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Abstract
Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used “slow” ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the “dauer” larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.
Highlights
ChR2 is a light-driven cation channel that enables fast photodepolarization of excitable cells in culture and in live animals ranging from Caenorhabditis elegans to primates [1,2,3,4,5,6]
As the proteins aggregated to a variable extent and we could not determine cell surface expression levels, these findings did not allow us to predict which protein may be best suited for long-term applications in C. elegans
Using C. elegans, we showed that: 1) photoactivation of slow ChR2 variants induces prolonged depolarization in body wall muscle (BWM), cholinergic and GABAergic neurons; 2) ChR2(C128S)-mediated depolarization induced by 1 s light lasts several minutes and requires about one order of magnitude less light than ChR2(H134R); 3) blue-light activated ChR2(C128S) can be inactivated by yellow light, enabling full temporal control, neurons can be switched ‘‘on’’ and ‘‘off’’; 4) photoactivation of ChR2(C128S) in command interneurons evokes long-term behavioral effects; and 5) using ChR2(C128S) even animal development can be altered
Summary
ChR2 is a light-driven cation channel that enables fast photodepolarization of excitable cells in culture and in live animals ranging from Caenorhabditis elegans to primates [1,2,3,4,5,6]. For long-term photodepolarization, e.g. to influence learning or neuron-controlled alternative developmental pathways, ChR2 is not suited: As it requires continuous illumination with blue light of high intensity ($1 mW/mm2) to keep the channel open a) phototoxicity may arise and b) intrinsic phototactic reactions of animals can occur that interfere with the studied behavior. These limitations may be overcome by the recently described ChR2(C128X) mutants [7,8,9]. Once in the open state, C128X mutants can be photoinactivated using green-yellow light, they are termed ‘‘step function opsins’’
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