Science is hard. How hard? Well, early in my PhD, I wanted to replicate some behavioral testing.

I was trained on the protocol and started testing a cohort of rats. There didn’t seem to be any problems, but when I unblinded the groups, everything was a mess.

Before, we had seen injured animals have low pain thresholds, and sham animals have high pain thresholds. Now, the groups collapsed toward the middle. Shams didn’t all recover and were indistinguishable from injury groups.

At first I assumed the weirdness was all my fault.

But then I talked to my thesis committee, read a LOT of papers, and asked scientists on twitter for help.

Turns out, stress & pain interact. So if animals are stressed, they can look like they’re in pain when doing behavioral testing. So what stresses out rats? A lot of things:

1) handling by an experimenter.

Now my rats get lots of cuddles and treats before testing so they’re not afraid of me. Any non-behavioral work is done by someone else (our “good cop/bad cop” protocol)

2) standing on wire bottom cages.

Now they also get time in the testing cages before any testing (and treats!) so the test cages aren’t scary.

3) testing males and females on the same day.

Pheromones make everything wonky, y’all

4) not having a roommate.

We used to single house post-op, because we were worried they would fight. Now they all have a cuddle buddy after one day of recovery.

5) testing at the wrong time of day.

Pain thresholds vary with the light/dark housing cycle (Minnett+ 2014 PLOSone). Thresholds are highest near the start of the dark cycle (and they’re also more active!). So now I test them in the evenings

6) seeing their friends during testing.

I painted the side of the testing cages black, so now they can’t see each other

7) smelling(?!) other animals’ pain.

Rodents might be able to “transfer” pain via olfactory cues (Smith+ 2016 Science Advances). It sounds bonkers, but I put filter tops on the cages just in case

8) testing cages being too close to the door(??!?!)

In short, if you know a behavioral neuroscientist, buy them a drink. If you are a behavioral neuroscientist, the first one’s on me.