Abstracts

Rationale: It is well known that limbic seizures associated with temporal lobe epilepsy are characterized by aberrant and unpredictable neuronal hyperexcitability. Failure of homeostatic plasticity (a process by which neurons maintain a physiologic balance between neuronal excitation and inhibition) is hypothesized to play a large role in epileptogenesis. However, the molecular basis of this important plasticity has yet to be elucidated. Homeostatic plasticity can be induced in primary dissociated neuronal culture. Prolonged blockade of neuronal activity by treating neurons with Tetrodotoxin (TTX, an inhibitor for voltage-gated sodium channels) leads to a homeostatic increase in synaptic transmission and intrinsic excitability. Conversely, prolonged enhancement of neuronal activity by treating neurons with bicuculline (GABAA receptor antagonist) results in a homeostatic decrease in synaptic transmission and intrinsic excitability. Recently this plasticity was shown to be dependent on transcription, suggesting that regulation of gene expression mediates its induction. Methods: To identify key molecular players of homeostatic plasticity, we performed cDNA microarray analysis on the rat dissociated hippocampal cultured neurons in which global homeostatic plasticity was induced by 48 hr treatment with 0.5 M TTX or 20 M bicuculline or control. Results: Our microarray analysis against rat whole genome Agilent array of 41012 genes revealed 1664 genes whose expression changed significantly upon TTX and bicuculline treatment. We observed over-representation of the genes involved in synaptic transmission , transmission of nerve impulses , and behavior . In particular, overrepresentation of genes involved in K+ ion transport caught our attention since most of them encode K+ ion channels that critically function to dampen neuronal excitability. TTX treatment repressed 11 out of 12 K+ channel genes whereas BC treatment induced 4 out of 7 K+ channel genes. We identified binding sites in these genes for transcription factors such as CREB and JUN. In particular, a significant bidirectional change was found for genes responsible for A-type potassium current, which mediates the duration of time between action potentials and thus serves as a critical brake for repetitive firing of action potentials. In addition to confirming our microarray data with real time PCR and western blotting, we are performing whole-cell patch clamp recording to determine the roles of activity-induced changes in K+ channel expression in homeostatic plasticity of intrinsic excitability. Conclusions: Together, our current findings suggest that activity-dependent expression of ion channels and their associated proteins contribute significantly to the stabilization of hippocampal neuronal excitability by serving as key proteins in the molecular mechanisms governing homeostatic plasticity. These predictions warrant further research, and may provide novel targets for the development of both preventative and therapeutic treatments for epilepsy and other hyperexcitability-associated diseases.