Mantoan, L; (2013) Neuronal Signalling Studied with Light-Activated Ion Channels to Target Interneurons, Entrain Hippocampal Gamma Oscillations and Suppress Epileptiform Activity. Doctoral thesis , UCL (University College London).

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Abstract

The versatility and the electrophysiological characteristics of the light-sensitive ion-channels channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR) make these optogenetic tools potent candidates in controlling neuronal firing in models of epilepsy and in providing insights into the physiology and pathology of neuronal network organization and synchronization. The experiments described in this thesis were designed to determine if the ChR2/NpHR system allows specific targeting and manipulation of interneuron activity in cortex and hippocampus, if it allows modulation of gamma oscillations in the hippocampal CA3 area, and if it constitutes a reliable toolbox enabling systematic analysis of epileptic neural circuits and a novel anti-epileptic treatment strategy that relies on optical activation of neurons to interrupt seizures. After successful generation of lentiviral constructs containing opsin genes driven by the interneuron-specific promoters glutamic acid decarboxylase (GAD) 67 and cholecystokinin (CCK) and the production of high-titre lentivirus, it was possible to demonstrate that both microbial opsins are expressed in neuronal cultures and rat motor cortex and hippocampus. Expression of the constructs, however, was not specific for interneurons and expression levels were low compared to the same opsins driven by the calcium calmodulin-binding kinase 2a (Camk2a) promoter: either fluorescence was only visible after immunofluorescent labelling or optical control of neural activity was not achievable despite visible fluorescence. In a separate set of experiments, stimulation of Camk2a–ChR2 with ramps of blue light induced oscillations in hippocampal area CA3. Oscillations entrained to modulated ramps over a wide range of frequencies with a frequency-dependent phase relationship. Finally, optical stimulation of halorhodopsin successfully reduced high frequency epileptic EEG activity in a tetanus toxin rat model of focal epilepsy. These results demonstrate that targeting opsins to interneurons with the GAD67 and CCK-promoters is not specific, that the CA3 network has properties that allow it to entrain and synchronize to input from the dentate gyrus, which may help explain how coherence between these two anatomically coupled networks arises, and finally, that optical inhibition of HF discharges with NpHR targeted to pyramidal neurons represents an exciting new tool to be pursued in models of epilepsy both to dissect epileptic networks and for the development of other optogenetic neuromodulation therapies.

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