Abstract
BackgroundThe light-gated cation channel channelrhodopsin-2 (ChR2) is a powerful tool for the optical induction of action potentials in neurons. Mutations of the cysteine 128 (C128) residue have been shown to greatly extend the lifetime of the conducting state of ChR2. However, until now, only subthreshold depolarizations have been reported from C128 mutants.Methods and FindingsHere we report the induction of long high-frequency spike trains by brief light pulses in ChR2(C128A)-transfected pyramidal cells in hippocampal slice culture. ChR2(C128A)-mediated spike bursts triggered expression of the immediate early gene c-fos in pyramidal neurons. Robust and cell-specific expression of c-Fos protein was detected after a single blue light pulse and depended on action potential firing, but not on synaptic activity. However, photocurrents diminished upon repeated stimulation and limited the number of action potential bursts that could be elicited.ConclusionsWe conclude that the C128A mutant is not suitable for chronic stimulation of neurons, but very useful for light-controlled induction of immediate early genes. This property of ChR2(C128A) could be harnessed to control the expression of proteins under control of the c-fos promoter with precise timing and single cell specificity.
Highlights
Optogenetic control of neuronal firing has become a widely used tool to manipulate the activity of single neurons or neuronal ensembles in vitro and in vivo [1,2,3,4]
We conclude that the C128A mutant is not suitable for chronic stimulation of neurons, but very useful for light-controlled induction of immediate early genes
To asses ChR2(C128A)-mediated currents, we recorded from pyramidal cells stimulated with 50 ms blue light pulses from a mercury arc lamp
Summary
Optogenetic control of neuronal firing has become a widely used tool to manipulate the activity of single neurons or neuronal ensembles in vitro and in vivo [1,2,3,4]. ChR2 mutants with greatly extended lifetimes of the open channel state have been generated These ‘bi-stable’ channelrhodopsins are based on point mutations at the C128 position and elicit long-lasting photocurrents that can be switched off by a green light pulse [6]. The C128A mutant has very interesting kinetics in that its open state outlast the activation light pulse by many orders of magnitude but it still spontaneously inactivates within an experimentally accessible time window. In their original publication, Berndt and colleagues used the C128A mutant to induce long and reversible subthreshold depolarizations in dissociated neurons and to sensitize cells to synaptic input [6]. Until now, only subthreshold depolarizations have been reported from C128 mutants
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