Hippocampal Paroxysmal Discharges Research Articles

Characterization of the intrahippocampal kainic acid model in female mice with a special focus on seizure suppression by antiseizure medications

Despite special challenges in the medical treatment of women with epilepsy, in particular preclinical animal studies were focused on males for decades and females have only recently moved into the focus of scientific interest. The intrahippocampal kainic acid (IHKA) mouse model of temporal lobe epilepsy (TLE) is one of the most studied models in males reproducing electroencephalographic (EEG) and histopathological features of human TLE. Hippocampal paroxysmal discharges (HPDs) were described as drug resistant focal seizures in males. Here, we investigated the IHKA model in female mice, in particular drug-resistance of HPDs and the influence of antiseizure medications (ASMs) on the power spectrum.After injecting kainic acid (KA) unilaterally into the hippocampus of female mice, we monitored the development of epileptiform activity by local field potential (LFP) recordings. Subsequently, we evaluated the effect of the commonly prescribed ASMs lamotrigine (LTG), oxcarbazepine (OXC) and levetiracetam (LEV), as well as the benzodiazepine diazepam (DZP) with a focus on HPDs and power spectral analysis and assessed neuropathological alterations of the hippocampus.In the IHKA model, female mice replicated key features of human TLE as previously described in males. Importantly, HPDs in female mice did not respond to commonly prescribed ASMs in line with the drug-resistance in males, thus representing a suitable model of drug-resistant seizures. Intriguingly, we observed an increased occurrence of generalized seizures after LTG. Power spectral analysis revealed a pronounced increase in the delta frequency range after the higher dose of 30 mg/kg LTG. DZP abolished HPDs and caused a marked reduction over a wide frequency range (delta, theta, and alpha) of the power spectrum.By characterizing the IHKA model of TLE in female mice we address an important gap in basic research. Considering the special challenges complicating the therapeutic management of epilepsy in women, inclusion of females in preclinical studies is imperative. A well-characterized female model is a prerequisite for the development of novel therapeutic strategies tailored to sex-specific needs and for studies on the effect of epilepsy and ASMs during pregnancy.

Trpm2 deficiency in microglia attenuates neuroinflammation during epileptogenesis by upregulating autophagy via the AMPK/mTOR pathway

Epilepsy is one of the most common neurological disorders. Neuroinflammation involving the activation of microglia and astrocytes constitutes an important and common mechanism in epileptogenesis. Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, non-selective cation channel that plays pathological roles in various inflammation-related diseases. Our previous study demonstrated that Trpm2 knockout exhibits therapeutic effects on pilocarpine-induced glial activation and neuroinflammation. However, whether TRPM2 in microglia and astrocytes plays a common pathogenic role in this process and the underlying molecular mechanisms remained undetermined. Here, we demonstrate a previously unknown role for microglial TRPM2 in epileptogenesis. Trpm2 knockout in microglia attenuated kainic acid (KA)-induced glial activation, inflammatory cytokines production and hippocampal paroxysmal discharges, whereas Trpm2 knockout in astrocytes exhibited no significant effects. Furthermore, we discovered that these therapeutic effects were mediated by upregulated autophagy via the adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in microglia. Thus, our findings highlight an important deleterious role of microglial TRPM2 in temporal lobe epilepsy.

Structural Analogs of the GABAkine KRM-II-81 Are Orally Bioavailable Anticonvulsants without Sedation.

To provide back-up compounds to support the development of the GABAA receptor (GABAAR) potentiator KRM-II-81, three novel analogs were designed: replacing the pyridinyl with 2'-Cl-phenyl (FR-II-60), changing the positions of the N and O atoms in the oxazole ring with addition of an ethyl group (KPP-III-34 and KPP-III-51), or substituting a Br atom for the ethynyl of KRM-II-81 (KPP-III-34). The compounds bound to brain GABAARs. Intraperitoneal administration of FR-II-60 and KPP-III-34 produced anticonvulsant activity in mice [maximal electroshock (MES)-induced seizures or 6 Hz-induced seizures], whereas KPP-III-51 did not. Although all compounds were orally bioavailable, structural changes reduced the plasma and brain (FR-II-60 and KPP-III-51) exposures relative to KRM-II-81. Oral administration of each compound produced dose-dependent increases in the latency for both clonic and tonic seizures and the lethality induced by pentylenetetrazol (PTZ) in mice. Since KPP-III-34 produced the highest brain area under the curve (AUC) exposures, it was selected for further profiling. Oral administration of KPP-III-34 suppressed seizures in corneal-kindled mice, hippocampal paroxysmal discharges in mesial temporal lobe epileptic mice, and PTZ-induced convulsions in rats. Only transient sensorimotor impairment was observed in mice, and doses of KPP-III-34 up to 500 mg/kg did not produce impairment in rats. Molecular docking studies demonstrated that all compounds displayed a reduced propensity for binding to α1His102 compared with the sedating compound alprazolam; the bromine-substituted KPP-III-34 achieved the least interaction. Overall, these findings document the oral bioavailability and anticonvulsant efficacy of three novel analogs of KRM-II-81 with reduced sedative effects. SIGNIFICANCE STATEMENT: A new non-sedating compound, KRM-II-81, with reduced propensity for tolerance is moving into clinical development. Three new analogs were orally bioavailable, produced anticonvulsant effects in rodents, and displayed low sensorimotor impairment. KPP-III-34 demonstrated efficacy in models of pharmacoresistant epilepsy. Docking studies demonstrated a low propensity for compound binding to the α1His102 residue implicated in sedation. Thus, three additional structures have been added to the list of non-sedating imidazodiazepine anticonvulsants that could serve as backups in the clinical development of KRM-II-81.

Long-term outcomes of classic and novel anti-seizure medication in a kainate-induced model of chronic epilepsy

ObjectiveIntrahippocampal injection of kainate (KA) is a reliable model of temporal lobe epilepsy (TLE) that replicates spontaneous recurrent seizures. Both electrographic seizures and electroclinical seizure (most generalized seizure) can be detected in KA model. Electrographic seizures such as high-voltage sharp waves (HVSWs) and hippocampal paroxysmal discharges (HPDs) are far more common and attracting much attention. A comprehensive study on the anticonvulsant effects of classic and novel antiseizure medications (ASMs) on spontaneous electroclinical seizures, especially during long-term treatment, is still lacking. Here, we evaluated the effects of six ASMs in this model on electroclinical seizures over eight weeks. MethodsUsing 24-hour continuous electroencephalographical (EEG) monitoring in free-moving mice, we tested the effectiveness of six ASMs (valproic acid, VPA; carbamazepine, CBZ; lamotrigine, LTG; perampanel, PER; brivaracetam, BRV; and everolimus, EVL) on the electroclinical seizures over eight weeks in the intrahippocampal kainate mouse model. ResultsVPA, CBZ, LTG, PER and BRV significantly suppressed electroclinical seizures in the early stages of treatment, but the mice gradually developed resistance to these drugs. Overall, the mean frequency of electroclinical seizures was not significantly lower during the 8-week treatment than that at baseline in any ASM-treated group. The individual responses to ASMs varied widely. ConclusionLong-term treatment with VPA, LTG, CBZ, PER, BRV and EVL did not relieve electroclinical seizures in this TLE model. Additionally, the window for screening new ASMs in this model should be set to at least 3 weeks to account for drug resistance.

Pronounced antiseizure activity of the subtype-selective GABAA positive allosteric modulator darigabat in a mouse model of drug-resistant focal epilepsy.

AimDarigabat is an α2/3/5 subunit‐selective positive allosteric modulator of GABAA receptors that has demonstrated broad‐spectrum activity in several preclinical models of epilepsy as well as in a clinical photoepilepsy trial. The objective here was to assess the acute antiseizure effect of darigabat in the mesial temporal lobe epilepsy (MTLE) mouse model of drug‐resistant focal seizures.MethodsThe MTLE model is generated by single unilateral intrahippocampal injection of low dose (1 nmole) kainic acid in adult mice, and subsequent epileptiform activity is recorded following implantation of a bipolar electrode under general anesthesia. After a period of epileptogenesis (~4 weeks), spontaneous and recurrent hippocampal paroxysmal discharges (HPD; focal seizures) are recorded using intracerebral electroencephalography. The number and cumulated duration of HPDs were recorded following administration of vehicle (PO), darigabat (0.3–10 mg kg−1, PO), and positive control diazepam (2 mg kg−1, IP).RESULTSDarigabat dose‐dependently reduced the expression of HPDs, demonstrating comparable efficacy profile to diazepam at doses of 3 and 10 mg kg−1.CONCLUSIONSDarigabat exhibited a robust efficacy profile in the MTLE model, a preclinical model of drug‐resistant focal epilepsy. A Phase II proof‐of‐concept placebo‐controlled, adjunctive‐therapy trial (NCT04244175) is ongoing to evaluate efficacy and safety of darigabat in patients with drug‐resistant focal seizures.

Miconazole exerts disease-modifying effects during epilepsy by suppressing neuroinflammation via NF-κB pathway and iNOS production

Neuroinflammation contributes to the generation of epilepsy and has been proposed as an effective therapeutic target. Recent studies have uncovered the potential effects of the anti-fungal drug miconazole for treating various brain diseases by suppressing neuroinflammation but have not yet been studied in epilepsy. Here, we investigated the effects of different doses of miconazole (5, 20, 80 mg/kg) on seizure threshold, inflammatory cytokines release, and glial cells activation in the pilocarpine (PILO) pentylenetetrazole (PTZ), and intrahippocampal kainic acid (IHKA) models. We demonstrated that 5 and 20 mg/kg miconazole increased seizure threshold, but only 20 mg/kg miconazole reduced inflammatory cytokines release, glial cells activation, and morphological alteration during the early post-induction period (24 h, 3 days). We further investigated the effects of 20 mg/kg miconazole on epilepsy (4 weeks after KA injection). We found that miconazole significantly attenuated cytokines production, glial cells activation, microglial morphological changes, frequency and duration of recurrent hippocampal paroxysmal discharges (HPDs), and neuronal and synaptic damage in the hippocampus during epilepsy. In addition, miconazole suppressed the KA-induced activation of the NF-κB pathway and iNOS production. Our results indicated miconazole to be an effective drug for disease-modifying effects during epilepsy, which may act by attenuating neuroinflammation through the suppression of NF-κB activation and iNOS production. At appropriate doses, miconazole may be a safe and effective approved drug that can easily be repositioned for clinical practice.

The Positive Allosteric Modulator of α2/3-Containing GABAA Receptors, KRM-II-81, Is Active in Pharmaco-Resistant Models of Epilepsy and Reduces Hyperexcitability after Traumatic Brain Injury.

The imidizodiazepine, 5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole (KRM-II-81), is selective for α2/3-containing GABAA receptors. KRM-II-81 dampens seizure activity in rodent models with enhanced efficacy and reduced motor-impairment compared with diazepam. In the present study, KRM-II-81 was studied in assays designed to detect antiepileptics with improved chances of impacting pharmaco-resistant epilepsies. The potential for reducing neural hyperactivity weeks after traumatic brain injury was also studied. KRM-II-81 suppressed convulsions in corneal-kindled mice. Mice with kainate-induced mesial temporal lobe seizures exhibited spontaneous recurrent hippocampal paroxysmal discharges that were significantly reduced by KRM-II-81 (15 mg/kg, orally). KRM-II-81 also decreased convulsions in rats undergoing amygdala kindling in the presence of lamotrigine (lamotrigine-insensitive model) (ED50 = 19 mg/kg, i.p.). KRM-II-81 reduced focal and generalized seizures in a kainate-induced chronic epilepsy model in rats (20 mg/kg, i.p., three times per day). In mice with damage to the left cerebral cortex by controlled-cortical impact, enduring neuronal hyperactivity was dampened by KRM-II-81 (10 mg/kg, i.p.) as observed through in vivo two-photon imaging of layer II/III pyramidal neurons in GCaMP6-expressing transgenic mice. No notable side effects emerged up to doses of 300 mg/kg KRM-II-81. Molecular modeling studies were conducted: docking in the binding site of the α1β3γ2L GABAA receptor showed that replacing the C8 chlorine atom of alprazolam with the acetylene of KRM-II-81 led to loss of the key interaction with α1His102, providing a structural rationale for its low affinity for α1-containing GABAA receptors compared with benzodiazepines such as alprazolam. Overall, these findings predict that KRM-II-81 has improved therapeutic potential for epilepsy and post-traumatic epilepsy. SIGNIFICANCE STATEMENT: We describe the effects of a relatively new orally bioavailable small molecule in rodent models of pharmaco-resistant epilepsy and traumatic brain injury. KRM-II-81 is more potent and generally more efficacious than standard-of-care antiepileptics. In silico docking experiments begin to describe the structural basis for the relative lack of motor impairment induced by KRM-II-81. KRM-II-81 has unique structural and anticonvulsant effects, predicting its potential as an improved antiepileptic drug and novel therapy for post-traumatic epilepsy.

Functional characterization of novel bumetanide derivatives for epilepsy treatment

Temporal lobe epilepsy (TLE) is the most common type of focal epilepsies, affecting approximately 35 million people worldwide. Despite the introduction of numerous novel antiepileptic drugs during the last decades, the proportion of patients with therapy-resistant TLE is still high. As an impaired cellular chloride homeostasis appears involved in disease pathophysiology, bumetanide, an antagonist to Na-K-Cl cotransporters, gained interest as potential therapeutic option. However, bumetanide induces a strong diuretic effect and displays poor penetration across the blood-brain barrier (BBB). To reduce these unwanted effects, we modified the already described BUM690 by exchanging the allyl-into a trifluoro-ethyl group to yield BUM532. Furthermore, we exchanged the nitrogen for oxygen in the trifluoro-ethyl group to yield BUM97.In the intrahippocampal kainic acid mouse model of TLE BUM532 ± phenobarbital (PB), bumetanide ± PB and PB alone significantly reduced hippocampal paroxysmal discharges (HPDs) but not spike trains. By contrast, treatment with BUM97 suppressed HPDs as well as spike trains dose-dependently, more pronounced compared to the other tested compounds and exerted a synergistic anticonvulsant effect with PB. Moreover, at higher doses BUM97 achieved long-lasting reduction of spike trains. In pentylenetetrazole-induced acute seizures only BUM532 combined with a sub-effective dose of PB increased the seizure threshold. No diuretic effects were observed at any dose of the three derivatives.Our data demonstrate the successful optimization of the pharmacological profile of bumetanide and the potential of the improved derivative BUM97 for the treatment of therapy-resistant TLE, in particular in combinatorial drug regimens with a GABA mimetic.

The Positive Allosteric Modulator of Alpha-2/3-Containing γ-Aminobutyric Acid Type A Receptors, KRM-II-81, Is Active in Pharmacoresistant Models of Epilepsy and in Human Epileptic Tissue

Abstract INTRODUCTION Epilepsy patients continue to suffer from the lack of efficacious medications. Recent attention has been directed toward the potential advantages of developing positive allosteric modulators of alpha-2/3-containing γ-aminobutyric acid type A (GABAA) receptors as antiepileptic drugs. A proof of principle has been reported with one such molecule in patients with photosensitive epilepsy. KRM-II-81 (5- (8-ethynyl-6- (pyridin-2-yl)-4H-benzo[f]imidazole[1,5-alpha][1,4]diazepin-3-yl)oxazole) is an orally-bioavailable compound recently designed for selectivity at alpha-2/3-containing GABAA receptors over the alpha-1-subtype involved in motor-impairing effects. KRM-II-81 has recently been reported to dampen seizure activity in rodents induced by acute and chronic seizure provocation. KRM-II-81 was often more efficacious than diazepam as an anticonvulsant while producing less motor impairment than diazepam. The reduced motor impact of KRM-II-81 is hypothesized to enable higher central target exposure and hence increased efficacy. METHODS The effects of KRM-II-81 were investigated in a mouse mesial temporal lobe model and a rat lamotrigine-resistant kindling model. We also explored the antiepileptic electrophysiological effects of KRM-II-81 in cortical slices from epileptic pediatric patients to help guide the development of novel compounds that might be valuable against antiepileptic drug-resistant epilepsies. RESULTS Mice with kainate-induced mesial temporal lobe seizures exhibited spontaneous recurrent hippocampal paroxysmal discharges (16.8 +/–2.5). KRM-II-81 significantly reduced the discharge frequency to 5.5 +/–1.4 after oral dosing at 15 mg/kg. KRM-II-81 also decreased convulsions in rats undergoing amygdala kindling in the presence of lamotrigine. In slices of epileptic cortex, KRM-II-81 produced a concentration-dependent dampening of network activity engendered by the GABAA receptor antagonist picrotoxin or the K+-channel modulator 4-aminopyridine. CONCLUSION This study provides increased levels of confidence regarding the unique anticonvulsant profile of KRM-II-81 and its potential as an improved antiepileptic drug. The data also help to solidify the veracity of alpha-2/3-containing GABAA receptors as a novel molecular target for antiepileptic drugs.

Transient use of a systemic adenosine kinase inhibitor attenuates epilepsy development in mice.

Over one-third of all patients with epilepsy are refractory to treatment and there is an urgent need to develop new drugs that can prevent the development and progression of epilepsy. Epileptogenesis is characterized by distinct histopathologic and biochemical changes, which include astrogliosis and increased expression of the adenosine-metabolizing enzyme adenosine kinase (ADK; EC 2.7.1.20). Increased expression of ADK contributes to epileptogenesis and is therefore a target for therapeutic intervention. We tested the prediction that the transient use of an ADK inhibitor administered during the latent phase of epileptogenesis can mitigate the development of epilepsy. We used the intrahippocampal kainic acid (KA) mouse model of temporal lobe epilepsy, which is characterized by ipsilateral hippocampal sclerosis with granule cell dispersion and the development of recurrent hippocampal paroxysmal discharges (HPDs). KA-injected mice were treated with the ADK inhibitor 5-iodotubercidin (5-ITU, 1.6mg/kg, b.i.d., i.p.) during the latent phase of epileptogenesis from day 3-8 after injury; the period when gradual increases in hippocampal ADK expression begin to manifest. HPDs were assessed at 6 and 9weeks after KA administration followed by epilepsy histopathology including assessment of granule cell dispersion, astrogliosis, and ADK expression. 5-ITU significantly reduced the percent time in seizures by at least 80% in 56% of mice at 6weeks post-KA. This reduction in seizure activity was maintained in 40% of 5-ITU-treated mice at 9weeks. 5-ITU also suppressed granule cell dispersion and prevented maladaptive ADK increases in these protected mice. Our results show that the transient use of a small-molecule ADK inhibitor, given during the early stages of epileptogenesis, has antiepileptogenic disease-modifying properties, which provides the rationale for further investigation into the development of a novel class of antiepileptogenic ADK inhibitors with increased efficacy for epilepsy prevention.

Model-guided control of hippocampal discharges by local direct current stimulation

Neurostimulation is an emerging treatment for drug-resistant epilepsies when surgery is contraindicated. Recent clinical results demonstrate significant seizure frequency reduction in epileptic patients, however the mechanisms underlying this therapeutic effect are largely unknown. This study aimed at gaining insights into local direct current stimulation (LDCS) effects on hyperexcitable tissue, by i) analyzing the impact of electrical currents locally applied on epileptogenic brain regions, and ii) characterizing currents achieving an “anti-epileptic” effect (excitability reduction). First, a neural mass model of hippocampal circuits was extended to accurately reproduce the features of hippocampal paroxysmal discharges (HPD) observed in a mouse model of epilepsy. Second, model predictions regarding current intensity and stimulation polarity were confronted to in vivo mice recordings during LDCS (n = 8). The neural mass model was able to generate realistic hippocampal discharges. Simulation of LDCS in the model pointed at a significant decrease of simulated HPD (in duration and occurrence rate, not in amplitude) for cathodal stimulation, which was successfully verified experimentally in epileptic mice. Despite the simplicity of our stimulation protocol, these results contribute to a better understanding of clinical benefits observed in epileptic patients with implanted neurostimulators. Our results also provide further support for model-guided design of neuromodulation therapy.

The intrahippocampal kainate mouse model of mesial temporal lobe epilepsy: Lack of electrographic seizure-like events in sham controls.

SummaryObjectiveThere is an ongoing debate about definition of seizures in experimental models of acquired epilepsy and how important adequate sham controls are in this respect. For instance, several mouse and rat strains exhibit high‐voltage rhythmic spike or spike‐wave discharges in the cortical electroencephalogram (EEG), which has to be considered when using such strains for induction of epilepsy by status epilepticus, traumatic brain injury, or other means. Mice developing spontaneous recurrent nonconvulsive and convulsive seizures after intrahippocampal injection of kainate are increasingly being used as a model of mesial temporal lobe epilepsy. We performed a prospective study in which EEG alterations occurring in this model were compared with the EEGs in appropriate sham controls, using hippocampal electrodes and video‐EEG monitoring.MethodsExperiments with intrahippocampal kainate (or saline) injections started when mice were about 8 weeks of age. Continuous video‐EEG recording via hippocampal electrodes was performed 6 weeks after surgery in kainate‐injected mice and sham controls, that is, at an age of about 14 weeks. Three days of continuous video‐EEG monitoring were compared between kainate‐injected mice and experimental controls.ResultsAs reported previously, kainate‐injected mice exhibited two types of highly frequent electrographic seizures: high‐voltage sharp waves, which were often monomorphic, and polymorphic hippocampal paroxysmal discharges. In addition, generalized convulsive clinical seizures were infrequently observed. None of these electrographic or electroclinical seizures were observed in sham controls. The only infrequently observed EEG abnormalities in sham controls were isolated spikes or spike clusters, which were also recorded in epileptic mice.SignificanceThis study rigorously demonstrates, by explicit comparison with the EEGs of sham controls, that the nonconvulsive paroxysmal events observed in this model are consequences of the induced epilepsy and not features of the EEG expected to be seen in some experimental control mice or unintentionally induced by surgical procedures.

Structural and functional changes during epileptogenesis in the mouse model of medial temporal lobe epilepsy.

An important issue in epilepsy research is to understand the structural and functional modifications leading to chronic epilepsy, characterized by spontaneous recurrent seizures, after initial brain insult. To address this issue, we recorded and analyzed electroencephalography (EEG) and quantitative magnetic resonance imaging (MRI) data during epileptogenesis in the in vivo mouse model of Medial Temporal Lobe Epilepsy (MTLE, kainate). Besides, this model of epilepsy is a particular form of drug-resistant epilepsy. The results indicate that high-field (4.7T) MRI parameters (T2-weighted; T2-quantitative) allow to detect the gradual neuro-anatomical changes that occur during epileptogenesis while electrophysiological parameters (number and duration of Hippocampal Paroxysmal Discharges) allow to assess the dysfunctional changes through the quantification of epileptiform activity. We found a strong correlation between EEG-based markers (invasive recording) and MRI-based parameters (non-invasive) periodically computed over the `latent period' that spans over two weeks, on average. These results indicated that both structural and functional changes occur in the considered epilepsy model and are considered as biomarkers of the installation of epilepsy. Additionally, such structural and functional changes can also be observed in human temporal lobe epilepsy. Interestingly, MRI imaging parameters could be used to track early (day-7) structural changes (gliosis, cell loss) in the lesioned brain and to quantify the evolution of epileptogenesis after traumatic brain injury.

The effects of carbamazepine in the intrahippocampal kainate model of temporal lobe epilepsy depend on seizure definition and mouse strain.

SummaryObjectiveMesial temporal lobe epilepsy (TLE) with hippocampal sclerosis is a predominant form of acquired epilepsy, characterized by recurrent simple and complex partial seizures that are often resistant to treatment. Mice developing spontaneous recurrent nonconvulsive and convulsive seizures after intrahippocampal injection of the excitotoxic glutamate agonist kainate are thought to represent a valuable model of mesial TLE. Epileptic electroencephalogram (EEG) activity recorded in this model from the kainate focus in the ipsilateral hippocampus is resistant to antiseizure drugs such as carbamazepine (CBZ). We compared the efficacy of CBZ in this model in two different mouse strains (FVB/N and NMRI). Furthermore, we evaluated whether changes in the definition of electrographic seizures affect the antiseizure efficacy of CBZ.MethodsAs in previous studies, two types of epileptic EEG activity were defined: high‐voltage sharp waves (HVSWs) and hippocampal paroxysmal discharges (HPDs). The characteristics of these paroxysmal EEG events in epileptic mice were compared with EEG criteria for nonconvulsive seizures in patients. For HVSWs, different spike frequencies, interevent intervals, and amplitudes were used as inclusion and exclusion criteria. In addition to CBZ, some experiments were performed with diazepam (DZP) and phenobarbital (PB).ResultsFemale epileptic FVB/N mice predominantly exhibited frequent HVSWs, but only infrequent HPDs or secondarily generalized convulsive seizures. Slight changes in HVSW definition determined whether they were resistant or responsive to CBZ. Male NMRI mice exhibited both HVSWs and HPDs. HVSWs were more resistant than HPDs to suppression by CBZ. Both types of epileptic EEG activity were rapidly suppressed by DZP and PB.SignificanceThe data demonstrate that focal electrographic seizures in the intrahippocampal kainate mouse model are less resistant than previously thought. Both mouse strain and the criteria chosen for definition of EEG seizures determine whether such seizures are drug‐resistant or ‐responsive.

Activation of GABAA receptors controls mesiotemporal lobe epilepsy despite changes in chloride transporters expression: In vivo and in silico approach

Mesiotemporal lobe Epilepsy (MTLE), the most frequent form of focal epilepsy, is often drug-resistant. Enriching the epileptic focus with GABA-releasing engineered cells has been proposed as a strategy to prevent seizures. However, ex vivo data from animal models and MTLE patients suggest that, due to changes in chloride homeostasis, GABAA receptor activation is depolarizing and partly responsible for focal interictal discharges and seizure initiation. To understand how these two contradictory aspects of GABAergic neurotransmission coexist in MTLE, we used an established mouse model of MTLE presenting hippocampal sclerosis and recurrent hippocampal paroxysmal discharges (HPDs) 30–40days after a unilateral injection of kainate in the dorsal hippocampus. We first showed that injections of GABAA receptor agonists either systemically or directly into hippocampus suppressed HPDs. Western-blotting and immunostaining revealed that levels of α1, α3 and γ2 GABAA receptor subunits were increased in epileptic mice, compared to saline controls, while levels of R1 and R2 GABAB receptor subunits but also NR1, NR2A and NR2B NMDA receptor subunits and GluR1 and GluR2 AMPA receptor subunits were decreased. In addition, we showed that the expression of the transporter NKCC1, which load neurons with chloride, was increased, whereas KCC2, a chloride extruder, was decreased and that HPDs were suppressed by injection of blockers of NKCC1. These different changes were integrated in a numerical model, and in silico simulations supported the notion that chloride imbalance impair local inhibitory control of pyramidal neurons' activity in this model of MTLE. However, our numerical model also suggested that lasting activation of these receptors restore physiological intracellular chloride concentrations and suppress HPDs. Overall, our study suggests that activation of GABAA receptor remains an effective antiepileptic strategy to suppress focal seizures in MTLE, and demonstrates that modeling and simulation studies provide new insights about the cellular and synaptic mechanisms of this disease.

Differential Effects of Antiepileptic Drugs on Focal Seizures in the Intrahippocampal Kainate Mouse Model of Mesial Temporal Lobe Epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most common form of drug-refractory epilepsy. Most of the morphological and electrophysiological features of human MTLE can be reproduced in a mouse by a unilateral intrahippocampal injection of kainate (MTLE mouse model). The effects of antiepileptic drugs (AEDs) on the occurrence of recurrent focal hippocampal seizures in this model remain to be specified. Here, we addressed the pharmacological reactivity of this model to the most commonly used AEDs. Using depth electroencephalographical (EEG) recordings, we tested the dose-response effects of acute injection of nine AEDs on the occurrence of hippocampal paroxysmal discharges (HPDs) as well as on ictal and interictal power spectra in the MTLE mouse model. Valproate, carbamazepine, and lamotrigine dose dependently suppressed HPDs and modified the general behavior and/or EEG activity. Levetiracetam and pregabalin suppressed HPDs at high doses but without any behavioral nor interictal EEG changes. Finally, phenobarbital, tiagabine, vigabatrin, and diazepam suppressed HPDs in a dose-dependent manner at doses devoid of obvious behavioral effects. The MTLE mouse model displays a differential sensitivity to AEDs with a greater efficacy of drug that facilitates GABAergic transmission. This model provides an efficient tool to identify new treatment for drug-resistant forms of focal epilepsies.

Significant effects of sex, strain, and anesthesia in the intrahippocampal kainate mouse model of mesial temporal lobe epilepsy.

The intrahippocampal kainate mouse model of mesial temporal lobe epilepsy is increasingly being used for studies on epileptogenesis and antiepileptogenesis. Almost all previous studies used male mice for this purpose, and no study is available in this or other models of acquired epilepsy that directly compared epileptogenesis in female and male rodents. Epidemiological studies suggest that gender may affect susceptibility to epilepsy and its prognosis; therefore, one goal of this study was to investigate whether sex has an influence on latent period and epileptogenesis in the intrahippocampal kainate model in mice. Another aspect that was examined in the present study was whether mouse strain differences in epileptogenesis exist. Finally, we examined the effects of different types of anesthesia (chloral hydrate, isoflurane) on kainate-induced status epilepticus (SE) and epileptogenesis. Continuous (24/7) video-EEG monitoring was used during SE and the 2 weeks following SE as well as 4-6 weeks after SE. In male NMRI mice with chloral hydrate anesthesia during kainate injection, SE was followed by a seizure-free latent period of 10-14 days if hippocampal paroxysmal discharges (HPDs) recorded from the kainate focus were considered the onset of epilepsy. Anesthesia with isoflurane led to a more rapid onset and higher severity of SE, and not all male NMRI mice exhibited a seizure-free latent period. Female NMRI mice differed from male animals in the lack of any clear latent period, independently of anesthesia type. Furthermore, HPDs were only rarely observed. These problems were not resolved by decreasing the dose of kainate or using other strains (C57BL/6, FVB/N) of female mice. The present data are the first to demonstrate marked sex-related differences in the latent period following brain injury in a rodent model of acquired epilepsy. Furthermore, our data demonstrate that the choice of anesthestic agent during kainate administration affects SE severity and as a consequence, the latent period, which may explain some of the differences reported for this model in the literature.

Activation of mTOR signaling pathway is secondary to neuronal excitability in a mouse model of mesio-temporal lobe epilepsy.

Recent studies in animal models have suggested that the mammalian target of rapamycin (mTOR) signaling pathway is involved in several features of mesio-temporal lobe epilepsy (MTLE), and that its inhibition could have therapeutic interests. However, it remains controversial whether mTOR activation is the cause or the consequence of MTLE. We previously showed in a mouse model of MTLE associated with hippocampal sclerosis that increased neuronal excitability and brain-derived neurotrophic factor (BDNF) overexpression contribute to the development of morphological features of this form of epilepsy. Here, we addressed whether mTOR activation promotes MTLE epileptogenesis via increasing neuronal excitability and/or BDNF expression or rather mediates neuroplasticity associated with hippocampal sclerosis. In mice injected intrahippocampally with kainate (1 nmol), we showed a biphasic increase of phospho-S6 (p-S6) ribosomal protein expression, the downstream product of the mTOR signaling pathway, in the dispersed granule cell layer (GCL) of the dentate gyrus with a second phase lasting up to 6 months. Chronic treatment with rapamycin suppressed p-S6 expression, granule cell dispersion and mossy fiber sprouting, but did not reduce cell loss, BDNF overexpression, glutamic acid decarboxylase (GAD)67 expression or the development of hippocampal paroxysmal discharges. Neuronal inhibition by midazolam (2 × 10 mg/kg, i.p.) abolished the increased expression of p-S6 in the dispersed GCL. Our data suggest that activation of the mTOR signaling pathway results from the increased neuronal excitation that develops in the GCL and may contribute to MTLE morphological changes. However, these data do not support the role of this pathway in the development of MTLE or its inhibition as a therapy for this form of epilepsy.

Long-term modifications of epileptogenesis and hippocampal rhythms after prolonged hyperthermic seizures in the mouse

Complex febrile seizures are often reported in the history of patients with mesio-temporal lobe epilepsy (MTLE) but their role in its physiopathology remains controversial. We postulated that prolonged hyperthermic seizures might, as a “single-hit”, modify the hippocampal rhythms, facilitate epileptogenesis and influence subsequent epilepsy when a second-hit already exists or subsequently occurs. To test this hypothesis, we examined the effects of hyperthermic seizures (30min at 40–41°C) at postnatal day 10 on hippocampal activity in C57BL/6J mice in comparison to their littermates in sham conditions (22°C), with or without another insult. Using local field potential, we observed an asymmetry in the hippocampal susceptibility to seize in hyperthermic conditions. When these mice were adult, an asymmetrical increase of low frequency power was also recorded in the hippocampus when compared to sham animals. Using two different “two-hit” protocols, no increase in seizures or hippocampal discharge frequency or duration was observed, either in mice with a genetic CA3 dysplasia (Dcx knockout), or in mice injected with kainate into the dorsal hippocampus at P60. However, in the latter condition, which is reminiscent of MTLE, the hyperthermic seizures accelerated epileptogenesis and decreased the power in the high frequency gamma band, as well as decreasing the coherence between hippocampi and the involvement of the contralateral hippocampus during hippocampal paroxysmal discharges. Our data suggest that a single episode of prolonged hyperthermic seizures does not induce per se, but accelerates epileptogenesis and could lead to an asymmetrical dysfunction in the hippocampal rhythmicity in both physiological and pathological conditions.

Expression of the AMPA receptor subunit GluR1 is associated with granule cell maturation in the dentate gyrus

Mesiotemporal lobe Epilepsy (MTLE), the most frequent form of focal epilepsy, is often drug-resistant. Enriching the epileptic focus with GABA-releasing engineered cells has been proposed as a strategy to prevent seizures. However, ex vivo data from animal models and MTLE patients suggest that, due to changes in chloride homeostasis, GABAA receptor activation is depolarizing and partly responsible for focal interictal discharges and seizure initiation. To understand how these two contradictory aspects of GABAergic neurotransmission coexist in MTLE, we used an established mouse model of MTLE presenting hippocampal sclerosis and recurrent hippocampal paroxysmal discharges (HPDs) 30–40 days after a unilateral injection of kainate in the dorsal hippocampus. We first showed that injections of GABAA receptor agonists either systemically or directly into hippocampus suppressed HPDs. Western-blotting and immunostaining revealed that levels of α1, α3 and γ2 GABAA receptor subunits were increased in epileptic mice, compared to saline controls, while levels of R1 and R2 GABAB receptor subunits but also NR1, NR2A and NR2B NMDA receptor subunits and GluR1 and GluR2 AMPA receptor subunits were decreased. In addition, we showed that the expression of the transporter NKCC1, which load neurons with chloride, was increased, whereas KCC2, a chloride extruder, was decreased and that HPDs were suppressed by injection of blockers of NKCC1. These different changes were integrated in a numerical model, and in silico simulations supported the notion that chloride imbalance impair local inhibitory control of pyramidal neurons' activity in this model of MTLE. However, our numerical model also suggested that lasting activation of these receptors restore physiological intracellular chloride concentrations and suppress HPDs. Overall, our study suggests that activation of GABAA receptor remains an effective antiepileptic strategy to suppress focal seizures in MTLE, and demonstrates that modeling and simulation studies provide new insights about the cellular and synaptic mechanisms of this disease.