Jan 23, 2025

Public workspaceMouse Habituation - Head Fixation into Tube

DOI
dx.doi.org/10.17504/protocols.io.rm7vzxd74gx1/v1
  • 1Allen Institute for Neural Dynamics
  • Allen Institute for Neural Dynamics
Avalon Amaya
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzxd74gx1/v1
Protocol Citation: Avalon Amaya, Jackie Swapp 2025. Mouse Habituation - Head Fixation into Tube. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxd74gx1/v1
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 25, 2024
Last Modified: January 23, 2025
Protocol Integer ID: 94174
Keywords: Rodent Head Fixation, Rodent Behavioral Habituation, Tube Enrichment
Abstract
This protocol describes the typical workflow for the habituation of a mouse for awake behavioral experimentation that utilizes tubes to hold mice 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 ad lib 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. 
Single house mice on diet or water restriction.
Ensure cleanliness of experimental environment.
Materials
Materials:

Tool / SupplyManufacturer / SupplierPart Number
70% Ethanol (Diluted in house)Sigma Aldrich459836
KimwipesKimberly - Clark ProfessionalS47299A
Liquid Reward - -
Nalgene® 125 ml PET Square Media Bottles, SterileCP Lab Safety342040-0125
1mL Luer-Lok Syringe Becton Dickinson309628
25G 5/8 Needle *Advantor89134-134
1000mL Nalgeneâ„¢ Straight-Sided Wide-Mouth Polycarbonate JarsFisher Scientific21161000
Ohaus® Scout® Portable BalanceSigma AldrichOH30253024
Mouse Tunnelsâ„¢, CertifiedBio-ServK3322
Lens WipesZeiss1011161-9-TV03
Cotton Swabs, double endedAdvantor89133-810
HEPA Replacement Filter for DC515DewaltDC5151H
18V Cordless 1/2 Gallon Wet/Dry Portable Vac (Tool Only)DewaltDC515B
Swiifer SweeperUlineH-1960
Swiffer Sweeper Pads - Wet ClothsUlineS-13901
Water BowlMade in-house0146-000-01
Cart CoverLife Science ProductsCC-34X19X38Z
Supplies can be substituted with their equivalent.

* = Optional


Personal Protective Equipment (PPE):

A
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 water restriction protocol below for starting and maintaining a mouse on water restriction:
Protocol
Mouse Water Restriction
NAME
Mouse Water Restriction
CREATED BY
Avalon Amaya

Day 1: Habituating a Mouse with Tubes
Day 1: Habituating a Mouse with Tubes
1w
1w
Handle mouse for 2-5 min.
5m
Scoop/cup mouse out of cage. Cup/cover mouse with hands for around 30 seconds to 1 minute.

Note
This act of cupping/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/uncover hands and let mouse explore the environment of your open hand for another 1-4 minutes.

Weigh mouse and return to cage.
Maintain mouse on water restriction if applicable (see Before Start section).
Optional
Return mice to holding room.
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 with Tubes
Day 2 : Habituating a Mouse with Tubes
1w
1w
Handle mouse for 2-5 min.
5m
Repeat Day 1 handling steps Go togo to step #1.1 - #1.2 .

Periodically touch the mouses headframe to habituate mouse to future head fixation.
Weigh mouse and return to cage.
Maintain mouse on water restriction if applicable (see Before Start section).
Return mice to holding room.
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 with Tubes
Day 3: Habituating a Mouse with Tubes
1w
1w
Handle mouse for 1-2 min, secure mouse to tube head fixation stage, and place mouse within designated behavioral training box for 5 minutes.
7m
Cup or scoop mouse out of cage.
Let mouse explore the environment of your hands for 1 minute. Periodically touch/grab the mouse's headframe to habituate mouse to head fixation.

Hands holding mouse headframe and tail.

While grabbing the mouse's headframe in one hand and the mouse's tail in the other (similar to image in step #5.2), secure mouse to tube head fixation stage. Slide tail end of mouses body into the tube and the headframe into the headframe clamp in a smooth motion.

Note
Do not force mouse body into the tube. Maintain typical mouse posture within your grasp (horizontal, straight, not stretched, etc.). If mouse is forcefully trying to escape your grasp, reset and cup mouse in hand before trying again.

Once secured, ensure tube height and anterior/posterior position are adjusted for the mouse's comfort.
  • All four paws touching tube.
  • Front paws holding front edge of tube (ideal but not required).
  • Head positioned so that mouth is past front paws/end of tube.


Place mouse secured to head fixation stage within designated behavioral training box for 5 minutes.

Note
Be mindful of lickspout position. Do not place mouse on top of lickspouts as it can hurt the mouse and/or cause the mouse to bend the spouts.


Computer model design of inside of behavior box.
Blue outline is behavior stage.
Red outline is lickspouts.

Image of mouse head fixed to stage and placed within behavior box. Tube is appropriately adjusted so that paws are grasping tube edge. Lickspouts are at an appropriate distance away from mouse face.

Remove mouse from behavior box and head fixation stage.
Weigh mouse and return to cage.
Maintain mouse on water restriction if applicable (see Before Start section).
Return mice to holding room.
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.
Day 4: Habituating a Mouse with Tubes
Day 4: Habituating a Mouse with Tubes
1w
1w
Handle mouse for 1-2 min, secure mouse to tube head fixation stage, and place mouse within designated behavioral training box for 10 minutes.
15m
Repeat Day 3 handling and head fixing steps Go togo to step #7.1 - #5.3 .

Place mouse secured to head fixation stage within designated behavioral training box for 10 minutes.
Remove mouse from box and head fixation stage.
Weigh mouse and return to cage.
Maintain mouse on water restriction if applicable (see Before Start section).
Return mice to holding room.
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.
Day 5: Habituating a Mouse with Tubes
Day 5: Habituating a Mouse with Tubes
1w
1w
Handle mouse for 1-2 min, secure mouse to tube head fixation stage, and place mouse within designated behavioral training box for 15 minutes.

15m
Repeat Day 3 handling and head fixing steps Go togo to step #7.1 - #5.3 .
Place mouse secured to head fixation stage within designated behavioral training box for 15 minutes.
Weigh mouse and return to cage.
Maintain mouse on water restriction if applicable (see Before Start section).
Return mice to holding room.
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.
Habituating a Mouse Quick Guide
Habituating a Mouse Quick Guide

Quick Guide:
Typical first month schedule of an experimental behavior mouse.

The intended use for this quick guide is to reference once familiarized with habituating a mouse.