Pilocarpine-induced Status Epilepticus Research Articles

Dynamic Expression of MicroRNAs (183, 135a, 125b, 128, 30c and 27a) in the Rat Pilocarpine Model and Temporal Lobe Epilepsy Patients.

Recently, microRNAs (miRNAs) are emerging as new regulators in the pathogenesis of temporal lobe epilepsy (TLE) and playing a major role in the inflammatory and immune processes. The aim of the present study was to evaluate the dynamic expression of brain-specific miR-183 and miR-135a, brain-enriched miR-125b and miR-128 and inflammation-related miR-30c and miR-27a. Status epilepticus evoked by pilocarpine administeration was used to induce epilepsy in rats. Quantitative polymerase chain reaction was performed on rat hippocampus 2 hours, 3 weeks and 2 months following pilocarpine-induced status epilepticus, representing the acute, latent, and chronic phases, respectively. Expression levels were also measured in hippocampus obtained from TLE patients and normal controls. In the rat model, miR-183, miR-135a and miR-125b were detected upregulated during the acute and chronic phases compared to controls, but not during the latent phase. miR-30c and miR-27a were upregulated in the acute and chronic phases of TLE, while in the latent phase miR-30c was downregulated and miR-27a was upregulated. On the other hand, miR-128 showed significantly downregulated in all phases of TLE development. In TLE patients, miR-183, miR- 135a, miR-125b, miR-30c and miR-27a were upregulated, whereas miR-128 was downregulated. Our study revealed upregulation of miR-183, miR-135a and miR-125b in the seizure-related phases and TLE patients, suggesting that all may provide a potential therapeutic approach for the treatment of TLE, whereas the dysregulation of miR-128, miR-30c and miR-27a may suggest different functions during the process of TLE development.

Hypothermia mitigates neurochemical alterations in rat's cerebral cortex during status epilepticus induced by pilocarpine.

Status epilepticus (SE) is a prolonged seizure activity associated with mortality and morbidity. SE is characterized by changes in neurotransmitter systems and oxidative stress that facilitate cellular damage. These alterations represent the neurochemical mechanisms underlying the initiation and progression of seizure activity and co-existing morbidity. In the present study, amino acid levels (glutamine, glutamate, GABA, aspartate, glycine and taurine) and oxidative stress parameters malondialdehyde (MDA), reduced glutathione (GSH) and nitric oxide NO) were determined in the cerebral cortex during SE induced by pilocarpine in rats. The study has also evaluated the effects of hypothermia, as a physical non-invasive tool, on neurotransmitters and oxidative stress alterations. The results obtained revealed that there are significant increases in glutamate, GABA, glycine and taurine and NO in the cortex of pilocarpinzed rats. Hypothermia pretreatment mitigated most of the alterations induced by pilocarpine and significantly decreased GABA concentration. These findings emphasize the involvement of extrahippocampal amino acid neurotransmitters in pilocarpine-induced SE and the ameliorative role played by hypothermia.

Kinetic study of benzyl [1-14C]acetate as a potential probe for astrocytic energy metabolism in the rat brain: Comparison with benzyl [2-14C]acetate

Brain uptake of [14C]acetate has been reported to be a useful marker of astrocytic energy metabolism. In addition to uptake values, the rate of radiolabeled acetate washout from the brain appears to reflect CO2 exhaustion and oxygen consumption in astrocytes. We measured the time–radioactivity curves of benzyl [1-14C]acetate ([1-14C]BA), a lipophilic probe of [1-14C]acetate, and compared it with that of benzyl [2-14C]acetate ([2-14C]BA) in rat brains. The highest brain uptake was observed immediately after injecting either [1-14C]BA or [2-14C]BA, and both subsequently disappeared from the brain in a single-exponential manner. Estimated [1-14C]BA washout rates in the cerebral cortex and cerebellum were higher than those of [2-14C]BA. These results suggested that [1-14C]BA could be a useful probe for estimating the astrocytic oxidative metabolism. The [1-14C]BA washout rate in the cerebral cortex of immature rats was lower than that of mature rats. An autoradiographic study showed that the washout rates of [1-14C]BA from the rat brains of a lithium–pilocarpine-induced status epilepticus model were not significantly different from the values in control rat brains except for the medial septal nucleus. These results implied that the enhancement of amino acid turnover rate rather than astrocytic oxidative metabolism was increased in status epilepticus.

Newly generated neurons at 2 months post-status epilepticus are functionally integrated into neuronal circuitry in mouse hippocampus

Emerging evidence has linked chronic temporal lobe epilepsy to dramatically reduced neurogenesis in the dentate gyrus. However, the profile of different components of neurogenesis in the chronically epileptic hippocampus is still unclear, especially the incorporation of newly generated cells. To address the issue, newly generated cells in the sub-granular zone of the dentate gyrus were labeled by the proliferation marker bromodeoxyuridine (BrdU) or retroviral vector expressing green fluorescent protein 2months after pilocarpine-induced status epilepticus. The newly generated neurons that extended axons to CA3 area or integrated into memory circuits were visualized by cholera toxin B subunit retrograde tracing, and detecting activation of BrdU+ cells following a recall of spatial memory test at the chronic stage of TLE. We found that the microenvironment was still able to sustain significant neuronal differentiation of newly generated cells at 2months post-status epilepticus time-point, and newly added neurons into granular cell layer were still able to integrate into neuronal circuitry, both anatomically and functionally. Quantified analyses of BrdU+ or Ki-67+ cells demonstrated that there was a reduced proliferation of progenitor cells and diminished survival of newly generated cells in the epileptic hippocampus. Both decreased levels of neurotrophic factors in the surrounding milieu and cell loss in the CA3 area might contribute the decreased production of new cells and their survival following chronic epilepsy. These results suggest that decreased neurogenesis in the chronically epileptic hippocampus 2months post status epilepticus is not associated with altered integration of newly generated neurons, and that developing strategies to augment hippocampal neurogenesis in chronic epilepsy might be protective.

Pilocarpine-induced status epilepticus in mice: A comparison of spectral analysis of electroencephalogram and behavioral grading using the Racine scale

Pilocarpine-induced status epilepticus (SE) is a widely used seizure model in mice, and the Racine scale has been used to index seizure intensity. The goal of this study was to analyze electroencephalogram (EEG) quantitatively using fast Fourier transformation (FFT) and statistically evaluate the correlation of electrographic seizures with convulsive behaviors. Simultaneous EEG and video recordings in male mice in a mixed genetic background were conducted and pilocarpine was administered intraperitoneally to induce seizures. The videos were graded using the Racine scale and the root-mean-square (RMS) power analysis of EEG was performed with Sirenia Seizure Pro software. We found that the RMS power was very weakly correlated with convulsive behavior induced by pilocarpine. Convulsive behaviors appeared long before electrographic seizures and showed a strong negative correlation with theta frequency activity and a moderate positive correlation with gamma frequency activity. Racine scores showed moderate correlations with RMS power across multiple frequency bands during the transition from first electrographic seizure to SE. However, there was no correlation between Racine scores and RMS power during the SE phase except a weak correlation with RMS power in the theta frequency. Our analysis reveals limitations of the Racine scale as a primary index of seizure intensity in status epilepticus, and demonstrates a need for quantitative analysis of EEG for an accurate assessment of seizure onset and severity.

P2X7 receptor-mediated PARP1 activity regulates astroglial death in the rat hippocampus following status epilepticus

Poly(ADP-ribose) polymerase-1 (PARP1) plays a regulatory role in apoptosis, necrosis, and other cellular processes after injury. Recently, we revealed that PARP1 regulates the differential neuronal/astroglial responses to pilocarpine-induced status epilepticus (SE) in the distinct brain regions. In addition, P2X7 receptor (P2X7R), an ATP-gated ion channel, activation accelerates astroglial apoptosis, while it attenuates clasmatodendrosis (lysosome-derived autophagic astroglial death). Therefore, we investigated whether P2X7R regulates regional specific astroglial PARP1 expression/activation in response to SE. In the present study, P2X7R activation exacerbates SE-induced astroglial apoptosis, while P2X7R inhibition attenuates it accompanied by increasing PARP1 activity in the molecular layer of the dentate gyrus following SE. In the CA1 region, however, P2X7R inhibition deteriorates SE-induced clasmatodendrosis via PARP1 activation following SE. Taken together, our findings suggest that P2X7R function may affect SE-induced astroglial death by regulating PARP1 activation/expression in regional-specific manner. Therefore, the selective modulation of P2X7R-mediated PARP1 functions may be a considerable strategy for controls in various types of cell deaths.

Ketogenic diet prevents epileptogenesis and disease progression in adult mice and rats

Epilepsy is a highly prevalent seizure disorder which tends to progress in severity and become refractory to treatment. Yet no therapy is proven to halt disease progression or to prevent the development of epilepsy. Because a high fat low carbohydrate ketogenic diet (KD) augments adenosine signaling in the brain and because adenosine not only suppresses seizures but also affects epileptogenesis, we hypothesized that a ketogenic diet might prevent epileptogenesis through similar mechanisms. Here, we tested this hypothesis in two independent rodent models of epileptogenesis. Using a pentylenetetrazole kindling paradigm in mice, we first show that a KD, but not a conventional antiepileptic drug (valproic acid), suppressed kindling-epileptogenesis. Importantly, after treatment reversal, increased seizure thresholds were maintained in those animals kindled in the presence of a KD, but not in those kindled in the presence of valproic acid. Next, we tested whether a KD can halt disease progression in a clinically relevant model of progressive epilepsy. Epileptic rats that developed spontaneous recurrent seizures after a pilocarpine-induced status epilepticus were treated with a KD or control diet (CD). Whereas seizures progressed in severity and frequency in the CD-fed animals, KD-fed animals showed a prolonged reduction of seizures, which persisted after diet reversal. KD-treatment was associated with increased adenosine and decreased DNA methylation, the latter being maintained after diet discontinuation. Our findings demonstrate that a KD prevented disease progression in two mechanistically different models of epilepsy, and suggest an epigenetic mechanism underlying the therapeutic effects.

Transcranial focal electrical stimulation reduces seizure activity and hippocampal glutamate release during status epilepticus.

Previously we demonstrated that noninvasive transcranial focal electrical stimulation (TFS) with sub-effective doses of diazepam reduces status epilepticus (SE)-induced neuronal damage. However, it was unclear if this neuroprotective effect is a consequence of the decrease in the glutamate release. The aim of the present study was to evaluate the effects of TFS on γ-Aminobutyric acid (GABA) and glutamate release in the hippocampus during pilocarpine-induced SE. After pilocarpine administration, the rats showed progressive behavioral changes that culminated in SE with a significant increase of GABA and glutamate (95 and 128% respectively), even more evident at the end of the experiment (120 and 182% respectively), 5 hours after pilocarpine injection and was associated with the prevalence of high-voltage rhythmic spikes and increased spectral power in the 4-90 Hz bands. The TFS application during the SE decreased the convulsive expression, the prevalence of high-voltage rhythmic spikes and spectral power in 4-8 Hz and 30-90 Hz bands. These effects were associated with lower release of GABA and glutamate in the hippocampus. These results support the anticonvulsive and neuroprotective effects induced by TFS.

Rapid changes in expression of class I and IV histone deacetylases during epileptogenesis in mouse models of temporal lobe epilepsy

A prominent role of epigenetic mechanisms in manifestation of epilepsy has been proposed. Thus altered histone H3 and H4 acetylation has been demonstrated in experimental models of temporal lobe epilepsy (TLE). We now investigated changes in the expression of the class I and class IV histone deacetylases (HDAC) in two complementary mouse TLE models. Unilateral intrahippocampal injection of kainic acid (KA) induced a status epilepticus lasting 6 to 24h, development of spontaneous limbic seizures (2 to 3days after KA injection) and chronic epilepsy, as revealed by telemetric recordings of the EEGs. Mice were killed at different intervals after KA injection and expression of HDAC mRNAs was investigated by in situ hybridization. We observed marked decreases in the expression of HDACs 1, 2 and 11 (by up to 75%) in the granule cell and pyramidal cell layers of the hippocampus during the acute status epilepticus (2 to 6h after KA injection). This was followed by increased expression of all class I HDAC mRNAs in all principal cell layers of the hippocampus after 12 to 48h. In the chronic phase, 14 and 28days after KA, only modest increases in the expression of HDAC1 mRNA were observed in granule and pyramidal cells. Immunohistochemistry using an antibody detecting HDAC2 revealed results consistent with the mRNA data and indicates also expression in glial cells on the injection side. Similar changes as seen in the KA model were observed after a pilocarpine-induced status epilepticus except that decreases in HDACs 2, 3 and 8 were also seen at the chronic 28day interval.The prominent decreases in HDAC expression during status epilepticus are consistent with the previously demonstrated increased expression of numerous proteins and with the augmented acetylation of histone H4. It is suggested that respective putative gene products could facilitate proconvulsive as well as anticonvulsive mechanisms. The increased expression of all class I HDACs during the “silent phase”, on the other hand, may be related to decreased histone acetylation, which could cause a decrease in expression of certain proteins, a mechanism that could also promote epileptogenesis. Thus, addressing HDAC expression may have a therapeutic potential in interfering with a status epilepticus and with the manifestation of TLE.

Role of the Mitochondrial Calcium Uniporter in Rat Hippocampal Neuronal Death After Pilocarpine-Induced Status Epilepticus.

The mitochondrial calcium uniporter (MCU) is reportedly involved in oxidative stress, apoptosis, and many neurological diseases. However, the role of the MCU in epilepsy remains unknown. In this study, we found that the MCU inhibitor Ru360 significantly attenuated neuronal death and exerted an anti-apoptotic effect on rat hippocampal neurons after pilocarpine-induced status epilepticus (SE), while the MCU activator spermine increased seizure-induced neuronal death and apoptosis. In addition, Ru360 decreased the level of seizure-induced reactive oxygen species (ROS) in mitochondria isolated from rat hippocampi. Moreover, Ru360 restored the altered mitochondrial membrane potential and cytochrome c (CytC) release in epileptic hippocampi. However, spermine treatment exerted an opposite effect on seizure-induced ROS production and mitochondrial membrane potential alteration and CytC release compared with Ru360 treatment. Altogether, the findings of this study suggest that MCU inhibition exerts a neuroprotective effect on seizure-induced brain injury possibly through the mitochondria/ROS/CytC pathway.

Status Epilepticus Induced Spontaneous Dentate Gyrus Spikes: In Vivo Current Source Density Analysis.

The dentate gyrus is considered to function as an inhibitory gate limiting excitatory input to the hippocampus. Following status epilepticus (SE), this gating function is reduced and granule cells become hyper-excitable. Dentate spikes (DS) are large amplitude potentials observed in the dentate gyrus (DG) of normal animals. DS are associated with membrane depolarization of granule cells, increased activity of hilar interneurons and suppression of CA3 and CA1 pyramidal cell firing. Therefore, DS could act as an anti-excitatory mechanism. Because of the altered gating function of the dentate gyrus following SE, we sought to investigate how DS are affected following pilocarpine-induced SE. Two weeks following lithium-pilocarpine SE induction, hippocampal EEG was recorded in male Sprague-Dawley rats with 16-channel silicon probes under urethane anesthesia. Probes were placed dorso-ventrally to encompass either CA1-CA3 or CA1-DG layers. Large amplitude spikes were detected from EEG recordings and subject to current source density analysis. Probe placement was verified histologically to evaluate the anatomical localization of current sinks and the origin of DS. In 9 of 11 pilocarpine-treated animals and two controls, DS were confirmed with large current sinks in the molecular layer of the dentate gyrus. DS frequency was significantly increased in pilocarpine-treated animals compared to controls. Additionally, in pilocarpine-treated animals, DS displayed current sinks in the outer, middle and/or inner molecular layers. However, there was no difference in the frequency of events when comparing between layers. This suggests that following SE, DS can be generated by input from medial and lateral entorhinal cortex, or within the dentate gyrus. DS were associated with an increase in multiunit activity in the granule cell layer, but no change in CA1. These results suggest that following SE there is an increase in DS activity, potentially arising from hyperexcitability along the hippocampal-entorhinal pathway or within the dentate gyrus itself.

Status epilepticus induction has prolonged effects on the efficacy of antiepileptic drugs in the 6-Hz seizure model.

Several factors may influence the efficacy of antiepileptic drugs (AEDs) in patients with epilepsy, and treatment resistance could be related to genetics, neuronal network alterations, and modification of drug transporters or targets. Consequently, preclinical models used for the identification of potential new, more efficacious AEDs should reflect at least a few of these factors. Previous studies indicate that induction of status epilepticus (SE) may alter drug efficacy and that this effect could be long-lasting. In this context, we wanted to assess the protective effects of mechanistically diverse AEDs in mice subjected to pilocarpine-induced SE in another seizure model. We first determined seizure thresholds in mice subjected to pilocarpine-induced SE in the 6-Hz model, 2 weeks and 8 weeks following SE. We then evaluated the protective effects of mechanistically diverse AEDs in post-SE and control animals. No major differences in 6-Hz seizure susceptibility were observed between control groups, while the seizure threshold of pilocarpine mice at 8 weeks after SE was higher than at 2 weeks and higher than in control groups. Treatment with AEDs revealed major differences in drug response depending on their mechanism of action. Diazepam produced a dose-dependent protection against 6-Hz seizures in control and pilocarpine mice, both at 2 weeks and 8 weeks after SE, but with a more pronounced increase in potency in post-SE animals at 2 weeks. Levetiracetam induced a potent and dose-dependent protection in pilocarpine mice, 2 weeks after SE, while its protective effects were observed only at much higher doses in control mice. Its potency decreased in post-SE mice at 8 weeks and was very limited (30% protection at the highest tested dose) in the control group. Carbamazepine induced a dose-dependent protection at 2 weeks in control mice but only limited effect (50% at the highest tested dose) in pilocarpine mice. Its efficacy deeply decreased in post-SE mice at 8 weeks after SE. Perampanel and phenytoin showed almost comparable protective effects in all groups of mice. These experiments confirm that prior SE may have an impact on both potency and efficacy of AEDs and indicate that this effect may be dependent on the underlying epileptogenic processes. This article is part of a Special Issue entitled "Status Epilepticus".

Endothelial NOS activation induces the blood–brain barrier disruption via ER stress following status epilepticus

The blood–brain barrier (BBB) maintains the unique brain microenvironment, which is separated from the systemic circulating system. Since the endoplasmic reticulum (ER) is an important cell organelle that is responsible for protein synthesis, the correct folding and sorting of proteins contributing to cell survivals, ER stress is a potential cause of cell damage in various diseases. Therefore, it would be worthy to explore the the relationship between the ER stress and BBB disruption during vasogenic edema formation induced by epileptogenic insults. In the present study, we investigated the roles of ER stress in vasogenic edema and its related events in rat epilepsy models provoked by pilocarpine-induced status epilepticus (SE). SE-induced eNOS activation induces BBB breakdown via up-regulation of GRP78 expression and dysfunction of SMI-71 (an endothelial BBB marker) in the piriform cortex (PC). In addition, caveolin-1 peptide (an eNOS inhibitor) effectively attenuated GRP78 expression and down-regulation of SMI-71. Taken together, our findings suggest that eNOS-mediated ER stress may participate in SE-induced vasogenic edema formation. Therefore, the modulation of ER stress may be a considerable strategy for therapy in impairments of endothelial cell function.

Increased extracellular levels of glutamate in the hippocampus of chronically epileptic rats

An increase in the release of excitatory amino acids has consistently been observed in the hippocampus during seizures, both in humans and animals. However, very little or nothing is known about the extracellular levels of glutamate and aspartate during epileptogenesis and in the interictal chronic period of established epilepsy. The aim of this study was to systematically evaluate the relationship between seizure activity and changes in hippocampal glutamate and aspartate extracellular levels under basal and high K+-evoked conditions, at various time-points in the natural history of experimental temporal lobe epilepsy, using in vivo microdialysis. Hippocampal extracellular glutamate and aspartate levels were evaluated: 24h after pilocarpine-induced status epilepticus (SE); during the latency period preceding spontaneous seizures; immediately after the first spontaneous seizure; in the chronic (epileptic) period. We found that (i) basal (spontaneous) glutamate outflow is increased in the interictal phases of the chronic period, whereas basal aspartate outflow remains stable for the entire course of the disease; (ii) high K+ perfusion increased glutamate and aspartate outflow in both control and pilocarpine-treated animals, and the overflow of glutamate was clearly increased in the chronic group. Our data suggest that the glutamatergic signaling is preserved and even potentiated in the hippocampus of epileptic rats, and thus may favor the occurrence of spontaneous recurrent seizures. Together with an impairment of GABA signaling (Soukupova et al., 2014), these data suggest that a shift toward excitation occurs in the excitation/inhibition balance in the chronic epileptic state.

Transcranial focal electrical stimulation reduces the convulsive expression and amino acid release in the hippocampus during pilocarpine-induced status epilepticus in rats.

The aim of the present study was to evaluate the effects of transcranial focal electrical stimulation (TFS) on γ-aminobutyric acid (GABA) and glutamate release in the hippocampus under basal conditions and during pilocarpine-induced status epilepticus (SE). Animals were previously implanted with a guide cannula attached to a bipolar electrode into the right ventral hippocampus and a concentric ring electrode placed on the skull surface. The first microdialysis experiment was designed to determine, under basal conditions, the effects of TFS (300 Hz, 200 μs biphasic square pulses, for 30 min) on afterdischarge threshold (ADT) and the release of GABA and glutamate in the hippocampus. The results obtained indicate that at low current intensities (<2800 μA), TFS enhances and decreases the basal extracellular levels of GABA and glutamate, respectively. However, TFS did not modify the ADT. During the second microdialysis experiment, a group of animals was subjected to SE induced by pilocarpine administration (300 mg/kg, i.p.; SE group). The SE was associated with a significant rise of GABA and glutamate release (up to 120 and 182% respectively, 5h after pilocarpine injection) and the prevalence of high-voltage rhythmic spikes and increased spectral potency of delta, gamma, and theta bands. A group of animals (SE-TFS group) received TFS continuously during 2h at 100 μA, 5 min after the establishment of SE. This group showed a significant decrease in the expression of the convulsive activity and spectral potency in gamma and theta bands. The extracellular levels of GABA and glutamate in the hippocampus remained at basal conditions. These results suggest that TFS induces anticonvulsant effects when applied during the SE, an effect associated with lower amino acid release. This article is part of a Special Issue entitled "Status Epilepticus".

Increased calcineurin expression after pilocarpine-induced status epilepticus is associated with brain focal edema and astrogliosis

Calcineurin plays an important role in the development of neuronal excitability, modulation of receptor's function and induction of apoptosis in neurons. It has been established in kindling models that status epilepticus induces brain focal edema and astrocyte activation. However, the role of calcineurin in brain focal edema and astrocyte activation in status epilepticus has not been fully understood. In this study, we employed a model of lithium-pilocarpine-induced status epilepticus and detected calcineurin expression in hippocampus by immunoblotting, brain focal edema by non-invasive magnetic resonance imaging (MRI-7T) and astrocyte expression by immunohistochemistry. We found that the brain focal edema was seen at 24 h after status epilepticus, and astrocyte expression was obviously seen at 7 d after status epilepticus. Meanwhile, calcineurin expression was seen at24 h and retained to 7 d after status epilepticus. A FK506, a calcineurin inhibitor, remarkably suppressed the status epilepticus-induced brain focal edema and astrocyte expression. Our data suggested that calcineurin overexpression plays a very important role in brain focal edema and astrocyte expression. Therefore, calcineurin may be a novel candidate for brain focal edema occurring and intracellular trigger of astrogliosis in status epilepticus.

Protein kinase C bidirectionally modulates Ih and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel surface expression in hippocampal pyramidal neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, particularly that of the HCN1 isoform, are enriched in the distal dendrites of hippocampal CA1 pyramidal neurons; these channels have physiological functions with respect to decreasing neuronal excitability. In the present study, we aimed to investigate phosphorylation as a mechanism controlling Ih amplitude and HCN1 surface expression in hippocampal principal neurons under normal physiological conditions. Tyrosine phosphorylation decreased Ih amplitude at maximal activation (maximal Ih ), without altering HCN1 surface expression, in two classes of hippocampal principal neurons. Inhibition of serine/threonine protein phosphatases 1 and 2A decreased maximal Ih and HCN1 surface expression in hippocampal principal neurons. Protein kinase C (PKC) activation irreversibly diminished Ih and HCN1 surface expression, whereas PKC inhibition augmented Ih and HCN1 surface expression. PKC activation increased HCN1 channel phosphorylation. These results demonstrate the novel finding of a phosphorylation mechanism, dependent on PKC activity, which bidirectionally modulates Ih amplitude and HCN1channel surface expression in hippocampal principal neurons under normal physiological conditions. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels attenuate excitability in hippocampal pyramidal neurons. Loss of HCN channel-mediated current (Ih ), particularly that mediated by the HCN1 isoform, occurs with the development of epilepsy. Previously, we showed that, following pilocarpine-induced status epilepticus, there are two independent changes in HCN function in dendrites: decreased Ih amplitude associated with a loss of HCN1 surface expression and a hyperpolarizing shift in voltage-dependence of activation (gating). The hyperpolarizing shift in gating was attributed to decreased phosphorylation as a result of a loss of p38 mitogen-activated protein kinase activity and increased calcineurin activity; however, the mechanisms controlling Ih amplitude and HCN1 surface expression under epileptic or normal physiological conditions are poorly understood. We aimed to investigate phosphorylation as a mechanism regulating Ih amplitude and HCN1 surface expression (i.e. as is the case for HCN gating) in hippocampal principal neurons under normal physiological conditions. We discovered that inhibition of either tyrosine phosphatases or the serine/threonine protein phosphatases 1 and 2A decreased Ih at maximal activation in hippocampal CA1 pyramidal dendrites and pyramidal-like principal neuron somata from naïve rats. Furthermore, we found that inhibition of PP1/PP2A decreased HCN1 surface expression, whereas tyrosine phosphatase inhibition did not. Protein kinase C (PKC) activation reduced Ih amplitude and HCN1 surface expression, whereas PKC inhibition produced the opposite effect. Inhibition of protein phosphatases 1 and 2A and activation of PKC increased the serine phosphorylation state of the HCN1 protein. The effect of PKC activation on Ih was irreversible. These results indicate that PKC bidirectionally modulates Ih amplitude and HCN1 surface expression in hippocampal principal neurons.

Effects of TRPV1 on the hippocampal synaptic plasticity in the epileptic rat brain.

Temporal lobe epilepsy is often presented by medically intractable recurrent seizures due to dysfunction of temporal lobe structures, mostly the temporomesial structures. The role of transient receptor potential vaniloid 1 (TRPV1) activity on synaptic plasticity of the epileptic brain tissues was investigated. We studied hippocampal TRPV1 protein content and distribution in the hippocampus of epileptic rats. Furthermore, the effects of pharmacologic modulation of TRPV1 receptors on field excitatory postsynaptic potentials have been analyzed after induction of long term potentiation (LTP) in the hippocampal CA1 and CA3 areas after 1 day (acute phase) and 3 months (chronic phase) of pilocarpine-induced status epilepticus (SE). A higher expression of TRPV1 protein in the hippocampus as well as a higher distribution of this channel in CA1 and CA3 areas in both acute and chronic phases of pilocarpine-induced SE was observed. Activation of TRPV1 using capsaicin (1 µM) enhanced LTP induction in CA1 region in non-epileptic rats. Inhibition of TRPV1 by capsazepine (10 µM) did not affect LTP induction in non-epileptic rats. In acute phase of SE, activation of TRPV1 enhanced LTP in both CA1 and CA3 areas but TRPV1 inhibition did not affect LTP. In chronic phase of SE, application of TRPV1 antagonist enhanced LTP induction in CA1 and CA3 regions but TRPV1 activation had no effect on LTP. These findings indicate that a higher expression of TRPV1 in epileptic conditions is accompanied by a functional impact on the synaptic plasticity in the hippocampus. This suggests TRPV1 as a potential target in treatment of seizure attacks.

Interictal spike frequency varies with ovarian cycle stage in a rat model of epilepsy

In catamenial epilepsy, seizures exhibit a cyclic pattern that parallels the menstrual cycle. Many studies suggest that catamenial seizures are caused by fluctuations in gonadal hormones during the menstrual cycle, but this has been difficult to study in rodent models of epilepsy because the ovarian cycle in rodents, called the estrous cycle, is disrupted by severe seizures. Thus, when epilepsy is severe, estrous cycles become irregular or stop. Therefore, we modified kainic acid (KA)- and pilocarpine-induced status epilepticus (SE) models of epilepsy so that seizures were rare for the first months after SE, and conducted video-EEG during this time.The results showed that interictal spikes (IIS) occurred intermittently. All rats with regular 4-day estrous cycles had IIS that waxed and waned with the estrous cycle. The association between the estrous cycle and IIS was strong: if the estrous cycles became irregular transiently, IIS frequency also became irregular, and when the estrous cycle resumed its 4-day pattern, IIS frequency did also. Furthermore, when rats were ovariectomized, or males were recorded, IIS frequency did not show a 4-day pattern. Systemic administration of an estrogen receptor antagonist stopped the estrous cycle transiently, accompanied by transient irregularity of the IIS pattern. Eventually all animals developed severe, frequent seizures and at that time both the estrous cycle and the IIS became irregular. We conclude that the estrous cycle entrains IIS in the modified KA and pilocarpine SE models of epilepsy. The data suggest that the ovarian cycle influences more aspects of epilepsy than seizure susceptibility.

Neurosteroidal modulation of in vitro epileptiform activity is enhanced in pilocarpine-treated epileptic rats

We employed field potential recordings in brain slices obtained from pilocarpine-treated epileptic (4–5weeks following a pilocarpine-induced status epilepticus) and age-matched, non-epileptic control (NEC) rats to establish the effects of the neurosteroid allotetrahydrodeoxycorticosterone (THDOC) on the epileptiform activity – including high frequency oscillations (HFOs; ripples: 80–200Hz, fast ripples: 250–500Hz) – induced by 4-aminopyridine (4AP) in piriform (PC) and entorhinal (EC) cortices. Both structures are highly susceptible to generate seizures and may also be involved in epileptogenesis. We found that THDOC application to pilocarpine-treated slices: (i) decreased interictal discharge frequency in PC while increasing it in EC; (ii) abolished ictal discharges in both areas in approx. one third of the experiments and reduced them in frequency and duration in the remaining experiments; and (iii) increased the occurrence of ripples and fast ripples associated to interictal events, and modified their pattern of occurrence during ictal discharges in both PC and EC. These effects were either weaker or absent in NEC tissue. Our results demonstrate that THDOC plays a structure-dependent modulatory role in epileptiform synchronization in the pilocarpine-treated epileptic rat brain where its actions are more pronounced than in NEC tissue. This evidence supports the application of neurosteroids as potential antiepileptic tools.