Dual Lickport Forced Choice Task

“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)”

“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)”

“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 were examined once a day for three days for signs of infection, lethargy, and grooming”

“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)”

“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”

“Relative humidity critically affects the animals' need of water and was kept at 40–50%, with little seasonal variations”

“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 were monitored daily for hydration, weight, ruffled fur, and movement”

“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”

“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”

“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 reported stimuli by licking in go/no-go tasks and also using a forced choice paradigm using a dual lickport”

“Water rewards are provided by different types of custom-made lickports that sense the movement of the tongue”

“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 showed no adverse health effects”