mediated by glycine receptors. Bath application of the glycinergic antagonist strychnine (0.3 μM) induced epileptiform discharges. We conclude from these results that in the immature hippocampus, activation of glycine receptors can mediate both pro- and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress epileptiform activity. In summary, our study elucidated the important role of glycine receptors in the control of neuronal excitability in the immature hippocampus.

Mechanisms underlying the neurotoxicity induced by glyphosate-based herbicide in immature rat hippocampus: Involvement of glutamate excitotoxicity

Previous studies demonstrate that glyphosate exposure is associated with oxidative damage and neurotoxicity. Therefore, the mechanism of glyphosate-induced neurotoxic effects needs to be determined. The aim of this study was to investigate whether Roundup® (a glyphosate-based herbicide) leads to neurotoxicity in hippocampus of immature rats following acute (30min) and chronic (pregnancy and lactation) pesticide exposure. Maternal exposure to pesticide was undertaken by treating dams orally with 1% Roundup® (0.38% glyphosate) during pregnancy and lactation (till 15-day-old). Hippocampal slices from 15 day old rats were acutely exposed to Roundup® (0.00005–0.1%) during 30min and experiments were carried out to determine whether glyphosate affects 45Ca2+ influx and cell viability. Moreover, we investigated the pesticide effects on oxidative stress parameters, 14C-α-methyl-amino-isobutyric acid (14C-MeAIB) accumulation, as well as glutamate uptake, release and metabolism. Results showed that acute exposure to Roundup® (30min) increases 45Ca2+ influx by activating NMDA receptors and voltage-dependent Ca2+ channels, leading to oxidative stress and neural cell death. The mechanisms underlying Roundup®-induced neurotoxicity also involve the activation of CaMKII and ERK. Moreover, acute exposure to Roundup® increased 3H-glutamate released into the synaptic cleft, decreased GSH content and increased the lipoperoxidation, characterizing excitotoxicity and oxidative damage. We also observed that both acute and chronic exposure to Roundup® decreased 3H-glutamate uptake and metabolism, while induced 45Ca2+ uptake and 14C-MeAIB accumulation in immature rat hippocampus. Taken together, these results demonstrated that Roundup® might lead to excessive extracellular glutamate levels and consequently to glutamate excitotoxicity and oxidative stress in rat hippocampus.

Inhibition of different GABA transporter systems is required to attenuate epileptiform activity in the CA3 region of the immature rat hippocampus

GABA transporters (GATs) are an essential element of the GABAergic system, which regulate excitability in the central nervous system and are thus used as targets for anticonvulsive therapy. However, in the immature nervous system the functions of the GABAergic system and the expression profile of GATs are distinct from the adult situation, obscuring to predict how different GAT isoforms influence epileptiform activity. Therefore we analyzed the effects of subtype specific GAT inhibitors on repetitive epileptiform discharges using field potential and whole-cell patch-clamp recordings in the CA3 region of hippocampal slices of immature (postnatal days 4-7) rats. These experiments revealed that inhibition of GAT-1 with either tiagabine (30 μM) or NO-711 (10 μM) exhibited only a minor anticonvulsive effect on repetitive epileptiform discharges. Blockade of GAT-2/3 with SNAP-5114 (40 μM) had no anticonvulsive effect, but significantly prolonged the decay of spontaneous GABAergic postsynaptic currents. In contrast, the combined application of 10 μM NO-711 and 40 μM SNAP-5114 blocked epileptiform activity in 33% of all slices and reduced the occurrence of epileptiform discharges by 54% in the remaining slices. In addition, the input resistance decreased by 10.5 ± 1.0% under this condition. These results indicate that both GAT-1 and GAT-2/3 are functional in the immature hippocampus and that only the combined inhibition of GAT 1-3 is sufficient to promote a considerable anticonvulsive effect. We conclude from these results that both GAT-1 and GAT-2/3 act synergistically to regulate the excitability in the immature hippocampus.

Elevated expression of pleiotrophin in pilocarpine-induced seizures of immature rats and in pentylenetetrazole-induced hippocampal astrocytes in vitro

Pleiotrophin (PTN) is a secreted extracellular matrix (ECM)-associated cytokine that has emerged as an important neuromodulator with multiple neuronal functions. In the present study, we detected and compared the dynamic expression of PTN in the hippocampus and adjacent cortex of immature rats with pilocarpine-induced epilepsy. Moreover, we also confirmed the results by examining PTN expression in hippocampal astrocytes cultured in the presence of pentylenetetrazole (PTZ). Immunohistochemistry showed faint immunostaining of PTN in the control hippocampus and adjacent cortex. Notably, PTN immunoreactivity began to increase in relatively small cells in the hippocampus and adjacent cortex at 2h and 3 weeks after seizures, and the labeling intensity reached the maximum level in the hippocampus and adjacent cortex at 8 weeks after seizures. Furthermore, we also found that PTZ treatment significantly reduced astrocytic viability in a dose-dependent manner and time-dependently increased expression levels of PTN in hippocampal astrocytes. In conclusion, our data suggest that increased expression of PTN in the brain tissues may be involved in epileptogenesis.

Expression Patterns of miR-124, miR-134, miR-132, and miR-21 in an Immature Rat Model and Children with Mesial Temporal Lobe Epilepsy

Mesial temporal lobe epilepsy (MTLE) is a particularly devastating form of human epilepsy with significant incidence of medical intractability. MicroRNAs (miRs) are small, noncoding RNAs that regulate the posttranscriptional expression of protein-coding mRNAs, which may have key roles in the pathogenesis of MTLE development. To study the dynamic expression patterns of brain-specific miR-124 and miR-134 and inflammation-related miR-132 and miR-21, we performed qPCR on the hippocampi of immature rats at 25days of age. Expressions were monitored in the three stages of MTEL and in the control hippocampal tissues corresponding to the same timeframes. A similar expression method was applied to hippocampi obtained from children with MTLE and normal controls. The expression patterns of miR-124 and miR-134 nearly showed the same dynamics in the three stages of MTLE development. On the other hand, miR-132 and miR-21 showed significant upregulation in acute and chronic stages, while in the latent stage, miR-132 was upregulated and miR-21 was downregulated. The four miRs were upregulated in hippocampal tissues obtained from children with MTLE. The significant upregulation of miR-124 and miR-134 in the seizure-related stages and children suggested that both can be potential targets for anticonvulsant drugs in the epileptic developing brains, while the different expression patterns of miR-132 and miR-21 may suggest different functions in MTLE pathogenesis.

Influence of propofol on hippocampus in developing brain: an experimental study

Anesthetic neurotoxicity and intraoperative cerebral neuroprotectioan is one of the important issues in modern anesthesiology. General anesthesia, in addition to its hypnotic effect, is considered to cause postoperative cognitive dysfunction as a manifestation of encephalopathy. Narcosis based on propofol is a “gold standard” of total intravenous anesthesia and is frequently used for surgery in patients of various ages. This experimental study investigates the effects of propofol on neuronal population in hippocampus of immature rats. In propofol-anesthezied rats within 30 min of exposition, a two-fold increase of altered hippocampal neurons was detected compared to control animals, however no neuronal cell death was observed. Intraoperative use of ethylmethylhydroxypyridine succinate ameliorates propofol-induced neuronal damagethat proves a neuroprotective effect of the drug tested.

Interleukin‐1β and microRNA‐146a in an immature rat model and children with mesial temporal lobe epilepsy

Increasing evidence indicates that neuroinflammation plays a critical role in the pathogenesis of mesial temporal lobe epilepsy (MTLE). The aim of this study was to investigate the dynamic expression of interleukin (IL)-1β as a proinflammatory cytokine and microRNA (miR)-146a as a posttranscriptional inflammation-associated microRNA (miRNA) in the hippocampi of an immature rat model and children with MTLE. To study the expression of IL-1β and miR-146a, we performed a reverse transcription polymerase chain reaction, Western blot, and real-time quantitative PCR on the hippocampi of immature rats at 11 days of age. Expression was monitored in the acute, latent, and chronic stages of disease (2 h and 3 and 8 weeks after induction of lithium-pilocarpine status epilepticus, respectively), and in control hippocampal tissues corresponding to the same timeframes. Similar expression methods were applied to hippocampi obtained from children with MTLE and normal controls. The expression of IL-1β and miR-146a in both children and immature rats with MTLE differs according to the stage of MTLE development. Both IL-1β and miR-146a are significantly up-regulated, but in opposite ways: IL-1β expression is highest in the acute stage, when expression of miR-146a is at its lowest level; miR-146a expression is highest in the latent stage, when IL-1β expression is at its lowest level. Both IL-1β and miR-146a are up-regulated in the chronic stage, but not as much as in the other stages. Our study is the first to focus on the expression of miR-146a in the immature rat model of lithium-pilocarpine MTLE and in children with MTLE. We have detected that the expression of proinflammatory cytokine IL-1β and posttranscriptional inflammation-associated miR-146a is variable depending on the disease stage. Furthermore, both IL-1β and miR-146a are up-regulated in immature rats and children with MTLE. Our findings elucidate the role of inflammation in the pathogenesis of MTLE in the immature rat model and children. Therefore, modulation of the IL-1β-miR-146a axis may be a novel therapeutic target in the treatment of MTLE.

Effect of Endothelin-1 on the Excitability of Rat Cortical and Hippocampal Slices In Vitro

Endothelin-1 (ET-1) is a neuroactive protein produced in most brain cell types and participates in regulation of cerebral blood flow and blood pressure. In addition to its vascular effects, ET-1 affects synaptic and nonsynaptic neuronal and glial functions. Direct application of ET-1 to the hippocampus of immature rats results in cerebral ischemia, acute seizures, and epileptogenesis. Here, we investigated whether ET-1 itself modifies the excitability of hippocampal and cortical circuitry and whether acute seizures observed in vivo are due to nonvascular actions of ET-1. We used acute hippocampal and cortical slices that were preincubated with ET-1 (20 μM) for electrophysiological recordings. None of the slices preincubated with ET-1 exhibited spontaneous epileptic activity. The slope of the stimulus intensity-evoked response (input-output) curve and shape of the evoked response did not differ between ET-1-pretreated and control groups, suggesting no changes in excitability after ET-1 treatment. The threshold for eliciting an evoked response was not significantly increased in either hippocampal or cortical regions when pretreated with ET-1. Our data suggest that acute seizures after intrahippocampal application of ET-1 in rats are likely caused by ischemia rather than by a direct action of ET-1 on brain tissue.

Expression of AMPA receptor subunits in hippocampus after status convulsion

The expression of 2-amino-3-(5-methyl-3-oxo-1, 2-oxazol-4-yl) propanoic acid receptor (AMPAR) subunits in the hippocampus of naive immature and adult rats (IRs, ARs) was investigated after status convulsion (SC). Seizures were induced in IRs and ARs with intraperitoneal injections of lithium and pilocarpine. Rats were killed at four time points (3h, 1day, 3days, and 7days) after SC. The proportion of apoptotic cells was quantified by Annexin V-FITC apoptosis detection. The location and type of apoptotic cells were assessed by using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. Immunoblotting techniques were used to demonstrate changes in AMPAR subunit expression. Severe seizures induced neuronal apoptosis in the hippocampus. The proportion of apoptotic cells in IRs was consistently lower than that in ARs after SC. The expressions of four AMPAR subunits in IRs were consistently lower than those in ARs before and after SC. SC for 1h inhibited the expression of glutamate receptors (GluR1-4) in the hippocampus of IRs and ARs and altered the subunit composition of AMPARs. GluR2 was the predominant AMPAR subunit in the hippocampus of normal ARs, while the GluR2/3 subunits were predominantly expressed 7days after SC. GluR3/4 subunits were mainly expressed in the hippocampus of normal IRs, which had the lowest levels of GluR2. Immature brain was more resistant to seizure-induced neural damage. The time course of reduction and recovery differed for each subunit and was dependent on developmental stage. The increased expression of GluR2 could confer early but transient protection in the immature brain after SC.

Phasic GABAA‐receptor activation is required to suppress epileptiform activity in the CA3 region of the immature rat hippocampus

Despite the consistent observation that γ-aminobutyric acid A (GABA(A) ) receptors mediate excitatory responses at perinatal stages, the role of the GABAergic system in the generation of neonatal epileptiform activity remains controversial. Therefore, we analyzed whether tonic and phasic GABAergic transmission had differential effects on neuronal excitability during early development. We performed whole cell patch-clamp and field potential recordings in the CA3 region of hippocampal slices from immature (postnatal day 4-7) rats to analyze the effect of specific antagonists and modulators of tonic and phasic GABAergic components on neuronal excitability. The GABAergic antagonists gabazine (3 μm) and picrotoxin (100 μm) induced epileptiform discharges, whereas activation of GABA(A) receptors attenuated epileptiform discharges. Under low-Mg(2+) conditions, 100 nm gabazine and 1 μm picrotoxin were sufficient to provoke epileptiform activity in 63.2% (n = 19) and 53.8% (n = 26) of the slices, respectively. Whole-cell patch-clamp experiments revealed that these concentrations significantly reduced the amplitude of phasic GABAergic postsynaptic currents but had no effect on tonic currents. In contrast, 1-μm 4,5,6,7-tetrahydroisoxaz-olo[5,4-c]-pyridin-3-ol (THIP) induced a tonic current of -12 ± 2.5 pA (n = 6) and provoked epileptiform discharges in 57% (n = 21) of the slices. We conclude from these results that in the early postnatal rat hippocampus a constant phasic synaptic activity is required to control excitability and prevent epileptiform activity, whereas tonic GABAergic currents can mediate excitatory responses. Pharmacologic intervention at comparable human developmental stages should consider these ambivalent GABAergic actions.