Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Open field test
• Procedure: Bottle choice test (lickometry): Boost
• Procedure: Food restriction
• Procedure: Operant conditioning - reversal learning
• Procedure: Operant conditioning - delay discounting
• Procedure: Bottle choice test (lickometry): Ethanol drinking in the dark
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Open field test	Open field chamber	Activity (beam breaks)
2	Bottle choice test (Boost)	Dual lickometer Scurry box	Number of licks (Boost consumed)
3	Food restriction	-	-
4	Reversal learning	Operant chamber	Number of trials needed to meet criteria (acquisition and reversal), number of nose pokes in correct flanking hole (acquisition and reversal), number of nose pokes in incorrect flanking hole (acquisition and reversal)
5	Delay discounting	Operant chamber	Scaling factor, side bias
6	Bottle choice test (ethanol): Drinking in the Dark	Dual lickometer Scurry box	Number of licks (ethanol consumed), preference for ethanol

Equipment and supplies

• Open field chamber (Med-Associates MED-OFAS-RSU; St Albans VT)
  • 43.2 x 43.2 x 30.5 cm
  • Fitted with infrared beams
  • Within sound attenuating cubicles measuring 66 x 52.7 x 55.9 cm at the interior with walls 1.9 cm thick
• Dual lickometer Scurry boxes (Model 808225, Lafayette Instruments)
  • 35.5 x 23.5 x 20 cm
  • Activity wheel removed
  • Fitted with a food hopper and two 50 mL sipper bottles
  • Licks are counted by a computer
• Dehumidifier (provided ambient background noise)
• Operant modular chambers (Model ENV-307W, Med Associates, Inc.)
  • Stainless steel grid floor (Model ENV-307W-GFW, Med Associates, Inc.)
  • Within a sound attenuating cubicle

Reagents and solutions

• 10% Alconox
• Boost solution (MedicalSupplyMI, Michigan)
• 20% ethanol

Procedure: Open field test

1. At 70 days of age, mice are assessed for locomotor response in a novel open field environment, during their light cycle.
2. Mice are transported to a separate testing room on a cart.
3. Each mouse is individually placed in the open field chamber.
4. Activity is recorded for 40 min, divided into eight 5-min bins.
5. The following day, the procedure is repeated and activity recorded.

Procedure: Bottle choice test (lickometry): Boost

1. On the day after the open field test (at 72-73 days of age), mice receive home cage exposure to a highly palatable chocolate-flavored Boost solution, made available in a plastic petri dish that is placed on the top of the bedding for 48h (solution refreshed at 24h time point).
2. At 74-80 days of age, mice are evaluated for Boost and water consumption for 7 consecutive days in daily 2h lickometry sessions. No food is provided during consumption testing.
3. All testing occurs during the light phase where each mouse is placed inside a dual lickometer Scurry box.
4. One bottle is filled with Boost solution and the other filled with water; the position of the two solutions relative to one another is counterbalanced pseudorandomly across the testing days.
5. The number of licks is divided by the body weight of the mouse to account for variability attributed to body weight differences.
6. After daily sessions are completed, mice are transported back to the colony room and returned to their home cages.

Procedure: Food restriction

1. Once lickometer testing is completed, mice are transitioned to a limited food access schedule.
2. Mice are weighed before food is removed, and that weight is recorded as their initial free feeding weight.
3. Mice are fed once a day in the early afternoon, with non-wild mice initially receiving 3 g of chow per mouse and wild-derived mice initially receiving 4 g of chow.
4. Mice are weighed daily and chow quantity provided per day is titrated until mice reach 80-85% (non-wild) or 83-88% (wild-derived) of their free feeding weights.
5. Once mice are approximately 85% of their free feeding weight, additional testing begins.
6. If at any point during the testing period, a mouse drops below 80% of their free feeding weight, their daily chow quantity is increased. If a mouse has two consecutive days being beneath 80%, they are temporarily returned to ad libitum access to food until their weight has recovered.
7. Once targeted reductions in body weight are achieved, half of the mice are randomly designated for evaluation using the operant discrimination/reversal learning procedure (below) and the other half designated for the delay discounting procedure (below).

Procedure: Operant conditioning - reversal learning

1. Mice are handled by their tails, either with gloves (non-wild) or forceps (wild-derived). Mice are removed from the operant box either by their tail (non-wild) or by inserting the red tube from their home cage into the box, and removing the tube when the mouse is inside (wild-derived).
2. Each mouse is sequentially tested in a series of programs; mice transition from program to program individually, as they meet criterion performance. Mice undergo the following programs:
   • Stage 1: Box habituation. House light and white noise are active; no reinforcements are provided; 1h session.
   • Stage 2: Magazine training. House light and white noise are active; 20-21 µL Boost is dispensed every 30 s; 1h session or after the mouse has received 50 rewards.
   • Stage 3: Operant conditioning-1. Mice are trained to insert their noses into the center nose-poke aperture (hole 3 of 5) for at least 100 ms. Session begins with illumination of the house light and activation of the white noise generator; 10 s later, aperture 3 of 5 is illuminated (illumination of the hole is extinguished each time the mouse initiates a response in this hole). A behavioral response that breaks the photocell in the aperture (usually, a nose poke) of at least 0, 100, or 200 ms (varied from trial to trial) is reinforced by the delivery of 20-21 µL of Boost solution. After each reinforce is retrieved, a new trial is initiated 1.5 s later (signaled by illumination of the center nose poke aperture). If a response is initiated but not sustained for the 0, 100, or 200 ms period, a time out period of 1 s occurs, during which time the central nose poke light and house light are extinguished. If a mouse does not respond in the center illuminated hole for 15 min, the center hole is baited with a Boost-saturated cotton swab. Daily sessions last up to 1h but are terminated if an individual mouse completes 50 schedules. Each mouse is tested daily on this stage until it receives at least 50 reinforcements in a single session, at which time it progresses to the next stage.
   • Stage 4: Operant conditioning-2. Mice are tested under the same basic conditions, except a minimum duration nose poke of 100 or 200 s is required to trigger reinforcement delivery. If a mouse does not respond in the center illuminated hole for 15 min, the center hole is baited with a Boost-saturated cotton swab. When the mouse completes 50 schedules in a single session, it progresses to Stage 5.
   • Stage 5: Operant conditioning-3. Mice are tested under the same basic conditions, except a minimum duration nose poke of 100, 200, or 300 s is required to trigger reinforcement delivery. If a mouse does not respond in the center illuminated hole for 15 min, the center hole is baited with a Boost-saturated cotton swab. When the mouse completes 50 schedules in a single session, it progresses to Stage 6.
   • Stage 6: Discrimination learning. Session onset is signaled by illumination of the house light and activation of the white noise generator. Trial onset is signaled by illumination of the center nose poke aperture. As in Stages 2-5, mice first complete an observing response into the center hole of 100 or 200 s duration. When this occurs, the two apertures flanking the central hole (holes 2 and 4) are next illuminated. A response into one of the two apertures (pseudorandomly assigned across strains) results in the delivery of a Boost reinforcer. Poking into the other hole, or not making any response within 30 s, triggers a time out, during which time the house light is extinguished. Responses into the reinforced hole are counted as correct trials; responses into the non-reinforced hole are counted as incorrect trials; no response after trial initiation is counted as an omission. Daily sessions of 1h are conducted until learning criteria are met; this includes a mouse completing at least 20 trials in a single session, and at least 80% running accuracy over the last 20 trials.
   • Stage 7: Reversal learning. Testing is nearly identical to that described in Stage 6, with the exception that the reinforcement contingencies associated with the two holes are switched. Testing progresses in daily sessions until mice once again meet the same learning criteria rule described above.
3. After reversal learning is completed, mice are slowly adjusted back onto a free-feeding schedule.

Procedure: Operant conditioning - delay discounting

1. Mice are handled by their tails, either with gloves (non-wild) or forceps (wild-derived). Mice are removed from the operant box either by their tail (non-wild) or by inserting the red tube from their home cage into the box, and removing the tube when the mouse is inside (wild-derived).
2. Mice are removed from their home cage and placed inside the operant box. Each mouse is sequentially tested in a series of programs; mice transition from program to program individually, as they meet criterion performance. Mice undergo the following programs:
   • Stage 1. Box habituation. House light and white noise are active; no reinforcements are provided; 1h session.
   • Stage 2. Magazine training. House light and white noise are active for the duration of the test, and 20-21 µL Boost is dispensed every 30 s; 1h session.
   • Stage 3. Lever press training - FR1. Session onset is signaled by illumination of the house light and activation of the white noise generator. On each trial, one lever (left or right) is inserted to the chamber and actuation of the lever by the mouse triggers delivery of 20-21 µL Boost. Across trials, the lever that is inserted (left or right) is pseudorandomly varied, such that each mouse actuated each lever roughly an equal number of times. Each session ends after 1h or after 60 reinforcements are obtained.
   • Stage 4: Lever press training - FR3. This procedure is nearly identical to Stage 3, except that 3 sequential responses on the inserted lever are required before the reinforcer is delivered. The program ends after 1h or after 60 rewards are obtained.
   • Stage 5: Trial initiation training. This procedure is nearly identical to Stage 4, except that the mouse is required to complete an observing response (nose poke response into the center hole (aperture 3 of 5) on the opposite side of the chamber in order to trigger insertion of a lever. Responses on that lever are still reinforced on an FR3 schedule. The program ends after 1h or after 60 rewards are obtained.
   • Stage 6: Continued trial training. This procedure is nearly identical to Stage 5, except that the program ends after 1.5h or after 80 rewards are obtained.
   • Stage 7. Side bias. Trials begin with both levers being presented, with a response on either on a FR3 schedule resulting in a delivery of 8-9 µL Boost. After a 10 s inter-trial interval, both levers are again presented, but only a response on the other lever is rewarded. A trial is counted if the mouse successfully presses the alternate lever. The program ends after 40 trials, or after 1.5h. The lever (right or left) on which each trial is initiated is recorded, and the dominant lever is considered the biased lever and is paired with the delayed lever.
3. After completing training, mice are placed on a randomized Latin square of 0, 3, 6, and 9 s. Three consecutive days of testing are done at each time. Reward is adjusted within trial and delay is adjusted between trials. Once the first Latin square is completed, mice receive a two-day break, and they undergo a second Latin square. Amount is measured for the immediate lever and the delayed lever and subtracted. The reward amount for each lever is averaged over the last 30 trials, and those values are averaged across the three days that delay is tested. These values are used to calculate a k-value for each animal: the scaling factor, or how much the subjective value of the reward is affected by the delay. The b-value is also recorded, which represents the animal's side bias, determined by dividing the average delayed amount by the average immediate amount for the 0 s delay.

Procedure: Bottle choice test (lickometry): Ethanol drinking in the dark

1. After 2 wks on free feed, mice are tested on a modified Drinking in the Dark procedure:
2. Mice are placed on a cart and moved to the Scurry testing room.
3. One bottle of water and one bottle of 20% ethanol are inserted into the Scurry boxes.
4. Mice are placed in the box for approximately 12h (in at 2000h and out at 0800h the next morning).
5. Mice are tested in three sessions over three days.
6. Food is available ad libitum during the trial in the food hopper.
7. Licks are recorded for each bottle.

Data collected by investigator

• Open field test
  • Locomotor activity (beam breaks)
• Bottle choice test
  • Number of Boost licks adjusted for body weight
  • Ratio of Boost licks to total licks adjusted for body weight
• Reversal learning
  • Number of trials needed to meet criteria (acquisition and reversal)
  • Number of hole pokes made in correct flanking hole (acquisition and reversal)
  • Number of hole pokes made in incorrect flanking hole (acquisition and reversal)
• Delay discounting
  • Scaling factor (k-value)
  • Side bias (b-value)
• Bottle choice test (Drinking in the Dark)
  • Ethanol consumption
  • Ethanol preference