Today I’ll be talking about the importance of optimising in vivo optogenetics frequency, having previously looked at the pulse on-times. All too often, I will see papers or talk to colleagues who use an unfeasible stimulation frequency for their in vivo optogenetics. For example, where I work in the hypothalamus, you often see stimulation at 20 Hz. And from my experience of patch clamping multiple neurone types in the hypothalamus, they just don’t fire that fast.
If you’re not an electrophysiologist, it might not be obvious, but action potentials are energetically expensive. So, neurones will only fire quickly if they need to. In fact, they will only be able to fire quickly if it is required for their function. Which it is for cognitive processing, but not for the much simpler processing required in many other brain regions.
Back to the beginning
As usual, first thing we do is go back to the early optogenetics publications from Karl Diesseroth. In their 2012 Nature Methods paper, Mattis et al. performed a thorough investigation of the opsins available at the time1. And, despite being a decade later, the data still stand, and are still very useful. I strongly recommend reading this paper for anyone who plans to perform optogenetic studies. It’s a huge paper with bags of useful info.
Mattis et al. measured spike fidelity, ie. the success rate of the cell to produce an action potential in response to a flash of light. They used a high light intensity, so there is no issue of there being not enough light to activate the opsin. Instead, the loss of fidelity comes from the neurone being unable to keep up. As I’ve mentioned before, the neurone needs to recover its membrane potential below a certain threshold or it won’t be able to trigger another action potential, so if you chronically overstimulate a neurone they become silenced.
I’ve shown here a comparison of ChR2h134r (also called ChR2R) and ChIEF (Figure 1A). The black lines show the spike fidelity to light pulses, and the grey lines show the fidelity to electrical pulses. Essentially, the grey lines show what the cell is intrinsically capable of, whereas the black lines show how it fares under optogenetic control. Notice how the ChR2h134r loses fidelity at 20 Hz, whereas ChIEF only loses it at 40 Hz. This is largely because of the “off kinetics”, which means that ChR2h134r takes a lot longer to close than ChIEF (Figure 1B). And it’s only after the opsin has closed that the cell can recover its membrane potential.
A self test
Luckily I have access to an electrophysiology rig, so I was able to test spiking fidelity my target neurones. Namely, AgRP neurones of the arcuate nucleus of the hypothalamus (Arc). I transfected AgRP neurones with ChR2h134r, cut ex vivo slices and patched using current clamp. I then flashed the neurone with increasing frequencies of 470 nm light at a high intensity (Figure 2).
As you can see, the cell responds nicely with big action potentials at low frequency stimulation. But the action potentials disappear even at 10 Hz. Remember that you really need the action potential to get the response you want, whether you are stimulating the soma or the terminal. Otherwise, you really don’t know what you’re doing to the neurone, although I strongly suspect you’ll be silencing the cells. Either way, I don’t recommend flashing faster than you are able to produce action potentials.
In fact, to demonstrate why you need to limit your flashing frequency, I’ve zoomed in on the 5 Hz flashing and aligned the electrical recording with a visual representation of the likely open/closed state of the ChR2 in those cells (Figure 3). I’ve drawn the light pulses and used the published τOFF to estimate the ChR2 channel close time1.
Now imagine that you have additional pulses in between the ones shown (1 extra for 10 Hz or 3 extra for 20 Hz). Between the slow closing of the ChR2 and the slow recovery of the neurone’s membrane potential, it’s easy to see why the neurone loses firing fidelity above 5 Hz.
An important message
One of the other tests done by Mattis et al. was to simply turn the blue light on continuously for 1 second in two different neurone types. A “regular-spiking” neurone fires one action potential before being silenced, whereas a “fast-spiking” neurone fires continuously through the illumination. The point here is that some neurones can fire at 200 Hz under optogenetic stimulation (mainly cortical neurones). And if your research involves them, you probably know that.
But every neurone I’ve ever investigated wasn’t capable of anything close to that rate. So please check the firing rate your neurone is actually capable of before deciding your in vivo optogenetics frequency. Or, if you are not able to do it or get a friend to, be very careful with your stimulation paradigm. And feel free to ask someone, it never hurts to ask for help.
1. Mattis et al. Nat Methods 9(2), 159-172 (2012) Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins
2 thoughts on “Optogenetic Stimulation Frequencies”
Nice post! I wonder if you have any thoughts about continuous vs. intermittent stimulation?
For example, can you stimulate neurons continuously at lower frequencies to ensure sufficient power? Or would you opt for an intermittent stimulation protocol to avoid neuron fatigue when stimulating for long periods of time, e.g. 1 s stimulation at 10 Hz, followed by 4 s off for 1 hour in vivo stimulation? How long can a neuron maintain firing at 10 Hz before neurotransmission is compromised?!
That’s a really important question that doesn’t get enough attention. I have mentioned this in a previous blog post (https://nicneuro.net/2021/06/18/optogenetics-stimulation-frequency/), but I think it bears repeating.
From my experience of patch clamping ChR2-expressing neurones in slice, there’s really no limit to how long you can stimulate them at 10 Hz. Therefore, I wouldn’t use “neurone exhaustion” as a reason to pulse stimulation, although I could understand wanting to limit thermal damage or phototoxicity. However, the researchers I know who do intermittent pulsing to limit phototoxicity use 20 Hz or even 30 Hz stimulation, which is much too fast for most neurone types.
When you pulse intermittent stimulation, the most likely outcome that you reduce the output from your target neurones. This may be counterproductive to obtaining a maximal physiological response. However, it may be that your target neurones naturally fire in this way.
Your goal when designing your opto stimulation pattern should be to mimic the natural firing dynamics as closely as possible. Some neurone types do fire intermittent pulses; my experience in the hypothalamus is that it is uncommon, and when they do pulse it is over longer timescales than 1:4 seconds.
My advice is to start with continuous stimulation, testing a range of frequencies (eg. 1, 5 and 10Hz), and then try intermittent pulsing if you want. It is possible to obtain different outcomes in this way; for example a colleague of mine found that 5 Hz stim of her neurone population increase blood glucose, but 20 Hz stimulation in the same animals induced escape behaviour. So try different paradigms and see what happens, but don’t be afraid of continuous stimulation.
Hope this helps,