Date of Award
Open Access Dissertation
J. Larry Durstine
Kainate receptors (KARs) are glutamate-gated ion channels that mediate synaptic transmission, modulate transmitter release, and mediate excitation in the brain. Potentiation in their function predisposes the hippocampus to hyperexcitability and seizures. These receptors are widely expressed throughout the central nervous system as tetramers composed of various combinations of GluK1-5 subunits. In the hippocampus (a brain region commonly associated with seizure initiation), GluK2-, GluK4-, and GluK5-containing receptors are highly expressed in the CA3 pyramidal cell layer, whereas GluK1 is barely detectable. The lack of pharmacological tools hinders identifying the functional contribution of each kainate receptor subtype in normal CA3 synaptic transmission. To address this critical obstacle, we used whole cell patch clamp electrophysiology on HEK-T 293 cells transfected with GluK2 homomers, GluK2/K4 or GluK2/K5 heteromers. We found that the drug ACET selectively inhibits GluK5 and GluK4 subunits, whereas the drug kynurenate is an antagonist at all kainate subunits but was more potent at GluK2 subunits. Furthermore, we were able to discover that binding of glutamate to either the two GluK2 subunits or two GluK4/K5 subunits in the heteromeric tetramer was sufficient to open the kainate receptor, albeit to a non-desensitizing current. However, glutamate binding to three or more subunits in the tetramer was sufficient to enable kainate receptor desensitization. Lastly, using field potential electrophysiology to stimulate and record KAR-mediated synaptic transmission (fEPSPs) at the mossy fiber – CA3 synapse, we found that perfusion of ACET was sufficient to entirely block KAR-mediate fEPSPs at the mossy fiber – CA3 synapse. These results suggest that 1) drugs ACET and kynurenate can be used as pharmacological tools to delineate the functional contribution of specific receptor subtypes, 2) kainate receptor activation and desensitization depends on the number of subunits bound to an agonist, and 3) KAR-mediated synaptic transmission at the mossy fiber – CA3 is conducted through heteromeric GluK4- or GluK5-containing KARs. Metabotropic receptors, such as muscarinic acetylcholine receptors (mAChRs) and dopamine receptors (DARs), can also alter the function of glutamate receptors and have been implicated in epilepsy. Muscarinic acetylcholine receptors also play a critical role in synaptic plasticity and neuronal excitability. There are five types of mAChR, M1-M5, all of which, except m5 mAChRs, are found at different levels of expression in area CA3 of the hippocampus where they are co-expressed with kainate receptors. Muscarinic receptors regulated the function of other glutamate receptors, but it is unknown whether they can interact with KARs. Dysfunctional interactions between KARs and muscarinic acetylcholine receptors (mAChRs) have been implied in neurological diseases, including temporal lobe epilepsy. For example, injection of a mAChR agonist (pilocarpine) in rodents induces prolonged seizures and epilepsy, which can be blocked by a KAR antagonist. Understanding how KARs and mAChRs interact may unlock novel therapies for epilepsy. Using field potential electrophysiology, we discovered that mAChR activation selectively depresses KAR-mediated fEPSPs at the mossy fiber – CA3 synapse. This mAChR depression of KAR fEPSPs is mediated through M1 mAChRs, but cannot be totally explained through PKC phosphorylation. Interestingly, M1 mAChR depression of KAR fEPSP goes away with aging, suggesting this phenomenon is developmentally regulated. Lastly, we investigated whether the dopaminergic system is altered in a chronic model of temporal lobe epilepsy. Similarly to mAChRs, DARs, specifically D1-like DARs, play a critical role in synaptic plasticity, neuronal excitability, and have been associated with seizure propagation. We demonstrated that D5 DARs, but not D1 DARs, expression is significantly depressed in the epileptic hippocampus. Furthermore, we found that dopamine clearance is reduced, while total dopamine content is unchanged in the epileptic brain compared to sham-treated controls. Taken together, we demonstrate the first steps toward discovering a novel interaction between KARs and mAChRs in the brain. Furthermore, we identified compensatory changes that occur in the dopaminergic system as a result of chronic temporal lobe epilepsy. These findings will provide potential targets for therapeutic interventions for patients with epilepsy.
Cooper, C. P.(2015). Metabotropic Receptor Modulation of Kainate Receptors in the Hippocampus. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/3239