Summary: Objective: The mouse 6 Hz model of psychomotor seizures is a well‐established and commonly used preclinical model for antiseizure drug (ASD) discovery. Despite its widespread use both in the identification and differentiation of novel ASDs in mice, a corresponding assay in rats has not been developed. We established a method for 6 Hz seizure induction in rats, with seizure behaviors similar to those observed in mice including head nod, jaw clonus, and forelimb clonus.Methods: A convulsive current that elicits these seizure behaviors in 97% of rats (CC₉₇) was determined using a Probit analysis. Numerous prototype ASDs were evaluated in this model using stimulus intensities of 1.5× and 2× the CC₉₇, which is comparable to the approach used in the mouse 6 Hz seizure model (e.g., 32 and 44 mA stimulus intensities). The ASDs evaluated include carbamazepine, clobazam, clonazepam, eslicarbazepine, ethosuximide, ezogabine, gabapentin, lacosamide, lamotrigine, levetiracetam, phenobarbital, phenytoin, rufinamide, tiagabine, topiramate, and sodium valproate. Median effective dose (ED₅₀) and median toxic (motor impairment) dose (TD₅₀) values were obtained for each compound.Results: Compounds that were effective at the 1.5 × CC₉₇stimulus intensity at protective index (PI) values >1 included clobazam, ethosuximide, ezogabine, levetiracetam, phenobarbital, and sodium valproate. Compounds that were effective at the 2 × CC₉₇stimulus intensity at PI values >1 included ezogabine, phenobarbital, and sodium valproate.Significance: In a manner similar to the use of the mouse 6 Hz model, development of a rat 6 Hz test will aid in the differentiation of ASDs, as well as in study design and dose selection for chronic rat models of pharmacoresistant epilepsy. The limited number of established ASDs with demonstrable efficacy at the higher stimulus intensity suggests that, like the mouse 6 Hz 44 mA model, the rat 6 Hz seizure model may be a useful screening tool for pharmacoresistant seizures.

Astrocyte expression of metabotropic glutamate receptor 5 (mGluR5) is consistently observed in resected tissue from patients with epilepsy and is equally prevalent in animal models of epilepsy. However, little is known about the functional signaling properties or downstream consequences of astrocyte mGluR5 activation during epilepsy development. In the rodent brain, astrocyte mGluR5 expression is developmentally regulated and confined in expression/function to the first weeks of life, with similar observations made in human control tissue. Herein, we demonstrate that mGluR5 expression and function dramatically increase in a mouse model of temporal lobe epilepsy. Interestingly, in both male and female mice, mGluR5 function persists in the astrocyte throughout the process of epileptogenesis following status epilepticus. However, mGluR5 expression and function are transient in animals that do not develop epilepsy over an equivalent time period, suggesting that patterns of mGluR5 expression may signify continuing epilepsy development or its resolution. We demonstrate that, during epileptogenesis, astrocytes reacquire mGluR5-dependent calcium transients following agonist application or synaptic glutamate release, a feature of astrocyte-neuron communication absent since early development. Finally, we find that the selective and conditional knock-out of mGluR5 signaling from astrocytes during epilepsy development slows the rate of glutamate clearance through astrocyte glutamate transporters under high-frequency stimulation conditions, a feature that suggests astrocyte mGluR5 expression during epileptogenesis may recapitulate earlier developmental roles in regulating glutamate transporter function.SIGNIFICANCE STATEMENTIn development, astrocyte mGluR5 signaling plays a critical role in regulating structural and functional interactions between astrocytes and neurons at the tripartite synapse. Notably, mGluR5 signaling is a positive regulator of astrocyte glutamate transporter expression and function, an essential component of excitatory signaling regulation in hippocampus. After early development, astrocyte mGluR5 expression is downregulated, but reemerges in animal models of temporal lobe epilepsy (TLE) development and patient epilepsy samples. We explored the hypothesis that astrocyte mGluR5 reemergence recapitulates earlier developmental roles during TLE acquisition. Our work demonstrates that astrocytes with mGluR5 signaling during TLE development perform faster glutamate uptake in hippocampus, revealing a previously unexplored role for astrocyte mGluR5 signaling in hypersynchronous pathology.

The discovery and development of novel antiseizure drugs (ASDs) that are effective in controlling pharmacoresistant spontaneous recurrent seizures (SRSs) continues to represent a significant unmet clinical need. The Epilepsy Therapy Screening Program (ETSP) has undertaken efforts to address this need by adopting animal models that represent the salient features of human pharmacoresistant epilepsy and employing these models for preclinical testing of investigational ASDs. One such model that has garnered increased interest in recent years is the mouse variant of the Intra-Amygdala Kainate (IAK) microinjection model of mesial temporal lobe epilepsy (MTLE). In establishing a version of this model, several methodological variables were evaluated for their effect(s) on pertinent quantitative endpoints. Although administration of a benzodiazepine 40 min after kainate (KA) induced status epilepticus (SE) is commonly used to improve survival, data presented here demonstrates similar outcomes (mortality, hippocampal damage, latency periods, and 90-day SRS natural history) between mice given midazolam and those that were not. Using a version of this model that did not interrupt SE with a benzodiazepine, a 90-day natural history study was performed and survival, latency periods, SRS frequencies and durations, and SRS clustering data were quantified. Finally, an important step towards model adoption is to assess the sensitivities or resistances of SRSs to a panel of approved and clinically used ASDs. Accordingly, the following ASDs were evaluated for their effects on SRSs in these mice: phenytoin (20 mg/kg, b.i.d.), carbamazepine (30 mg/kg, t.i.d.), valproate (240 mg/kg, t.i.d.), diazepam (4 mg/kg, b.i.d.), and phenobarbital (25 and 50 mg/kg, b. i.d.). Valproate, diazepam, and phenobarbital significantly attenuated SRS frequency relative to vehicle controls at doses devoid of observable adverse behavioral effects. Only diazepam significantly increased seizure freedom. Neither phenytoin nor carbamazepine significantly altered SRS frequency or freedom under these experimental conditions. These data demonstrate that SRSs in this IAK model of MTLE are pharmacoresistant to two representative sodium channel-inhibiting ASDs (phenytoin and carbamazepine) and partially sensitive to GABA receptor modulating ASDs (diazepam and phenobarbital) or a mixed-mechanism ASD (valproate). Accordingly, this model is being incorporated into the NINDS-funded ETSP testing platform for treatment resistant epilepsy.

Summary: Objective: Cognitive comorbidities are increasingly recognized as an equal (or even more disabling) aspect of epilepsy. In addition, the actions of some antiseizure drugs (ASDs) can impact learning and memory. Accordingly, the National Institute of Neurological Disorders and Stroke (NINDS) epilepsy research benchmarks call for the implementation of standardized protocols for screeningASDs for their amelioration or exacerbation of cognitive comorbidities. Long‐term potentiation (LTP) is a widely used model for investigating synaptic plasticity and its relationship to learning and memory. Although the effects of someASDs onLTPhave been examined, none of these studies employed physiologically relevant induction stimuli such as theta‐burst stimulation (TBS). To systematically evaluate the effects of multipleASDs in the same preparation using physiologically relevant stimulation protocols, we examined the effects of a broad panel of existingASDs onTBS‐inducedLTPin areaCA1 of in vitro brain slices, prepared in either normal or sucrose‐based artificial cerebrospinal fluid (ACSF), fromC57BL/6 mice.Methods: Coronal brain slices containing the dorsal hippocampus were made using either standard or sucrose‐basedACSF. Recordings were obtained from four slices at a time using theScientificaSlicemaster high throughput recording system. Slices exposed toASDs were paired with slices from the opposite hemisphere that served as controls. Field excitatory postsynaptic potentials (fEPSPs) were recorded, and allASDs were applied to slices by bath perfusion for 20 min prior to the induction stimulus.LTPwas induced byTBSor by high‐frequency stimulation (HFS). The followingASDs were examined: 100 μM phenobarbital (PB), 80 μM phenytoin (PHT), 50 μM carbamazepine (CBZ), 600 μM valproate (VPA), 60 μM topiramate (TPM), 60 μM lamotrigine (LTG), 100 μM levetiracetam (LEV), 10 μM ezogabine (EZG), and 30 μM tiagabine (TGB).Results: Among voltage‐gated sodium channel inhibitors,CBZsignificantly attenuatedTBS‐inducedLTP,PHTattenuated bothTBS‐inducedLTPand post–tetanic potentiation (PTP), andLTGfailed to affectLTPbut did attenuatePTP.ASDs that modulate γ‐aminobutyric acid (GABA)ergic synaptic transmission, such asPBandTGB, significantly attenuatedLTPin brain slices prepared in sucrose‐basedACSFbut not standardACSF. Third generationASDs, such asLEVandTPM, did not affectLTPinACSF‐ or sucrose‐prepared brain slices. AlthoughEZGfailed to affectLTP, it did significantly attenuatePTPunder both slicing conditions.VPAfailed to affectLTPin areaCA1, both inC57BL/6 mice and Sprague‐Dawley rats, usingTBSorHFS. However,VPAdid attenuateTBS‐inducedLTPin the dentate gyrus (DG).Significance: The results of experiments describe herein provide a comprehensive summary of the effects of many commonly usedASDs on short‐ and long‐term synaptic plasticity while, for the first time, using physiologically relevantLTPinduction protocols and slice preparations from mice. Furthermore, methodologic variables, such as brain slice preparation protocols, were explored. These results provide comparative knowledge ofASDeffects on synaptic plasticity in the mouse hippocampus and may ultimately contribute to an understanding of the differences in the cognitive side effect profiles of ASDs and the prediction of cognitive dysfunction associated with novel investigationalASDs.