Optogenetic approaches to the study of hippocampal long-term plasticity

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.