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.


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5 responses

18 07 2008
spencer

“I was under the impression that ChR2 has a low conductance for calcium…”

ChR2 conducts a significant amount of calcium. See figure 1g in the original paper by Nagel et al. 2003:
http://www.pnas.org/content/100/24/13940.full

22 07 2008
andrewhires

Spencer,
Figure 1g of Nagel is of an oocyte in 80mM calcium at -100mV, pH 9. What’s the fractional calcium current under physiologically relevant conditions? Again, my memory is hazy, but I think I’ve seen data (unpublished?) that the calcium component is quite small during AP generation. If someone could provide a careful published reference of the fractional Ca current under those conditions, I’d be most appreciative.

28 07 2008
andrewhires

I conferred with an expert on channelrhodopsin who has personally measured it’s fractional conductances. Calcium conductance of the channel under physiologically relevant conditions is very small. It might be optically detectable with highly overexpressed ChR2 under ideal conditions, but these expression levels will be toxic to neurons.

28 11 2008
Graham Ellis-Davies

Again, I wonder will those GECI ever record activity?
Does activity = single AP?

28 11 2008
andrewhires

Neither TN-XXL or D3cpv can do consistent 1AP detection in vivo, despite what some papers might suggest. We are working on improved GECIs at Janelia, and I’m confident that we will reach the consistent 1AP in vivo level before too long. I’ll leave it at that for now🙂

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