Optical tools to dissect synaptic changes underlying epilepsy A prevailing hypothesis about epilepsy contends that neural circuits become overexcitable because of a pathological imbalance between synaptic excitation and inhibition. However, many questions remain about whether this is the dominant principle of epileptiform activity, and whether the imbalance comes about because excitatory synapses are bolstered, because inhibitory synapses are weakened, or both. These issues are challenging to approach, in part because conventional recordings of network activity do not allow the strength of individual types of synaptic input to be readily resolved or dissected. To overcome such difficulties, we are engaged in developing new approaches that use genetically encoded optical indicators to track the contributions of different kinds of presynaptic terminal. We have constructed a new optical probe for vesicle fusion, called sypHTomato, which fluoresces in the red when synaptic vesicles fuse and release neurotransmitter. We are currently generating a mouse that will express sypHTomato within specific types of neurons under control of genetically targetable enzyme, Cre recombinase. SypHTomato can be used in conjunction with existing green probes such as synaptopHluorin or GCaMP3. This will enable independent and simultaneous monitoring at multiple types of synapses, be they excitatory, generically inhibitory, or inhibitory neurons of a particular subclass;it will also allow synaptic activity to be tracked along with action potential firing. We will validate and optimize this two-color system, using neural networks of increasing complexity and relevance to epilepsy. Optical recordings will be performed in conjunction with advanced methods for electrical recording currently in use within the lab. Probes for monitoring synaptic activity will be co-expressed in conjunction with light-sensitive proteins such as Channelrhodopsin-2 and Halorhodopsin to allow manipulation of selected synaptic inputs to a circuit while monitoring the output. In this way, the activity of specific synapses can be assessed during the development of interictal and ictal activity in brain slices and that activity can be further enhanced or turned off by appropriate illumination as tests of their causative role. As proof-of-principle, we will clarify the changes in synaptic input that favor or restrain the genesis of epileptiform bursts in select experimental models of epilepsy. Our molecular reagents, animals and technical approaches will be freely available to epilepsy investigators and to the scientific community at large. The reporter strategy can be easily integrated with existing lines of mice that serve as animal models of human epilepsy. Thus, powerful optical approaches to elucidate the underpinnings of epileptiform activity can be readily put to use in a wide range of mutational and experimental settings, thereby leveraging recent advances in the genetics of epilepsy.
Epilepsy is a severe and debilitating human disorder arising from defects in neuronal circuit activity. This project develops new, genetically targeted optical indicators of neuronal synaptic transmission that epilepsy researchers can use to monitor shifts in excitatory and inhibitory neurotransmission during healthy and epileptiform neuronal activity. We propose specific experiments to use these new tools to test important hypotheses regarding which cell types and synapses are most critical in controlling the transition from healthy to epileptiform activity in the brain. Understanding these fundamental questions in the field may inform future studies in developing treatments for these highly prevalent and debilitating disorders.
|Rosenberg, Evan C; Tsien, Richard W; Whalley, Benjamin J et al. (2015) Cannabinoids and Epilepsy. Neurotherapeutics 12:747-68|
|Cohen, Samuel M; Tsien, Richard W; Goff, Donald C et al. (2015) The impact of NMDA receptor hypofunction on GABAergic neurons in the pathophysiology of schizophrenia. Schizophr Res 167:98-107|
|Li, Yulong; Tsien, Richard W (2012) pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity. Nat Neurosci 15:1047-53|