Hi all.
Recently, I encountered a problem regarding the optogenetic stimulation experiments.
In our study, we are using the blue laser (473 nm) to optogenetically stimulate parvalbumin (PV) neurons at 40 Hz (gamma frequency) in the mouse basal forebrain. According to the principles of optogenetics, the neurons that do not express light-sensitive opsins (ex. ChR2) are not supposed to be responsive to optogenetic light delivery. However, we discovered that even in the control virus (GFP)-injected group and the Sham (no virus injection) group, the 40 Hz response was observed when the blue laser was delivered at 40 Hz, just as in the ChR2-injected group (although this response was weaker compared to the ChR2-injected group). Other stimulation conditions were the same in all groups. (The laser intensity was 70mW/mm^2 in all trials.)
We hypothesized that this can be due to heat-inducing effect of optogenetic light delivery into neural tissues according to our literature survey, since the blue light is also known to induce heating of nearby tissues, which can actually induce changes in neuronal activities. However, we also questioned that if this non-specific stimulation was due to heat induction, how can the temperature increase during the stimulation interval induce neuronal firing at 40 Hz just as in optogenetic stimulation, rather than non-specific firing at random frequencies (i.e. Can the temperature change of neurons take place every 25 ms?).
Has anyone ever faced the similar issues?
I wonder if there could be other possible explanations or causes for this phenomenon.
I would also like to inquire if there are any references or resources that may deal with this problem.
How did you record the response of the cells to light?? In vitro slice electrophysiology ??
We implanted an optical fiber (200 um core) with the tip targeting the basal forebrain (BF) in freely moving PV-Cre and B6(WT) mice. During the light delivery, we recorded the cortical EEG signals via EEG headmount device that was chronically implanted on the mouse skull.
We used the same protocol in this paper: https://www.pnas.org/content/112/11/3535
Can you exclude that the rhythm is induced via retinal stimulation? How much power are your output at the tip of the light guide?
As I remember there was a study published in e-neuro showing that light alone is able to induce plastic changes. I don't remember if they also checked for the induction of any rhythm.
Niels Niethard,
I haven't thought about the effect of retinal stimulation, but I suppose there could be a possibility since the light was given in a dark room. The light power was approximately 3.14 mW.
Can you share the title of the article or the link for that information?
I appreciate your advice.
Now I found the paper, here you go:
https://www.eneuro.org/content/6/5/ENEURO.0085-19.2019
First, I would try to reduce any leakage of light from the implant. But even if there is no leakage basal forbrain is also not to far from the eyes and you might stimulate the retina via scatter light through the tissue.
yes, I agree with Niels. As a control, you could do the same experiment in a group of mice not expressing the ChR2 and see if similar EEG activity is there while shining light through the ferrule.
oh. correct.. my mistake...
So, can we say if we subtract the control case EEG activity from ChR2 activity, that could be considered as specifically ChR2 evoked activity?
I guess between animals that would be impossible. But you could stimulate right after injecting the virus when ChR2 is not yet expressed... but I don't think that this makes sense... I would check for retinal stimulation first I think this is most likely the reason.
Manish Prajapat & Niels Niethard ,
Thank you very much for your advice.
Can you lower your laser intensity to the point where you still see the effect in your ChR2 group and not the control group?
I’m not sure where you are calculating your intensity but IIRC only 1-2 mW/mm2 at the cell is required for ChR2 activation. So factor in how far away you implant your fibers and figure out your irradiance at your targeted population.
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