Abstracts

Seizures occur in nearly 90% of patients with tuberous sclerosis complex (TSC). While cortical tubers are believed to participate in seizure genesis, epilepsy has also been reported in TSC patients and mouse models lacking tubers. Therefore, a fundamental question is whether loss of TSC gene function, in the absence of tuber formation, result in a hyperexcitable phenotype. To address this question, we have initiated studies to knockdown TSC gene function in larval zebrafish using morpholino-based technology. Here we present electrophysiological evidence for synaptic hyperexcitability following knockdown of zebrafish TSC1. The cDNA sequences of the zebrafish TSC genes (TSC1a, TSC1b, and TSC2) were obtained by performing BLAST searches against the zebrafish genome. Sequences were confirmed by RT-PCR and DNA sequencing. Antisense morpholino oligonucleotides were designed near the start codon of the mRNA sequences in order to block translation of the transcripts. TSC1 and control morpholinos were injected into one-cell stage zebrafish embryos at a concentration of 0.5-1.0 mM. For electrophysiological recordings, morphant and control larvae (3 days postfertilization) were immobilized in 1.2% agarose. Under visual guidance, a glass microelectrode was placed in the optic tectum and a bipolar stimulating electrode was placed on the contralateral eye. Voltage records were filtered, digitized and stored on a PC computer running Axoclamp software. In some experiments, normal solution was replaced with media containing 5 mM pentylenetetrazole (PTZ). Under baseline recording conditions, TSC1 morphants exhibited a mild hyperexcitable phenotype. Spontaneously occurring epileptiform-like discharge (0.6 - 3 sec in duration; [sim]0.1 Hz) was observed in 80% of morphants tested. Stimulation of the contralateral eye evoked multiple field excitatory postsynaptic potential (fEPSP) responses in 4 of 5 morphants. Spontaneous bursts and multiple fEPSP responses were not observed in controls. Exposure to a low concentration of PTZ (5 mM) elicited more robust epileptiform-like discharge (1.6 - 8.7 sec in duration; [sim]0.6 Hz) in 100% of morphants tested. In controls, 5 mM PTZ elicited no activity (n = 5) or short duration ([sim]1 sec) low frequency bursting (n = 3). Our study demonstrates that TSC1 knockdown in a relatively simple brain structure can result in hyperexcitability. This suggests that cortical tuber formation may not be required for seizure genesis. Furthermore, this model provides a unique opportunity to examine early embryonic events that may contribute to disease phenotype. Future experiments aimed toward understanding the cellular and molecular mechanisms of seizures in TSC1 morphants will likely lead to a better understanding of the disease and may reveal novel therapeutic targets. (Supported by Klingenstein Fund.)