Optogenetic Mapping of Adult Newborn Neuron Projections

Over 2 million people in the United States have experienced unprovoked seizures or been diagnosed with epilepsy. In approximately 25% of cases, seizures are refractory to medical therapies. Inability to effectively treat epilepsy reflects a lack of understanding of the basic mechanisms of this disorder. Temporal lobe epilepsy (TLE) is associated with alterations in hippocampal circuits including cell loss and reactive plasticity. In the mammalian brain, there is continual generation of new neurons in a few key brain regions throughout adult-hood. This process, referred to as adult neurogenesis, represents a form of experience-dependent plasticity that is believed to support normal brain function. Brain insults including seizures and epileptic lesions, traumatic brain injury, and stroke are associated with increases in hippocampal adult neurogenesis. Abnormal integration of adult-born neurons within hippocampal circuitry may provide a substrate for hyper-excitable circuits that cause seizures. Epilepsy is associated with an emergence of dentate granule cells (DGCs) that display abnormal dendritic fields and project to unexpected targets. Many of these abnormal cells express genetic markers of adult-born neurons and this expression parallels spontaneous seizure frequency and severity in experimental TLE. Blockade of adult neurogenesis reduces spontaneous seizure expression in an animal model of post-traumatic epilepsy. Despite these reports, the functional connections that adult-born neurons form with downstream targets remain uncharacterized. Quantitative anatomical studies have provided evidence for the types of connections formed by adult-born neurons, however this work cannot directly establish the existence of functional signaling within these circuits. This proposal aims to develop a new technique to selectively label and stimulate newly-born neurons in the adult brain and then use this technique to assess functional outputs of hippocampal adult-born neurons in a chronic mouse model of TLE. Work here will selectively target expression of channelrhodopsin (ChR2) in adult-born progenitor cells based on their expression of the transcriptional activator GLi1 (Gli1 Kruppel family member) using Gli1-Cre mice. Gli1-Cre mice will be administered with a Cre-inducible Adeno-associated virus (DIO-AAV) with double floxed reverse cassettes containing channelrhodopsin (ChR2) and the fluorescent report mCherry (abbreviation: ChR2-mCherry; construct: pAAV-Ef1a-DIO-hChR2(H134R)-mCherry-WPRE-pA). Blue-light stimulation parameters will be used to activate adult-born neurons and drive signaling to their post-synaptic targets. Whole-cell patch recordings will be performed on DGCs in hippocampal slices from Gli1-Cre mice that have received injections of the ChR2-mCherry construct to characterize functional projections formed by adult-born neurons during the development of epilepsy. Understanding of how adult-born neurons incorporate into neural networks and signal during normal and disease states will help define their relevance as therapeutic targets and will also provide new context for evaluation of clinically available drugs that have documented effects on adult-born cells.