Live Chat: Technical Considerations for Optogenetic Experiments

[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

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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.

[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?

[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)?

[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

[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.

[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.

[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.

[julia.lemos]

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

[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!

[lynn.liang]

We are trying to stimulate through the 40X objective.

[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.

[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.

[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:

osapublishing.org

Fabrication, characterization and applications of flexible vertical InGaN...

Flexible vertical InGaN micro-light emitting diode (micro-LED) arrays have been fabricated and characterized for potential applications in flexible micro-displays and visible light communication. The LED epitaxial layers were transferred from initial...

Scott

[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

[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?

[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?

[nedloh]

Hi All,

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

[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?

[Jaadeja]

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

[Jaadeja]

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

[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.

[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?

[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?

[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.

[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.

[Jaadeja]

Thank you.

[scott_owen]

Hi Kathia,

If removal of back-propagating action potentials is critical for your experiment, you could potentially consider trying local drug delivery (e.g. TTX or lidocaine) to silence cell bodies during your terminal stimulation, but this would be technically challenging to get right.

[bargadi]

Thank you for your reply. Earlier studies have shown peak activation for eNpHR3.0 at 590 nm, while a peak activation at 532 nm for eArchT3.0 (although some studies used the same wavelength for both). So when choosing the wavelength when ordering a laser, would it be better to choose one of these two, or somewhere in the middle (around 561), which give a relatively high efficiency for both?

[scott_owen]

It sounds like you are also interested in using blue light to silence neurons? There are some tools out there to do that (e.g. http://science.sciencemag.org/content/344/6182/409) but they are not as widely used as eNpHR or eArchT

[KathiaRamirez]

Thanks, yes we stimulate the terminals during an instrumental task.

[KathiaRamirez]

It could be done with a cannule to infuse the drug?

[julia.lemos]

You can further limit the light path by putting a beveled tip on top of the fiber. However, I think if you wanted really sparse stimulation of neurons, you’d have to combine limiting the coverage with dilution of the ChR2 virus. Try serially diluting the viral vector to get sparse expression.

[nedloh]

Hi Lynn,

Many of the scopes in my current lab have an iris in front of the light stimulation source in the microscope. We typically constrict this down as much as possible to get spot sizes on the order of several hundred microns.I would check some of the features of your microscope to see if this is possible.

CC

[aabdullah]

Question submitted by email:

What are your thoughts on reducing light-induced artifacts in recording electrodes for in vivo experiments?

[KathiaRamirez]

I’ve another question…
Given that many axons provide synapses in an enpassant manner beafore reaching their final target it is sometimes useful to inhibit that synapses without inhibiting the target . What could be a good approach to do this?

[scott_owen]

Exactly, if you infuse the drug over cell bodies or axonal projections (but well away from the terminal stimulation site) you could use ChR2 to drive terminal release while using the drug to block back-propagating action potentials.

[sands1m]

Hello KathiaRamirez,
It may be you are replying to my post. I’m interested in trying to get an infusion cannula that’s connected so that it can be right alongside where the fiber optic fiber ends in dorsal striatum. Have you heard of anything like that available commercially?

[scott_owen]

The first thing to do is make sure your fiber is not pointed directly at your electrodes. After that, it gets to be a little bit trial-and-error and it’s best to try out a variety of probes from different vendors. In our hands different probes can vary immensely in light artifacts.

If light artifacts seem to be growing over time when you re-use a probe, consider cleaning the probre with trypsin (just make sure you do a really good job of rinsing off the trypsin before you use the probe again)

[bargadi]

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?

[Katinka Stecina]

Any suggestions for good papers, resources talking about the dissipation of light/energy in live tissue- about 1-2mm below surface in brain or spinal cord? How could I be convinced that I deliver sufficient light to stimulate (working with channelrhodopsin2).

[nedloh]

I have found that the difference in efficacy of inhibition at those different wavelegnths is minimal. I would honestly just choose whichever light source is cheaper with the most power. Remember that you can make up for off-peak activation with more power (within reason of course, if you cook your sample you won’t be doing opto!). For whatever reason, it is difficult to find cheap 590 lasers with significant power, but 532 you get much more power for your dollar. Both halorhodopsin and arch are typically very powerful inhibitors. If those wavelengths at reasonable power don’t work, I would first troubleshoot expression.

[julia.lemos]

The con is that the inhibitory channelrhodopsin doesn’t appear to be commercially available. You may have to contact the developers (Deisseroth et al.) directly and arrange an MTA, etc.

[scott_owen]

Hi Katinka

This one is my go-to for light dissipation:

ncbi.nlm.nih.gov

An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology.

AM Aravanis, LP Wang, F Zhang, LA Meltzer, MZ Mogri, MB Schneider and K Deisseroth, Journal of neural engineering, Sep 2007

Neural interface technology has made enormous strides in recent years but stimulating electrodes remain incapable of reliably targeting specific cell types (e.g. excitatory or inhibitory neurons) within neural tissue. This obstacle has major scientific and clinical implications. For example, there is intense debate among physicians, neuroengineers and neuroscientists regarding the relevant cell types recruited during deep brain stimulation (DBS); moreover, many debilitating side effects of DBS likely result from lack of cell-type specificity. We describe here a novel optical neural interface technology that will allow neuroengineers to optically address specific cell types in vivo with millisecond temporal precision. Channelrhodopsin-2 (ChR2), an algal light-activated ion channel we developed for use in mammals, can give rise to safe, light-driven stimulation of CNS neurons on a timescale of milliseconds. Because ChR2 is genetically targetable, specific populations of neurons even sparsely embedded within intact circuitry can be stimulated with high temporal precision. Here we report the first in vivo behavioral demonstration of a functional optical neural interface (ONI) in intact animals, involving integrated fiberoptic and optogenetic technology. We developed a solid-state laser diode system that can be pulsed with millisecond precision, outputs 20 mW of power at 473 nm, and is coupled to a lightweight, flexible multimode optical fiber, approximately 200 microm in diameter. To capitalize on the unique advantages of this system, we specifically targeted ChR2 to excitatory cells in vivo with the CaMKIIalpha promoter. Under these conditions, the intensity of light exiting the fiber ( approximately 380 mW mm(-2)) was sufficient to drive excitatory neurons in vivo and control motor cortex function with behavioral output in intact rodents. No exogenous chemical cofactor was needed at any point, a crucial finding for in vivo work in large mammals. Achieving modulation of behavior with optical control of neuronal subtypes may give rise to fundamental network-level insights complementary to what electrode methodologies have taught us, and the emerging optogenetic toolkit may find application across a broad range of neuroscience, neuroengineering and clinical questions.

Also be aware that light dissipation means heating:

ncbi.nlm.nih.gov

Modeling the Spatiotemporal Dynamics of Light and Heat Propagation for In Vivo Optogenetics.

JM Stujenske, T Spellman and JA Gordon, Cell reports, Jul 21 2015

Despite the increasing use of optogenetics in vivo, the effects of direct light exposure to brain tissue are understudied. Of particular concern is the potential for heat induced by prolonged optical stimulation. We demonstrate that high-intensity light, delivered through an optical fiber, is capable of elevating firing rate locally, even in the absence of opsin expression. Predicting the severity and spatial extent of any temperature increase during optogenetic stimulation is therefore of considerable importance. Here, we describe a realistic model that simulates light and heat propagation during optogenetic experiments. We validated the model by comparing predicted and measured temperature changes in vivo. We further demonstrate the utility of this model by comparing predictions for various wavelengths of light and fiber sizes, as well as testing methods for reducing heat effects on neural targets in vivo.

And that heating can cause all sorts of unpredictable off-target effects

[nedloh]

Neuronexus might have a solution you can custom order, but the easiest solution (not very elegant, but should work) might just be to carefully glue a fiberoptic to hypodermic tubing.

[julia.lemos]

Are you able to use an optrode (combination of optogenetic fiber and in vivo ephys electrode) to measure light induced spiking in vivo?_

[dklorig]

I’ve had good luck with 35um formvar coated tungsten cut at an angle so that the exposed surface of the electrode is pointing away from the fiber. You can repeat your stimulus protocol post-mortem to quantify the magnitude of the artifact if any.

[Katinka Stecina]

Hi Scott, many thanks - I will have to digest these!! I have other stimuli to activate cells - so I know that I have them alive in the vicinity of my electrode (optopatch configuration but extracellular fields I am recording) but having sufficient light delivery has been my main concern with an intended excitatory-stimulus via the ChR2 channels.

[Katinka Stecina]

No real desire to introduce a bigger “object” into my tissue- many neurons just die off with that approach… but I am not sure if there is a way to measure output with the optopatch-like configuration (i.e. light probe is within the recording glass capillary above the thin, tapered portion). Any thoughts?

[bargadi]

Great, thank you for your help!

[Katinka Stecina]

ok re-reading this - yes, It is what I use (I thought you meant to ask if I could use a combination of many electrodes side-by-side)… sorry, but yes, that is what I use and it is a thin fibre (my concern number one)…