Optogenetic approaches to the study of hippocampal long-term plasticity

Abstract

Synaptic plasticity is one of the cellular mechanisms thought to underlie learning and memory formation. Enormous progress has been made in the last two decades concerning the molecular mechanisms of plasticity induction and expression for both long-term potentiation (LTP) and long-term depression (LTD). LTP and LTD, together with so-called homeostatic plasticity that keeps overall activity levels near a certain set point, are experimental models for the processes that are thought to enable neuronal circuits to adapt to changing requirements and to store information. Electrophysiological approaches allow inducing synaptic plasticity with high reliability, which is ideal to study the precise molecular pathways involved in changing synaptic strength. However, it remains unclear how stable these changes in synaptic strength are. A better understanding of the long-term stability of synaptic plasticity will be crucial to better understand the relationship between synaptic plasticity and memory formation. The present thesis consists of three main parts. In the first part we explore the resolution limits of optogenetic stimulation, which relies on the activation of the light-gated ion channel channelrhodopsin-2 (ChR2) by blue light. In the second part we characterize the photocycle of an engineered ChR2 variant with very slow channel kinetics and show that light-induced firing can alter gene expression in stimulated neurons. In the third part we present a novel class of ChR2 variants that enormously improves the reliability of optogenetic neuronal stimulation and will allow delivering plasticity-inducing stimuli to genetically targeted neurons in a non-invasive manner. Part I: Spatial resolution of ChR2 activation We investigate the spatial resolution of ChR2 excitation by one-photon activation using focal laser illumination. Interestingly, resolution in hippocampal slice culture and dissociated hippocampal cells is best at minimal light intensities. At high light intensities, focal saturation of excitation and increased out-of-focus ChR2 activation degrade spatial selectivity of channel stimulation. We show that a trade-off between photocurrent amplitude and the local specificity of ChR2 activation determines the spatial precision of optical action potential (AP) induction. Furthermore, local stimulation allows to induce APs with more physiological shapes that wide-field illumination. Part II: Photocycle of bi-stable channelrhodopsins and effect of light-controlled firing on immediate early gene expression The so-called bi-stable channelrhodopsins have open channel state lifetimes of seconds to minutes. We show that the photocycle of the ChR2(C128A) variant is branched. Accumulation of desensitized channel in a long-lived non-conducting state leads to progressive reduction of photocurrent amplitudes. Vigorous burst firing can be elicited by ChR2(C128A) activation even with minimal light intensities, but the number of bursts is limited by photocurrent run-down. Finally, we show that high-frequency AP firing mediated by the C128A mutant can induced c-Fos expression in a cell-autonomous manner, which may be exploited to identify light-responsive neurons or to induce expression of foreign proteins under control of the c-fos promoter with precise timing and single cell specificity. Part III: High-efficiency channelrhodopsins for high-frequency spiking and optical control of synaptic plasticity Optogenetic control of synaptic plasticity has been hindered by the large cell-to-cell variability in the reliability of optical AP induction. We characterize the novel ChR2(T159C) mutation that dramatically increases photocurrents. When introduced in a wild-type background, the TC mutation generates very large photocurrents and sensitizes neurons to very low light intensities. Because TC can trigger several APs in response to a single light pulse, we combined the TC mutation with the previously reported E123T mutation to increase channel speed. ChR2(E123T/T159C), or simply ET/TC, combines large photocurrents with rapid channel kinetics and allows triggering single APs with high reliability up to 60 Hz. In contrast to currently used channelrhodopsins, the rapid ET/TC kinetics are preserved even at depolarized membrane potentials, which speeds up membrane repolarization after AP firing and allows high-frequency spiking even during plateau depolarizations in pyramidal neurons. In conclusion, the novel TC variants will greatly improve the reliability of optogenetic plasticity induction and enable us to investigate the long-term fate of changes in synaptic strength.