Dear Polina,

We've experienced the same problem with out ceiling lights. When we got the lights down to the required luminous flux (which was around 400 lx).

What you ideally want is scattered light, so no direct light sources that could influence the behaviour of the animals. One way to do this is to cover the ceiling lights with linen or cotton sheets. It this something that would work for you?

Regarding the flickering; I'm not 100% sure, but I believe that the optical nerve of the rat works at a frequency in a way that they would register a flickering light, where we humans don't see flickering. But this is a shot in the dark (pun intended).

Let me know if something is unclear or you need further help!

Maurice

We run watermaze testing in Lister hooded rats, using Noldus Ethovision software (e.g., McGarrity et al., 2017, Cereb Cortex; https://academic.oup.com/cercor/article/27/9/4447/3056475/Hippocampal-Neural-Disinhibition-Causes?guestAccessKey=a82e271a-29d9-498c-86e2-7ecf15e5374b).

The use of indirect lighting will help, e.g. up-lights pointing towards the ceiling with the light being reflected down. The type of bulbs is less critical - we use standard halogen bulbs from the super market. However, you should also make sure that you achieve a consistent, reproducible and high (about 200 lx over water surface) ambient light level, as this will affect the rats' performance.

In addition, you may want to look at the 'Detection settings' in Ethovision. 'Dynamic subtraction' with 'Detection' set to 'Darker than background' typically works well with Lister hooded rats and the water made opaque with latex or white children's paint. However, detection setting may occasionally require adjustment, until detection is reliable. People from Noldus or your local supplier can probably also help with this. 

Dear Polina, the best way to resolve the light reflection problems is to avoid direct illumination on the pool. Try to use, instead of the ceiling lights, a simple floor uplight lamp, keeping the illumination level low.

First of all, many thanks for sharing your experience! It is very helpful for me.

Dear Maurice, I've already tried to use the white bed sheets as a "diffuser" on the ceiling, but the result is still not optimal (reflections on the water diminished but not eliminated). So we don't have another choice that set up-lights around the maze. And indeed I agree with you, aggressive ceiling light could affect rats' behaviour or stress levels in the maze. 

Thank you for this idea that rats can detect flickering light. We don't see it but our camera does. I can try to change the settings on it to minimise flickering on the video. However, even if I manage to do that, it still can potentially affect rats' behaviour. 

Dear Tobias, we have installed 3 up-lights around the maze and the light is reflecting from white ceiling and walls so it's indirect. And we always control the brightness on the platform as ~450 lx. So you have any flickering on your videos using halogen bulbs from supermarket? People from Tracksys/Noldus UK told us that the system failed to track because of the flickering. I think that also our Detection settings were not optimal and I will ask their help when correct lightening will be settled.

Are the rats ok after swimming in painted water? Do you dry them and put in their home cage after each trial?

Could you please PM me the model of your bulbs / lamps if possible? Thank you in advance!

Best wishes,

Polina

Dear Polina,

I assume you're using a digital colour camera, right? Noldus standard supply consists of the infrared setup. You don't get the colour videos, but it should be less stressful for the animals, I assume.

Regarding the paint; that would depend on the paint you're using, of course. We used white latex (with the use of black mice and a black pool background) from the local DIY store and we dried the mouse after each trial.

We did 4 trials in one day; first two trials mouse A, 2 trials mouse B, followed by 2 trials mouse A and 2 trials mouse B. After each two trials we placed the mouse underneath a heat lamp to prevent a drop in body temperature, since the temperature of the water in the MWM was around 21 degrees Celsius (= 70 degrees F). I would suggest to do the same with rats. Preferably in a cage with some bedding from the home cage (to mimmic the smell, reducing stress compared to a new cage, and preventing the home cage of becoming wet).

Maurice

Dear Polina,

The light bulbs are standard halogen bulbs, 100 W, as you can buy them in any supermarket.

The children's paint is also from the supermarket, and doesn't harm the rats (or experimenter). Latex is also fine with the rats based on my experiments, but there is the problem that many people have a latex allergy.

We place rats onto a towel in a plastic transport box between trials.

All the best,

Tobias

Tobias Bast Maurice Overzier Polina E. Panchenko Raffaele d'Isa

I second the floor lamp. You can also buy fancy diffusers, or accomplish this with a frosted shower curtain as well. You want to make sure that you have an even number of lights around the water maze so that the animals do not use them as directional markers.

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