Head-Fixed Behavioral Training

“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.”

“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) 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, was removed from the dorsal skull with a single cut. The remaining gelatinous periostium was removed with small scissors.”

“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 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.”

“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 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.”

“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 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 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 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, 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.”

“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.”

“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)”