Building a Custom Elevated Plus Maze to Conduct Behavioral Testing in Mice

Our Project

Hi, I am Brinkley Morse, a rising senior at the University of Texas at Austin studying Biochemistry and Computer Science. This summer, I am working with Shannon Ronca, Ph.D., and Alex Sandweiss M.D. Ph.D. on building a custom elevated plus maze to help study the behavior of mice with NMDA Receptor Encephalitis (NMDARE). Our group is in the process of optimizing a mouse model for NMDARE so we can better understand the neuropathology and immunobiological mechanism of disease. NMDARE is an antibody-mediated neurological condition caused by one's immune system attacking NMDA receptors in neurons in the brain and spinal cord. NMDA receptors play a critical role in neural plasticity, which refers to the adaptability of our brains as we change and grow. These receptors are also believed to be heavily involved in the process of memory formation.

Although the etiology of this condition is far from fully understood, it has been positively associated with a rare type of tumor (called a teratoma) and with Herpes Simplex Virus-1 infection. The rest of the occurrences of this disease are idiopathic, meaning their cause is unknown.

Patients with NMDARE can experience psychosis, delusions, seizures, loss of memory, and loss of ability to speak among other many potential neurological symptoms. During the acute phase of the disease, patients form no memories of their experience, therefore upon recovery, they have no recollection of their several-month-long illness. Complete recovery can take up to three years. Some patients may never fully recover.

We are interested in optimizing an animal model of disease to better understand its underlying pathology. To do so, we will evaluate markers of mouse cognition upon triggering the disease in our model. One such cognitive test, elevated plus maze, evaluates memory and anxiety-like behaviors. In brief, an elevated plus-shaped apparatus (see picture) with two open arms and two closed arms forces an animal to choose exploration versus shelter. We measure the amount of time spent in each arm and compare the duration between healthy mice and diseased mice. Mice displaying anxiety-like behavior will spend more time in enclosed arms, avoiding open arms. Conversely, less anxious rodents venture into open arms more frequently.

The walls of the maze are typically made of opaque material, such as acrylic or PLA. The arms and supports tend to be made of stainless steel or aluminum. The dimensions of the arms and the maze itself can vary depending on the species of rodent being tested.

For our project, we plan to use store-bought materials and 3D printing to build our maze.

Why did we need to make our own?

Working with HSV-1 requires that we conduct all of our mouse studies in an ABSL-2 biosafety cabinet, which comes with one major drawback: lack of space. A standard elevated plus maze would take up too much space in an ABSL-2 cabinet, blocking ventilation and making any experiment extremely difficult (if not impossible) to conduct.

Additionally, the maze must be thoroughly cleaned after each use to avoid contamination. Our maze must be easy to disassemble to aid the sanitization process.

Finally, name-brand mazes can run you thousands of dollars. Why buy when you can build?

See the receipt above for a complete list of parts and their respective prices. Additionally, we used roughly 2 kg of opaque black PLA filament (for printing the walls). All aluminum parts were purchased from Framing Technology Incorporated.

We also used the following tools/programs/products:

1. Screwdriver
2. 3D Printer (We used a Prusa i3 Mk2)
3. Fusion 360 Modeling Software
4. Cura (or any other slicing program such as Slic3r)
5. Gauze pads
6. Glue Stick
7. Isopropyl Alcohol

Luckily for us, the elevated plus maze is a pretty simple structure. Building the "plus" and its stand out of aluminum is pretty straightforward, but making/mounting the wall is a bit more complicated. It would be somewhat difficult to find aluminum hardware that fits our exact needs on the internet and having custom stock milled would be expensive. The easiest option was to 3D print our walls and mounts, so that's what we did. We used Fusion 360 to create models of each component.

After measuring the completed "plus", we settled on dimensions of 230 x 40 x 174 mm for the wall insert and 230 x 40 x 24 mm for the wall mount.

The inserts needed to have a strong connection to the mounts but also had to be easily removable for cleaning. For these reasons, we went with a slotted design, wherein the wall would have four prongs that would slide into the mount. The screw holes for the mount were placed in between the slots (see screenshots for clarity). Lastly, we didn't want any gaps in the wall, so we ensured that each wall overlapped with its counterpart.

Once we settled on this design, we exported the mesh as a .stl file and got ready to print.

We decided to use Cura for our slicing program because of scaling issues that we faced in Slic3r. The walls needed to be durable so we used 20% grid infill, which took almost 2 days to print. Once sliced, we saved the files to an SD card and started printing using a Prusa i3 MK2.

If you do not have access to a 3D printer, there are a number of online services you can use to have these parts made and shipped to your doorstep.

To prep your printer, spray down the printer bed with alcohol and wipe it away with a gauze pad or two. Once the bed is clean and dry, coat it with a thin layer of glue using your glue stick. This will ensure that the first layers of your print will adhere to the bed well, preventing warping mid-print.

Hopefully, the pictures above give a pretty good idea of how the "plus" is assembled, but I have written out specific building instructions as best I can.

To begin, start by assembling the "plus" component before attaching the stands. Place the long aluminum bar on your workbench with the slots facing upward. Locate the center of this bar and determine where you would like to attach the two shorter arms.

Next, affix both 8-hole connection plates to the 4-hole plate by inserting two screws through each half of the 4-hole plate and into two holes at the end of each 8-hole plate. Utilize these four screws to mount these plates to the center of the largest bar.

Proceed by threading eight screws through T-nuts and inserting eight T-nuts into the slots in the shorter arms. Align each of the slotted T-nuts with the holes of the 8-hole plates. Securely fasten the screws to the plate, following the configuration depicted in the picture illustrating the bottom of the maze.

Cover the ends of the long aluminum bar with caps.

Now that the "plus" component has been constructed, it is time to attach the stands. Retrieve four aluminum bars, four gusset brackets, eight connection screws, 16 T-nuts, and four gusset caps.

Thread eight screws through T-nuts and place one T-nut into each short bar.

Attach each gusset to a short bar using one screw. Repeat this process for all four bars.

Mount each bar onto the "plus" by using another screw along with a T-nut.

The pictures above should give a pretty good idea of how to mount the printed parts to the "plus", but I have written out instructions below just in case. The Installing_Walls.mp4 video may also give you a good idea of how the mounts are installed and how to slide the walls into place.

First, you'll want to screw the wall mounts into the "plus". Pick two opposing arms (it doesn't matter which two) and slide two T-nuts into the slots in the aluminum arms.

Hold the mount against the side of the arm and screw it in using the connection screws. You will need three screws per mount. Repeat this process for each mount.

Now that the mounts are attached, simply slide the wall inserts in. If the prongs on the inserts are rough/don't fit smoothly, you can use a Dremel with a sanding band to quickly remove excess plastic. Make sure you were proper protective equipment when sanding (safety glasses and a mask). You are now done!

Congratulations! You have built your own maze, saving hundreds of dollars and picking up valuable design/fabrication skills in the process. If you end up using our design on your project, we would love to hear what kind of research you are doing. Please feel free to reach out in the comments for questions. Now for a bonus, check out this video of one of our mice exploring the maze.