In the last grant cycle, we characterized, validated and published a unique in vitro tool for the study of epileptogenesis - hippocampal organotypic slice cultures that develop spontaneous seizures after 1 week in vitro. We used this preparation to blindly screen over 500 drug-concentration combinations for activity in chronic, post-traumatic epilepsy at speeds that are orders of magnitude faster than any other therapeutic testing strategy for chronic epilepsy. We then used double-blind in vivo EEG testing in the kainate model of chronic epilepsy to confirm the anticonvulsant effect of a lead compound, celecoxib. A key innovation that made these speeds possible was the use of lactate and lactate dehydrogenase (LDH) levels in spent media as assays for seizure burden and cell death, respectively. In searching for the cause of the increase in lactate, we found a persistent neuronal membrane leak that increases cytoplasmic sodium and calcium (? Nai & Cai) days before histochemical evidence of cell death. New data indicate that COX2 induction leads to translocation of Bax, a canonical mitochondrial permeabilizing protein, to the cytoplasmic membrane, where it forms pores that admit Na+ and Ca2+. We propose to test the following pathophysiology: traumatic or ictal injury induces Ca2+-dependent Bax translocation to the cytosolic membrane, where it creates a progressive Ca2+ and Na+ leak that should kill the neuron. However, neurons survive for some time due to their uniquely high ion transport capacity. The ion transport consumes a lot of ATP, and lactate production is a consequence of ATP generation. Progression of the leak eventually leads to membrane depolarization, which may contribute to ictogenesis, and cell death. We will test these ideas by correlating seizures, lactate, and ATP production in Aim 1.
In Aim 2 we will establish the nature of the membrane leak.
In Aim 3 we will evaluate the consequences of the membrane leak on ATP production, membrane potential, ictogenesis, and cell death. We propose to use cell-type specific expression of ratiometric, fluorescent reporters of Na+, Ca2+, ATP, NADH, lactate, caspase and membrane potential in the organotypic slice model, together with multiphoton and custom- built low-light, wide-field microscopes to address these questions at temporal and spatial resolutions that have not previously been feasible.
These investigations will 1) provide estimates regarding the amount of seizure activity that is injurious to neurons 2) identify a progressive membrane leak that may contribute to the persistence of epilepsy 3) establish anticonvulsant mechanisms and therapies that are unique to chronic epilepsy 4) provide the capacity to rapidly screen promising drugs for activity in chronic epilepsy.
