Materials & Equipment Checklist
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Gather these items before starting the experiment. Check off items as you prepare.
Equipment14
Materials13
Software4
Brody Lab, Princeton University
Myers Lab, Janelia
Janelia Farm Research Campus
Janelia Farm Research Campus
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Protocol Steps
1
Surgical preparation and anesthesia
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
View evidence from paper
“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)”
2
Scalp preparation and anesthesia
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
View evidence from paper
“The scalp was cleaned with 70% ethanol and betadine. Marcaine (50 µl 0.5% solution) was injected under the scalp for topical anesthesia”
3
Skull exposure and preparation
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
View evidence from paper
“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”
4
Head bar attachment
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
View evidence from paper
“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”
5
Optional viral gene transfer
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.
View evidence from paper
“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”
6
Post-operative analgesia and monitoring
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.
3 days post-operative monitoring
Note: Water restriction should not begin until at least 3 days after surgery for full recovery
View evidence from paper
“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”
7
Head-fixation apparatus assembly
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.
View evidence from paper
“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”
8
Lickport setup and positioning
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.
View evidence from paper
“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”
9
Water restriction initiation
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.
Continuous during behavioral training period
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.
View evidence from paper
“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”
10
Daily health monitoring during water restriction
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.
Daily throughout water restriction period
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.
View evidence from paper
“All mice undergoing water restriction were monitored daily for hydration, weight, ruffled fur, and movement”
11
Behavioral training initiation
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).
Hundreds of trials per behavioral session
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.
View evidence from paper
“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”
12
Water delivery during behavioral sessions
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.
Throughout behavioral training period
Note: Behavioral performance correlates with the degree of water restriction
View evidence from paper
“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)”
13
Supplemental water for viral transduction
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.
2 days pre-surgery
Note: This is an optional step only if viral injection is planned during training rather than during initial head bar surgery
View evidence from paper
“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)”
Subjects / Specimens
- Species
- mouse
- Strain
- C57BL/6J (referenced for water consumption baseline)
- Age
- 2-6 months old
- Sex
- typically males, females showed similar performance
- Weight
- 23-30g pre-restriction; stabilized at approximately 80% of initial weight after water restriction
Mice housed singly in cages with tunnels and bedding material in reverse light cycle room