Real-time place preference/aversion apparatus: a proposal/idea

Experimental Techniques Animal Behavior and Models
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Like everyone else who does preclinical pain research, I’m always trying to think about better, more objective, more predictive, and higher throughput assays for assessing mouse nociceptive behavior.

While the problems with our bread-and-butter assays (von Frey, Hargreaves, acetone drop, hot plate, etc.) are recognized by all, the reason these assays predominate still is because the alternatives are not much better.

Operant assays such as conditioned place preference/aversion have been used increasingly to assess cognitive/emotional factors of pain, but these assays take a lot of time and training.
What’s nice about CPP/CPA is that they can be partially automated and more objective: you put a mouse in a chamber, use video tracking software, and out pops data. The training takes a lot of user input, but the monitoring is automated and user-independent.

In contrast to the conditioned assays, real-time aversion/preference assays don’t capture a rodent’s memory of a past experience, but rather report on the current aversion/preference that it feels toward a real-time stimulus. This setup is used most now in optogenetic experiments, where light stimulation drives some place preference in the present moment.

In the pain world, people have used peltier floors to drive temperature preference in real time. But mechanical pain is one of the major modalities we test, and there doesn’t seem to be a good real-time preference assay for this.

So the idea I’m proposing is the following:

  • Make two chamber without any contextual cues ( clear boxes)
  • Make the floor in one box some kind of material that does not elicit an aversion during in naive animals, but causes aversion after injury/inflammation.: This is the most challenging part I’d say. Possible candidates for flooring are (1) sandpaper of different grit types (2) some fiber such as cotton or wool that can elicit allodynia, (3) synthetic material such as astroturf.
  • Monitor behavior using video tracking, or even better, some homemade sensors (beam-breaks) that can be controlled via Arduino or something. This would help with the expense and throughput.

I think the precedence for such an assay setup are there. We use cotton or wool or whatever to elicit dynamic allodynia when applied by an experimenter. Why not if it is part of the floor?

What are your thoughts? Any ideas for the flooring? And what key considerations am I missing here?

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Clever idea. I like using sticky-back velcro hook for creating a noxious surface. We use this in hand therapy for desensitization. It comes in 1" wide rolls, or wider, You could cut it, create paths, for example hook next to something soft like moleskin, or use it to cover a chamber floor. Because it is adhesive, it can’t be kicked or dug out of place.

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Great suggestion @Donnakennedy! Il give that a try. The adhesive part is great. And good to know it works in humans. Is it innocuous to normal control patients?

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Sorry, I’ve used it clinically, and it is commonly used for this purpose. But I haven’t studied if it is innocuous in controls. All of my evidence is clinical and anecdotal, apologies!

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I’ve had a very similar idea that I have been trying to work on, and I agree that finding the flooring that can produce allodynia only in injury/inflammation is difficult. It does not need to be perfect though in my opinion, like say 70-30% split in neurtal vs aversive as long as you get enough behavior to look at shifts. Anyway, this paper by Jeff Martin at Wake looks at some different (not in terms of substance but height/spacing) flooring for real time exploration. http://www.ncbi.nlm.nih.gov/pubmed/27544012

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@LegakisL Nice! Thanks for the new paper and for sharing your experience. Did it work? Are you using this apparatus? I’m going to try the Velcro suggestion and see what happens. Also, the Martin paper was on rats, which is not what I’m working with. I’ll keep you posted.

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I have not tried it yet, I am working with rats though. I am considering astroturf or lego-made surface. I have tried coldplates but saw no difference in chemotherapy treated and vehicle rats. Let me know if you make the box and what results you get. I will also consider the velcro surface

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An update

I tried a simple setup. A 6 x 12 x 6 plexiglass box with one side having 36 grit sandpaper, and the other just the smooth plexiglass. I took 3 mice (naive, C57 males) and let them hang out in the box for 30 mins. No habituation or conditioning. I just put them in there. I used the Any-maze tracking software to assess the duration.

IMG_1354.JPG644×859 91.3 KB

The setup

Result: The mice preferred the sandpaper side by a large marging. About 1200 s (sandpaper) to 600s (smooth). That’s exactly the opposite of what I expected. (and the variation was very low).

Possible interpretations:

  1. The gritty feel of the sandpaper is not bothersome to them, but actually is pleasurable? Perhaps it feels more like the corncob bedding that they are used to in their home cages. A smooth plastic floor is more unfamiliar to them than gritty sandpaper.
  2. Maybe they like the red color of the sandpaper? Red is evidently not visible to mice, so perhaps they perceived it as a darker area?

To see if the mice would feel differently afterwards, I did a 4 ug injection of capsaicin into the left hindpaw and then waited 2 hours. I then returned them to the apparatus. Surprisingly, there was no difference at all. Same 2:1 preference.

The number of animals here was small, but the effect was striking enough for me to believe it.

Takeaways and next steps:

  1. Find new flooring. Sandpaper (36-grit), rather than being aversive, is actually preferred at baseline and after capsaicin. I’m going to try the velcro hook next.
  2. I think the contrast between smooth and rough was too great, and hence the mice showed strong preference for the more familiar flooring. I think maybe having two difference grit sandpapers on each side might have been better, so that you’re not assessing gritty vs. smooth, but gritty vs. more gritty. The smooth maybe was just too alien to the mice.
  3. This won’t be easy :slight_smile:

@LegakisL @ram_kandasamy @jmogil @fmoehring @Donnakennedy @YawarJQ

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Alex,

Those results are certainly surprising, thank you for sharing!

Could be due to the nature of the mechanical stimulus for sandpaper which relies on friction to be aversive. It’s possible due to the size of the mice that it there is not enough movement across the grain and its acting more like a gritty surface they are more comfortable with. Interested to see what the velcro does!

-Luke

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Interesting!
I wonder if the plexiglass is so slick that it decreases stability and proprioception, (sort of like a dog running and skidding across a slick floor) so themice prefer the texture better?

Other thought, looking at your photo, is if the sandpaper end of the box is always by the wall, is that end of the box preferable for some reason? Might it seem more protective if it is closer to the wall and not as exposed? Would you get the same result if the cage ends were reversed?

I mentioned that in hand therapy we use textures and immersion in particles for desensitization. In the kit pictured, the most noxious immersion particle is small Lego pieces. What could be more irritating than stepping on Lego?!?

Good luck!

Donna

Donna Kennedy
NIHR Clinical Doctoral Research Fellow

Pain Research Group
Imperial College London
Chelsea & Westminster Campus
369 Fulham Road
London SW10 9NH
Telephone: 0208 746 8424
email: d.kennedy@imperial.ac.uk

CAPS Study phone: 07714 051654

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The results are intriguing. The points made by Luke and Donna are good ones.

Is it possible to create a conflict-type of real-time place preference? I’m drawing from the paper below, which uses an operant system. However, parts of it could potentially be applied.