The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

Laboratory animal care was according to Public Health Service (PHS) Policy on the Humane Care and Use of Laboratory Animals. All procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Institutional Animal Care and Use Committee of the Johns Hopkins University approved the protocol and all procedures. Johns Hopkins also maintains accreditation of their program by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC).

1. Animals

1. Use adult male Long Evans rats (approximately 10 – 12 weeks old at the start of behavioral training), although other strains are suitable, including inbred rats.
2. Upon arrival in the colony, house rats singly in approved caging and rooms, due to the food restriction needs described below. Acclimate animals to the housing situation as well as handling for several days prior to starting the food restriction procedure (approximately 7 days).
3. Provide rat chow ad libitum for 1 – 2 weeks to allow rats to achieve a "free-feeding" weight. Once at this weight, restrict food access to reduce rats' weights, over 1 – 2 weeks, to 85 – 90% of the free-feeding weight. Maintain animals at 85 – 90% of their free-feeding weights during behavioral training and testing. Start with 15 – 18 g of food per day and adjust this amount based on whether or not a rate is maintaining its target weight.
   NOTE: Food restriction normally requires approval of one's institutional ACUC or other regulatory body prior to starting the restriction.
4. Weigh animals daily and record the weight on weight sheets. Ensure weighing is done at the same time and on the same scale each day prior to the start of any behavioral training or feeding.
   NOTE: Only start pretraining (below) when the animals have achieved their target weights. This practice allows for immediate corrections to food amounts for animals that are either under or over their target weights. It also provides a method to determine if rats are maintaining weights at 24-h intervals. Also avoid weighing rats at different times of the day; it will result in dramatic differences in weight, incorrect feedings, and poor behavioral performances.
5. Provide fresh water at all times in the home cage by the use of glass or plastic bottles with rubber stopper tops and sipper tubes.
6. When sessions are not conducted (e.g., weekends, holidays), feed the rats their normal allotment of rat chow to maintain weight stability. Weigh rats to determine if more food is needed to maintain weight during these times.

2. Equipment and Software

1. Use standard modular operant chambers equipped with one nose poke key, a house light, and a pellet dispenser (i.e., feeder).
2. Place the modular nose poke key on either side of the centrally located pellet dispenser; ensuring that this placement remains consistent in other operant chambers and across studies. Here, the nose poke key is on the left hand side of the pellet dispenser.
3. Place the modular house light high on the back wall of the chamber so as to not interfere with other behaviors.
4. Use rat food pellets (e.g., Noyes 45-mg pellets or similar) for reinforcement. The feeder model determines food pellet size, but most rat feeders use 45-mg food pellets (mouse feeders typically use 20-mg food pellets). Use standard chow pellets, but other pellets can be purchased (e.g., sucrose) for different experimental manipulations.
5. Control stimulus presentations, nose poke response inputs, reinforcement delivery, and data collection via an interface connected to a computer. Contact the authors for specific information regarding behavioral programs written with a programming language specifically designed for behavioral testing (see Table of Materials and Reagents).
   NOTE: It is important to record independent and dependent variables on a trial-by-trial basis, including animals' response types (e.g., correct responses, premature responses, misses), the response latency, and number of trials presented. When using different strains, sexes or species, these variables can be easily altered to maximize training in almost any type of rodent.

3. Pretraining

1. Assign each animal to an operant chamber where it will be tested each day, and at approximately the same time of day, throughout the experiment.
2. Weigh the rat and place it in its specific operant chamber with food pellets in the pellet dispenser. Place 10 – 20 food pellets in the receptacle for the rat to eat.
3. For pretraining, set the following parameters to low, fixed values: foreperiod value = 2 s, timeout value = 2 s, limited hold = 10 s.
4. Start the computer program to control presentation of the house light, nosepoke key illumination, and automatic food pellet delivery.
   NOTE: It is best to have the behavioral program set to a level that allows for frequent reinforcements for the majority of responses produced by the animal, such that the nose poke key is illuminated for a relatively long duration of time (e.g., 10 s). This increases the likelihood that the rat will poke the key while it is illuminated, resulting in the delivery of a food reinforcer by the computer program.
5. Once the rat finishes the food pellets, start the shaping process by reinforcing the rat when it approaches the food receptacle or the nose poke key. Here, manually shape each rat, which means that an experimenter watches the rat's behavior (by leaving the sound attenuating cubicle doors open) and delivers pellets from the feeder as quickly as possible after the rat emits the desired behavior. Deliver pellets from a button on the feeder itself, through a hand-held switch that is wired to the feeder, or through the computer software used to control the behavioral contingencies by clicking on the appropriate output on the interface.
6. Using the method of shaping by successive approximations, provide a food reinforcer for behavior changes that are successive approximations to the final desired behavior²³. For example, once the rat is reliably retrieving food pellets from the food receptacle, start reinforcing only movement towards or around the nose poke key, and no longer reinforce approaches to the food receptacle. Here, shape rats to move to the left after retrieving a food pellet, since the nose poke key is located to the left of the food receptacle in the operant chambers.
   NOTE: Even though the experimenter is delivering food reinforcers during the beginning of pretraining, the computer program is still running and controlling the house light, nosepoke key, and feeder. If a rat pokes the key, the computer program will reinforce it automatically. Experimenter-reinforced behaviors are only those leading the rat to poke the illuminated key, such as sniffing the key or around it, moving the head near the key or towards it, etc.
7. Determine the amount of food earned during the pretraining session by counting the number of pellets delivered manually plus any pellets delivered automatically by the computer software (i.e., pellets delivered because the rat poked the illuminated key). Multiply this number by 0.045 g to determine total food earned (e.g., 30 pellets x 0.045 g = 1.35 g).
8. Remove the rat and place it back in its home cage. Subtract, by hand, the amount of food earned (see 3.7) in the behavioral session from the rat's daily food allotment. Feed the remainder of the food allotment to the rat after the behavioral training session. Feed at a minimum of 30 min after the conclusion of the behavioral session to avoid home cage food reinforcement immediately after nonproductive sessions.
9. Clean the operant chamber daily with a gentle disinfectant (e.g., soap and water). Use 70% EtOH sparingly, given that it can damage the operant chambers. Check the nose poke key daily in order to maintain proper working condition and clean with disinfectant when needed, since a key that sticks or gets locked in place will disrupt an animal's performance if not repaired quickly.
10. Shape each rat in this manner until the rat emits 40 – 50 correct computer-reinforced (not manually or experimenter-reinforced) responses on the illuminated key, retrieves each food pellet, and returns to and pokes the nose poke key without additional shaping. Typically, rats require approximately 2 – 3 30-min sessions to reach these criteria (see Figure 2).
    NOTE: Manual delivery of food pellets is not needed after the rat is reliably poking the key and receiving food reinforcement delivered by the computer program.

4. rPVT Training

1. Once shaped, start the rat at a fixed, relatively short foreperiod interval and TO value (e.g., 2 s) on the next 30-min session until the rat completes 8 out of 10 correct trials. For each of these trials during the training, illuminate the nose poke key for a period of time long enough for a rat to make a response. Here, keep the key illuminated for 9 seconds; this response window is also called the limited hold (LH).
2. Use a relatively short time out (TO; e.g., 2 s) as a punishment for premature responses, or responding prior to the illumination of the nose poke key. During the TO, rats have no opportunity to earn food. Here, the TO and foreperiod value are the same duration during these training sessions and change at the same rate.
3. Once the rat completes 8 out of 10 correct trials, increase the foreperiod and TO by 0.1 s (i.e., to 2.1), while shortening the LH by 0.1 s (i.e., to 8.9). It is best to use a computer program that can automatically make these adjustments during a session.
   NOTE: When training mice on the rPVT, a longer ITI is employed since mice require more time to consume food pellets. Here, use a 30 s ITI for training and testing male C57BL/6J. Additionally, since a mouse may weigh ~ 20 g, it will consume significantly fewer pellets during a session. Therefore, shorten a typical session by having it end when a maximum of 50 pellets are earned; the criterion for increasing the foreperiod and TO is also changed to be 4 out of 5 correct trials before increasing the foreperiod and TO by 0.1 s.
4. Once the 30 min session is over, record the final foreperiod the rat reached during the training session (e.g., 4.2 s), either in a spreadsheet or by hand on a data sheet, and the amount of food earned, which is calculated by multiplying the number of correct trials times 0.045 g (e.g., 100 correct trials x 0.045 g = 4.5 g). Subtract this food amount from the rat's daily allotment and feed the rat the remaining food in the home cage.
   NOTE: The daily data files record the final foreperiod reached by each rat. The calculation to determine the amount of food earned in the session can be done automatically by the computer program or by hand using the equation above. Food earned is subtracted by hand from each rat's the daily food allotment.
5. On the following day, start the 30 min training session with a foreperiod that is 300 ms lower than the value the rat ended on in the previous session. For example, if the rat ended at 4.2 s, this session starts at 3.9 s. Continue increasing the foreperiod by 0.1 s as the rat completes 8 out of 10 correct trials at each foreperiod, until rats reach a foreperiod of 10 s. In this way, rats will experience all of the possible foreperiods.
   NOTE: When rats reach a foreperiod of 9.7 – 10 s, the TO remains at 8 s and the LH is 1.5 s.
6. At the end of each session, check the rat's daily performance during this time by examining the percentage of correct trials, premature trials, and misses. Rats commonly maintain an average of 40 – 90% correct responses during these training sessions, with the average percent correct increasing steadily across training. Here, export the daily data for each rat to a spreadsheet that calculates these percentages in order to track daily session performance.
7. Once the rat reaches a foreperiod of 10 s, start the next subsequent 30 min session with the foreperiods presented at random intervals, starting with foreperiods between 7 – 10 s. Ensure that the TO and LH remain at 8 and 1.5 s, respectively. Here, the computer program controlling the behavioral contingencies automatically switches from presenting ascending foreperiods to variable foreperiods once each rat reaches the 8 out of 10 criterion at the 10 s foreperiod.
8. Once the rat maintains at least 70% correct with randomly presented foreperiods between 7 – 10 sec, change the program to present foreperiods at random intervals, starting with foreperiods between 5 – 10 s. Examine each rat's daily performance during this time because this change often happens within a session.
9. After the rat maintains 70% correct, change the program again to present the full range of foreperiods (3 – 10 s) at random intervals during the 30 min session. In a manner similar to 4.8, examine each rat's daily performance during this time because this change often happens within a session.
   NOTE: The computer software controlling the behavioral contingencies automatically makes these changes based on each rat's performance (e.g., 4.7 – 4.9). It is important to only make the change in 4.9 when a rat's success rate is high (>70% correct), since rats frequently adopt the strategy of responding randomly in time, instead of properly waiting and attending to the stimulus. If a rat's success rat is low, repeat 4.7 – 4.9.
10. For the Acquisition Criteria, define the baseline performances as stable when, on 4 out of 5 daily sessions within a week, rats' percentage of correct responses is 75% or greater during a session and the percentage of premature responses is less than 30%.

5. Data Analysis for Basic Response Measures

1. To assess behavioral performances during acquisition, use repeated-measures ANOVAs, with the repeated factor of either Session, Time on Task Value, or Foreperiod Value for each performance measure described below.
   NOTE: Here, the studies use both within- and between-subjects designs, where we compare all animals to their own baselines after an experimental condition and compare experimental animals to control animals following the same manipulation. When stable behavioral baselines are achieved, use of within-subjects designs will limit the number of rats needed.
2. Define premature responses as responses prior to the onset of the key light or within the first 150 ms of the key light being on.
3. Define correct responses as responses made after the key light comes on, with a minimal latency greater than 150 ms, and a maximal latency of the response window length; here, 1,500 ms.
4. Define misses (or omissions) as trials on which the subject fails to make a response.
5. Define response latencies as the elapsed time (in ms) from light onset to depression of the nose poke key; also called reaction time (RT).
6. Define total trials as the number of premature responses plus correct responses plus misses.
   NOTE: In human PVT performances, premature responding and lapses in attention can be quite rare, and investigators most often report only the actual raw frequencies of these measures. However, this is not the case in performances with rats. To allow for comparisons between different versions of the rPVT in the literature, rats' performance measures are presented here as a percentage of total trials. Use of a computer program that can record and label each of these items on a trial-by-trial basis for subsequent analysis is preferred, since rats will complete over 200 trials per 30 min session and will often meet the criteria to move to another step in the training procedure within a session.

6. Additional Computed Performance Measures

1. For a 30 min behavioral session, define lapses as correct responses in which the RT was greater than twice the mean reaction time for each rat's session (done after a session); also include missed trials in the lapse calculation.
   NOTE: In the human PVT, lapses are typically defined as reaction times (RT) > 500 ms⁴. For rodents, it is best to perform post-hoc comparisons to each rat's mean RT – acquired from the trial-by-trial analysis described above – instead of comparing them to a threshold value, due to the variability in RTs from individual rats. This method is similar to lapses in other published versions of the rPVT^15,16. Here and in other published versions, lapses also include missed trials (lapses = responses with RTs > twice the mean RT for a session, plus misses) because missed trials in the human PVT are recorded as lapses, as well.
2. Determine the estimated false alarm rate by calculating the percentage of premature responses that occur during the actual 3 – 10 s interval when a stimulus could have appeared.
   NOTE: This measure uses a subset of premature responses that occur only during a time when the stimulus could occur, i.e., premature responses that occur within the 3 – 10 s foreperiod window. This measure does not include premature responses prior to the 3 s because the stimulus light would never be illuminated during that time. Here, this subset of premature responses is automatically sorted from all premature responses in a spreadsheet computer program.