). Interestingly, an unexpected variation in potency of drugs became evident when different mouse strains were compared in the 6-Hz model. Despite the lack of relevant pharmacokinetic differences, the responsiveness of NMRI (Naval Medical Research Institute) mice to levetiracetam and phenytoin exceeded that of C57Bl/6J mice (Leclercq and Kaminski, 2015). These findings suggest that strain comparisons may provide information about the molecular mechanisms of differences in pharmacodynamics and drug resistance. Based on these data and its ease of use, it was decided to implement the mouse 6-Hz model (in CF-1 [Carworth Farms] mice) as an early screening tool and "high-hurdle" seizure assay in the identification phase of the pharmacoresistant epilepsy workflow of the ETSP (Kehne et al., 2017;Löscher, 2017a) (Fig. 2).
Recently, it has been suggested that a rat 6-Hz paradigm might serve as an alternate model (Metcalf et al., 2017). The availability of a comparable model in a different species can be of particular relevance considering species differences in pharmacodynamics and pharmacokinetics. Moreover, the rat 6-Hz screening model can provide important guidance for dose selection and study design for subsequent drug candidate Drug-Resistant Epilepsy assessment in chronic rat models. With head nodding, jaw clonus, and forelimb clonus, behavioral seizure patterns proved to be comparable to "psychomotor" seizures described in the mouse model (Metcalf et al., 2017). Metcalf et al. (2017) assessed the efficacy of 16 standard ASDs in this new rat seizure model. At the highest stimulus intensity, i.e., twice the convulsive current that elicits seizures in 97% of the rats, compounds that were effective with a protective index.1 comprised ezogabine, phenobarbital, and sodium valproate (Metcalf et al., 2017).
As an alternate to electrical seizure induction, the administration of chemoconvulsants has always been a mainstay of seizure model generation. However, the fact that chemoconvulsants induce seizures by interfering with different neurotransmitter systems (e.g., GABA, glutamate, glycine) will necessarily lead to different efficacy of drugs against chemoconvulsantinduced seizures, depending on the mechanism of action of the drug and the chemoconvulsant to be tested. For instance, allylglycine modulates GABAergic neurotransmission by weak inhibition of glutamate decarboxylase, resulting in the induction of electrographic and behavioral seizure events.  compared the pharmacoresponsiveness of allylglycine-induced seizures in mice and larval zebrafish. While diazepam and valproate protected against chemically induced seizures, levetiracetam, phenytoin, and topiramate exhibited only a limited protective effect. The fact that the cross-species validation indicated a comparable responsiveness profile of the allylglycine model in zebrafish led the authors to conclude that the zebrafish model might serve as a high-throughput model of treatment-resistant seizures.
In contrast to acute seizure models, chronic rodent models may better reflect the situation following epilepsy manifestation with a multitude of molecular, cellular, and network alterations. Kindling paradigms are based on repeated seizure induction resulting in a progressively increasing seizure severity and duration and a persistent lowering of seizure threshold. Repeated 50-Hz corneal stimulations in mice resulted in a fully kindled state with a good responsiveness to most of the ASDs tested in this paradigm (Matagne and Klitgaard, 1998;Rowley and White, 2010). As these data indicated that the model provides a sensitive screening tool, the paradigm has been integrated in the identification phase of the ETSP (Fig. 2).
In this context, it of interest that Leclercq et al. (2014) addressed the hypothesis that a corneal kindling paradigm with twice daily 6-Hz stimulations (3 seconds, 44 mA) in mice may provide a better tool concerning robustness and responsiveness. The authors reported a kindling progression with seizure activity evolving in severity and duration in response to repeated stimulations, finally resulting in a reproducible induction of generalized seizure activity (Leclercq et al., 2014). A direct comparison with a traditional 50-Hz corneal kindling paradigm revealed that the 6-Hz model proved to be advantageous regarding the persistence of the kindled state (Leclercq et al., 2014). Moreover, the ASDs  clonazepam, valproate, carbamazepine, and levetiracetam all showed a relatively lower potency in the 6-Hz than the 50-Hz kindling paradigm (Leclercq et al., 2014). Thus, as concluded by the authors, the new 6-Hz corneal kindling model may serve as a new tool for selection of ASDs targeting DRE (Leclercq et al., 2014). Considering the relevance of chronic models for the selection process of new drug candidates, it has been decided to additionally implement further chronic models in the differentiation phase of the ETSP (Fig. 2).
The lamotrigine-resistant kindling model in rats was first described by Postma et al. (2000). In this paradigm, exposure to lamotrigine during kindling acquisition with repeated electrical stimulations of the amygdala results in a fully kindled state characterized by a poor drug responsiveness to lamotrigine and some other ASDs (Postma et al., 2000;Srivastava et al., 2013). Interestingly, ASD resistance in this model proved to extend from lamotrigine to carbamazepine, phenytoin, and topiramate (Postma et al., 2000;Srivastava et al., 2013). In contrast, valproate exerted potent anticonvulsant effects in lamotrigine-resistant kindled rats. More recently, Metcalf et al. (2019) described the dose response effects of numerous ASDs in this model. Five sodium channel blockers (eslicarbazepine, lacosamide, lamotrigine, phenytoin, and rufinamide) were either not efficacious or effective only at doses that were not well-tolerated in this model. Similarly, topiramate and levetiracetam were not effective at the doses tested, indicating that pharmacoresistance is not limited to sodium channel blockers. In contrast, compounds targeting either GABA receptors (clobazam, clonazepam, phenobarbital) or GABA-uptake proteins (tiagabine) produced dose-dependent efficacy against convulsive seizures. Similarly, ezogabine and valproate were also highly effective. Compounds acting to modulate Ca 2+ channels (ethosuximide, gabapentin) showed differential activity. Taking into account that ASD exposure in combination with seizure elicitation results in the poor responsiveness of lamotrigine-resistant kindled rats, the failure of these animals to respond to selected ASDs may also reflect contingent or cross-tolerance.
More recently, efforts have been made to establish and characterize a comparable model in mice (Koneval et al., 2018). Therefore, the rat protocol was adapted to the 60-Hz corneal-kindling mouse model. Lamotrigine exposure during the kindling phase did not affect kindling progression (Koneval et al., 2018). However, fully kindled mice failed to respond to lamotrigine, carbamazepine, retigabine, and valproate (Koneval et al., 2018). Interestingly, drug resistance in these kindled mice proved to be associated with more pronounced kindling-associated behavioral alterations with increased hyperexcitability and anxiety-associated behavior (Koneval et al., 2018). The paradigm has been suggested as a moderate-throughput platform, which can be applied for early compound selection (Koneval et al., 2018).
Seizures in kindling paradigms are elicited by repeated electrical or chemical stimulation. This approach allows a precise determination of an impact of a test compound on seizure threshold and seizure characteristics at threshold stimulation. Spontaneous seizures only occur following several weeks to months of repeated stimulation, thus rendering it impractical to study drug effects on generation of spontaneous seizures in kindling paradigms. This is in contrast to post-SE models, in which electrical or chemical induction of SE results in the manifestation of epilepsy with spontaneous recurrent seizures. One of the available models is standing out with a high nonconvulsive seizure frequency, which renders the model suitable for rapid screening of ASD candidates. The model is induced by intrahippocampal kainate injection in mice, which results in a limbic SE followed by the development of spontaneous recurrent electrographic and electroclinical seizures. Though generalized convulsive seizures occur occasionally in this model, nonconvulsive electrographic seizures recorded in the Cornu Ammonis sector 1 (CA1) region typically occur multiple times per hour (Lévesque and Avoli, 2013;Löscher, 2016). Pharmacoresponsiveness of the paradigm has been intensely studied. Riban et al. (2002) reported a resistance of electrographic seizures to different ASDs, including phenytoin, carbamazepine, and valproate. Further support for a poor drug responsiveness of focal electrographic seizures in the intrahippocamal kainate model of mesial temporal lobe epilepsy (TLE) came from a recent comprehensive study, which confirmed resistance to phenytoin and carbamazepine (Klein et al., 2015). Although the group observed only moderate effects of valproate and phenytoin, they demonstrated a relevant response to phenobarbital and diazepam (Klein et al., 2015). In line with these studies, Duveau et al. (2016) reported that valproate, carbamazepine, and lamotrigine suppressed electrographic seizures only at high doses