All experiments were conducted in accordance with the National Institutes of Health guidelines and were approved by the Temple University Institutional Animal Use and Care Committee. This protocol was developed with adult (~60 days of age when starting the study) male and female Sprague-Dawley rats (Charles River).

1. Materials for Touchscreen SAT

1. Ensure that a sound attenuation cubicle surrounds each touchscreen chamber and that chambers are equipped with a house light, tone generator, camera positioned above the chamber, and a pellet dispenser with feeder reward area (that can be illuminated) on the wall opposite the touchscreen.
2. Use a feeder reward area with a large aperture if cannula or electrodes implants are planned. Use 45 mg reward pellets that have been sifted with a flour sifter to reduce dust, which can clog the feeder.
3. Fit each chamber with a black acrylic mask (3.175 mm thick, matte) to limit response areas on the touchscreen. Prepare the mask with a central circle opening (28.58 mm diameter, 107.95 mm from the bottom, centered between the left and right side) for the light stimulus and two square response areas (28.57 mm x 28.57 mm) each positioned below and off center (one 85.73 mm from the left side and one 85.73 mm from the right side, and both 34.93 mm from the bottom). A depiction of the mask can be found in the publication, Wicks, et al.¹⁹.
4. Place the mask in front of the touchscreen with matte finish positioned towards the chamber.

2. Schedule Design for Touchscreen SAT

NOTE: The below protocol details how to set the parameters for the shaping and testing schedules for touchscreen SAT. All these schedules are also available upon request. Table 1 depicts the schedule parameters.

1. Nose Poke Shaping parameters.
   1. Set program so that no signals are presented and the houselight remains off.
   2. Set the feeder reward area to illuminate to indicate the presence of a pellet and for this light to turn off following pellet retrieval as determined by the feeder port infrared beam.
   3. Set program so that nose-pokes in either response area are rewarded with food pellets under a fixed-ratio 1 (FR1) reinforcement schedule (i.e., each appropriate response is rewarded). However, set the program so that if the side differential for nose-pokes is five or greater, the rat must poke the non-favored side to receive a reward to prevent the development of a side bias.
   4. Set the session to end after 40 min or 120 rewarded pokes. Advance subjects to Training Schedule 1 if they complete 120 nose-pokes in 40 min for two consecutive days.
2. Description of shared parameters for Training Schedule 1, Training Schedule 2, and SAT.
   1. Program training schedules so that the houselight remains on to increase attentional demands.
   2. Program each session so that it consists of 162 trials that can be divided into three blocks of 54 trials per block with the intertrial interval (ITI) set at 9 ± 3 s.
   3. Ensure that signal and non-signal trials are pseudorandomized such that the same number of signal and non-signal trials occur in each of the three blocks.
   4. Set the program to deliver a food pellet and turn on the food port light automatically at the start of each session. When an infrared beam break indicates that the rat has removed its head from the reward area, set the program to turn off the food port light and begin the first ITI.
   5. Following each trial (i.e., signal or non-signal), set the program to indicate the 4-s response window with a 200 ms, 70 dB tone (3 kHz).
   6. If the rat correctly responds, set the program to initiate the ITI when the rat removes its head from the reward area. If the rat incorrectly responds, set the ITI to begin following the response. If the rat omits (i.e., makes no response), set the ITI to begin after the termination of the response window.
   7. For counterbalancing, develop two versions of these schedules, the first with the right response area designated for hits (Version A) and the second with the left response area designated for hits (Version B).
3. Training Schedule 1 parameters.
   1. Set all signal durations to 500 ms.
   2. On signal trials, set the program to simultaneously illuminate the response area designated for hits at 50% intensity, along with the signal, to draw the rat's attention to the correct response area.
   3. Keep the designated area for correct rejections unlit on non-signal trials.
   4. Set the program so that incorrect responses trigger correction trials in which the previous trial type (i.e., signal or non-signal) is presented again. Ensure that a correct response or four consecutive incorrect responses ends the series of correction trials. Do not count correction trials towards the 162 total trials required for task completion.
   5. Advance subjects to Training Schedule 2 if they have at least 70% hits, at least 70% correct rejections, and less than 20% omissions for three consecutive days.
4. Training Schedule 2 parameters.
   1. Set all signal durations to 500 ms.
   2. Unlike Training Schedule 1, set the program to keep the response area for hits unlit on signal trials.
   3. Unlike Training Schedule 1, do not have the program execute correction trials.
   4. Keep all other parameters the same as in Training Schedule 1.
   5. Advance subjects to SAT if they have at least 70% hits, at least 70% correct rejections, and less than 20% omissions for three consecutive days.
5. SAT parameters.
   1. Set three signal durations: 500 ms, 50 ms, and 25 ms.
   2. Set the program to present the variable signals in a pseudorandom fashion, such that the three signal durations are presented an equal number of times during each one of the three trial blocks.
   3. Define criteria performance as at least 70% hits at the 500 ms signal duration, at least 70% correct rejections, and less than 20% omissions for three consecutive days.

Table 1: Training Schedules and Average Acquisition Time for Each Schedule.

6. Distractor SAT.
   1. Keep parameters for Blocks 1 and 3 identical to SAT.
   2. Program the houselight so that it flashes at a frequency of 0.5 Hz during Block 2.
   3. When analyzing data, compare performance between Blocks 1, 2, and 3 to analyze performance before, during, and after the distractor within the same session.
7. Data extraction program.
   1. Extract the following data for SAT and distractor SAT broken down by trial block and each signal duration (for VI and percent hits):
      1. Calculate the percentage of hits by dividing the number of hits by the combined number of hits and misses and then multiplying by 100.
      2. Calculate the percentage of correct rejections by dividing the number of correct rejections by the combined number of correct rejections and false alarms and then multiplying by 100.
      3. Calculate the vigilance index (VI), an overall measure of attentional performance, using the following formula: where h and f are the proportion of hits on signal trials and false alarms on non-signal trials, respectively. The values of VI range from -1 to +1 with a value of 0 indicating that the rat is unable to distinguish between signal and non-signal trials. Do not include omitted trials in the VI measure.
      4. Calculate the percentage of omissions by determining the percentage of trials where no response occurred.

3. Procedure for Running Rats in Touchscreen SAT

1. Reverse light/dark cycle considerations
   NOTE: We test our rats during their dark cycle, but see the discussion section for considerations regarding testing in the light cycle.
   1. For testing during the dark cycle, begin behavior testing at least 30 min after the start of the dark cycle.
   2. Once the equipment is set up, keep the behavioral testing room in darkness during all training and testing procedures to avoid interfering with the rats' sleep/wake cycle.
   3. Cover rat cages with black towels during transport to minimize light exposure.
   4. Use headlights with red lights turned on for hands-free work in the dark without disrupting the light/dark cycle.
2. Food restriction procedures and schedule assignment
   1. Don appropriate personal protective equipment (PPE; e.g., gloves, mask, bonnet, lab coat, shoe covers, etc.) prior to handling the rats.
   2. When rats arrive to the colony room, house rats individually or in groups. Most rats in our studies are eventually implanted with cannulae, which necessitates individual housing.
   3. Handle rats daily for 5 min per day, 5 days prior to the start of the study.
   4. Prior to food restriction, record each rat's free feeding weight and calculate their target weight at 85% of their free feeding weight.
   5. Food restrict rats for 3 days before beginning shaping.
   6. Prior to shaping, pre-expose rats to 15 food reward pellets in their home cage.
   7. After the initial food restriction, adjust daily food amounts as needed to maintain performance on the attention task and ~85% of each rat's free-feeding body weight allowing for slight weight gain over time (see guidelines²⁵). Prior to starting a new schedule, restrict rats more than usual to increase motivation.
   8. On the day(s) of a week when rats do not perform behavior, increase their food by at least 1.0 g to make-up for the calories that they would have received while being rewarded during the behavioral task.
   9. Record each rat's weight after their session is completed.
   10. Do not feed rats until at least 30 min after completing their session to avoid an association between behavioral testing cessation and feeding time. If rats are group housed, feed each rat separately for 1 h, then put rats back into the group housing condition.
   11. Prior to the start of the study, randomly assign each rat to one of the two versions of the training schedules (A or B) and maintain them on their designated version throughout the training and SAT procedures.
3. Daily running procedure
   1. Don appropriate PPE.
   2. Power up operant chambers and the computer.
   3. Check that the pellet dispensers are filled with pellets and that the mask is inserted in front of the touchscreen.
   4. Ensure that each box is functioning properly by verifying that the feeder is properly dispensing food and the screen, lights, and tone are working. A simple box test schedule can be written for this purpose and ours is available upon request.
   5. Load the correct schedule in each box, paying close attention to the appropriate version (i.e., A or B). Input relevant information regarding animal ID, sex, experimental condition, user initials, and other notes if needed (e.g., infusion day 1).
   6. Retrieve rats from colony room and put them in the appropriate box.
   7. Start the session.
   8. Watch performance via the video camera set-up at the start of the session for several minutes to ensure that everything is functioning properly. Check performance periodically.
   9. Once a session is complete, record summary data (e.g., percent hits, percent CRs, percent omissions) and any issues in the notebook.
   10. Wipe each chamber with 10% ethanol and replace the bedding in between sets of animals.
   11. After running the final set of rats for the day, clean the chambers thoroughly with 10% ethanol, ensuring that the floor grid is as clean as possible, and replace the bedding.
   12. Back up data on a cloud server and extract any critical data.
   13. Power down equipment.
   14. Run animals at the same time in the same boxes each day.
   15. Run rats 5 – 6 days a week (we run our rats 6 days a week).

4. Troubleshooting the Behavior

NOTE: It is normal for performance to drop when rats are transitioned to the next shaping phase (i.e., Nose Poke Shape to Training Schedule 1, Training Schedule 1 to Training Schedule 2, Training Schedule 2 to SAT). However, if performance does not improve over time or if a rat was performing well and then suddenly stops performing well, most often the issue is with food restriction.

1. Troubleshooting food restriction issues
   1. If food restriction is a suspected issue, watch the rat perform the task to help diagnose where the problem lies and check the omission numbers, particularly across the session.
   2. If a rat has high omissions, especially if they increase in Block 3, and sits calmly in the box not performing, reduce daily food amount by 1 g and monitor the situation to determine if performance is improved.
   3. If a rat has high omissions, impatiently pokes the screen, and runs back and forth to the food port throughout the session, increase daily food amount by 1 g and monitor the situation to determine if performance is improved.
   4. In cases where it is difficult to tell whether a rat is over- or underfed, increase food amount for 2 days and monitor performance. If performance does not improve or gets worse, decrease food amount for 2 days and monitor performance
   5. If performance does not improve after adjusting food restriction, move the rat to an easier scheduled as detailed next.
2. Troubleshooting performance at different schedules of training
   NOTE: Drop a poorly performing rat if its acquisition time exceeds two standard deviations above the mean number of days that it takes an animal of its same sex to reach criteria on that schedule. We have made suggestions based on our data for cut-offs for Sprague-Dawley rats, but these numbers may need to be adjusted for different strains.
   1. Training Schedule 1: For Training Schedule 1, drop Sprague-Dawley males if they cannot acquire after 36 days and drop females if they have not acquired after 48 days.
   2. Training Schedule 2: If a rat's performance on Training Schedule 2 stagnates below criteria for 12 days in a row and a food restriction issue has been ruled out, move the rat back down to Training Schedule 1 for a minimum of 5 days or until it reaches criteria. If the rat does not reach criteria on training Schedule 1 after 12 days, drop the rat from the study. If the rat passes Training Schedule 1 and is moved back to Training Schedule 2, but does not improve after 36 and 42 days for males and females, respectively, after initially starting Training Schedule 2, then drop the rat from the study.
   3. SAT: If a rat's performance on SAT stagnates below criteria for 12 days in a row and a food restriction issue has been ruled out, move the rat back down to Training Schedule 1 for a minimum of 5 days or until they reach criteria. If the rat does not reach criteria on Schedule 1 in 12 days, drop the rat from the study. If the rat passes Training Schedule 1 and is moved back to SAT, but does not improve after 41 and 28 days for males and females, respectively, after initially starting SAT, drop the rat from the study.
3. Troubleshooting other issues
   1. If a rat develops a side bias (i.e., consistently scoring >70% hits and <40% on correct rejections, or vice versa) while on Training Schedule 2 or SAT, move the rat to Training Phase 1, which has correction trials that encourage the rat to respond to both response areas. Keep the rat at Training Schedule 1 for a minimum of 5 days or until they reach criteria. Drop the rat from the study if criteria on training Schedule 1 is not met after 12 days.
   2. Consult with the veterinary staff immediately if a rat stops eating, develops porphyrin, or otherwise is showing signs of pain and distress.