Purdue University Graduate School
Milder Dissertation Manuscript-properly formatted-120522.pdf (18 MB)


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posted on 2023-02-03, 13:32 authored by Patrick Clement MilderPatrick Clement Milder, James A. Marrs


Approximately 30% of patients with epilepsy do not achieve adequate seizure control through current anti-seizure drugs (ASD) and treatment methods. Therefore, a critical need exists to efficiently screen ASDs to enhance our ability to tailor treatment protocols and improve patient outcomes. The zebrafish pentylenetetrazol (PTZ) seizure model has become an increasingly popular screening paradigm for novel ASDs. Here, we present an optimized PTZ assay to improve reliability and reproducibility based on work in our laboratory. This optimized assay improves robustness in our screening of anti-seizure drugs (topiramate, lamotrigine, carbamazepine and GS967). These findings show that electroencephalogram (EEG) and calcium sensitive GFP from fusion protein (GCaMP) assays largely correlate with the behavioral findings, helping us connect physiological and behavioral responses to ASDs. Genetic epilepsy syndromes, like voltage gated sodium channel SCN2A and SCN8A pathogenic variants, are often poorly controlled by current medications. Our optimized assay relied on a fast and precise zebrafish seizure model using mRNA overexpression of hSCN2A and hSCN8A variants including: hSCN2A R1882Q and R853Q and hSCN8A R1872Q. All three pathogenic variants increased seizure activity, and the ASDs significantly decreased this seizure activity. This mRNA overexpression assay can be used to quickly evaluate seizure activity induced by pathogenic variants in voltage gated sodium channel genes and test ASDs to determine efficacy. In a separate study, we tested if the addition of the human SCN2A sodium channel could potentially rescue the loss of the zebrafish scn1Lab gene. Our GCaMP assay data indicates that this loss was successfully rescued. Cumulatively, these findings can be used to improve the screening of novel ASDs and treatments for patients with refractory epilepsy.


Degree Type

  • Doctor of Philosophy


  • Biological Sciences

Campus location

  • Indianapolis

Advisor/Supervisor/Committee Chair

Dr. James A. Marrs

Additional Committee Member 2

Dr. Anthony J. Baucum II

Additional Committee Member 3

Dr. Brian R. Overholser

Additional Committee Member 4

Dr. Theodore R. Cummins

Additional Committee Member 5

Dr. Teresa L. Mastracci

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