As you probably know, carbon dioxide (CO2) is the most popular anesthesia for fly pushing. It immobilizes flies quickly and flies wake up shortly after being removed from anesthesia seemingly unharmed. However, CO2 is a stress odor to Drosophila (Matheson & Parsons, 1973; Matheson & Arnold, 1973; Suh et al, 2004) and flies will avoid it, depending on the context (Turner & Ray, 2009; Siju et al, 2014). Elevated exposure of CO2 suppresses immune response (Helenius et al, 2009) and long-term exposure reduces longevity (Poon et al, 2010; Shen et al, 2020).
I know some in the field feel that cold anesthesia is less stressful for flies, but I’m not entirely convinced because it’s less studied than CO2 anesthesia and cold-shock is also a stress response (Burton et al, 1988). It disrupts memory formation if given shortly after memory formation (Bouroliti & Skoulakis, 2020a, 2020b) and probably affects a number of other behaviors.
How long can I leave my flies anesthetized? The way I think about this is the sooner you can get them off the CO2, the better. Using CO2 or ice or a cold plate - I’d aim for about 2-3 minutes and recommend not leaving them on the pad for more than 5 minutes. You will probably notice that if they are on the CO2 pad for 10 minutes or longer, it takes them much longer to wake up. We don't have any controlled studies showing that 10+ minutes affects behavior, but in the lab we tend to use the 'better safe than sorry' approach.
Pro-tip: Make sure you bubble CO2 through water to humidify the gas. Otherwise the flies will dry out (aka dessicate), and it also makes them stick to the pad and to each other because of static. You can build a simple bubbler yourself (more on this in another post).
How long should I wait after anesthetization to perform behavior experiments? I’d recommend to plan to run your behavior experiments two or more days after collecting anesthetized flies. We typically collect flies that are one-to-three days old (depending on the type of experiment we are doing), then let them hang out in their collected vials in the incubator for a couple of days before doing behavior. We have performed behavior on flies 24 hours after anesthetization but generally try to avoid this and allow more time for recovery. We try not to perform experiments on flies that have been anesthetized the same day as the behavior experiment. As mentioned above, we have no definitive data that this affects fly behavior but I always figure it’s better to be safe than sorry.
That being said, for those that need to tether or head-fix flies, you can’t avoid using same-day anesthetized flies. My recommendation is to limit the time you are anesthetizing the flies to as short a time as possible and make sure you include enough detail in your methods that everyone who reads your paper knows what you did so it can be reproduced.
Do you have any other recommendations? I’d love to hear them!