Undergraduate Research and Creativity

URECA

2007-2008

Abalone Settlement Bioassay for Anti-epileptic Substance Screening

Farzan Gorgani, Farrukh Jafari, Stony Brook Medical School,
David Anschel, Department of Neurology;
and Bradley Peterson, School of Marine and Atmospheric Sciences

Epilepsy is the most prevalent neurological illness which affects people of all ages. Approximately one third of all cases (~1,000,000 people in USA) are refractory to current medications. Anti-epileptic drug (AED) screening depends largely upon animal models of epilepsy. Most of these assays involve rats, with other mammals utilized to a lesser extent, but do not perfectly model human epilepsy. Additionally, they are costly, time consuming, and labor intensive. The present proposal involves the development of a novel assay using a marine mollusk which can potentially address the above shortcomings of currently employed techniques. The hypothesis of the study I plan on working on is that a bioassay utilizing marine mollusk (Haliotis) larval settlement will be effective for the identification of anti-epileptic substances.

In particular, the larval development of Haliotis rufescens (red abalone) has been the subject of much research. After fertilization the larvae are free floating. Several days later they stop swimming, sink to the bottom and attach to a substrate in order to begin the process of metamorphosis which will eventually result in the mature phenotype. This process has been termed "settlement". It was discovered that settlement can be induced by γ-aminobutyric acid (GABA). Since then, researchers from all over the world have demonstrated that in addition to GABA, several other substances promote or inhibit Haliotis larvae settlement. Remarkably, the biochemistry of Haliotis (primarily H. rufescens) settlement has much in common with that of epilepsy.This includes the fact that several substances of various different chemical structures all have analogous effects upon abalone larval settlement and the prevention of seizures including GABA, K+, Ca++, Bromides and thyroxine, many of which will be subject to study in our assay.

Adult H. rufescens (Abulones Cultivados, Ensenada BC Mexico) will be used to obtain competent swimming larvae using standard techniques of fertilization following hydrogen peroxide induction of male and female spawning. We will then produce a dose-response curve to abalone larval settlement using GABA. The method consists of filling small glass vials with filtered sea water (15? C) and approximately 150 swimming larvae (8-10 days post fertilization). GABA will then be added to individual vials at the following concentrations: 1 x 10^-4 M, 1 x 10^-5 M, 1 x 10^-6 M, and 1 x 10^-7 M. Penicillin G and dihydrostreptomycin (150 ppm) will be utilized in all vials. The percent larva settled will then be compared across various doses.

Assay concentrations will cover a broad range centered about serum or CNS concentrations known or suspected to be effective for preventing seizures. For the initial study we will test gabapentin, levetiracetam, topiramate, pentobarbital, thyrotropin-releasing hormone, and midazolam.

The Research Foundation of The State University of New York funded this research. It took place in the neurology department at The Health Science Center in Stony Brook University, but our facilities have moved to Blue Point marine labs.