A Novel Optogenetic Tool to Image Calcium in The Tripartite Synapse during Epileptogenesis

Abstract

In the past few decades, a revolution in our understanding of brain function has occurred based on demonstrations that astrocytes play critical roles in synaptic physiology. These findings led to the concept of the which redefines the synapse to be comprised of not only the pre- and postsynaptic neuronal elements, but also the astrocyte processes that interact with them. Likewise, temporal lobe epilepsy (TLE) is a seizure disorder that affects the structure and function of networks of both neurons and astrocytes. One of the hallmark findings in TLE is the profound change in astrocyte structure and gene expression, a process called astrogliosis, throughout the brain regions involved in seizure generation. Using a well-established rat model of TLE, our lab recently demonstrated that astrocytes begin to express kainate receptor subunits during the development of TLE, and this increased expression persists throughout chronic epilepsy, suggesting that this pathway may play a role in the development of hyperexcitable circuits. However, the functional consequences of changes in reactive astrocytes and their impact on tripartite synapse function are not known. To facilitate imaging experiments at the tripartite synapse, I developed a novel genetic tool that uses a fluorescent reporter system to label astrocytes with tdTomato and neurons with Cerulean. This plasmid also includes the genetically encoded calcium-indicating protein, Lck-GCaMP6f, enabling the monitoring of calcium transients in the fine processes of all transfected cells. I expressed this novel tool in the rat brain with in utero electroporation and characterized expression throughout development using immunohistochemistry for markers of astrocytes and neurons. I demonstrated the utility of this tool to investigate functional subcellular Ca2+ signals in both astrocytes and neurons. This tool was used in experiments that showed that Ca2+ signaling is altered during the development of temporal lobe epilepsy in the rat kainic acid-induced model of status epilepticus. Spontaneous Ca2+ events in the processes of reactive astrocytes exhibited longer interevent intervals and longer duration of events compared to astrocytes from healthy tissue. I also found that increased protein expression of kainate receptors translated to functional expression of kainate receptors on a subset of reactive astrocytes following kainic acid-induced SE, suggesting a pathway that may be involved in pathological neuron-glial signaling contributing to the development of epilepsy. Taken together, these results indicate that alterations in Ca2+ signaling in astrocytes during the development of TLE may have important consequences on function at the tripartite synapse. This dissertation lays the groundwork for future studies in the tripartite synapse and points to a novel pathway that may be involved in pathological neuron-astrocyte signaling that contributes to hyperexcitability during epileptogenesis.