This project is designed to identify and quantify the molecular mechanisms of NMDA receptor proteins and their subunit coassemblies that are necessary and/or sufficient for hyperexcitability of physiologically-verified (EEG) epileptic human cortical seizures. The most frequent drug-resistant neocortical seizures occur in human epileptics with cortical dysplasia. This serious seizure disorder occurs in approximately 20% OF all epileptics and is associated with the most severe social and educational retardations compared to the other focal or generalized epilepsies. Most of these cortical dysplasias can be surgically removed and in some cases seizures are reduced or eliminated. Surgical success however cannot be predicted from routine histopathologic analysis of the resected cortex. Continued seizures requiring medication occur in about 50% of cases. By contrast, sophisticated immunocytochemistry on NMDA receptors has now revealed upregulation of the NR2 subunits, and their coexpression with NR1 subunits in epileptic but not non-epileptic cortex. This proposal is designed to uncover the mechanisms by which NMDA receptors generate hyperexcitability by examining freshly resected epileptic cortex (documented by preoperative cortical recordings of the EEG seizure onset). Quantitative comparisons will be made in each patient's """"""""epileptic"""""""" and """"""""non-epileptic"""""""" cortex (no EEG seizure onsets). These parallel studies will uncover differences in NMDA receptor composition and function in: 1) NMDA receptor protein subunit gene products NR2A, B and their coassemblies with NR1 A-H )splice variants); 2) double-labeled NR1-NR2 immunofluorescence on single dysplastic neurons; 3) coimmunoprecipitation blots for NR1-NR2 antibodies; 4) Northern blots (with mRNA hybridization tests for NR2A, B, and NR1 splice variants A-H; 5) quantitative in situ hybridization to confirm Northern blot mRNAs; and 6) in vitro slice and dissociated neuron electrophysiology with field potential, patch clamp recordings, and selective pharmacologic blockade of NMDA receptor subunits. These multidisciplinary protein, molecular, and pharmaco-physiologic analyses will provide new information about the mechanisms of epilepsy and may suggest novel approaches to designing new drugs. These drugs should selectively act on hyperexcitable dysplastic neurons that have unique heteromeric coassemblies of NR2 and NR1 subunits not found on """"""""non-epileptic"""""""" cortical neurons. Specific receptor-targeted drugs would avoid general nervous system depression and should provide more effective management of epilepsy in cortical dysplasia.
