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 discriminate sensory stimuli and report responses by licking in go/no-go paradigms, with focus on whisker-based tactile behaviors and object localization tasks
This is a Go/No-Go Licking Task protocol using mouse as the model organism. The procedure involves 13 procedural steps, 14 equipment items, 13 materials. Extracted from a 2014 paper published in PLoS ONE.
Model and subjects
mouse • C57BL/6J (referenced for water consumption baseline) • typically males, females showed similar performance • 2-6 months old • 23-30g pre-restriction; stabilized at approximately 80% of initial weight after water restriction
Study window
~1.4 week study window
Core workflow
Surgical preparation and anesthesia • Scalp preparation and anesthesia • Skull exposure and preparation
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 covered with petroleum jelly. Anesthesia is adjusted to 1-1.5% during surgery to achieve approximately 1/second breathing rate.
Note: All procedures follow Janelia Farm IACUC-approved protocols using standard aseptic procedures
“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)”
Scalp is cleaned with 70% ethanol and betadine. Marcaine (50 µl 0.5% solution) is injected under the scalp for topical anesthesia. Ketofen (5 mg/kg) is injected subcutaneously and buprenorphine (0.05 mg/kg) is injected intraperitoneally.
Note: Ketofen is a non-steroidal anti-inflammatory drug; buprenorphine is an opioid analgesic
“The scalp was cleaned with 70% ethanol and betadine. Marcaine (50 µl 0.5% solution) was injected under the scalp for topical anesthesia”
A flap of skin approximately 1 cm² is removed from the dorsal skull with a single cut. The gelatinous periosteum is 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 glue.
Note: Careful preparation ensures strong adhesion of head bar to skull
“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 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. The head bar links the skull rigidly to the behavioral apparatus.
Note: Two types of head bars are available: extended head bar for maximal stability (fitted to dorsal skull shape) 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 needed, a small hole is drilled into the skull using a dental drill with FG 1/4 drill bit. Virus is introduced using a fine glass injection pipette (tip diameter 15-20 µm, beveled to 20-30 µm outer diameter). The pipette is lowered into the brain region of interest and viral suspension is injected slowly at 10 nL per minute. Approximately 30 nL of AAV (approximately 10^12 titer) is sufficient to transduce neurons in a 500 µm diameter column of neocortex.
Note: Beveling the pipette is critical to penetrate dura without dimpling cortex, reducing tissue damage. This step is optional and performed during head bar surgery.
“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”
Following surgery, buprenorphine (0.1 mg/kg) is administered once. Ketoprofen (5 mg/kg) is administered once a day for two days as an analgesic to reduce inflammation. Animals are examined once a day for three days for signs of infection, lethargy, and grooming.
Note: Water restriction should not begin until at least 3 days after surgery for full recovery
“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”
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 (1⅝ 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: The head bar is typically about 30 mm above the bottom of the body tube. The caddy allows convenient head-fixation outside the apparatus and rapid, consistent placement into the apparatus.
“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”
Water rewards are provided by custom-made lickports that sense tongue movement. Electrical lickports are activated by tongue contact with the steel nozzle. Optical lickports are activated by interruptions in the light path between an LED and phototransistor. The lickport position is critical: 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 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 to maintain optical path clarity.
“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”
Water restriction is started after mice recover from surgery (at least 3 days post-surgery). Mice are housed singly in cages containing tunnels and bedding material in a reverse light cycle room. Dry food (Rodent diet 5053) is continuously available. One ml of water is dispensed manually into bowls attached to cage walls at consistent times of day. Mice consume this water within minutes, corresponding to approximately 35% of ad libitum water consumption for C57BL/6J mice.
Note: Relative humidity is kept at 40-50% with little seasonal variation, as it critically affects water need. Housing in small groups of siblings is also possible.
“Following full and complete recovery from a previous surgery (at least three days post surgery), mice were placed on a water restriction schedule in preparation for behavioral conditioning”
All mice undergoing water restriction are monitored daily for hydration, weight, ruffled fur, and movement. Pre-restriction body weight is typically 23-30 g for 2-6 month old males. 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 system.
Note: Body weight stabilizes at approximately 80% of initial weight after about 10 days of water restriction. Health scores of 1-2 typically reflect slightly reduced activity.
“All mice undergoing water restriction were monitored daily for hydration, weight, ruffled fur, and movement”
After 10 days of water restriction when body weight stabilizes at approximately 80% of initial weight, mice are trained to discriminate sensory stimuli using operant conditioning. Head-fixed mice report stimuli by licking in go/no-go tasks. Delay epochs lasting a second or more are used to separate sensation (e.g., tactile exploration) and action (licking).
Note: In some cases mice learn to discriminate sensory stimuli in a few trials within the first behavioral session. Training and behavioral testing occur mainly during the dark phase.
“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”
On days when behavioral experiments are carried out, mice typically obtain all of their water during performance in the behavior apparatus (approximately 1 ml water per day). On other days, including weekends and holidays, mice receive 1 ml water per day.
Note: Behavioral performance correlates with the degree of water restriction
“On days when behavioral experiments were carried out, mice typically obtained all of their water during performance in the behavior apparatus (approximately 1 ml water per day)”
If viral transduction is necessary during training, water should be supplemented for 2 days prior to surgery at 3-4 ml water per day, as viral transduction efficiency can be low in water-restricted mice.
Note: This is an optional step only if viral injection is planned during training rather than during initial head bar surgery
“As viral transduction efficiency can be low in water restricted mice, water should be supplemented for 2 days prior surgery (3–4 ml water per day)”
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 discriminate sensory stimuli and report responses by licking in go/no-go paradigms, with focus on whisker-based tactile behaviors and object localization tasks
Objective
Head-fixed mice discriminate sensory stimuli and report responses by licking in go/no-go paradigms, with focus on whisker-based tactile behaviors and object localization tasks
Subjects
From papermouse • C57BL/6J (referenced for water consumption baseline) • typically males, females showed similar performance • 2-6 months old • 23-30g pre-restriction; stabilized at approximately 80% of initial weight after water restriction
Cohort notes
From paperMice housed singly in cages with tunnels and bedding material in reverse light cycle room
Surgical preparation and anesthesia
Scalp preparation and anesthesia
Skull exposure and preparation
Head bar attachment
Licking behavior (go/no-go responses)
From paperNot explicitly detailed in methods section.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Discrimination accuracy for sensory stimuli
From paperNot explicitly detailed in methods section.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Reaction time to stimuli
From paperNot explicitly detailed in methods section.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Number of trials completed per session
From paperNot explicitly detailed in methods section.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Licking behavior (go/no-go responses)
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
Discrimination accuracy for sensory stimuli
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
Reaction time to stimuli
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
Number of trials completed per session
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
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
Preprocessing / cleaning
Not explicitly detailed in methods section.
Scoring or quantification
Quantify the primary readouts for this experiment: Licking behavior (go/no-go responses); Discrimination accuracy for sensory stimuli; Reaction time to stimuli; Number of trials completed per session.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Licking behavior (go/no-go responses), Discrimination accuracy for sensory stimuli, Reaction time to stimuli, Number of trials completed per session.
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
Surgical preparation and anesthesia • 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)”
Scalp preparation and 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”
Skull exposure and preparation • Protocol step
“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”
Head bar attachment • Protocol step
“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”
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Kopf Instruments
Harvard Apparatus
FG 1/4
custom-made
custom-made
Thorlabs
Thorlabs • KB3X3
SurgiVet; Smiths Medical
Jet Repair Acrylic
McMaster • P/N 8486K433
Brody Lab, Princeton University
Myers Lab, Janelia
Janelia Farm Research Campus
Janelia Farm Research Campus
3 items with ReplicateScience direct pages
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