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Brief review: Simultaneous two-color optogenetics using novel probes (Klapoetke et al., 2014)

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Rhodopsin-transducin

Cartoon structure of rhodopsin – Wikipedia Commons

To catch up on the field of optogenetics, here is a primer and here is an update on some new stuff.  Also refer to the literature (Mattis et al., 2011; Fenno et al., 2011).  This post assumes a working understanding of Optogenetics, Electrophysiology and some genetics.

A recent paper out of MIT (Ed Boyden lab) identifies two new probes in the ever growing quest to find improve optogenetic tools that will allow for greater spectral separation of activation/excitation wavelengths.  Some of the major challenges for optogenetics research are:

  • genetically expressing opsins, or other light sensitive molecules in model organisms/specific cells
  • finding the right probe that addresses a specific need (i.e. high frequency stimulation, or activate a specific Gi/o pathway)
  • delivering light of a specific wavelength to a particular target in tissue and/or specific cell types
  • spectrally separating a single probe (i.e. ChR2 activated at 470nm) from an imaging probe (i.e. GCamp3, or even the new RCamp which can be activated by blue light)
  • finding two or more probes to express that will not “cross-talk”- such that excitation wavelength of probe 1 will not activate probe 2

Optogenetics allows for very precise control of cell excitability and/or signaling pathways and researchers continue to push the limit of the existing tools and pharmacological agents.  For instance, if studying the interaction of interneurons in the hippocampus area CA1 it would be beneficial to be able to simultaneously activate CCK interneurons while optically inhibiting PV interneurons, or vice versa.  There are currently methods of blocking one versus the other (machR agonist carbachol for instance activates CCK only) but to rapidly be able to control the activity of these neurons would be of great interest in studying the details of synaptic transmission of individual or small groups of neurons (worth noting is these types of on/off, two wavelength probes exist for other applications such as PIF-2.

In this new paper (part of Dr. Klapoetke’s PhD work) Klapoetke and colleagues begin with transcriptome sequencing of 127 species of alga (http://www.onekp.com/) to identify two new target opsins.  The goal is finding one that is activated only by blue light and a second that is activated primarily by some other wavelength (red or green) without interfering with the first opsin (so they can be used together in a single experiment without cross talk).  In their search they identified Chronos (blue and green light drivable channelrhodopsin) and Chrimson (red-shifted activated channelrhodopsin), which we will consider here.  As always, I intend this to be a brief overview for those looking for new things in the field, and for the experimental details see the methods, supplemental methods and 21 (yes, twenty-one) supplemental figures!

By Victor Blacus (SVG version of File:Electromagnetic-Spectrum.png) [CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

By Victor Blacus (SVG version of File:Electromagnetic-Spectrum.png) [CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0)%5D, via Wikimedia Commons

From the 127 species/transcriptomes, Klapoetke and colleagues isolated 61 channelrhodopsins which they express in HEK cells and do basic electrophysiology to determine which are best suited as far as peak current, peak wavelength of excitation, and kinetics.  They then chose a variety of the “best” 61 and express these in cultured neurons (co-transfected with tdTomato for identification; see Figure 1).  In the end, they identified two that were prime candidates:  one from Chloromans subdivsa (CsChR) and one from Chlamydomonas noctigama(CnChR), or Chronos and Chrimson, respectively.

Chronos and Chrimson were then put through a battery of functional and physiological testing.  In cultured neurons each were exposed to different intensities of light, pulse frequencies, etc,  and were characterized next to two well known channelrhodopsins (ChR2, C1V1TT) (Figure 2).  The results show that Chrimson is excited at 625 nm (spectral peak at 590nm, 45nm more red than other ChRs and activation as far as 720nm in drosophila, see their discussion for more on this), and with a single site mutagenesis (K176R mutant) ChrimsonR is a red-light activated probe with fast, reliable on/off kinetics.  In contrast, Chronos is peak activated at ~500nm, but with good excitation at 470nm (with very good sensitivity to blue light (<.05 mW/mm^2)) where Chrimson is not optimally activated even at 1 mW/mm^2 (Figure 4-c, 4-e).  There is some excitation of Chrimson at higher light levels of the blue light (~470nm, >1 mW/mm^2) as well as fast spiking (Chronos is the “fastest” Channelrhodopsin to date and high frequency stimulation appears to result in charge accumulation and activation of Chrimson)  suggesting that a combination of spectral separation, as well as precise control of light intensity, will allow for activation of Chronos, or Chrimson under the ideal experimental conditions (they comment that Chrimson cells can show sub-threshold depolarizations throughout the blue light spectrum).

Figure 5 from Klapoetke et al 2014 - Nature Methods - DOI:10.1038/NMETH.2836

Figure 5 from Klapoetke et al 2014 – Nature Methods – DOI:10.1038/NMETH.2836

Key to this new approach is independent optical excitation of distinct populations of neurons in a acute cortical slice preparation (Figure 5).  Specifically, they use 470nm blue light (0.3 mW/mm^2) to activate only Chronos, while using 625nm red light (1-4 mW/mm^2) to activate only Chrimson (Figure 5-b-d).  They show that downstream of a Chronos or Chrimson expressing neuron they can illicit specific postsynaptic currents depending on whether they deliver 470nm light, or 625nm light (figure 5-f, k, l).

These new tools add to an ever growing optogenetic toolkit and offer an exciting opportunity for precision control of two types of neurons simultaneously with spectral separation.  Additionally, the red-shifted excitation spectrum of Chrimson may lend well to researchers who seek to excite deeper in tissue or live animals, since longer wavelengths tend to scatter less and penetrate deeper.

I urge those interested to read the entire paper as there is a ton of stuff I skipped over (such as optimal excitation frequency, comparative blue light sensitivity, etc).   As with any new tools/probes, I’m very interested to see how these fair in the hands of other labs/collaborators, especially as people move to other live organisms (c. elegans) and in vivo in rodents.  As researchers demand greater and greater spatio-temporal control of optogenetic proteins and signaling pathways it’s quite clear that those identifying and developing these new tools are up to the challenge.

 

 

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Written by Michael Mohammadi

March 31, 2014 at 05:17

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