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Live Chat: Technical Considerations for Optogenetic Experiments


aabdullah
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I’m not totally clear on the design of your experiment. Are you transplantinc cells? Or trying to use opsins to manipulate the activity of a class of neurons already in the brain?

We typically do a channelrhodopsin virus injection at the same time that we do the fiber implant, so it’s all done in a single surgery. If you need a cannula, you could do a cannula implant, inject virus through the cannula, then use a fiber threaded down through the cannula to do fiber-optic stimulation. Threading the fiber through the cannula is not a surgery and can be done with the awake animal immediately before a behavioral session. This is much trickier than the more standard optical fiber/ferrule approach, though, because optical fibers can break off in the cannula if the animal twitches, and when that happens there is no recourse.

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Katinka Stecina

Also, I would like to know, if there have been any papers discussing the expected latencies for action potential generation via ChR2 stimulation in vivo? I know it all depends on a lot of factors, but ranges I have seen are soooo varied and maybe there has been ground-work done on this but I am not aware of it. If you have any reference suggestions, I would greatly appreciate those too!

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It will probably be difficult to get an absolute measure of the power you’re putting down with the optopatcher, but measuring without the pipette attached will at least give you an upper bound. Aside from that I would try just carefully approaching a power meter with the pipette attached to get another measure.

Another way to restrict your light with the optopatcher (or at least direct it) is to paint the pipette with something opaque (black nail polish is common) up to the tip. This might help with measuring/stimulation.

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John Carlo Combista

Good Day! I only have a few knowledge about Optogenetics because I am still new to the field of neuroscience and still a medical student but since my interest is towards Neurodevelopmental Disorders, I would like to ask if Optogenetics can be used to do some behavioral experiments in ASD mouse model. What will be the best parameters to be used for the experiment. I apologize for a very shallow question. Thank you very much.

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Katinka Stecina

Could you please, recommend where to get a power meter from? As you can see, I am just getting started with these…

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Exactly, I’m interested in the tools using blue light, however I see they are not as widely used. What are the Cons for using these tools compared to the more common inhibitory opsins?

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This paper (figure 4) might be what you’re looking at–shows the delay from axon stimulation to the impulse reaching the soma

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Hi Katinka,

As you point out, there are a huge number of factors in play. One end of the range is found here in our paper with MSNs that are very slow (and batches of virus that were not as efficient as current virus is):

A second end of the specturm is here with cortical interneurons that respond very rapidly:

I think one way to think about this would be to assume that for monosynaptic activation, latencies can be anywhere from sub-millisecond to a few milliseconds depending on ChR2 expression levels, light powers and membrane dynamics, but the variance should be considerably lower for monosynaptic activation than for disynaptic activation. If you suspect your data set is contaminated by some di-synaptic units, I would plot a distribution of the event variance against a distribution of the latency to see if events cluster in that space.

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Yes, you can use optogenetics for to do behavioral experiments in ASD mouse models. There have been several papers that have used optogenetics to reverse or emulate ASD-related phenotypes in mouse models.

For example, if you find that in a Fragile X model, you have a dysfunction in the synaptic strength of glutamatergic inputs into a specific region and that this is accompanied with a reduction in social interaction or increased stereotypy…you can use optogenetics both to try and reverse these effects in the disease model or emulate these effects in a control WT animal. You can restrict expression to one type of input to get a better handle on which specific glutamatergic inputs are being disrupted.

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Katinka Stecina

Since you mentioned - virus, I was also wondering what would be the difference if I have switched to another excitatory opsin from ChR2? Would ChETAs better, for example, for the in vivo work?

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You could try co-expressing ChR2 and eNpHR, using blue light to stimulate ChR2 terminals in your target region, and red light to stimultaneously suppress en passant synapses in any off-target regions you are concerned about

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Katinka Stecina

This is something I can implement, thanks very much for this suggestion and for the papers.
soo much out there :slight_smile:

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Chr2 is typically my workhorse. With Cheta the upside with it is that the currents are much faster, the downside (at least with the versions I worked with–perhaps they have updated in recent years) is less total charge so while you might get fast currents, you might not activate your cell enough to spike it.

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Honestly, none of us have tried the blue light inhibitory channels, since it hasn’t been required for our experiments, and when they were first released it wasn’t easy to get our hands on them. They may be more readily available now. Many of these opsins traffic unpredictably in specific cell types, which may affect the popularity of the tool, since efficacy is a product of expression, trafficking and conductivity. That’s just conjecture, though, because I haven’t spoken with anyone who has tried the ChloCs specifically.

If it helps, it is possible to drive eNpHR or eArchT with blue light, but efficiency is lower than with green or amber.

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