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aabdullah

Live Chat: Technical Considerations for Optogenetic Experiments

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aabdullah

Live Chat: Technical Considerations for Optogenetic Experiments

Thursday, August 16, 1–2 p.m. EDT

Learn the key technical issues that arise in the design and interpretation of optogenetics experiments in Module 7 of the Optogenetics Training Series.

After reviewing the Module 7 materials, submit questions from August 1 – August 16 to the Module 7 faculty in the discussion thread below. On Thursday, August 16, from 1–2 p.m. EDT, they will answer your questions live right here in the Community. You may also email your questions to digitallearning@sfn.org.

Live Chat Facilitators


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     Chris Chen             Scott Owen            Julia Lemos


Visit the rest of the Optogenetics Training Series forum for all eight modules to share your insights and best practices, ask questions, and engage with other training series’ participants.

If you are unable to log in, you can email your questions for the facilitators to digitallearning@sfn.org.

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KathiaRamirez

Do you have any recommendation to reduce the backpropagation (specific constructs, fiber localization, ligth intensity, etc) when we try to stimulate cortical terminals in the striatum?

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bargadi

Hi, what is the optimal wavelength for activating inhibitory opsins such as eArchT3.0 and eNpHR3.0? I’ve seen in the literature wavelengths ranging from green light (532nm) to yellow/red light (590 nm). We would like a wavelength that could optimally activate both opsins. Additionally, is there a different opsin that can be inhibited by blue light (473 nm)?

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Sampurna Chakrabarti

Hi,
We would like to optogenetically stimulate knee-innervating neurons specifically in freely moving mice. As far as I can see, there are no implantable devices that are small enough for that and transdermal activation might not reach the deep-seated joint nerves. I would appreciate any suggestions.
Thanks
Sam

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aabdullah

If you are unable to log in during the live chat, you can send your questions to digitallearning@sfn.org and we will post them in the thread.

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lynn.liang

Hi, do you have any suggestions to reduce the light diffraction in ex vivo optogenetics? We would like to optogenetically stimulate ChR2 within a small area in cortex with laser which integrated into the microscope, but the scope is too big even the laser power is 0.5 mW.

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julia.lemos

Hello Everyone!

This is Dr. Julia Lemos from the Department of Neuroscience at the University of Minnesota. I am happy to answer your questions on technical considerations for optogenetic experiments.

Thank you for participating in the live chat.

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julia.lemos

Are you trying to stimulate through the objective or with a fiber?

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scott_owen

Hi all! This is Scott Owen, with the Gladstone Institute at UCSF. Thank you for all your questions and looking forward to a good discussion!

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lynn.liang

We are trying to stimulate through the 40X objective.

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julia.lemos

At the moment, the best thing you can do to reduce backpropagation is to try and titrate your stimulus intensity, width and number of pulses so that you evoke terminal release w/o generating APs somatodendritically. We also recommend using inhibitory opsins within your behavioral task to compliment your excitatory opsin results.

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julia.lemos

This method is useful for when you want a lot of coverage, but you could look into stimulating with a fiber instead. This reduces the light defraction. You can order fibers from Thor (the same as what you get with in vivo experiments), cut off the end and polish the tip. This can be attached to either an LED or laser. You can set configure it the same way you would an electrical stim.

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scott_owen

Hi Sam,

This sounds like a really tough problem, especially if you think that transdermal activation cannot reach deep enough for your purposes. Optical fibers that are used for intracranial stimulation are probably not a good solution, because I assume you need the knee joint to flex. Have you looked into micro-LEDs at all? It’s not a technology I have used, but there is at least some potential for local light delivery powered by flexible implants.

for example:

Scott

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nedloh

Hi bagardi,

In general the activation wavelengths for the inhibitory opsins are very broad. You can check the original papers for this–while the peak of these guys is usually around 590 like you note, they can be activated even up to 640 (at 20% of the peak efficiency) or at 470.

So while there is a peak activation for all of these, going far off peak can still give you some inhibition of the neurons. Whether this is good enough for your experiments will depend on how much expression you get and how much light you can/want to put into your sample. If you are going this route, I would suggest testing these different wavelengths in your neurons to be absolutely sure you get what you want.

CC

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aabdullah

Question submitted by email:

My question is about the idea of combining optogenetics with neurochemical measurement. I render transplanted neuron-like stem cells optogenetically sensitive to channelrhodopsin prior to transplantation and I’m interested in measuring their selective and phasically controlled contributions to ongoing behavioral actions in vivo. I’m trying to get a good combination that allows precision stimulation that can go alongside measurement tools such as microdialysis or other such tools. This involves cannula alongside where the fiber optic cable will be implanted and the ability to do the surgery on the animal with minimal intervention and repeated surgery. Any thoughts?

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Jaadeja

What type of training does one need to use optogenetics? If we would like to use this technique should we employ a neurophysiologist or can someone with a strict anatomical background be trained to use the technique?

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nedloh

Hi All,

This is Chris Chen @ the Neurobiology Dept in Harvard Medical School. Happy to be part of the discussion.

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julia.lemos

What specific question are you trying to answer? Anyone can employ optogenetics as a technique. The training requirements depend on what the measurements and endpoints are?

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Jaadeja

Ok just wanted to know since this is very new to me and it sounds more like neurophysiology

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Jaadeja

By the way anyone can perform immunocytochemistry but some expertise is required for interpretation

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scott_owen

This sounds like a technically challenging experiment. Do the optogenetic stimulation and microdialysis have to take place at the same time? If not, you could implant a cannula chronically. For the first half of the experiment you can thread an optical fiber through the cannula. For the second half you could remove the optical fiber to do microdialysis.

If both microdialysis and optogenetics have to take place concurrently, this gets more complicated. I have had some luck with “home-built” solutions epoxying an optical fiber to a microdialysis probe for local drug delivery, but it never worked as consistently as I would have liked and I am not aware of any commercial solutions to this problem.

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nedloh

Hi Kathia,

This is a huge problem with all activation experiments. If you activate the axon enough to spike, you’re going to get some backpropagation, activation of collaterals, etc. I think Julia’s suggestion to complement your excitatory opsin’s results with an inhibitory opsin is critical. Is this experiment in the context of behavior?

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lynn.liang

Thanks for your advice! Do you know the the minimum coverage when stimulate with the fiber? Is that possible to stimulate a single cell? Do you think using soma restricted ChR2 will help?

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julia.lemos

Let me rephrase. Obviously training is required for any technique, but people use optogenetics to look at functional neuroanatomy with cfos IHC, synaptic plasticity and behavior. If your endpoint is measuring EPSCs, you need someone who has training in electrophysiology in order to conduct the experiment. If your endpoint is locomotor activity, you need someone who is trained in running the behavioral paradigm. Optogenetics can be added on top of those techniques.

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sands1m

Hi, Scott. Right now I’ve got data from cells that are producing responses to the luminopsin-driven channelrhodopsin but I really wanted to get the fiber optic thing going. I will start off just stimulating the cells without measuring the neurochemistry and confirming matters after the fact. Right now the idea of getting cells put in there through some sort of cannula alongside a fiber optic implant would be useful since I don’t want to end up having to do multiple surgeries.

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