How you do optogenetic stimulation depends on what your experimental goals are, and on your experimental preparation. If the goal is to trigger a train of action potentials, it might not be best to do a prolonged depolarization. if you depolarize a neuron too much or too long, sodium channels inactivate, which suppresses spiking. By turning the light off, it allows the membrane to repolarize (briefly), so sodium channels can deinactivate, and be ready to go when the next light pulse comes along. My guess is that the details would depend on the type of neuron, what kinds of potassium channels they express, etc.

Also, channelrhodopsin itself may require some recovery time. The current tends to decay during the light pulse, losing efficacy at driving action potentials, and recovers when the light is off. The rate and degree of decay depends a lot on the variant of channelrhodopsin.

You may be able to reduce the laser power, as you suggest, but if you fall below spike threshold, that won’t help. It may be worthwhile to record from your cell of interest to verify your stimulation paradigm will work as you want it to.

-Matthew

Dear Wei Xu ,

You cannot use continuous laser irradiation in optogenetic techniques, even with low power. This is because it would cause the nerve cells to receive excessive energy over time and result in thermal damage. Pulsed irradiation allows the cells to cool down and reduce thermal damage during the short intervals between pulses.

Best regards

I think Matthew and Elias are both answering the question, but from different perspectives.

Matthew is talking about activating neurones eg. with ChR2; then you’re looking at (relatively) fast pulsing on the order of 10 Hz. The goal here is to drive action potentials at the rate/pattern that they would fire naturally. You need to be careful not too pulse too quickly, as that also leads to inhibition in the same way Matthew explains for prolonged activation.

Elias is talking about limiting the light damage, which is particularly relevant when using inhibitory opsins. In this case, the ideal situation would likely be continuous light stimulation, but like Elias says that will lead to tissue warming and cellular damage. In theory, you could drop the light power to limit damage, but then you will drop below the activation threshold for the opsin and nothing will happen. Therefore, we do slow pulses (on the order of 0.1-1 Hz); for example, light on for 5 seconds, then off for 5 seconds. This compromise provides prolonged opsin activation while limiting tissue damage. Be careful not to do fast pulsing of inhibitory opsins, as you can induce reflex action potentials.

I have written about this exact issue on my blog, feel free to check it out and hopefully it will help answer the question in more detail:

https://nicneuro.net/2022/05/24/in-vivo-optogenetics-frequency/