We are trying to standardize our mouse pain behavior assays to what other people do

Hi Alex and co.

we have had a couple behavior assay questions that came up in our lab meeting and we were wondering what other labs do because we want to standardize our procedures as best as we can to what the majority in the field does. I know we touched on some of those topics before but I wanted to see what you have decided to do for these questions (like the male and female mice problem)

  1. Do you ever test males and females in the same room on the same day? Or do you always separate them? Have you tested them one after the other in the same room but not having them in there at the same time?
    and 2) what do you call a response for the von Frey up-down Assay, our lab actually calls the flare of the toes a response, but we are now wondering if this is really the right thing to do, because technically it is not a paw withdrawal response.

We would appreciate any insights you might have for us.

Hi @fmoehring

Ahh behavior testing… So ambiguous. So variable. But necessary (probably).
If you look back, I started a thread with @jmogil about standardizing behavior assays.

It’s probably the right thing, but there would need to be consensus about whose way is the right way. I think at best we can do right now is take stock of what the predominant way is in the literature (and through conversation) and do that. Maybe we’ll all be wrong together, but we’ll be systematically wrong (or right).

Anyway, to your specific questions.

  1. Testing males and females same time
    Since the NIH now strongly encourages (requires?) male and females in studies, how we do we do this? Separate males and females across time and space, or test them at the same time?

I asked this question a few months ago for exactly this reason.

People feel both ways about it. There is some concern about smells or vocalizations from one sex to the other that might have an effect. If a male experimenter can affect analgesia, why not male mice onto females?

I talked with @jmogil at NAPS about this issue. He mentioned that his lab has addressed this question. Perhaps he can chime in about their conclusion.

Right now, I personally am testing them in the same room at the same time, but on different von Frey racks separated by a few feet. I figure the time of day and their overall behavioral state adds more variability than the inter-sex effects, and so I’d rather test everybody at the same time than the spread males and females across time and space. That’s just my take. Others may feel strongly that they should be separated. Hopefully someone can argue for or against those choices here.

  1. What is a von Frey response?

This is another huge area of variability. I’ve seen some people report toe spread as a response, as you do. But for me, a response is a complete lifting of the paw off the floor.

The thing is, even the paw withdrawal, is complex. As anyone who has done the VF assay knows, there are various kinds of lifts, as well as other behaviors that accompany the lift. Sometimes the lift is just quick and subtle. Sometimes, especially with higher force fibers, there is a repetitive up-down that is really strong. It sounds like vibration (tap-tap-tap). Other times, there is a lift and a holding of the paw above the body for some time. And then other times, there can be a lift and then a lick or a bite. It’s complex, and all these behaviors probably mean something different.

What is a paw withdrawal saying anyway? Is it pain? Or annoyance? Or tickle?

I think your fellow MCW colleague Quinn Hogan persuasively made this argument (what is a paw withdrawal and what does it say about pain) in a paper more than 10 years ago:


I’ve wondered how the field responded. Clearly the VF assay is still widely used assay, but has many limitations. We need to do better and come up with more informative and reproducible assays. Smart people have tried and are trying. It’s not an easy problem. That’s why we still fall back on the imperfect but still useful VF assay.

Anyway, to the specific question, I personally do not call toe spreading a positive response, although I see it often.

One thing I would encourage is for people to not just look at the VF response as yes-no, but also look at quality. I did this recently with some of my optogenetic work. I marked responses and non-responses, but also I made a scoring system, similar to what people do with cotton brush or acetone drop.

0 = Simple paw lift
1 = Paw lift above the body, prolonged
2 = Bite or lick

I based this somewhat on the scoring system for dynamic allodynia as described in Duan et al. (2014)

it doesn’t require any more work than you’re already doing, so might as well get as much information as possible from your testing.

Hopefully other, more experienced behaviorists such as @ram_kandasamy, @jmogil and @taves can say more. But this is my take.

I agree with Alex, I do not take the toe spreading as a positive response, I use a lift as a positive.