Seizures associated with medial temporal lobe epilepsy (TLE) damage structures in the limbic region of the brain, including the hippocampus, entorhinal cortex (EC), and amygdala. The EC actually decreases in volume in patients with TLE, and layer III specifically experiences heavy neuronal loss. Why the neurons in layer III, but hot layer II or layer V, are so susceptible to damage from seizures is not understood. The layer III neurons show an increase in dendritic excitability as soon as twenty-four hours following the induction of seizures, and changes in the hyperpolarization current, Ih, partially contribute to the change in excitability. However, the contribution of another dendritic current that is also modified by seizures, the A-type voltage-gated potassium current (la), has not been investigated. In CA1 pyramidal neurons the la is down-regulated by TLE seizures, resulting in an increase in intrinsic excitability. We want to determine if changes in the la contribute to the seizure-induced changes in the intrinsic excitability of layer III neurons. Additionally, to probe for differences or similarities among layers II, III and V, we want to investigate whether neurons in the EC layers II and V express la and la channel subunits. Thus, our specific goals are first to characterize the la and the la channel subunits, Kv4.2 and Kv4.3 proteins, in the major cell types of layers II, III and V of the EC. Next, we will generate mouse models of TLE and repeat the same experiments to identify the changes induced by seizures. To measure the la directly is very difficult, but because the la decreases the amplitude of back-propagating action potentials (bAPs), we will measure the amplitude of bAPs in control conditions and in the presence of an inhibitor of la, and use it as an indirect measure of la. This will be done in pyramidal neurons in EC layers III and V, but because the dominant cell type in layer II is stellate neurons, and their dendrites are too small for recordings, we will use whole-cell, voltage-clamp recordings to measure the la. To characterize the distribution of the la channel subunits, Kv4.2 and KV4.3, we will do immunohistochemistry experiments. In order to quantify the expression levels of the Kv4.2 and Kv4.3 proteins in the EC we will do western blot experiments. Additionally, we will conduct quantitative, single-cell RT-PCR experiments to quantify the expression level of Kv4.2 and Kv4.3 mRNA in individual neurons.
The results of these experiments will add to the understanding of the cause of increased neuronal excitability, in entorhinal cortex, a hallmark of temporal lobe epilepsy, one of the most common types of adult epilepsy. Also, Alzheimer's and schizophrenia affect EC neurons, so characterizing intrinsic currents in the neurons might be useful in the study of the diseases as well.
