Kainate Mouse Model Research Articles

A comparison of the antiepileptogenic efficacy of two rationally chosen multitargeted drug combinations in a rat model of posttraumatic epilepsy

Post-traumatic epilepsy (PTE) is a recurrent and often drug-refractory seizure disorder caused by traumatic brain injury (TBI). No single drug treatment prevents PTE, but preventive drug combinations that may prophylax against PTE have not been studied. Based on a systematic evaluation of rationally chosen drug combinations in the intrahippocampal kainate (IHK) mouse model of acquired epilepsy, we identified two multi-targeted drug cocktails that exert strong antiepileptogenic effects. The first, a combination of levetiracetam (LEV) and topiramate, only partially prevented spontaneous recurrent seizures in the model. We therefore added atorvastatin (ATV) to the therapeutic cocktail (TC) to increase efficacy, forming “TC-001”. The second cocktail – a combination of LEV, ATV, and ceftriaxone, termed “TC-002” – completely prevented epilepsy in the mouse IHK model. In the present proof-of-concept study, we tested whether the two drug cocktails prevent epilepsy in a rat PTE model in which recurrent electrographic seizures develop after severe rostral parasagittal fluid percussion injury (FPI). Following FPI, rats were either treated over 3–4 weeks with vehicle or drug cocktails, starting either 1 or 4–6 h after the injury. Using mouse doses of TC-001 and TC-002, no significant antiepileptogenic effect was obtained in the rat PTE model. However, when using allometric scaling of drug doses to consider the differences in body surface area between mice and rats, PTE was prevented by TC-002. Furthermore, the latter drug cocktail partially prevented the loss of perilesional cortical parvalbumin-positive GABAergic interneurons. Plasma and brain drug analysis showed that these effects of TC-002 occurred at clinically relevant levels of the individual TC-002 drug components. In silico analysis of drug-drug brain protein interactions by the STITCH database indicated that TC-002 impacts a larger functional network of epilepsy-relevant brain proteins than each drug alone, providing a potential network pharmacology explanation for the observed antiepileptogenic and neuroprotective effects observed with this combination.

Probing hippocampal stimulation in experimental temporal lobe epilepsy with functional MRI.

Electrical neurostimulation is currently used to manage epilepsy, but the most effective approach for minimizing seizure occurrence is uncertain. While functional MRI (fMRI) can reveal which brain areas are affected by stimulation, simultaneous deep brain stimulation (DBS)-fMRI examinations in patients are rare and the possibility to investigate multiple stimulation protocols is limited. In this study, we utilized the intrahippocampal kainate mouse model of mesial temporal lobe epilepsy (mTLE) to systematically examine the brain-wide responses to electrical stimulation using fMRI. We compared fMRI responses of saline-injected controls and epileptic mice during stimulation in the septal hippocampus (HC) at 10 Hz and demonstrated the effects of different stimulation amplitudes (80-230 μA) and frequencies (1-100 Hz) in epileptic mice. Motivated by recent studies exploring 1 Hz stimulation to prevent epileptic seizures, we furthermore investigated the effect of prolonged 1 Hz stimulation with fMRI. Compared to sham controls, epileptic mice showed less propagation to the contralateral HC, but significantly stronger responses in the ipsilateral HC and a wider spread to the entorhinal cortex and septal region. Varying the stimulation amplitude had little effect on the resulting activation patterns, whereas the stimulation frequency represented the key parameter and determined whether the induced activation remained local or spread from the hippocampal formation into cortical areas. Prolonged stimulation of epileptic mice at 1 Hz caused a slight reduction in local excitability. In this way, our study contributes to a better understanding of these stimulation paradigms.

ArcKR expression modifies synaptic plasticity following epileptic activity: Differential effects with invitro and invivo seizure-induction protocols.

Pathological forms of neural activity, such as epileptic seizures, modify the expression pattern of multiple proteins, leading to persistent changes in brain function. One such protein is activity-regulated cytoskeleton-associated protein (Arc), which is critically involved in protein-synthesis-dependent synaptic plasticity underlying learning and memory. In the present study, we have investigated how the expression of ArcKR, a form of Arc in which the ubiquitination sites have been mutated, resulting in slowed Arc degradation, modifies group I metabotropic glutamate receptor-mediated long-term depression (G1-mGluR-LTD) following seizures. We used a knock-in mice line that express ArcKR and two hyperexcitation models: an invitro model, where hippocampal slices were exposed to zero Mg2+, 6 mM K+; and an invivo model, where kainic acid was injected unilaterally into the hippocampus. In both models, field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 region of hippocampal slices in response to Schaffer collateral stimulation and G1-mGluR-LTD was induced chemically with the group 1 mGluR agonist DHPG. In the invitro model, ArcKR expression enhanced the effects of seizure activity and increased the magnitude of G1-mGluR LTD, an effect that could be blocked with the mGluR5 antagonist MTEP. In the invivo model, fEPSPs were significantly smaller in slices from ArcKR mice and were less contaminated by population spikes. In this model, the amount of G1-mGluR-LTD was significantly less in epileptic slices from ArcKR mice as compared to wildtype (WT) mice. We have shown that expression of ArcKR, a form of Arc in which degradation is reduced, significantly modulates the magnitude of G1-mGluR-LTD following epileptic seizures. However, the effect of ArcKR on LTD depends on the epileptic model used, with enhancement of LTD in an invitro model and a reduction in the kainate mouse model.

Involvement of the contralateral hippocampus in ictal-like but not interictal epileptic activities in the kainate mouse model of temporal lobe epilepsy.

Animal and human studies have shown that the seizure-generating region is vastly dependent on distant neuronal hubs that can decrease duration and propagation of ongoing seizures. However, we still lack a comprehensive understanding of the impact of distant brain areas on specific interictal and ictal epileptic activities (e.g., isolated spikes, spike trains, seizures). Such knowledge is critically needed, because all kinds of epileptic activities are not equivalent in terms of clinical expression and impact on the progression of the disease. We used surface high-density electroencephalography and multisite intracortical recordings, combined with pharmacological silencing of specific brain regions in the well-known kainate mouse model of temporal lobe epilepsy. We tested the impact of selective regional silencing on the generation of epileptic activities within a continuum ranging from very transient to more sustained and long-lasting discharges reminiscent of seizures. Silencing the contralateral hippocampus completely suppresses sustained ictal activities in the focus, as efficiently as silencing the focus itself, but whereas focus silencing abolishes all focus activities, contralateral silencing fails to control transient spikes. In parallel, we observed that sustained focus epileptiform discharges in the focus are preceded by contralateral firing and more strongly phase-locked to bihippocampal delta/theta oscillations than transient spiking activities, reinforcing the presumed dominant role of the contralateral hippocampus in promoting long-lasting, but not transient, epileptic activities. Altogether, our work provides suggestive evidence that the contralateral hippocampus is necessary for the interictal to ictal state transition and proposes that crosstalk between contralateral neuronal activity and ipsilateral delta/theta oscillation could be a candidate mechanism underlying the progression from short- to long-lasting epileptic activities.

Canonical Wnt activator Chir99021 prevents epileptogenesis in the intrahippocampal kainate mouse model of temporal lobe epilepsy

The Wnt signaling pathway mediates the development of dentate granule cell neurons in the hippocampus. These neurons are central to the development of temporal lobe epilepsy and undergo structural and physiological remodeling during epileptogenesis, which results in the formation of epileptic circuits. The pathways responsible for granule cell remodeling during epileptogenesis have yet to be well defined, and represent therapeutic targets for the prevention of epilepsy. The current study explores Wnt signaling during epileptogenesis and for the first time describes the effect of Wnt activation using Wnt activator Chir99021 as a novel anti-epileptogenic therapeutic approach.Focal mesial temporal lobe epilepsy was induced by intrahippocampal kainate (IHK) injection in wild-type and POMC-eGFP transgenic mice. Wnt activator Chir99021 was administered daily, beginning 3 h after seizure induction, and continued up to 21-days. Immature granule cell morphology was quantified in the ipsilateral epileptogenic zone and the contralateral peri-ictal zone 14 days after IHK, targeting the end of the latent period. Bilateral hippocampal electrocorticographic recordings were performed for 28-days, 7-days beyond treatment cessation. Hippocampal behavioral tests were performed after completion of Chir99021 treatment.Consistent with previous studies, IHK resulted in the development of epilepsy after a 14 day latent period in this well-described mouse model. Activation of the canonical Wnt pathway with Chir99021 significantly reduced bilateral hippocampal seizure number and duration. Critically, this effect was retained after treatment cessation, suggesting a durable antiepileptogenic change in epileptic circuitry. Morphological analyses demonstrated that Wnt activation prevented pathological remodeling of the primary dendrite in both the epileptogenic zone and peri-ictal zone, changes in which may serve as a biomarker of epileptogenesis and anti-epileptogenic treatment response in pre-clinical studies. These findings were associated with improved object location memory with Chir99021 treatment after IHK.This study provides novel evidence that canonical Wnt activation prevents epileptogenesis in the IHK mouse model of mesial temporal lobe epilepsy, preventing pathological remodeling of dentate granule cells. Wnt signaling may therefore play a key role in mesial temporal lobe epileptogenesis, and Wnt modulation may represent a novel therapeutic strategy in the prevention of epilepsy.

Low frequency stimulation for seizure suppression: Identification of optimal targets in the entorhinal-hippocampal circuit

BackgroundMesial temporal lobe epilepsy (MTLE) with hippocampal sclerosis (HS) is a common form of drug-resistant focal epilepsy in adults. Treatment for pharmacoresistant patients remains a challenge, with deep brain stimulation (DBS) showing promise for alleviating intractable seizures. This study explores the efficacy of low frequency stimulation (LFS) on specific neuronal targets within the entorhinal-hippocampal circuit in a mouse model of MTLE. ObjectiveOur previous research demonstrated that LFS of the medial perforant path (MPP) fibers in the sclerotic hippocampus reduced seizures in epileptic mice. Here, we aimed to identify the critical neuronal population responsible for this antiepileptic effect by optogenetically stimulating presynaptic and postsynaptic compartments of the MPP-dentate granule cell (DGC) synapse at 1 Hz. We hypothesize that specific targets for LFS can differentially influence seizure activity depending on the cellular identity and location within or outside the seizure focus. MethodsWe utilized the intrahippocampal kainate (ihKA) mouse model of MTLE and targeted specific neural populations using optogenetic stimulation. We recorded intracranial neuronal activity from freely moving chronically epileptic mice with and without optogenetic LFS up to 3 h. ResultsWe found that LFS of MPP fibers in the sclerotic hippocampus effectively suppressed epileptiform activity while stimulating principal cells in the MEC had no impact. Targeting DGCs in the sclerotic septal or non-sclerotic temporal hippocampus with LFS did not reduce seizure numbers but shortened the epileptiform bursts. ConclusionPresynaptic stimulation of the MPP-DGC synapse within the sclerotic hippocampus is critical for seizure suppression via LFS.

Reproducible network changes occur in a mouse model of temporal lobe epilepsy but do not correlate with disease severity

Studying the development of brain network disruptions in epilepsy is challenged by the paucity of data before epilepsy onset. Here, we used the unilateral, kainate mouse model of hippocampal epilepsy to investigate brain network changes before and after epilepsy onset and their stability across time.Using 32 epicranial electrodes distributed over the mouse hemispheres, we analyzed EEG epochs free from epileptic activity in 15 animals before and 28 days after hippocampal injection (group 1) and in 20 animals on two consecutive days (d28 and d29, group 2). Statistical dependencies between electrodes were characterized with the debiased-weighted phase lag index. We analyzed: a) graph metric changes from baseline to chronic stage (d28) in group 1; b) their reliability across d28 and d29, in group 2; c) their correlation with epileptic activity (EA: seizure, spike and fast-ripple rates), averaged over d28 and d29, in group 2.During the chronic stage, intra-hemispheric connections of the non-injected hemisphere strengthened, yielding an asymmetrical network in low (4–8 Hz) and high theta (8–12 Hz) bands. The contralateral hemisphere also became more integrated and segregated within the high theta band. Both network topology and EEG markers of EA were stable over consecutive days but not correlated with each other.Altogether, we show reproducible large-scale network modifications after the development of focal epilepsy. These modifications are mostly specific to the non-injected hemisphere. The absence of correlation with epileptic activity does not allow to specifically ascribe these network changes to mechanisms supporting EA or rather compensatory inhibition but supports the notion that epilepsy extends beyond the sole repetition of EA and impacts network that might not be involved in EA generation.

On-demand low-frequency stimulation for seizure control: efficacy and behavioural implications.

Mesial temporal lobe epilepsy (MTLE), the most common form of focal epilepsy in adults, is often refractory to medication and associated with hippocampal sclerosis. Deep brain stimulation represents an alternative treatment option for drug-resistant patients who are ineligible for resective brain surgery. In clinical practice, closed-loop stimulation at high frequencies is applied to interrupt ongoing seizures, yet has (i) a high incidence of false detections; (ii) the drawback of delayed seizure-suppressive intervention; and (iii) limited success in sclerotic tissue. As an alternative, low-frequency stimulation (LFS) has been explored recently in patients with focal epilepsies. In preclinical epilepsy models, hippocampal LFS successfully prevented seizures when applied continuously. Since it would be advantageous to reduce the stimulation load, we developed a protocol for on-demand LFS. Given the importance of the hippocampus for navigation and memory, we investigated potential consequences of LFS on hippocampal function. To this end, we used the intrahippocampal kainate mouse model, which recapitulates the key features of MTLE, including spontaneous seizure activity and hippocampal sclerosis. Specifically, our online detection algorithm monitored epileptiform activity in hippocampal local field potential recordings and identified short epileptiform bursts preceding focal seizure clusters, triggering hippocampal LFS to stabilize the network state. To probe behavioural performance, we tested the acute influence of LFS on anxiety-like behaviour in the light-dark box test, spatial and non-spatial memory in the object location memory and novel object recognition test, as well as spatial navigation and long-term memory in the Barnes maze. On-demand LFS was almost as effective as continuous LFS in preventing focal seizure clusters but with a significantly lower stimulation load. When we compared the behavioural performance of chronically epileptic mice to healthy controls, we found that both groups were equally mobile, but epileptic mice displayed an increased anxiety level, altered spatial learning strategy and impaired memory performance. Most importantly, with the application of hippocampal LFS before behavioural training and test sessions, we could rule out deleterious effects on cognition and even show an alleviation of deficits in long-term memory recall in chronically epileptic mice. Taken together, our findings may provide a promising alternative to current therapies, overcoming some of their major limitations, and inspire further investigation of LFS for seizure control in focal epilepsy syndromes.

Differential vulnerability of neuronal subpopulations of the subiculum in a mouse model for mesial temporal lobe epilepsy.

Selective loss of inhibitory interneurons (INs) that promotes a shift toward an excitatory predominance may have a critical impact on the generation of epileptic activity. While research on mesial temporal lobe epilepsy (MTLE) has mostly focused on hippocampal changes, including IN loss, the subiculum as the major output region of the hippocampal formation has received less attention. The subiculum has been shown to occupy a key position in the epileptic network, but data on cellular alterations are controversial. Using the intrahippocampal kainate (KA) mouse model for MTLE, which recapitulates main features of human MTLE such as unilateral hippocampal sclerosis and granule cell dispersion, we identified cell loss in the subiculum and quantified changes in specific IN subpopulations along its dorso-ventral axis. We performed intrahippocampal recordings, FluoroJade C-staining for degenerating neurons shortly after status epilepticus (SE), fluorescence in situ hybridization for glutamic acid decarboxylase (Gad) 67 mRNA and immunohistochemistry for neuronal nuclei (NeuN), parvalbumin (PV), calretinin (CR) and neuropeptide Y (NPY) at 21 days after KA. We observed remarkable cell loss in the ipsilateral subiculum shortly after SE, reflected in lowered density of NeuN+ cells in the chronic stage when epileptic activity occurred in the subiculum concomitantly with the hippocampus. In addition, we show a position-dependent reduction of Gad67-expressing INs by ∼50% (along the dorso-ventral as well as transverse axis of the subiculum). This particularly affected the PV- and to a lesser extent CR-expressing INs. The density of NPY-positive neurons was increased, but the double-labeling for Gad67 mRNA expression revealed that an upregulation or de novo expression of NPY in non-GABAergic cells with a concomitant reduction of NPY-positive INs underlies this observation. Our data suggest a position- and cell type-specific vulnerability of subicular INs in MTLE, which might contribute to hyperexcitability of the subiculum, reflected in epileptic activity.

TGF-β Activated Kinase 1 (TAK1) Is Activated in Microglia After Experimental Epilepsy and Contributes to Epileptogenesis

Increasing evidence suggests that inflammation promotes epileptogenesis. TAK1 is a central enzyme in the upstream pathway of NF-κB and is known to play a central role in promoting neuroinflammation in neurodegenerative diseases. Here, we investigated the cellular role of TAK1 in experimental epilepsy. C57Bl6 and transgenic mice with inducible and microglia-specific deletion of Tak1 (Cx3cr1CreER:Tak1fl/fl) were subjected to the unilateral intracortical kainate mouse model of temporal lobe epilepsy (TLE). Immunohistochemical staining was performed to quantify different cell populations. The epileptic activity was monitored by continuous telemetric electroencephalogram (EEG) recordings over a period of 4 weeks. The results show that TAK1 was activated predominantly in microglia at an early stage of kainate-induced epileptogenesis. Tak1 deletion in microglia resulted in reduced hippocampal reactive microgliosis and a significant decrease in chronic epileptic activity. Overall, our data suggest that TAK1-dependent microglial activation contributes to the pathogenesis of chronic epilepsy.

LINCs Are Vulnerable to Epileptic Insult and Fail to Provide Seizure Control via On-Demand Activation.

Temporal lobe epilepsy (TLE) is notoriously pharmacoresistant, and identifying novel therapeutic targets for controlling seizures is crucial. Long-range inhibitory neuronal nitric oxide synthase-expressing cells (LINCs), a population of hippocampal neurons, were recently identified as a unique source of widespread inhibition in CA1, able to elicit both GABAA-mediated and GABAB-mediated postsynaptic inhibition. We therefore hypothesized that LINCs could be an effective target for seizure control. LINCs were optogenetically activated for on-demand seizure intervention in the intrahippocampal kainate (KA) mouse model of chronic TLE. Unexpectedly, LINC activation at 1 month post-KA did not substantially reduce seizure duration in either male or female mice. We tested two different sets of stimulation parameters, both previously found to be effective with on-demand optogenetic approaches, but neither was successful. Quantification of LINCs following intervention revealed a substantial reduction of LINC numbers compared with saline-injected controls. We also observed a decreased number of LINCs when the site of initial insult (i.e., KA injection) was moved to the amygdala [basolateral amygdala (BLA)-KA], and correspondingly, no effect of light delivery on BLA-KA seizures. This indicates that LINCs may be a vulnerable population in TLE, regardless of the site of initial insult. To determine whether long-term circuitry changes could influence outcomes, we continued testing once a month for up to 6 months post-KA. However, at no time point did LINC activation provide meaningful seizure suppression. Altogether, our results suggest that LINCs are not a promising target for seizure inhibition in TLE.

Granule Cell Layer Morphology and Wnt Signaling in Temporal Lobe Epilepsy

Background and Hypothesis: Temporal lobe epilepsy (TLE) is the most common human seizure disorder and can develop after neurologic insults such as trauma or infection. No treatment exists to prevent the development of epilepsy during this critical period. Epileptogenesis is characterized by pathological neuronal network remodeling in the hippocampal dentate gyrus (DG). Previously, we found that Wnt pathway signaling is dysregulated in the kainate (KA) mouse model of TLE, such that Wnt antagonism exacerbated epileptogenic DG remodeling. We hypothesize that Wnt agonism will mitigate pathological DG remodeling of the granule cell layer (GCL) during epileptogenesis.Project Methods: TLE was induced by unilateral intrahippocampal KA injection in POMC-eGFP transgenic mice, while controls received saline. Mice received injections of vehicle or Wnt agonist Chir99021 daily. eGFP+ immature dentate granule cells were characterized by confocal microscopy. GCL width and immature dentate granule neuronal migration in the ictal/ipsilateral and peri-ictal/contralateral DG were quantified. Quantitative analyses were performed to compare means of the 4 groups.Results: We found that GCL width significantly increased in the ictal zone 2 weeks after seizure induction in both KA groups and was not mitigated by Chir treatment. Immature dentate granule cell migration also increased in the ictal zone in the KA groups and was not altered by Chir treatment. In the peri-ictal zone, GCL width and cell migration were unchanged across KA and saline control mice.Conclusion and Potential Impact: The Wnt agonist Chir99201 did not appear to alter GCL morphology in control or KA mice. It is likely that Wnt signaling may impact neuronal functioning rather than morphology in DG remodeling, and this will be explored through future electrophysiological studies. The Wnt pathway remains a potential therapeutic target in the prevention of the development of epilepsy.

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.

Integration of the CA2 region in the hippocampal network during epileptogenesis.

The CA2 pyramidal cells are mostly resistant to cell death in mesial temporal lobe epilepsy (MTLE) with hippocampal sclerosis, but they are aberrantly integrated into the epileptic hippocampal network via mossy fiber sprouting. Furthermore, they show increased excitability in vitro in hippocampal slices obtained from human MTLE specimens or animal epilepsy models. Although these changes promote CA2 to contribute to epileptic activity (EA) in vivo, the role of CA2 in the epileptic network within and beyond the sclerotic hippocampus is still unclear. We used the intrahippocampal kainate mouse model for MTLE, which recapitulates most features of the human disease including pharmacoresistant epileptic seizures and hippocampal sclerosis, with preservation of dentate gyrus (DG) granule cells and CA2 pyramidal cells. In vivo recordings with electrodes in CA2 and the DG showed that EA occurs at high coincidence between the ipsilateral DG and CA2 and current source density analysis of silicon probe recordings in dorsal ipsilateral CA2 revealed CA2 as a local source of EA. Cell-specific viral tracing in Amigo2-icreERT2 mice confirmed the preservation of the axonal projection from ipsilateral CA2 pyramidal cells to contralateral CA2 under epileptic conditions and indeed, EA propagated from ipsi- to contralateral CA2 with increasing likelihood with time after KA injection, but always at lower intensity than within the ipsilateral hippocampus. Furthermore, we show that CA2 presents with local theta oscillations and like the DG, shows a pathological reduction of theta frequency already from 2 days after KA onward. The early changes in activity might be facilitated by the loss of glutamic acid decarboxylase 67 (Gad67) mRNA-expressing interneurons directly after the initial status epilepticus in ipsi- but not contralateral CA2. Together, our data highlight CA2 as an active player in the epileptic network and with its contralateral connections as one possible router of aberrant activity.

Reactive microglia are the major source of tumor necrosis factor alpha and contribute to astrocyte dysfunction and acute seizures in experimental temporal lobe epilepsy.

Extensive microglia reactivity has been well described in human and experimental temporal lobe epilepsy (TLE). To date, however, it is not clear whether and based on which molecular mechanisms microglia contribute to the development and progression of focal epilepsy. Astroglial gap junction coupled networks play an important role in regulating neuronal activity and loss of interastrocytic coupling causally contributes to TLE. Here, we show in the unilateral intracortical kainate (KA) mouse model of TLE that reactive microglia are primary producers of tumor necrosis factor (TNF)α and contribute to astrocyte dysfunction and severity of status epilepticus (SE). Immunohistochemical analyses revealed pronounced and persistent microglia reactivity, which already started 4 h after KA-induced SE. Partial depletion of microglia using a colony stimulating factor 1 receptor inhibitor prevented early astrocyte uncoupling and attenuated the severity of SE, but increased the mortality of epileptic mice following surgery. Using microglia-specific inducible TNFα knockout mice we identified microglia as the major source of TNFα during early epileptogenesis. Importantly, microglia-specific TNFα knockout prevented SE-induced gap junction uncoupling in astrocytes. Continuous telemetric EEG recordings revealed that during the first 4 weeks after SE induction, microglial TNFα did not significantly contribute to spontaneous generalized seizure activity. Moreover, the absence of microglial TNFα did not affect the development of hippocampal sclerosis but attenuated gliosis. Taken together, these data implicate reactive microglia in astrocyte dysfunction and network hyperexcitability after an epileptogenic insult.

Initiation of Experimental Temporal Lobe Epilepsy by Early Astrocyte Uncoupling Is Independent of TGFβR1/ALK5 Signaling.

Blood–brain barrier (BBB) dysfunction following brain insults has been associated with the development and progression of focal epilepsy, although the underlying molecular mechanisms are not fully elucidated yet. Activation of transforming growth factor beta (TGFβ) signaling in astrocytes by extravasated albumin impairs the ability of astrocytes to properly interact with neurons, eventually leading to epileptiform activity. We used the unilateral intracortical kainate mouse model of temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) to gain further insights into the role of BBB leakage in status epilepticus (SE)-induced epileptogenesis. Immunohistochemical examination revealed pronounced albumin extravasation already 4 h after SE induction. Astrocytes were virtually devoid of albumin immunoreactivity (IR), indicating the lack of uptake by this time point. Inhibition of the TGFβ pathway by the specific TGFβ receptor 1 (TGFβR1) kinase inhibitor IPW-5371 did not prevent seizure-induced reduction of astrocytic gap junction coupling. Thus, loss of coupling, which is thought to play a causative role in triggering TLE-HS, is most likely not mediated by extravasated albumin. Continuous telemetric EEG recordings and video monitoring performed over a period of 4 weeks after epilepsy induction revealed that inhibition of the TGFβ pathway during the initial phase of epileptogenesis slightly attenuated acute and chronic epileptiform activity, but did not reduce the extent of HS. Together, these data indicate that albumin extravasation due to increased BBB permeability and TGFβ pathway activation during the first hours after SE induction are not significantly involved in initiating TLE.

Vulnerability of cholecystokinin-expressing GABAergic interneurons in the unilateral intrahippocampal kainate mouse model of temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is characterized by recurrent spontaneous seizures and behavioral comorbidities. Reduced hippocampal theta oscillations and hyperexcitability that contribute to cognitive deficits and spontaneous seizures are present beyond the sclerotic hippocampus in TLE. However, the mechanisms underlying compromised network oscillations and hyperexcitability observed in circuits remote from the sclerotic hippocampus are largely unknown. Cholecystokinin (CCK)-expressing basket cells (CCKBCs) critically participate in hippocampal theta rhythmogenesis, and regulate neuronal excitability. Thus, we examined whether CCKBCs were vulnerable in nonsclerotic regions of the ventral hippocampus remote from dorsal sclerotic hippocampus using the intrahippocampal kainate (IHK) mouse model of TLE, targeting unilateral dorsal hippocampus. We found a decrease in the number of CCK+ interneurons in ipsilateral ventral CA1 regions from epileptic mice compared to those from sham controls. We also found that the number of boutons from CCK+ interneurons was reduced in the stratum pyramidale, but not in other CA1 layers, of ipsilateral hippocampus in epileptic mice, suggesting that CCKBCs are vulnerable. Electrical recordings showed that synaptic connectivity and strength from surviving CCKBCs to CA1 pyramidal cells (PCs) were similar between epileptic mice and sham controls. In agreement with reduced CCKBC number in TLE, electrical recordings revealed a significant reduction in amplitude and frequency of IPSCs in CA1 PCs evoked by carbachol (commonly used to excite CCK+ interneurons) in ventral CA1 regions from epileptic mice versus sham controls. These findings suggest that loss of CCKBCs beyond the hippocampal lesion may contribute to hyperexcitability and compromised network oscillations in TLE.

Long term evolution of fast ripples during epileptogenesis

Objective. Fast ripples (FRs) have received considerable attention in the last decade since they represent an electrophysiological biomarker of the epileptogenic zone (EZ). However, the real dynamics underlying the occurrence, amplitude, and time-frequency content of FRs generation during epileptogenesis are still not well understood. This work aims at characterizing and explaining the evolution of these features. Approach. Intracortical electroencephalographic signals recorded in a kainate mouse model of temporal lobe epilepsy were processed in order to compute specific FR features. Then realistic physiologically based computational modeling was employed to explore the different elements that can explain the mechanisms of epileptogenesis and simulate the recorded FR in the early and late latent period. Main results. Results indicated that continuous changes of FR features are mainly portrayed by the epileptic (pathological) tissue size and synaptic properties. Furthermore, the microelectrodes characteristics were found to dramatically affect the observability and spectral/temporal content of FRs. Consequently, FRs evolution seems to mirror the continuous pathophysiological mechanism changes that occur during epileptogenesis as long as the microelectrode properties are taken into account. Significance. Our study suggests that FRs can account for the pathophysiological changes which might explain the EZ generation and evolution and can contribute in the treatment plan of pharmaco-resistant epilepsies.

Medial septal GABAergic neurons reduce seizure duration upon optogenetic closed-loop stimulation.

Seizures can emerge from multiple or large foci in temporal lobe epilepsy, complicating focally targeted strategies such as surgical resection or the modulation of the activity of specific hippocampal neuronal populations through genetic or optogenetic techniques. Here, we evaluate a strategy in which optogenetic activation of medial septal GABAergic neurons, which provide extensive projections throughout the hippocampus, is used to control seizures. We utilized the chronic intrahippocampal kainate mouse model of temporal lobe epilepsy, which results in spontaneous seizures and as is often the case in human patients, presents with hippocampal sclerosis. Medial septal GABAergic neuron populations were immunohistochemically labelled and were not reduced in epileptic conditions. Genetic labelling with mRuby of medial septal GABAergic neuron synaptic puncta and imaging across the rostral to caudal extent of the hippocampus, also indicated an unchanged number of putative synapses in epilepsy. Furthermore, optogenetic stimulation of medial septal GABAergic neurons consistently modulated oscillations across multiple hippocampal locations in control and epileptic conditions. Finally, wireless optogenetic stimulation of medial septal GABAergic neurons, upon electrographic detection of spontaneous hippocampal seizures, resulted in reduced seizure durations. We propose medial septal GABAergic neurons as a novel target for optogenetic control of seizures in temporal lobe epilepsy.

Deletion of the Na-K-2Cl cotransporter NKCC1 results in a more severe epileptic phenotype in the intrahippocampal kainate mouse model of temporal lobe epilepsy

Increased neuronal expression of the Na-K-2Cl cotransporter NKCC1 has been implicated in the generation of seizures and epilepsy. However, conclusions from studies on the NKCC1-specific inhibitor, bumetanide, are equivocal, which is a consequence of the multiple potential cellular targets and poor brain penetration of this drug. Here, we used Nkcc1 knockout (KO) and wildtype (WT) littermate control mice to study the ictogenic and epileptogenic effects of intrahippocampal injection of kainate. Kainate (0.23 μg in 50 nl) induced limbic status epilepticus (SE) in both KO and WT mice with similar incidence, latency to SE onset, and SE duration, but the number of intermittent generalized convulsive seizures during SE was significantly higher in Nkcc1 KO mice, indicating increased SE severity. Following SE, spontaneous recurrent seizures (SRS) were recorded by continuous (24/7) video/EEG monitoring at 0-1, 4-5, and 12-13 weeks after kainate, using depth electrodes in the ipsilateral hippocampus. Latency to onset of electrographic SRS and the incidence of electrographic SRS were similar in WT and KO mice. However, the frequency of electrographic seizures was lower whereas the frequency of electroclinical seizures was higher in Nkcc1 KO mice, indicating a facilitated progression from electrographic to electroclinical seizures during chronic epilepsy, and a more severe epileptic phenotype, in the absence of NKCC1. The present findings suggest that NKCC1 is dispensable for the induction, progression and manifestation of epilepsy, and they do not support the widely held notion that inhibition of NKCC1 in the brain is a useful strategy for preventing or modifying epilepsy.