UPDATE : Bi-Directional Optogenetic Control

26 03 2010

The Deissseroth lab has released an updated version of their optical neuronal silencing gene Natronomonas halorhodopsin. In Molecular and Cellular Approaches for Diversifying and Extending Optogenetics, Gradinaru et al review current optogenetic methodology, and introduce eNpHR3.0-2A-ChR2, a genetic vector whose expression allows both action potential silencing and firing via illumination. This vector uses post-translational cleavage (via cis-acting hydrolase elements) of the 2A peptide to coexpress channelrhodopsin and halorohdopsin at high levels via a single promoter. The use of 2A provides a more balanced level of relative expression compared to the traditional strategy of using an IRES site, though differing degradation rates of the two proteins cause expression to not be truly stoichiometric.

eNpHR3.0 has superior cellular membrane expression

The improved eNpHR 3.0 contains additional trafficking sequences that greatly reduce expression in intracelluar compartments.  This results in enhanced surface expression a 20-fold increase in photocurrents over eNpHR1 and large, near-nanoampere currents at modest 3.5mW/mm^2 light intensities.  The paper implies superior performance over the Boyden group’s Arch optogenetic silencer technology, but shows no head to head data.  As always, testing both in your own system is the best way to evaluate their relative merits.

Activation spectrum for eNPAC (left), and for ChR2(H134R) (right, blue) and eNpHR3.0 (right, yellow) alone. Maximum eNPAC steady-state excitation was 567 ± 49 pA at 427 nm (n = 9), 62% of the value for ChR2(H134R) alone (916 ± 185 pA; n = 5). Similarly, maximum eNPAC inhibition was 679 ± 109 pA at 590 nm (n = 9), 61% of the value for eNpHR3.0 alone (1110 ± 333 pA; n = 4). Output power density for peak eNpHR3.0 current values was 3.5–5 mW/mm2 (3.5 mW/mm2 at 590 nm).

Gradinaru, V., Zhang, F., Ramakrishnan, C., Mattis, J., Prakash, R., Diester, I., Goshen, I., Thompson, K., & Deisseroth, K. (2010). Molecular and Cellular Approaches for Diversifying and Extending Optogenetics Cell DOI: 10.1016/j.cell.2010.02.037


Tang, W., Ehrlich, I., Wolff, S., Michalski, A., Wolfl, S., Hasan, M., Luthi, A., & Sprengel, R. (2009). Faithful Expression of Multiple Proteins via 2A-Peptide Self-Processing: A Versatile and Reliable Method for Manipulating Brain Circuits Journal of Neuroscience, 29 (27), 8621-8629 DOI: 10.1523/JNEUROSCI.0359-09.2009


Ultrafast optogenetic control with ChETA

19 01 2010

The Deisseroth and Hegemann groups have just published a newly engineered channelrhodopsin, ChETA, in the Nature Neuroscience paper, Ultrafast optogenetic control.  Gunaydin et al rationally targeted mutations to the opsin pocket of channelrhodopsin-2 to increase the speed of channel deactivation/closing. ChETA provides higher fidelity optical control of spiking at high expression levels or firing frequencies (up to 200Hz!) and eliminates plateau potentials during sustained spike trains.

ChETA improves spike train fidelity

ChETA clearly provides higher precision in optical control of spiking, particularly at high spike rates.  However, a big problem limiting some in vivo channelrhodopsin use has been insufficient conductance. Some groups have sought to increase single channel conductance, but this approach can lead to increased ChR toxicity and/or spurious spikes. At first glance, increasing deactivation rates, and thus decreasing single channel current from a brief light pulse, seems to make life MORE difficult for situations where light and conductance levels are limiting. ChETA produces a very low number of successful spikes at 1ms illumination (Fig 3f), as compared to ChR2. ChETA response peaks within 1ms but requires 2ms illumination and >10Hz trains to induces spikes more reliably than ChR2. Is this due to a decreased peak single channel conductance in ChETA or just the activation/deactivation rate differences?  I couldn’t find a direct single-channel conductance comparison in the paper.

ChETA requires a longer light pulse than ChR2 to generate spikes.

A reduced conductance might not be a bad thing though. ChETA’s increased deactivation rate might make less toxic to cells, allowing a higher expression level, which would compensate for a reduced single channel current flow. It all depends on what causes ChR2 toxicity.  Is toxicity caused by a non-illuminated leak current or something else?  Is the deactivation rate correlated with a leak current and/or toxicity?  I would love to see a quantitative comparison of expression level and toxicity between wt ChR2  and ChETA.  Maybe our readers can post their experiences with it in the coming weeks.

Deisseroth is on fire

29 04 2009

Is there any biology lab hotter that Karl Deisseroth’s right now?  In the last TWO WEEKS he’s authored

3 Nature papers

2 Science papers

and a PLOS One for icing.


He’s the Xander Cage of neuroscience, having just triggered an avalanche, he manages to move quickly enough to stay ahead as his pursuers get mowed down by the rapidly accelerating barrage of papers.  Over the next year, we are going to see hundreds of ChR2 papers coming out, making it really hard to stand out from the crowd.

Non-desensitizing Channelrhodopsin

12 03 2009

One of the problems with Channelrhodopsin-2 is that it desensitizes during continuous illumination or at high illumination frequencies. This limits the application of ChR2 to systems where high spike rates are not used to code information.

Desensitization of various Channelrhodopsins

Desensitization of various Channelrhodopsins

In Characterization of Engineered Channelrhodopsin Variants with Improved Properties and Kinetics, John Lin reports a new Channelrhodopsin, ChIEF, that has increased single channel conductance, faster kinetics and much less desensitization. This allows much higher rates of action potential generation. The in vitro results look great!

Success of action potential firing for ChR2 and ChIEF for various frequencies

Success of action potential firing for ChR2 and ChIEF for various frequencies

However, ChR2 has a relatively small effective expression window.  Too little expression and you cannot drive spiking.  Too high and the cell gets sick.  Tuning this expression is finicky. For ChIEF, it remains to be seen how the lack of desensitization, which might cause additional current leak in the resting state, effects this expression window in vivo. It certainly looks like its worth testing in your system of choice.

Deep & local Channelrhodopsin-2 two-photon activation

17 07 2008

An interesting paper on two-photon activation of channelrhodopsin-2 is out in Biophysical Journal. In In-depth activation of ChR2 sensitized excitable cells with high spatial resolution using two-photon excitation with near-IR laser microbeam, Mohanty et. al show cellular activation with a fast-scanning two-photon laser.

Action potential generation from Channelrhodopsin-2 with a two-photon beam has been difficult to achieve, presumably due to the small activation volume of the 2p spot. They show similar calcium transients in response to 2p stimulation as with one-photon stimulation. As depth increases, the one-photon response attenuates faster than the two-photon. Unfortunately, the supplemental info with  electrophysiology traces are not yet online.  Presumably, they are generating action potentials, but I’d like to see the raw data.  Interestingly, they also show calcium increases when the laser stays in once place.  This would imply that local depolarization causes local voltage-gated calcium channels to open, or that calcium is getting through the ChR2. I was under the impression that ChR2 has a low conductance for calcium, though this study by Caldwell et. al, in press for JBC, uses ChR2 specifically for its calcium permeability.

I’m not sure what to make of the first paper. Are they really able to fire action potentials with two-photon stimulation, at depth?  Or are the calcium traces they are seeing simply the result of localized calcium flux.  I’ll followup once the Supplemental Data becomes available.  Still worth a look if this is the sort of thing you are interested in.