Audiogenic Seizure Mouse Model
Objective: To assess seizure protection by LEV derivatives in an audiogenic mouse model of epilepsy and correlate binding affinity to SV2A with anticonvulsant efficacy
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Equipment1
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Materials4
Software1
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Protocol Steps
Prepare brain membranes and synaptic vesicles
Isolate brain membranes and purified synaptic vesicles from wild-type and SV2A knockout mice for binding studies
Note: Samples prepared from both wild-type and knockout mice to determine necessity of SV2A for LEV binding
View evidence from paper
“Brain membranes and purified synaptic vesicles from mice lacking SV2A do not bind a tritiated LEV derivative”
Perform photoaffinity labeling
Conduct photoaffinity labeling of purified synaptic vesicles to determine apparent molecular mass of LEV-binding site
Note: Binding site identified as approximately 90 kDa molecular mass
View evidence from paper
“photoaffinity labeling of purified synaptic vesicles confirms that it has an apparent molecular mass of ≈90 kDa”
Test LEV binding to SV2A in fibroblasts
Express SV2A in fibroblasts and test binding of LEV and related compounds to determine sufficiency of SV2A for LEV binding
Note: No binding observed to related isoforms SV2B and SV2C, confirming specificity to SV2A
View evidence from paper
“LEV and related compounds bind to SV2A expressed in fibroblasts, indicating that SV2A is sufficient for LEV binding. No binding was observed to the related isoforms SV2B and SV2C”
Measure binding affinities of LEV derivatives
Determine binding affinities of a series of LEV derivatives to SV2A in fibroblasts and to the LEV-binding site in brain tissue
Note: High degree of correlation observed between fibroblast and brain binding affinities
View evidence from paper
“there is a high degree of correlation between binding affinities of a series of LEV derivatives to SV2A in fibroblasts and to the LEV-binding site in brain”
Assess seizure protection in audiogenic mouse model
Test LEV derivatives in audiogenic seizure model to evaluate anticonvulsant efficacy and correlate with SV2A binding affinity
Note: Strong correlation found between SV2A affinity and seizure protection ability
View evidence from paper
“there is a strong correlation between the affinity of a compound for SV2A and its ability to protect against seizures in an audiogenic mouse animal model of epilepsy”