Development and pharmacologic characterization of the rat 6 Hz model of partial seizures

Abstract

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 × CC₉₇ stimulus intensity at protective index (PI) values >1 included clobazam, ethosuximide, ezogabine, levetiracetam, phenobarbital, and sodium valproate. Compounds that were effective at the 2 × CC₉₇ stimulus 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.

Keywords: Animal models; Antiseizure drugs; Pharmacoresistant seizures; Psychomotor seizures.

Figure 1

Electroconvulsive currents experienced by rodents being stimulated in constant-voltage mode obey Ohm’s-law and can be used to determine rodent resistances. A. Circuit diagram illustrating experimental design. B. Representative trace of a 6 Hz stimulation waveform, sampled at 250 kHz, as measured across a 100 Ohm resistor in series with a male Sprague Dawley rat (100–120g, N=6–8 per group). Scale bar represents 1 V and 1 second. C–D. Stimulation currents (C) and mouse resistances (D) as a function of the stimulation voltage experienced by CF-1 mice (20–30 g). Each point represents the mean ± SEM (N=6 per group). Solid lines represent linear regression curves fit to the data. E–F. Stimulation currents (E) and mouse resistances (F) as a function of the stimulation voltage experienced by Sprague Dawley rats. Each point represents the mean ± SEM (N=6 per group). Solid lines represent linear regression curves fit to the data.

Figure 2

Example of electrographic activity from a convulsive seizure induced using 60V stimulation (1.5CC97, Grass S48 stimulator, constant voltage stimulation). A. 3 minutes of EEG activity surrounding a single stimulation. The time of stimulation is marked by a blue line. B. The time-frequency power normalized across frequency bands of the EEG from part (A) demonstrates the changing high-powered frequency content during epileptiform discharges inherent to a 6 Hz-induced convulsive seizure (arbitrary units). C. An enlargement of the EEG activity from the part in (A) highlighted by a red line. D. The time-frequency power normalized across frequency bands of the EEG from part (C) (arbitrary units). The behavioral aspects of this example seizure are demonstrated in the video as Figure S1. (N=6 per group, 250–350g at testing).