Jan 25, 2025
Version 2
Mouse Habituation - Head Fixation on Disk V.2
- 1Allen Institute for Neural Dynamics
- Allen Institute for Neural Dynamics
Protocol Citation: Avalon Amaya, Jackie Swapp 2025. Mouse Habituation - Head Fixation on Disk. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkojmxv5r/v2Version created by Avalon Amaya
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 26, 2024
Last Modified: January 25, 2025
Protocol Integer ID: 118030
Keywords: Rodent Head Fixation, Rodent Behavioral Habituation
Abstract
This protocol describes the typical workflow for the habituation of a mouse for awake behavioral experimentation that utilizes a disk during head fixation.
Mouse habituation is a common technique utilized for mice undergoing behavioral experimentation and/or head fixation. Habituation allows the mouse to acclimate to new environments, handlers, and handling techniques, lowering the mouse's stress.
Habituation timeline summary: Mice are habituated for a total of 5 days, involving daily handling and increasing durations of head fixation in the behavior enclosure. Mice can be either water restricted or on Adlib water during habituation. In days 1 and 2 mice are habituated to handling, and on days 3-5 head fixing is introduced.
Guidelines
Only perform this procedure in accordance with IACUC and veterinary requirements.
Utilize proper PPE when handling mice. Prioritize handling the mouse over the cage to minimize risk of mouse jumping/falling. Keep mouse cage covered when not attending to mouse. Follow IACUC and veterinary requirements for handling mice.
Mice under water restriction should be closely monitored for health concerns due to the effects of dehydration. All mice should receive 1.0mL of water daily. Follow IACUC and veterinary requirements for water restricting mice.
Animal Behavior:
Utilize a 12-hour light cycle.
Mice should have access to ad-lib food and water until placed on diet/liquid restriction.Â
Single house mice on diet or water restriction.
Ensure cleanliness of experimental environment.
Materials
Materials:
| Tool / Supply | Manufacturer / Supplier | Part Number | |
| 70% Ethanol (Diluted in house) | Sigma Aldrich | 459836 | |
| Kimwipes | Kimberly - Clark Professional | S47299A | |
| Liquid Reward | - | - | |
| Nalgene® 125 ml PET Square Media Bottles, Sterile | CP Lab Safety | 342040-0125 | |
| 1mL Luer-Lok Syringe | Becton Dickinson and Company | 309628 | |
| 25G 5/8 Needle * | Advantor | 89134-134 | |
| 1000mL Nalgeneâ„¢ Straight-Sided Wide-Mouth Polycarbonate Jars | Fisher Scientific | 21161000 | |
| Ohaus® Scout® Portable Balance | Sigma Aldrich | OH30253024 | |
| Mouse Igloo® and Fast Tracs, Certified | Bio-Serv | K3327 | |
| HEPA Replacement Filter for DC515 | Dewalt | DC5151H | |
| 18V Cordless 1/2 Gallon Wet/Dry Portable Vac (Tool Only) | Dewalt | DC515B | |
| Swiifer Sweeper | Uline | H-1960 | |
| Swiffer Sweeper Pads - Wet Cloths | Uline | S-13901 | |
| Water Bowl | Made in-house | 0146-000-01 | |
| Cart Cover | Life Science Products | CC-34X19X38Z | |
| Wheel Foam (MC® Solid Grip Black 18 in. x 4 ft.) | Kittrich | 04F-187110-06 | |
| Precision Screwdriver Hex 1/16th | WIHA | 084705263137 |
Supplies can be substituted with their equivalent.
* = Optional
Equipment:
| Material | Part Number | |
| Behavior Stage 1 (Behavior Platform, Behavior Training, Visual Behavior, Hotshoe Version 2) AND | 0107-032-00 | |
| Behavior Stage 2 (Passive Behavior Shroud Assembly ) | 0312-300-00 |
All equipment can be substituted with their equivalent.
Key:
AND = Including the tool/supply in row below.
Personal Protective Equipment (PPE):
| Suggested PPE | |
| Gloves | |
| Disposable lab coat | |
| Disposable face mask | |
| Scrubs | |
| Biohazard sharps disposal container | |
| Biohazard waste disposal container |
Utilize PPE in accordance with IACUC and veterinary requirements.
Safety warnings
Personal Protective Equipment (PPE) should be used at all times while operating this protocol.
Ethics statement
Research focused rodent behavior must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee(s).
This protocol has been approved by the Allen Institute Animal Care and Use Committee (IACUC).
PHS Assurance : D16-00781
AAALAC : Unit 1854
Before start
Reference protocol below for specific start-up and take down procedures for behavioral training hardware and software:
Amaya, A. M., Swapp, J., Groblewski, P. A. (ND). Using behavioral training clusters. protocols.io. Unpublished. https://www.protocols.io/view/using-behavioral-training-clusters-c8vbzw2n
Reference water restriction protocol below for starting and maintaining a mouse on water restriction:
Protocol
NAME
Mouse Water Restriction
CREATED BY
Avalon Amaya
Day 1: Habituating a Mouse on a Disk
Day 1: Habituating a Mouse on a Disk
Handle mouse for 2-5 min.
Scoop/cup mouse out of cage. Cup/cover mouse with hands for around 30 seconds to 1 minute.
Note
This act of cuping/covering the mouse acts to "calm" the mouse down and to prevent mouse from immediately jumping out of hands.
Hands cupping mouse over cage.
Uncup/cover hands, and let mouse explore the environment of your open hand for another 1-4 minutes.
Weigh mouse and return back to cage.
Maintain mouse on water restriction if applicable (See above section "Maintaining Water Restriction").
Clean and disinfect behavior room.
Use 70% Ethanol spray and Kimwipes to clean weigh boats and surface areas.
Wipe lickspouts with alcohol wipes.
Sweep floors.
Day 2: Habituating a Mouse on a Disk
Day 2: Habituating a Mouse on a Disk
Handle mouse for 2-5 min.
Repeat Day 1 handling steps go to step #1.1 - #1.2 .
Periodically touch the mouses headframe to habituate mouse to future head fixation.
Maintain mouse on water restriction if applicable (see Before Start section).
Clean and disinfect behavior room.
Use 70% Ethanol spray and Kimwipes to clean weigh boats and surface areas.
Wipe lickspouts with alcohol wipes.
Sweep floors.
Day 3: Habituating a Mouse on a Disk
Day 3: Habituating a Mouse on a Disk
Handle mouse for 1-2 min, secure mouse to head fixation stage, and place mouse within designated behavioral training box for 5 minutes.
Cup or grab mouse out of cage by the tail.
Let mouse explore the environment of your hands for 1 minute. Periodically touch the mouses headframe to habituate mouse to head fixation.
Hands holding mouse head frame and tail over cage.
While grabbing the mouses headframe in one hand and the mouse's tail in the other. Secure mouse to disk head fixation stage.
Stage, screws, and model of attached headframe.
A & B labels for securing points of contact in operational order.
Adjust disk height to allow mice to run/walk at a natural and comfortable gait.
Knob for disk height adjustment
Place mouse secured to head fixation stage within designated behavioral training box for 5 minutes.
Computer model design of inside of behavior box with behavior stage.
Remove mouse from box and head fixation stage.
Maintain mouse on water restriction if applicable (see Before Start section).
Clean and disinfect behavior room.
Use 70% Ethanol spray and Kimwipes to clean weigh boats and surface areas.
Wipe lickspouts with alcohol wipes.
Sweep floors.
Day 4: Habituating a Mouse on a Disk
Day 4: Habituating a Mouse on a Disk
Handle mouse for 1-2 min, secure mouse to head fixation stage, and place mouse within designated behavioral training box for 10 minutes.
Repeat Day 3 handling and head fixing steps go to step #5.1 - #5.4 .
Place mouse secured to head fixation stage within designated behavioral training box for 10 minutes.
Remove mouse from box and head fixation stage.
Maintain mouse on water restriction if applicable (see Before Start section).
Clean and disinfect behavior room.
Use 70% Ethanol spray and Kimwipes to clean weigh boats and surface areas.
Wipe lickspouts with alcohol wipes.
Sweep floors.
Day 5: Habituating a Mouse on a Disk
Day 5: Habituating a Mouse on a Disk
(Passive Behavior):
Handle mouse for 1-2 min, secure mouse to head fixation stage, and place mouse within designated behavioral training box for 10 minutes.
Repeat Day 4 steps go to step #7 - #8.3 .
(Active Behavior):
Handle mouse for 1-2 min, secure mouse to head fixation stage, place mouse within designated behavioral training box, and run 15 minute training session.
Open MouseDirector and load your username and the mouse ID onto its designated behavioral training box.
Repeat Day 3 steps go to step #5.1 - #5.5
Use lickspout controller to find Mouse Offset.
Mentioned in protocol Using Behavioral Training Clusters, section Aligning Lickspout
Amaya, A. M., Swapp, J., Groblewski, P. A. (ND). Using behavioral training clusters. protocols.io. Unpublished. https://www.protocols.io/view/using-behavioral-training-clusters-c8vbzw2n
Confirm script regimen and the mouse ID.
Press "start session" to begin auto train session.
Confirm that script has begun via camera view (typically static grating images).
Monitor mouse behavior. If not licking or if tongue is not able to make contact with lickspout, move lickspout closer to mouth.
Once session is completed, press the "safe" button on Mouse DIrector to retract the lickspout.
Remove mouse from box and head fixation stage.
Weigh mouse.
Return mouse to cage.
Click "Session Data" tab on Mouse Director.
Enter mouse weight in "Post-Session: " text box.
Session Data tab in Mouse Director.
Post-Session weight and Supplemental water calculation boxes outlined in red.
Session Data tab and Finalize button outlined in green.
Note
Mouse baseline weight and restriction percentage should already be associated with the mouse ID if water restricted through Waterlog. See water restriction protocol in above section.
Provide mouse with amount of supplemental water as written in "Supplemental:" box.
Note
If the supplemental water text box displays 0.00mL, ensure mouse has received at least 1.0mL of water for the session.
Press the "Finalize" to save session data.
Clean and disinfect behavior room.
Use 70% Ethanol spray and Kimwipes to clean weigh boats and surface areas.
Wipe lickspouts with alcohol wipes.
Sweep floors.
Clean
Clean
Clean and disinfect behavior room.
Use 70% Ethanol spray and Kimwipes to clean weigh boats and surface areas.
Note
Make sure to remove all fecal matter from behavior boxes.
Wipe lickspouts with alcohol wipes.
Sweep floors.
Mop floors once a week at the end of the day once all mice have left the room.
Note
Make sure there are no animals present in room. Mop when room has sufficient time to air out.
Vacuum behavior boxes once a month at the end of the day once all mice have left the room.
Note
Use HVAC certified vacuum. Make sure no animals are present in room.
Habituating a Mouse Quick Guide
Habituating a Mouse Quick Guide
Quick Guide:
Typical first 2-3 week schedule of an experimental behavior mouse.
The intended use for quick guide is to reference once familiarized with habituating a mouse.