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, 4 (12), e8185

Temporal Control of Immediate Early Gene Induction by Light

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Temporal Control of Immediate Early Gene Induction by Light

Philipp Schoenenberger et al. PLoS One.

Abstract

Background: The 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 findings: Here 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.

Conclusions: 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. 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ChR2(C128A) photocurrents in hippocampal pyramidal neurons.
(A) Rat organotypic slice culture co-transfected with ChR2(C128A) and cytoplasmic RFP. (B) Light-induced currents in voltage-clamped pyramidal cells (n = 52). Cells were stimulated using blue 50 ms light pulses (8.4 mW), current measurements were performed in the presence of 1 µM TTX, 10 µM NBQX, and 10 µM bicuculline. (C) Repeated stimulation of pyramidal neurons with high (8.4 mW for 50 ms; left) or low (0.05 mW for 1000 ms; right) light intensity. Blue bars indicate light stimulation pulses. (D) Photocurrent reduction with repeated stimulation at high or low light intensity (n = 6, 3, resp.). (E) Population comparison of photocurrents in non-stimulated cells (ctrl, n = 10) or cells stimulated in the incubator prior to photocurrent measurement (50 ms blue LED pulses at 90 s intervals, n = 6, 5, 18, 10). Photocurrents in cells stimulated for 4 d fully recovered within 24 h in the dark (n = 9). (F) No photocurrent reduction was observed in 4 d stimulated cells expressing wt ChR2 (n = 8) compared to non-stimulated control cells (n = 11).
Figure 2
Figure 2. Photocurrent reduction can be alleviated by green switch-back pulses.
(A) Neurons were stimulated with two blue pulses spaced 2.5 min apart. After a 2.5 min (gray, n = 10) or 15 min (black, n = 4) recovery period photocurrent reduction was comparable (p>0.5). (B) Following excitation by a blue light pulse, ChR2(C128A) photocurrents could be inactivated by a green switch-back pulse. Inactivation efficiency was 90.6±1.2% (n = 24). (C) Left: Photocurrent reduction was markedly diminished when activated channel was inactivated by a green switch-back pulse after a 3 s interval (black line). Application of a green light pulse after 20 s or 60 s was less effective in preventing current reduction (n = 3, 3). Gray line: Current reduction with single blue pulses for comparison. Right: Quantification of photocurrent reduction relative to the previous stimulation pulse without green switch-back (gray line) or with a green light pulse applied 3 s after blue light stimulation (black line). The green box indicates stimulation trials following a trial terminated with a green pulse. (D) Minimal model for the ChR2(C128A) photocycle, modified after Berndt et al. . (E) Photocurrent reduction with repeated stimulation was also observed in ChR2(C128T) and ChR2(C128S) mutants.
Figure 3
Figure 3. Light-induced spike trains in pyramidal neurons.
(A) Spike trains triggered by four subsequent blue stimulation pulses. Depolarization was allowed to revert to baseline between stimulations. Right: Instantaneous frequency of spike trains. (B) Analysis of light-triggered spike trains. The number of spikes, maximal depolarization, and initial frequency decreased with repeated stimulation. Insert in first panel shows cell firing 355 APs after first stimulation pulse. Gray lines represent 12 individual cells, average is shown in black. (C) Light-induced depolarizations were interrupted by a green switch-back pulse after 20 s. Traces depict two subsequent spike trains. (D) The reduction in spike numbers and maximal depolarizations was comparable to experiments without green switch-back pulse (n = 10).
Figure 4
Figure 4. Analysis of light-triggered activity in current-clamped cells.
(A) Sample traces of a cell entering depolarization block after the first stimulation pulse. Note the pronounced spike amplitude attenuation. The second pulse induced a smaller depolarization and a series of APs. (B – D) Spike train parameters. Cells were classified based on their response to the first stimulation pulse. Train firing: >30 APs, n = 6. Depolarization block, n = 7. Subthreshold depolarization, n = 4. Sparse firing: 1–30 APs, n = 5. (E) Spike trains in a pyramidal neuron stimulated with very low or high light intensity. Analysis of the instantaneous firing frequency reveals a similar rapid frequency drop under both conditions.
Figure 5
Figure 5. Cell-attached recordings of light-induced spike trains.
(A) Sample trace. Cell-autonomous activity was isolated by bath-application of NBQX. (B) In the majority of cells the number of spikes decreased with repeated stimulation (8/11 cells). Asterisks indicate cells presumably entering depolarization block after initial stimulation pulses. (C) Top: Spike raster for cell with long delay to first spike (10.75 ms). Bottom: Spike raster for cell with short delay to first spike (4.25 ms) presumably entering depolarization block. (D) Top: Long spike train containing 394 APs. Bottom: Spike train in current-clamped cell (355 APs) for comparison.
Figure 6
Figure 6. Cell-specific c-Fos induction by light-triggered spike trains.
(A) Immunostaining for endogenous c-Fos in CA1 area of rat hippocampal slice cultures under basal conditions (top) and 2 h after stimulation by extracellular application of 50 mM K+ (bottom). (B) c-Fos induction in a pyramidal neuron expressing ChR2(C128A) and cytosolic RFP after 10 stimulation pulses. Cultures were fixed 2 h after stimulation and stained for c-Fos. (C) c-Fos signal in non-stimulated cells (n = 27) and at different time points after light stimulation (n = 18, 24, 18, 25, 21). Green shaded bar indicates mean±SD of non-stimulated control cells. c-Fos induction was significant at all time points (p<0.01). (D) Significant c-Fos induction was observed after a single blue light pulse (n = 17, 15, 14, 21, 19, 11). Dashed line indicates average c-Fos signal after 50 mM K+ stimulation. (E) Light-triggered c-Fos induction was blocked by TTX, but not by NBQX or CPP. *: p<0.01 compared to control. (F) Green fluorescence in live organotypic slice cultures (CA3) from fosGFP transgenic mice under basal conditions (top) and 4 h after brief stimulation with 50 mM K+ (bottom). (G) Light-induced GFP expression in fosGFP reporter mouse neurons expressing ChR2(C128A) and RFP. (H) Light-induced GFP expression was quantified 4 h after light stimulation (10 pulses). Scale bars in (A,B,F,G): 10 µm.

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