Source Paper
Zengcai V. Guo, S. Andrew Hires, Nuo Li, Daniel H. O'Connor, Takaki Komiyama et al.
PLoS ONE • 2014
The mouse is an increasingly prominent model for the analysis of mammalian neuronal circuits. Neural circuits ultimately have to be probed during behaviors that engage the circuits. Linking circuit dynamics to behavior requires precise control of sensory stimuli and measurement of body movements. Head-fixation has been used for behavioral research, particularly in non-human primates, to facilitate precise stimulus control, behavioral monitoring and neural recording. However, choice-based, perceptual decision tasks by head-fixed mice have only recently been introduced. Training mice relies on motivating mice using water restriction. Here we describe procedures for head-fixation, water restriction and behavioral training for head-fixed mice, with a focus on active, whisker-based tactile behaviors. In these experiments mice had restricted access to water (typically 1 ml/day). After ten days of water restriction, body weight stabilized at approximately 80% of initial weight. At that point mice were trained to discriminate sensory stimuli using operant conditioning. Head-fixed mice reported stimuli by licking in go/no-go tasks and also using a forced choice paradigm using a dual lickport. In some cases mice learned to discriminate sensory stimuli in a few trials within the first behavioral session. Delay epochs lasting a second or more were used to separate sensation (e.g. tactile exploration) and action (i.e. licking). Mice performed a variety of perceptual decision tasks with high performance for hundreds of trials per behavioral session. Up to four months of continuous water restriction showed no adverse health effects. Behavioral performance correlated with the degree of water restriction, supporting the importance of controlling access to water. These behavioral paradigms can be combined with cellular resolution imaging, random access photostimulation, and whole cell recordings.
Objective: Head-fixed mice locate a vertical pole near their heads using active whisker movements to discriminate tactile spatial features in a whisker-based tactile object localization task
This is a Whisker-Based Tactile Object Localization protocol using mouse as the model organism. The procedure involves 18 procedural steps, 15 equipment items, 12 materials. Extracted from a 2014 paper published in PLoS ONE.
Model and subjects
mouse • C57BL/6J (referenced for water consumption baseline) • typically males, though females showed similar performance • 2-6 months old • 23-30g pre-restriction body weight for 2-6 months old males • Not specified in extracted text
Study window
~1.4 week study window
Core workflow
Pre-surgical preparation • Anesthesia adjustment and monitoring • Scalp preparation and local anesthesia
Primary readouts
Key equipment and reagents
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Mice aged 2-6 months (typically males) are deeply anesthetized with 2% isoflurane in O2 and mounted in a stereotaxic apparatus. Mice are kept on a thermal blanket and eyes are covered with petroleum jelly.
Note: Standard aseptic procedures used throughout
“Mice (∼2–6 months old, typically males) were deeply anesthetized with 2% isoflurane (by volume in O2; SurgiVet; Smiths Medical) and mounted in a stereotaxic apparatus (Kopf Instruments)”
During surgery, anesthesia levels are adjusted to 1-1.5% to achieve approximately 1/second breathing rate in mice.
Note: Breathing rate used as indicator of appropriate anesthesia depth
“During the surgery, the anesthesia levels were adjusted to 1–1.5% to achieve ∼1/second breathing rate in mice”
Scalp is cleaned with 70% ethanol and betadine. Marcaine (50 µl of 0.5% solution) is injected under the scalp for topical anesthesia.
Note: Aseptic technique maintained
“The scalp was cleaned with 70% ethanol and betadine. Marcaine (50 µl 0.5% solution) was injected under the scalp for topical anesthesia”
Ketofen (5 mg/kg) is injected subcutaneously as a non-steroidal anti-inflammatory drug. Buprenorphine (0.05 mg/kg) is injected into the intraperitoneal cavity as an opioid analgesic.
Note: Administered before surgical incision
“Ketofen (non-steroidal anti-inflammatory drug, 5 mg/kg) was injected subcutaneously and buprenorphine (opiod analgesic, 0.05 mg/kg) was injected into the intraperitoneal cavity”
A flap of skin approximately 1 cm² is removed from the dorsal skull with a single cut. The remaining gelatinous periosteum is removed with small scissors.
Note: Careful dissection to minimize tissue damage
“A flap of skin, approximately 1 cm², was removed from the dorsal skull with a single cut. The remaining gelatinous periostium was removed with small scissors”
The skull is cleaned and dried with sterile cotton swabs. The bone is scraped with a scalpel or slowly turning dental drill for better bonding with the glue.
Note: Proper surface preparation is critical for head bar adhesion
“The skull was cleaned and dried with sterile cotton swabs. The bone was scraped with a scalpel or slowly turning dental drill for better bonding with the glue”
The exposed skull is covered with a thin layer of cyanoacrylic glue. The head bar is positioned directly onto the wet glue. Dental acrylic (Jet Repair Acrylic) is added to cover the glue and cement the head bar in position.
Note: Head bar links skull rigidly to behavioral apparatus. Two types available: extended head bar for maximal stability or minimal head bar (22.3 × 3.2 mm) for large brain access
“The exposed skull was covered with a thin layer of cyanoacrylic glue. The head bar was positioned directly onto the wet glue. Dental acrylic (Jet Repair Acrylic) was added to cover the glue and cement the head bar in position”
If viral transduction is desired, a small hole is drilled into the skull using a dental drill with FG 1/4 drill bit. A fine glass injection pipette (tip diameter 15-20 µm, beveled to outer diameter 20-30 µm) is lowered into the brain region of interest. Approximately 30 nL of AAV (approximately 10^12 titer) is injected slowly into the parenchyma at 10 nL per minute.
Note: Beveling is critical to penetrate dura without dimpling cortex. Sufficient to transduce neurons in 500 µm diameter column of neocortex. Can be performed during head bar surgery or later during training
“Using a dental drill with an FG 1/4 drill bit, a small hole was drilled into the skull. The virus was introduced using a fine glass injection pipette (tip diameter approximately 15–20 µm) beveled to a sharp tip (outer diameter, 20–30 µm)”
Following surgery, buprenorphine (0.1 mg/kg) is administered once. Ketoprofen (5 mg/kg) is administered once daily for two days as an analgesic to reduce inflammation.
Note: Different dosage than intra-operative buprenorphine
“Following the surgery, buprenorphine (0.1 mg/kg) was administered once. Ketoprofen (5 mg/kg) was administered once a day for two days as an analgesic”
Animals are examined once daily for three days following surgery for signs of infection, lethargy, and grooming behavior.
Note: Health assessment critical for animal welfare
“Animals were examined once a day for three days for signs of infection, lethargy, and grooming”
Following full and complete recovery from surgery (at least three days post-surgery), mice are placed on a water restriction schedule. Mice are housed singly in cages containing tunnels and bedding material in a reverse light cycle room. Relative humidity is maintained at 40-50%.
Note: Housing in small groups of siblings is also possible. Relative humidity critically affects water need
“Following full and complete recovery from a previous surgery (at least three days post surgery), mice were placed on a water restriction schedule. Mice were housed singly in cages containing tunnels and bedding material, in a reverse light cycle room”
One ml of water is dispensed manually into bowls attached to cage walls at consistent times daily. Dry food (Rodent diet 5053) is continuously available. On behavioral testing days, mice obtain all water during apparatus performance (approximately 1 ml/day). On non-testing days including weekends and holidays, mice receive 1 ml water per day.
Note: Approximately 35% of ad libitum water consumption for C57BL/6J mice. Mice consume dispensed water within minutes
“One ml of water was dispensed manually into bowls which were attached to the inside walls of individual cages, at consistent times of day. Mice consumed this water within minutes”
All mice undergoing water restriction are monitored daily for hydration, weight, ruffled fur, and movement. If mice drop below 70% of pre-restriction weight, or show signs of dehydration or pain, detailed health assessment is performed using a health score.
Note: Pre-restriction body weight typically 23-30g for 2-6 months old males. Health scores 1-2 reflect slightly reduced activity
“All mice undergoing water restriction were monitored daily for hydration, weight, ruffled fur, and movement. If mice drop below 70% of pre-restriction weight, or if mice show signs of dehydration or pain, their health is assessed in more detail”
After ten days of water restriction, body weight stabilizes at approximately 80% of initial weight.
Note: This is the point at which mice are ready for behavioral training
“After ten days of water restriction, body weight stabilized at approximately 80% of initial weight”
The head bar wings are seated into notches in a stainless steel holder and fixed with clamps and thumbscrews. The mouse body is inserted into an acrylic body tube (1⅜ inch inner diameter; McMaster P/N 8486K433) with the mouse head extending out and front paws gripping the tube edge or ledge. The holder and body tube are attached to a caddy assembled from standard optomechanical components (Thorlabs). The caddy is fixed to the behavior box using magnetic kinematic bases (Thorlabs KB3X3).
Note: Head bar typically about 30 mm above bottom of body tube. Caddy allows convenient head-fixation outside apparatus and rapid placement inside
“For head-fixation, the wings of the head bar are seated into notches in a stainless steel holder and fixed with a pair of clamps and thumbscrews. The mouse body is inserted into an acrylic 'body tube' (1⅜ inch i.d.; McMaster; P/N 8486K433)”
Water rewards are provided through custom-made lickports (electrical or optical). Lickport position is critical: typically started 0.5 mm below the lower lip and 5 mm posterior to the tip of the nose. During training, the lickport is typically moved away from the mouth to discourage compulsive licking.
Note: Electrical lickports are more robust but can introduce electrophysiological artifacts. Optical lickports require regular cleaning. If lickport too close, mouse licks compulsively; if too far, mouse misses rewards
“We typically start with the lickport 0.5 mm below the lower lip, and 5 mm posterior to the tip of the nose. During training the lickport typically is moved away from the mouth to discourage compulsive licking”
After water restriction and head-fixation acclimation, mice are trained to discriminate sensory stimuli using operant conditioning. Head-fixed mice report stimuli by licking in go/no-go tasks and using forced choice paradigm with dual lickport. Delay epochs lasting a second or more separate sensation (e.g., tactile exploration) and action (licking).
Note: Mice can learn to discriminate sensory stimuli in few trials within first behavioral session. High performance maintained for hundreds of trials per session
“Head-fixed mice reported stimuli by licking in go/no-go tasks and also using a forced choice paradigm using a dual lickport. Delay epochs lasting a second or more were used to separate sensation (e.g. tactile exploration) and action (i.e. licking)”
Mice are trained to locate a vertical pole near their heads using active whisker movements. This is an active sensation behavior where mice move their whiskers intelligently to collect information about object location. High-speed imaging reveals whisker movements underlying discrimination.
Note: Task measures tactile spatial perception and sensorimotor integration. Changes in whisker shape from contact report mechanical inputs to somatosensory system
“we have trained head-fixed mice to locate an object (a vertical pole) near their heads with their whiskers. This is by construction an active sensation behavior: mice have to move their whiskers in an intelligent manner to collect information about the world”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
Head-fixed mice locate a vertical pole near their heads using active whisker movements to discriminate tactile spatial features in a whisker-based tactile object localization task
Objective
Head-fixed mice locate a vertical pole near their heads using active whisker movements to discriminate tactile spatial features in a whisker-based tactile object localization task
Subjects
From papermouse • C57BL/6J (referenced for water consumption baseline) • typically males, though females showed similar performance • 2-6 months old • 23-30g pre-restriction body weight for 2-6 months old males
Sample count
From paperNot specified in extracted text
Cohort notes
From paperMice housed singly in cages with tunnels and bedding material in reverse light cycle room
Pre-surgical preparation
Anesthesia adjustment and monitoring
Scalp preparation and local anesthesia
Systemic pain management
Whisker position and movement during tactile exploration (measured via high-speed imaging)
From paperNot explicitly described in extracted methods section
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Whisker shape changes from object contact
From paperNot explicitly described in extracted methods section
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Licking behavior (go/no-go responses or forced choice responses)
From paperNot explicitly described in extracted methods section
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Behavioral performance accuracy across trials
From paperNot explicitly described in extracted methods section
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Whisker position and movement during tactile exploration (measured via high-speed imaging)
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Whisker shape changes from object contact
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Licking behavior (go/no-go responses or forced choice responses)
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Behavioral performance accuracy across trials
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
Preprocessing / cleaning
Not explicitly described in extracted methods section
Scoring or quantification
Quantify the primary readouts for this experiment: Whisker position and movement during tactile exploration (measured via high-speed imaging); Whisker shape changes from object contact; Licking behavior (go/no-go responses or forced choice responses); Behavioral performance accuracy across trials.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Whisker position and movement during tactile exploration (measured via high-speed imaging), Whisker shape changes from object contact, Licking behavior (go/no-go responses or forced choice responses), Behavioral performance accuracy across trials.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Zengcai V. Guo et al. (2014). Procedures for Behavioral Experiments in Head-Fixed Mice. PLoS ONE
Pre-surgical preparation • Protocol step
“Mice (∼2–6 months old, typically males) were deeply anesthetized with 2% isoflurane (by volume in O2; SurgiVet; Smiths Medical) and mounted in a stereotaxic apparatus (Kopf Instruments)”
Anesthesia adjustment and monitoring • Protocol step
“During the surgery, the anesthesia levels were adjusted to 1–1.5% to achieve ∼1/second breathing rate in mice”
Scalp preparation and local anesthesia • Protocol step
“The scalp was cleaned with 70% ethanol and betadine. Marcaine (50 µl 0.5% solution) was injected under the scalp for topical anesthesia”
Systemic pain management • Protocol step
“Ketofen (non-steroidal anti-inflammatory drug, 5 mg/kg) was injected subcutaneously and buprenorphine (opiod analgesic, 0.05 mg/kg) was injected into the intraperitoneal cavity”
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Kopf Instruments
Harvard Apparatus
FG 1/4
McMaster • P/N 8486K433
Thorlabs • KB3X3
Thorlabs
custom-made
custom-made
SurgiVet; Smiths Medical
Brody Lab, Princeton University
Myers Lab, Janelia
Janelia
Janelia
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