Optogenetics Guide

Optogenetics is an important technique in modern neuroscience, with more than 1000 publications per year. However, it is challenging to get right, and there is plenty of misinformation out there. My optogenetics guide explains how to make optogenetics work. Hopefully, I can help you sidestep the snake oil salesmen and optimise your studies.

Optogenetics publications per year.
The increasing number of optogenetics publications per year. Source: pubmed.gov

This optogenetics guide will cover key aspects of an opto study, from planning through to analysis and troubleshooting. I’ll also include some thoughts on animal welfare during optogenetics studies.

For an excellent and indepth review of optogenetics, check out the Optogenetics Primer by Yizhar et al.

The basics

Optogenetics involves the use of light to control the activity of cells expressing light-responsive channels or receptors. There are a number of important aspects to optogenetics:

  • Cellular specificity – like chemogenetics, this technique requires the ectopic expression of a genetically modified gene in your cells of interest. Using conditional expression methods, such as cre and/or targeted AAV microinjections, means you can be extremely specific in which population you target.
  • Temporal specificity – unlike traditional pharmacology or chemogenetics, which require the administration of a chemical that needs to be absorbed, transported, metabolised, excreted etc, optogenetics uses light, which is there and gone in a… um… flash. Add to that the fast responses of opsins, and you can get high temporal specificity. This expands the possible experiments you can perform.
  • Location specificity – the spread of light through a tissue is limited by geometric spread, scattering and absorption.
  • Connections – opsins are expressed in the membrane, including at the nerve terminal. If you flash the a terminal site, you can activate (or inhibit) neurotransmitter release. In this way, optogenetics allows you to probe neuronal circuits.

A beginner’s guide

If you are new to in vivo optogenetics and are unsure how to begin, I suggest the following basic protocol:

  1. Buy these fibres, cut to length by scoring with a ruby blade and pulling, then test the light power output connected to your optogenetics setup.
  2. Input your power values (in mW) into the depth calculator, which will let you know your effective stimulation depth.
  3. I recommend starting with ChR2(h134r), either using AAV or a transgenic cross.
  4. Implant a fibre directed at your ChR2-expressing population (aim for half your stimulation depth away from the fibre tip) and stimulate at 10 Hz with 10 ms flashes.
  5. Assemble other lab members/friends/family to witness your glory!

Planning an optogenetics study

There are many ways to structure an optogenetics study, each one more complicated than the last. I am going to use the most common (and probably the simplest), which involves expression of an opsin in a specific population of neurones, and activation of that opsin in the soma or neurone terminals to drive a behavioural or physiological response in mice.

Anything more complex requires a great deal of thought, planning and validation, and is beyond the scope of this optogenetics guide. However, if you are interested in taking your studies up a notch but are unsure about any aspect of it, please post your query in the forum, or get in touch for a consultation.

When planning an in vivo optogenetics study, I find there are five key things to get right:

  1. Using the correct opsin
  2. Getting good opsin expression
  3. Proper illumination of target region
  4. Having the correct opto stimulation paradigm
  5. Behaviour/physiology measurement

1. Picking your opsin

Opsins are light-sensitive channels or receptors, and form the basis of all optogenetics. However, there is a long list of available opsins for use in optogenetics, so how do you know which to use in your experiment?

Beginners’ opsin

The “bread-and-butter” opsin that you’ll see most frequently in the literature is channerhodopsin-2 (ChR2), variant h134r. This seems to be due to a number of factors in addition to the “everyone uses it, so will I” attitude:

  • It’s responsive to blue light in the region 450-470 nm, which is the easiest to get bright lasers and LED’s for
  • The h134r mutation gives it a high photocurrent, while keeping a low irradiance threshold (~1 mW/mm^2) for activation
  • The off-time (~18 ms) is reasonable and allows for 10 Hz neuronal stimulation with little effort

More advanced opsin choices

If you want to do something other than simple activation at 10 Hz, you will likely want to investigate a different opsin. When picking an opsin, there are three key requirements:

  • Stimulation wavelength – typical colours are blue (470 nm), green (520 nm) and orange (595 nm)
  • Light sensitivity – the threshold irradiance I mention frequently, 1 mW/mm^2 for ChR2
  • Opsin channel speed – the time it takes for the opsin to close and allow your neurone to recover, typically in the 10-20 ms range

You might already have the equipment, in which case your wavelength of light is likely to be predetermined. If not, I would suggest picking an opsin in the blue range, as these are the easiest to get bright LED’s/lasers for. I’ve put together a list of opsins you might want to use depending on your specific application:

Key aspectBlue responsiveGreen responsiveOrange/Red responsive
Fast closingChronosChRGRvf-Chrimson
Good all-rounderChR2 (h134r)C1V1 (t/t)Chrimson
High sensitivityChRMine
Long termStep-function opsin
InhibitorystGtACR2eArchT3.0eNpHR3.0 or Jaws

Ok, that’s still a profusion of options, so I’ve distilled it to 3 top choices:

  • Normal activation: ChR2(h134r)
  • Fast activation: Chronos
  • Inhibition: eArchT3.0

As a rule, increasing the sensitivity will decrease the response time, and vice versa. So only pick a fast-responsive opsin such as Chronos if you really need fast action potentials (ie. 20 Hz or faster). Otherwise, you’re just making life harder for yourself for no reason.

Finally, there are a couple of interesting options that have appeared recently for neuronal inhibition in the UV range (~ 405 nm):

2. Expressing your opsin

Next we come to actually expressing the opsin. There are 2 common methods for ensuring opsin expression in neurones: crossing with an opsin reporter such as Ai32, or injecting a virus, usually an adeno-associated virus (AAV).

Using a reporter mouse

This is the easy option, although it comes with a serious caveat around the expression of your chosen cre line. I would urge caution if you want to go down this route.

For example, say you wanted to express ChR2 in AgRP neurones, you could simply cross the AgRP-cre mouse with the Ai32 reporter. The Ai32 has a ChR2-eYFP downstream of a floxed stop codon, which means that the presence of cre will switch on ChR2 expression.

Schematic of transgenic mouse breeding for optogenetics.
Crossing a cre-driver line with a ChR2 reporter produces offspring with ChR2 expressed in your target cells.

Certainly within the brain, AgRP gene expression is limited to a localised population within the arcuate nucleus of the hypothalamus. This makes it a valid choice to use a ChR2 reporter in this way. However, if you have ectopic expression during development, or expression in regions outside your region of interest, you need to use a targeted expression method.

Injecting an AAV

Performing stereotaxic surgery to inject an AAV is a complex and time-consuming process, but can be necessary to answer your scientific question. These days it’s easy and (reasonably) cheap to buy good quality AAV’s. I’ve used the following suppliers:

My recommended supplier is Addgene, because they have a huge selection of available viruses, they’re quick and easy to buy from, and the viruses are always fantastic quality. In fact, Addgene have been improving their viral preps to the point that the titre is too high, and we end up diluting a fair bit.

However, it can be daunting trying to pick the correct AAV, so my suggestion for anyone starting out is to get the cre-dependent ChR2(H134R)-mCherry in serotype AAV8. This one I’ve found to give fantastic transfection. Specific details (such as titre and volume injection) will need to be validated in your model. If you are unsure which AAV to purchase, or want help with any other aspect of you study, please post the question with as many details as you feel comfortable sharing on the forum.

3. Illuminating target region

Aside from the odd corner case, illumination will come from a laser or LED, usually connected to an implanted optic fibre in the mouse’s brain. Personally, I prefer LED’s because they are so easy to use. The only drawback with LED’s is that the light scatters, so it can be challenging to get sufficient light through your implanted fibre. I recommend getting these 0.22 NA fibres, regardless of whether you are using lasers or LED’s. Please check out my Effective Stimulation Power calculator and relevant blog post for more info about what NA fibre to use.

What brightness do I need for in vivo optogenetics?

When it comes to activating an opsin, the relevant value is the irradiance. Irradiance is light power (mW) over a given surface area (mm^2), which therefore determines how much power remains after the light scatters. Each opsin will have a certain irradiance of light above which you get robust activation. I call this the irradiance threshold. For example, ChR2(H134R) has an irradiance threshold of 1 mW/mm^2.

As you move away from the fibre tip, the irradiance drops rapidly due to geometric spread and tissue scattering. Once the irradiance drops below the threshold, you will no longer reliably activate the opsin. This is the effective stimulation depth.

Irradiance loss at increasing distance from tip of optic fibre.
Decreasing irradiance from tip of optic fibre, and the irradiance threshold of ChR2.

Here is a visual representation of the loss of action potentials as you move beyond the effective stimulation depth:

Decreasing irradiance and loss of action potentials with distance from fibre tip.
Visual representation of decreasing irradiance and loss of action potentials with distance from fibre tip.

For most opsins you need irradiance of 1-5 mW/mm^2. Given light spread and scatter in the brain, this means you need 5-15 mW of light out the end of your optic fibre. As a rule, the only LED systems you can buy that reach this level of brightness are the blue 470 nm ones.

For more details head over to my Effective Stimulation Power calculator. Always check the power figures published by the manufacturers (it also helps to check the power used by people who have published using those systems).

Lasers vs LED’s

I have not used lasers for in vivo optogenetics, due to the complexity and hazardous nature of laser systems. LED’s are just so much easier to set up and use, but there are situations that you need a laser to get sufficient power. This is because of the scattering nature of LED light.

Laser vs LED light scattering into an optic fibre
Coherent laser light is easily focused down an optic fibre compared to highly scattering LED light.

Because LED light scatters, I find it useful to think of the number of “connections” between the LED and your target. The more connections you have, the more light you will lose along the way, so the more powerful the LED needs to be:

Number of optical connections in LED systems for in vivo optogenetics.
There are 4 ways to connect an LED to a moving mouse; the fewer optical connections the less powerful the LED needs to be.

Which in vivo optogenetics system should I buy?

This is one of the most important questions when embarking on your optogenetics journey. It really determines what kind of experiments you can do, and how successful they are likely to be. The easy option, which is what I did back in 2016, was to buy a complete setup from one of the optogenetics equipment manufacturers.

Here are some example setups you can buy:

1. Plexbright LED’s on commutator

This is the system I bought back in 2016 when I first started in vivo optogenetics. It’s a 2-connection rotary LED system. You can wire in up to two LED’s on the commutator, which are easily interchangeable with different colours. The blue 470 nm LED’s have good brightness (7.5 mW from 200 um 0.22 NA cannula), but the other colours are not as bright. Incorporating other behavioural equipment modules is not easy.

Plexbright LED's on commutator setup for in vivo optogenetics.

2. Prizmatix ultra-bright LED

A collaborator has this system, and I’ve used it as well. It’s a 3-connection desktop LED. The blue 470 nm LED’s are very bright (10 mW from 200 um 0.22 NA cannula). They are controllable by TTL input, which makes them much more modular and customisable than the Plexbright commutator.

Prizmatix LED setup for in vivo optogenetics.

In a recent blog post, I validated the power output from commercially available systems to activate a range of opsins in vivo. Here is a summary of the typical effective stimulation depth from 4 big suppliers:

Validating LED's for in vivo optogenetics.
Effective stimulation depth (mm) for appropriate LED’s for each opsin, according to the manufacturer’s published power outputs.

It is also possible to buy modular parts of these setups, and mix-and-match, including with open source kit. More complex setups let you coordinate optogenetic stimulation with behaviours (eg. nosepoking or place preference); more details further down the page.

I have started doing personal reviews of some of the available in vivo optogenetics systems. If you need help designing a setup, please head over to the forum or send me a message and we can organise a consultation.

Fibre implantation

Probably the most technically challenging aspect of in vivo optogenetics, so I would highly recommend in-person training before doing this yourself. When it comes to targeting the fibre, I always make sure that I have more than 1 mm of effective stimulation depth, and aim the fibre 0.5 mm away from my region of interest.

If you plan to do bilateral stimulation, you will likely need to angle the fibres. This is not difficult to do. Just head over to my angled coordinates calculator.

4. Optogenetics stimulation paradigm

The optogenetics stimulation paradigm is crucial to the functioning of your study. You need to think about the flash frequency and ontimes. As a general rule of thumb, these are the stimulation frequencies I would use:

  • For stimulatory opsins (eg. ChR2) – 10 Hz flashing with with 10 ms flash ontime
  • For inhibitory opsins (eg. ArchT) – on for 5 seconds then off for 5 seconds

Obviously, these are only starter protocols, and I would suggest doing a “positive control” stimulation to begin to find what works best in your situation. For example, measuring food intake during stimulation of AgRP neurones.

For inhibitory opsins, you don’t want to do high frequency flashing, as you can induce reflex action potentials. The ideal situation would be having the light constantly on, but then you run the risk of heating up the tissue and causing damage and/or altering behaviour of the neurones.

Be wary of flashing too fast and causing chronic depolarisation and inhibition:

Optogenetics spike fidelity at increasing flash frequencies.
Optogenetic spike fidelity in an AgRP neurone during patch clamping.

If you need inspiration, check out what others have done in the literature for similar cell types as you are interested in. For more information, check out this blog post, head over to the forum, or request a consult.

5. Measuring physiology/behaviour

This aspect of in vivo optogenetics is far too broad for me to give full details in this guide. It is important to time the stimulation based on the behaviour or physiology outcomes you are investigating. This is definitely a time to check the literature (there’s no shame in copying), or head over to the forum. Here are some examples that I have done:

  • Measuring food intake, body temperature (using implanted telemetry) and locomotor activity (using IR beam breaks) upon activating the neurones with ChR2, both during 10 Hz stimulation and after 30-minute pre-stim
  • Measuring valence by real-time place preference – stimulate ChR2 at 10 Hz while mouse is on one side of a 2-chamber field
  • Measuring aversion by pairing ChR2 stimulation with a particular flavour

Analysing an optogenetics study

work in progress

Troubleshooting optogenetics experiments

work in progress

Ethical considerations

work in progress

© NicNeuroNet June 2022

This optogenetics guide is a work in progress. If there is information you think is missing, or you have a question or comment, please send a message using the form below. I really do welcome any and all feedback. Alternatively, head on over to the neuroscience forums and post your question there. I will answer if I can; if not, I will endeavour to find someone who can answer to provide a response.

%d bloggers like this: