Habituation Protocol for rodent Von Frey assay?


How and how long do you habituate animals for nociceptive assay testing?

For mice

In our lab, typically we bring the mice from the animal facility, either in their home cages or a transport vessel, and then put the mice in the testing apparatus, which consists of individual plexiglass containers on a mesh rack. We usually let the mice spend several hours (>2, although variable). We do this for two days, with no behavioral testing on those two habituation days. Then, on the third day, we bring the mice into the behavioral room as above, and let them habituate for at least two hours, which is about the time it takes for them to quiet down. Then we perform the VF assay. That measure is then the baseline measure.

Some people I’ve spoken to don’t do the multiple days of habituation before testing, and just test on the first day after a few hours of habituation to the room. Others whom I’ve spoken with say that they bring the mice into the room, let them get quiet (2-3 hours) and then perform VF assay over a series of days (2-3 days), taking the last day’s measure as the baseline. The reason for this multi-day testing is because the baseline value varies over time, becoming more constant as days go on.

What do you think?


I have previously exposed rats to the von Frey containers without collecting any data in order to get the rats used to the apparatus. This seems to be a good approach and may reduce any confounds due to the stress of removing the animal from its home cage and obtaining numbers on the same day. I haven’t come across the multi-day testing approach; however, I imagine repeated testing has issues on baseline acquisition in and of itself.

Two important factors that haven’t been discussed are: 1) the size of the von Frey containers; 2) testing in sound-attenuating chambers. First, the smaller the von Frey container, the quicker rats become calm. Second, all of my testing is now conducted in large sound-attenuating chambers. I highly recommend this approach if it is feasible, as it allows the rats to settle down much faster and provides a (relatively) calm environment.

The variability in the habituation protocol itself is shocking, as each lab has its own unique way of testing mice/rats.


Don’t use mice but for my rat VF procedure. We don’t use sound attenuating chambers but I might look into that. Right now what I do is take two days of baseline measurements before beginning a test session on a separate day. I then let the rats (SD) habituate for at least 15 minutes. We begin with 2g and move up accordingly applying the filament to the glaborous skin of the paw for 5secs for no withdrawal or less if a withdrawal response is observed. I may also implement an extra day to acclimate with the chamber with no filament presentation but I have not noticed really any variability in response of control animals (our max filament is 15g)


@ram_kandasamy That’s an interesting point about the sound-attenuating chambers and the container size. Do you have a commercial source for such chambers, or are they homemade? Maybe a picture? You’re right about the variability in habituation protocols. Everyone does it differently. And same goes for the assays themselves. No wonder we have a hard time reproducing each others results. Thanks for sharing your insights.


The sound attenuating chambers are the size of a small bedroom, where we do all of our behavioral experiments. This is a pretty good representation of our rooms: http://www.governmentauctions.org/uploaded_images/booth-784484.jpg

I do want to add that Luke’s approach is how most people do the von Frey test using multiple filaments - but we’ve adopted an electronic von Frey anesthesiometer that is a little easier to use (in my opinion). The technique is more-or-less the same.

This is a good topic because although we focus on pain assays (von Frey, Hargreaves, etc.) as the source for the lack of translation of analgesics, the methods underlying these tests (which don’t really measure clinically relevant endpoints) vary considerably between labs and certainly don’t help translation either.


Here are some more questions for discussion about habituation.

  1. Should you keep the mice in the behavior containers for a defined period (say 2 hours exactly), or just let them hang out all day on the rack? I ask because in a lot of method sections, you see people say "mice were habituated for at least 2 hours, which implies that it may be more.
  2. How many days of habituation before you start testing?
  3. Do you put the same mouse in the same container every time (say you have 16 spaces in a von Frey setup), or do you mix it up every time?
  4. Do you wash the containers between sessions?


chiming in on this thread, as I’ve been troubleshooting our von Frey recently:

we use virgin SD rats, 4-5 months old

  1. We give them 20 minutes day of to acclimate to the testing cages. I just did some baseline testing, and a couple seemed really antsy, so I may give them more time on the rack to settle before testing. The only concern I have with longer acclimation is that the animals sometimes go to sleep during testing–I’m hoping some sugary cereal and maybe smaller testing cages will keep them up!

  2. I habituate the rats to handling (20 minutes in a towel in the crook of my arm) and VF cages (20 minutes, no testing) at least 4 consecutive days before baseline. This cohort, I acclimated for 8 days, as they were a little bit older, so they were less comfortable being handled.

  3. I put the same rat in the same container, with the hopes of it becoming somewhat of a “second home cage” (and reducing stress!)

  4. Blue pads are thrown out after each testing session, and containers and tabletops wiped down with disinfectant spray. I don’t want any stress hormonal/olfactory stimuli from earlier sessions, and can’t use ethanol on plexiglass.

Also, while testing, I’ve been trying out giving the rats cereal to keep them still and distracted while testing. I still get positive responses, so it doesn’t seem to be interfering with measurements, but I’m curious what others think about this?