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 perform forced choice discrimination tasks using dual lickports to report stimulus perception and measure perceptual decision-making
This is a Dual Lickport Forced Choice Task protocol using mouse as the model organism. The procedure involves 25 procedural steps, 14 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, females showed similar performance • 2-6 months old • 23-30g pre-restriction; stabilized at approximately 80% of initial weight after water restriction
Study window
~3 day study window
Core workflow
Pre-surgical preparation • Anesthesia adjustment and monitoring • Scalp preparation and anesthesia
Primary readouts
Key equipment and reagents
Verified items
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Mice aged 2-6 months (typically males) are deeply anesthetized with 2% isoflurane in O2 and mounted in stereotaxic apparatus. Mice are kept on thermal blanket with eyes 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)”
Anesthesia levels adjusted to 1-1.5% to achieve approximately 1/second breathing rate in mice during surgery
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 cleaned with 70% ethanol and betadine. Marcaine (50 µl of 0.5% solution) injected under scalp for topical anesthesia
Note: Topical anesthesia applied before incision
“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) injected subcutaneously and buprenorphine (0.05 mg/kg) injected into intraperitoneal cavity for pain management
Note: Non-steroidal anti-inflammatory and opioid analgesics administered
“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 dorsal skull with single cut. Gelatinous periosteum removed with small scissors
Note: Careful dissection to expose clean skull surface
“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”
Skull cleaned and dried with sterile cotton swabs. Bone scraped with scalpel or slowly turning dental drill for better bonding with glue
Note: Surface preparation critical for adhesive bonding
“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”
Exposed skull covered with thin layer of cyanoacrylic glue. Head bar positioned directly onto wet glue. Dental acrylic added to cover glue and cement 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”
Small hole drilled into skull using dental drill with FG 1/4 drill bit. Fine glass injection pipette (tip diameter 15-20 µm, beveled to 20-30 µm outer diameter) used to introduce virus into brain region of interest
Note: Beveling critical to penetrate dura without dimpling cortex. Approximately 30 nL of AAV (10^12 titer) sufficient to transduce neurons in 500 µm diameter column of neocortex
“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)”
Pipette containing virus lowered into brain region of interest. Viral suspension injected slowly into parenchyma at 10 nL per minute
Note: Slow injection rate minimizes tissue damage
“Viral suspension is injected slowly into the parenchyma (10 nL per minute)”
Following surgery, buprenorphine (0.1 mg/kg) administered once. Ketoprofen (5 mg/kg) administered once daily for two days as analgesic to reduce inflammation
Note: Post-operative pain management protocol
“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 examined once daily for three days for signs of infection, lethargy, and grooming
Note: Health monitoring critical for animal welfare
“Animals were examined once a day for three days for signs of infection, lethargy, and grooming”
If viral transduction during training is necessary, water should be supplemented for 2 days prior to surgery at 3-4 ml water per day
Note: Viral transduction efficiency can be low in water-restricted mice
“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)”
Mice allowed to recover fully from surgery before water restriction begins
Note: Full recovery essential before behavioral training
“Water restriction was started after mice recovered from surgery (at least three days after surgery)”
Mice housed singly in cages containing tunnels and bedding material in reverse light cycle room. Housing in small groups of siblings also possible
Note: Reverse light cycle room used; training and testing occur mainly during dark phase
“Mice were housed singly in cages containing tunnels and bedding material, in a reverse light cycle room”
Relative humidity maintained at 40-50% with little seasonal variation
Note: Humidity critically affects animals' water needs
“Relative humidity critically affects the animals' need of water and was kept at 40–50%, with little seasonal variations”
Following full recovery from surgery (at least 3 days post-surgery), mice placed on water restriction schedule. Dry food continuously available (Rodent diet 5053). One ml of water dispensed manually into bowls attached to cage walls at consistent times of day
Note: Mice consume 1 ml water within minutes; corresponds to approximately 35% of ad libitum water consumption for C57BL/6J mice
“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. Dry food was continuously available (Rodent diet 5053). One ml of water was dispensed manually into bowls”
All mice undergoing water restriction monitored daily for hydration, weight, ruffled fur, and movement
Note: Pre-restriction body weight typically 23-30 g for 2-6 month old males
“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 show signs of dehydration or pain, detailed health assessment performed and summarized in health score
Note: Health scores 1-2 typically reflect slightly reduced activity
“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: Weight stabilization indicates readiness for behavioral training
“After ten days of water restriction, body weight stabilized at approximately 80% of initial weight”
Wings of head bar seated into notches in stainless steel holder and fixed with pair of clamps and thumbscrews. Mouse body inserted into acrylic body tube (1⅝ inch inner diameter) with head extending out and front paws gripping tube edge or ledge
Note: Head bar typically about 30 mm above bottom of body tube. Caddy with mouse can be placed into apparatus rapidly and consistently
“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”
Lickport position relative to mouse is critical parameter. Typically start with lickport 0.5 mm below lower lip and 5 mm posterior to tip of nose. During training, lickport typically moved away from mouth to discourage compulsive licking
Note: If lickport too close, mouse might lick compulsively; if too far, mouse might miss rewards and become discouraged
“We typically start with the lickport 0.5 mm below the lower lip, and 5 mm posterior to the tip of the nose”
Head-fixed mice trained to discriminate sensory stimuli using operant conditioning. Mice report stimuli using forced choice paradigm with dual lickport. Delay epochs lasting a second or more separate sensation and action (licking)
Note: In some cases mice learned to discriminate sensory stimuli in few trials within first behavioral session. Mice performed variety of perceptual decision tasks with high performance
“Head-fixed mice reported stimuli by licking in go/no-go tasks and also using a forced choice paradigm using a dual lickport”
Water rewards provided by custom-made lickports that sense tongue movement. Electrical lickports activated by tongue contact with steel nozzle. Optical lickports activated by interruptions in light path between LED and phototransistor
Note: Electrical lickports more robust but can introduce electrophysiology artifacts. Optical lickports require regular cleaning
“Water rewards are provided by different types of custom-made lickports that sense the movement of the tongue”
On days when behavioral experiments conducted, mice typically obtain all water during performance in behavior apparatus (approximately 1 ml water per day). On other days including weekends and holidays, mice receive 1 ml water per day
Note: Water restriction maintained throughout training
“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)”
Up to four months of continuous water restriction monitored for health effects
Note: Four months continuous water restriction showed no adverse health effects. Behavioral performance correlated with degree of water restriction
“Up to four months of continuous water restriction showed no adverse health effects”
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 perform forced choice discrimination tasks using dual lickports to report stimulus perception and measure perceptual decision-making
Objective
Head-fixed mice perform forced choice discrimination tasks using dual lickports to report stimulus perception and measure perceptual decision-making
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
Pre-surgical preparation
Anesthesia adjustment and monitoring
Scalp preparation and anesthesia
Systemic pain management
Licking behavior (left vs. right lickport choice)
From paperNot explicitly described in methods section provided.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Discrimination accuracy between sensory stimuli
From paperNot explicitly described in methods section provided.
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 stimulus presentation
From paperNot explicitly described in methods section provided.
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 described in methods section provided.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Licking behavior (left vs. right lickport choice)
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 between 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 stimulus presentation
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 described in methods section provided.
Scoring or quantification
Quantify the primary readouts for this experiment: Licking behavior (left vs. right lickport choice); Discrimination accuracy between sensory stimuli; Reaction time to stimulus presentation; 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 (left vs. right lickport choice), Discrimination accuracy between sensory stimuli, Reaction time to stimulus presentation, 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
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 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
Thorlabs • KB3X3
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
Estimated: $100.60
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