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: Training head-fixed mice to perform perceptual decision tasks using operant conditioning with water restriction motivation, with focus on whisker-based tactile discrimination behaviors
This is a Head-Fixed Behavioral Training protocol using mouse as the model organism. The procedure involves 15 procedural steps, 15 equipment items, 13 materials. Extracted from a 2014 paper published in PLoS ONE.
Model and subjects
mouse • C57BL/6J • 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
~2 day study window
Core workflow
Pre-surgical preparation • Anesthesia adjustment and scalp preparation • Local anesthesia and systemic analgesia administration
Primary readouts
Key equipment and reagents
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Mice aged 2-6 months old (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: All procedures follow protocols approved by Janelia Farm IACUC. Standard aseptic procedures used.
“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). Mice were kept on a thermal blanket (Harvard Apparatus) and their eyes were covered with a thin layer of petroleum jelly.”
Anesthesia levels are adjusted to 1-1.5% to achieve approximately 1/second breathing rate. Scalp is cleaned with 70% ethanol and betadine.
Note: Breathing rate monitoring is critical for proper anesthesia depth
“During the surgery, the anesthesia levels were adjusted to 1–1.5% to achieve ∼1/second breathing rate in mice. The scalp was 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: Multiple routes of analgesia administration for comprehensive pain management
“Marcaine (50 µl 0.5% solution) was injected under the scalp for topical anesthesia. 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 removal of periosteum is necessary for proper skull preparation
“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 glue.
Note: Proper skull surface preparation is critical for secure head bar attachment
“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 selection depends on experimental requirements: 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 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. 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 of pipette is critical to penetrate dura without dimpling cortex. 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 (outer diameter, 20–30 µm). Viral suspension is injected slowly into the parenchyma (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 the neocortex.”
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.
Note: Animals are examined once a day for three days for signs of infection, lethargy, and grooming
“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 to reduce inflammation.”
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 (1 5/8 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 is typically positioned about 30 mm above the bottom of the body tube. Caddy allows convenient head-fixation outside apparatus and rapid, consistent placement.
“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' (1 5/8 inch i.d.; McMaster; P/N 8486K433). The holder and body tube in turn are attached to a caddy. The caddy is fixed to the behavior box using magnetic kinematic bases (e.g. Thorlabs, KB3X3).”
Water rewards are provided by custom-made lickports that sense tongue movement. Electrical lickports are activated by tongue contact with steel nozzle (more robust but can introduce electrophysiology artifacts). Optical lickports are activated by light path interruption between LED and phototransistor (require regular cleaning). Lickport position is critical: typically start with lickport 0.5 mm below lower lip and 5 mm posterior to tip of nose. During training, lickport is moved away from mouth to discourage compulsive licking.
Note: Lickport position adjustment is important for behavioral performance. Bottom of body tube is coated with aluminum foil for electrical contact with electric lickports.
“Water rewards are provided by different types of custom-made lickports that sense the movement of the tongue. Electrical lickports are activated by the tongue making contact with the steel nozzle of the lickport. Optical lickports are activated by interruptions in the light path between an LED and a phototransistor. We typically start with the lickport 0.5 mm below the lower lip, and 5 mm posterior to the tip of the nose.”
Water restriction is started after mice recover from surgery (at least three 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.
“Water restriction was started after mice recovered from surgery (at least three days after surgery). Mice were housed singly in cages containing tunnels and bedding material, in a reverse light cycle room. Dry food was continuously available (Rodent diet 5053). One ml of water was dispensed manually into bowls which were attached to the inside walls of individual cages, at consistent times of day.”
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 ten days of water restriction. Up to four months of continuous water restriction showed no adverse health effects.
“All mice undergoing water restriction were monitored daily for hydration, weight, ruffled fur, and movement. The pre-restriction body weight is typically in the range 23–30 g for 2–6 months old males. 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 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 and using forced choice paradigm with dual lickport. Mice can learn to discriminate sensory stimuli in a few trials within the first behavioral session.
Note: Training occurs mainly during dark phase. Behavioral performance correlates with degree of water restriction.
“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.”
Mice perform perceptual decision tasks with delay epochs lasting a second or more to separate sensation (e.g., tactile exploration) and action (licking). Mice achieve high performance for hundreds of trials per behavioral session.
Note: Tasks can be combined with cellular resolution imaging, random access photostimulation, and whole cell recordings
“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.”
If viral transduction is necessary during training period, 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 for experiments requiring viral injection during training
“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.
Training head-fixed mice to perform perceptual decision tasks using operant conditioning with water restriction motivation, with focus on whisker-based tactile discrimination behaviors
Objective
Training head-fixed mice to perform perceptual decision tasks using operant conditioning with water restriction motivation, with focus on whisker-based tactile discrimination behaviors
Subjects
From papermouse • C57BL/6J • 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
Pre-surgical preparation
Anesthesia adjustment and scalp preparation
Local anesthesia and systemic analgesia administration
Skull exposure
Behavioral performance in perceptual decision tasks (accuracy, reaction time)
From paperNot explicitly described in methods section provided
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Licking behavior (frequency, timing, compulsive licking)
From paperNot explicitly described in methods section provided
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Body weight during water restriction (target 80% of initial weight)
From paperNot explicitly described in methods section provided
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Health indicators (hydration status, fur condition, movement, signs of infection)
From paperNot explicitly described in methods section provided
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Behavioral performance in perceptual decision tasks (accuracy, reaction time)
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 (frequency, timing, compulsive licking)
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
Body weight during water restriction (target 80% of initial weight)
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
Health indicators (hydration status, fur condition, movement, signs of infection)
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 methods section provided
Scoring or quantification
Quantify the primary readouts for this experiment: Behavioral performance in perceptual decision tasks (accuracy, reaction time); Licking behavior (frequency, timing, compulsive licking); Body weight during water restriction (target 80% of initial weight); Health indicators (hydration status, fur condition, movement, signs of infection).
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Behavioral performance in perceptual decision tasks (accuracy, reaction time), Licking behavior (frequency, timing, compulsive licking), Body weight during water restriction (target 80% of initial weight), Health indicators (hydration status, fur condition, movement, signs of infection).
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). Mice were kept on a thermal blanket (Harvard Apparatus) and their eyes were covered with a thin layer of petroleum jelly.”
Anesthesia adjustment and scalp preparation • Protocol step
“During the surgery, the anesthesia levels were adjusted to 1–1.5% to achieve ∼1/second breathing rate in mice. The scalp was cleaned with 70% ethanol and betadine.”
Local anesthesia and systemic analgesia administration • Protocol step
“Marcaine (50 µl 0.5% solution) was injected under the scalp for topical anesthesia. 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.”
Skull exposure • 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.”
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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
Jet Repair Acrylic
Brody Lab, Princeton University
Myers Lab, Janelia
Janelia
Janelia
3 items with ReplicateScience direct pages
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