Design and Methods - Seizure Threshold Screen for Epilepsy Mutations
Detecting seizure mutants and modifier genes using ECT screens - Our approach to detecting seizure threshold mutants will evolve over time as Screens 1-4 below. Screens 1 and 2 are complementary, and the more selective Screens 3 and 4 are contingent on prior results. For example, for AED breakthrough mutants we do not yet know if it will be more efficient to test mutants from Screens 1-2 for AED selectivity, or to employ an AED selective screen up-front. Relevant decisions will be based on the number and type of mutants from Screens 1-2. All primary screens will be done at JAX.
Screen 1 (yr 1-2). Minimal or maximal seizure mutants - HF electroshock is performed using corneal electrodes and a local anaesthetic in saline. With proper application, eye irritation and damage does not occur [149]. Current settings will be at the estimated CC0 (critical current for 0% of the population) for minimal seizures (299 Hz sq wave, 1.6 pulse width, 0.2 s, 5 mA {fem.} 7 mA {male}).
Screen 2 (yr 2-3). Partial seizure mutants - LF electroshock will be performed as above except with its own stimulator settings for CC0 in B6 mice (6 Hz, 0.2 pw, 3.0 s, 9 mA {fem} 11 mA {male)).
Screen 3 (yr 3-4). AED "breakthrough" mutants - For the companion RFA, we will have characterized strain thresholds to phenytoin, ethosuximide, valproate and levetiracetam. The rationale for their selection is based on differences in clinical profile and mechanism of action [150, 151]. We will begin with phenytoin as it is a common AED with demonstrated refractory patients [132]. After a time course on 20 C57BL/6J mice of each sex and a dose-response at the optimal timepoint, various levels of efficacy including ED100 will be calculated [152]. The screen is for mutants which "breakthrough" treatment and experience seizures at 2x CC97 at the respective ED100.
Screen 4 (yr 4-5). Modifiers - Homozygotes K+ channel (Kcna1) null mutants, made by collaborator B. Tempel [137], have spontaneous seizures from 3 wks of age, but no other gross abnormalities. His characterization of neuroexcitability of hippocampal slices, with reduced threshold for action potential generation resulting in periodic rather than chronic seizures, shows that sensitized screens using Kcna1 will be fruitful. Further, heterozygotes have lower threshold to PTZ and fluorothyl seizures, and we showed their sensitivity to electroshock. The semidominance allows us to exploit heterozygotes for detecting modifiers: the "test" allele need not be present during mutagenesis. We will screen for alleles that: 1) lower ECT further than that of Kcna1 +/-, or 2) raise ECT (protective at CC100). These efforts will be complimented with further characterization, classifying mutants as Kcna1-specific, affecting K+ flux or Kcna1 mRNA levels, vs. general. The screen will also produce new functional Kcna1 alleles. Kcna1 mice are now in importation at JAX.
Further characterization of new seizure mutants - Three further major issues need be addressed to characterize new seizure mutants: 1) its generality; susceptibility to other stimuli or to spontaneous seizures, 2) knowing the AED profile, 3) determining brain regions involved. For each mutant we will respectively determine sensitivity to chemoconvulsants or spontaneous seizures, sensitivity to AED and brain regions involved.
Intravenous pentylenetetrazol (PTZ) infusion - is a sensitive parametric method for assessing seizure threshold [147]. PTZ is a non-competitive GABAA antagonist which lowers neuronal inhibition. Unlike excitotoxins, PTZ does not cause cell death and is less likely to induce secondary epilepsies. Also, PTZ need not need be meta-bolized for use and has a rapid turnover. Whether the variable is a given AED dose or a genetic mutation, with PTZ infusion it does not have to block a seizure but only delay or accelerate its onset: correlated increases or decreases of the amount of PTZ required to induce a seizure event provides a quantitative measure of its neuro-excitatory potential. In addition, a quantifiable endpoint can be obtained with only 8-10 animals per group [147].
Electrocorticography (EEG) - is a standard for validating a convulsive seizure, detecting relatively silent petit-mal seizures and also detecting inter-ictal events indicating states of neuroexcitability.
Methods - Will essentially follow published methods [153], recording 1-7 days post surgery for up to 24 hr. For AED profiles, mutants will be treated with a standard dose of one of four AEDs and at varying times after treatment given sufficient current for a threshold seizure (at CC97 for B6). Twenty mice per time point is the smallest number which, in our experience, will minimize the probability that potentially useful AEDs are rejected from further consideration. A dose-response is then done: 40 animals (5 doses x 8 per dose) from each group will be pretreated with increasing AED until either full protection against a given endpoint is seen when stimulated at CC97, or motor impairment is noted. Median effective (ED50) or motor deficit (TD50) doses will then be calculated. When an AED blocks the seizure endpoint at CC97, we will assess the ability of a maximally effective dose to elevate seizure threshold. Eight mice per dose are the minimum required to facilitate results that are statistically significant (p < 0.05) using log-probit methods [152]. Brain uptake of 2-deoxy-D-[1-14C]glucose (2-DG) is a simple method used in animal studies, mostly rats (including electro-shock) and mice, to reveal brain regions with high metabolic rates and therefore active during seizure and other states [154, 155]. Mice will be injected iv with 3 µCi of 2-D[1-14C]G and stimulated to seizure 5 minutes later. A typical experiment in the HF model consists of at least three groups: sub-threshold stimulated, stimulated to minimal seizure, stimulated to maximal seizure. Mice are sacrificed, brains removed and snap frozen, embedded, serially-sectioned and alternate sections exposed to X-ray film for 2 wks with standards, or counterstained.
