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.


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6 responses

19 01 2010
at

stupid question, but could you point out some references which claim that chr2 expressed at high levels causes toxicity?

19 01 2010
Chris

First things first: I think this is a great step forwards and can’t wait to see what people will be doing with this new tool. But given the title of the paper (and this post), I can’t help and wonder:

Where is this inflation of terminology coming from?

For decades “ultrafast” referred to events on the time scale of pico- and femtoseconds. Now the authors are selling a optophysiological switch on the 5 to 15 millisecond scale as “ultrafast”. Besides misusing an established term, it also poses a problem for the not too distant future: The kinetics of ChETA still leave room for another improvement on the same scale. What will that improvement be called? In holding with Manfred Eigen: “Damn ultrafast optogenetics, indeed”?

Just my little bickering😉

Chris

19 01 2010
andrewhires

There certainly is a reasonable window of effectiveness with ChR2 in many systems, but excessive expression can make cells sick. In drosophila particularly, getting enough light through the cuticle makes ChR2 stimulation tough. Unfortunately, that’s not what most publications are going spend figure panels on.

D. Zimmermann, A. Zhou, M. Kiesel, K. Feldbauer and U. Terpitz et al., Effects on capacitance by overexpression of membrane proteins, Biochem. Biophys. Res. Commun. 369 (2008), pp. 1022–1026

“Functional Channelrhodopsin-2 (ChR2) overexpression of about 104 channels/lm2 in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (Cm). The Cm increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large Cm of 1.15 ± 0.08 lF/cm2 in ChR2-expressing cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (0.79 ± 0.02 lF/cm2). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger Cm (0.85 ± 0.07 lF/cm2) as compared to non-expressing control cells (0.70 ± 0.03 lF/cm2). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins.”

Gradinaru, K.R. Thompson, F. Zhang, M. Mogri and K. Kay et al., Targeting and readout strategies for fast optical neural control in vitro and in vivo, J. Neurosci. 27 (2007), pp. 14231–14238

“In general, sufficient levels of functional expression (which may depend on gene delivery method, copy number, promoter strength, and time after initiation of gene expression) must be obtained to either potentiate or inhibit spiking. However, care must be taken not to express the opsins at excessive levels, because overexpression of membrane proteins can lead to toxicity and loss of membrane integrity in a wide variety of systems.”

19 01 2010
Graham

@Chris: I had the same comment you made from a reviewer of my recent paper that is now online in Nature Methods. However, the term “ultra-fast” MUST predate lasers. Thus, why should this term now be reserved for this small area of science? It is not logical. It turns out each area has to allowed to use terms as they think fit. Cell biologists use the letters “Rb” to mean what, the element rubidium? I don’t think so.

Andrew, thanks for your great weblog! It is always most informative.

20 01 2010
Chris

@Graham:

I can assure you that I wasn’t that reviewer (otherwise I wouldn’t have raised the point here🙂. I just whet my appetite on the abstract of your paper — sounds pretty nifty.

The term “ultra-fast” is of course not just used for lasers; biophysics and spectroscopy are other examples. As protein engineering is interdisciplinary, I think the term should be used a bit more conservatively. Especially given the point that without doubt there will be future improvements to the kinetics of ChR and I was only half kidding with the question of what that will be referred to. I really does remind me of Manfred Eigen’s little anecdote🙂

Nonetheless: as I said besides the title, I liked the paper a lot.

7 02 2010
John Y. Lin

Hi Andrew,
Regarding your comment about the on-time of ChETA (1ms vs 2ms). The characterisation of E123T mutation at the first part of the paper is based on ChR2+ E123T only, which has the on-rate ~0.9ms. However, the variant that was tested in the neurons was ChR H134R + E123T, which may actually has a slower on-rate that E123T (I don’t think the combined mutant was characterised in this paper).
One major comment I have about the paper would be the lack of parallel dose-response comparisons between ChR2 and ChR2+E123T. As the light intensity would ultimately affects the amount of ‘transient peak’/’plateau’ ratio, on-rate of the channel and the speed of transient decay. Measurements of these variables at one single light intensity can be misleading.
It may also be possible to just reduce the expression of ChR2 to achieve higher spiking rate, as the membrane resistance of fast spiking interneuron is generally very high, that a low expression is sufficient to achieve spiking without the depolarisation block and allow for faster hyperpolarisation below threshold. The figures indicates a high expression level in the interneurons.
Well, I may be biased though………….

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