Animal Behavior Guide

Here are some tips to for using the miniscopes to run experiments in freely moving mice.

Experimental timeline

Plan you experiments schedule accordingly. Here is a typical timeline for getting mice ready for experiments in freely moving mice.

Week 1: virus injection surgery

Week 2: GRIN lens implant surgery

Week 3: recovery

Week 4: base plate surgery + acclimation to experimenters/handling

Week 5: habituation to wearing miniscopes & setting imaging focal plane

Week 6: begin experiment!

Acclimation to experimenters/Handling

We typically wait at least a day after base plate surgery to begin handling. We recommend that mice become well acclimated to the experimenters prior to mounting miniscopes on awake moving mice. It is important that the mice feel comfortable with the experimenters as stress during the mounting of the miniscope during the experiment can cause adverse effects for the mice and thus, experiment. After gently handling the animals a few times, acclimate the mice to getting something placed on and off their heads by gently taking the protective caps on and off their base plate (that is cemented on the mice's heads). The experimenters should be able to quickly remove the cap without much struggle from the animal before continuing onto the habituation phase.

Habituation to wearing miniscopes

Once the mice (and experimenters) are comfortable with the cap removal, habituate the mice to wearing the miniscopes while freely moving. To habituate, one experimenter should calmly hold the mouse and remove the cap, while the second experimenter replaces the cap with the miniscope. The miniscope should easily snap into place with the magnets. The second experimenter should gently hold the sides of the base plate with one hand while securing the miniscope with the set screw with the other hand. Especially during the first time, the animal may struggle. If the animal is struggling too much, let go and calm the animal. If the animal resists and struggles TOO much while you are holding onto the base plate, it can lead to the dental cement holding the base plate to come off the skull. When the animal is calm, try to hold the base plate and insert the set screw to secure the miniscope again. Once the miniscope is secured, let the animal explore your habituation environment of choice. We typically just let them move freely around in their home cage (even if there are other mice in the cage). Depending on your experimental demands, you will need to habituate to different levels of movement. If the experiment only requires mice to walk around and explore, we recommend habituating for at least 3 days for 10 minutes each day. If the experiment requires the mice to run swiftly (e.g. down a linear track), then we recommend habituating for at least 5 days for 10-30 minutes each day.

Setting imaging focal plane & LED intensity

We recommend setting the focal plane of imaging and LED intensity prior to the experimental day. This can be done during the habituation sessions. Move the focus slider along the neck of the body of the miniscope until you've reached your optimal focal plane of the brain. To set the focal plane, tighten the set screw into the focus slider until the focus slider is secure. Be careful of over-tightening, as the screw can make indents into to the plastic material of the neck of the body. Once the focal plane is set, do not change it for the duration of your experiment if you need to image the same set of cells across sessions. Typically, we have found that for some mice, they can use the same miniscope set at a certain focal plane. For other mice, they need the miniscope to be set to a different height.

During this session, you may also want to find the optimal level of LED intensity for imaging. Make sure that the there is enough blue light to see both the firing of the cells as well as some blood vessels to be used as landmarks to align frames. We recommend taking a video during the habituation session with your settings and analyzing the video to make sure the settings are optimal prior to the start of the experiment. Also, take a snapshot of the brain that you will use to reference for all future recording sessions. That way, you can begin your experiment with confidence and ease!

Starting an experiment

Here is an example checklist that can be used.

• Check if there is enough space on the hard drive for the data files that will be collected throughout the experiment. If you are using a behavioral camera, don't forget to include that as well.
• Check and clean the imaging sensor before screwing it on the focus slider. If there is dust or dirt, use lens paper and alcohol to gently clean the sensor. This typically only needs to be done once prior to securing onto the focus slide. If the imaging sensor is being removed between sessions, make sure to screw it back onto the focus slide in the same orientation!
• Connect the miniscope cable with the DAQ, connect the USB 3.0 cable with the DAQ and computer, and open the Miniscope Control application on the computer.
• Make sure the 3 lights on the DAQ turn on and the red light on the miniscope turns on.
• Make sure that the miniscope is flush against the base plate or as much as possible and the miniscope is not tilted and the set screw is not in the way.
• Compare the live image you see with the snapshot of the same region you took during the habituation. Try to match it up as best as possible. The blood vessels, contrast, brightness should all look similar. If it doesn't then troubleshoot as to why it is different.
• Gently tighten the set screw to secure the miniscope onto the base plate. Stop once the screw makes contact with the plastic of the miniscope. Over tightening will make an indentation into the plastic body and cause damage. To check if the miniscope is secure, GENTLY wiggle and tug on the miniscope while attached to the base plate while watching the imaging of the the brain. Moving the miniscope should not cause movement of the image on the video.
• Make sure to have enough slack on the cable so the mice can move freely but not so much that the mice can chew on the cable. If you're not using a commutator, attend to the possible twisting of the cables while the animals are moving.
• Check to see that the write speed of the computer exceeds the FPS collected.
• After the trial, visually inspect that data folder to make sure the data was correctly collected.
• Back up your data!!!