Psychomotor Vigilance Task Performance During and Following Chronic Sleep Restriction in Rats

Transcription

1 pii: sp PVT PERFORMANCE AND CHRONIC SLEEP RESTRICTION IN RATS Psychomotor Vigilance Task Performance During and Following Chronic Sleep Restriction in Rats Samuel Deurveilher, PhD 1 ; Jacquelyn E. Bush, BSc 1 ; Benjamin Rusak, PhD 2,3,4 ; Gail A. Eskes, PhD 2,3,5 ; Kazue Semba, PhD 1,2,3 Departments of 1 Medical Neuroscience, 2 Psychology and Neuroscience, 3 Psychiatry, 4 Pharmacology, and 5 Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Study Objectives: Chronic sleep restriction (CSR) impairs sustained attention in humans, as commonly assessed with the psychomotor vigilance task (PVT). To further investigate the mechanisms underlying performance deficits during CSR, we examined the effect of CSR on performance on a rat version of PVT (rpvt). Design: Adult male rats were trained on a rpvt that required them to press a bar when they detected irregularly presented, brief light stimuli, and were then tested during CSR. CSR consisted of 100 or 148 h of continuous cycles of 3-h sleep deprivation (using slowly rotating wheels) alternating with a 1-h sleep opportunity (3/1 protocol). Measurements and Results: After 28 h of CSR, the latency of correct responses and the percentages of lapses and omissions increased, whereas the percentage of correct responses decreased. Over h of CSR, all performance measures showed partial or nearly complete recovery, and were at baseline levels on the first or second day after CSR. There were large interindividual differences in the magnitude of performance impairment during CSR, suggesting differential vulnerability to the effects of sleep loss. Wheel-running controls showed no changes in performance. Conclusions: A 28-h period of the 3/1 chronic sleep restriction (CSR) protocol disrupted performance on a sustained attention task in rats, as sleep deprivation does in humans. Performance improved after longer periods of CSR, suggesting allostatic adaptation, contrary to some reports of progressive deterioration in psychomotor vigilance task performance during CSR in humans. However, as observed in humans, there were individual differences among rats in the vulnerability of their attention performance to CSR. Keywords: individual differences, neurobehavioral impairment, PVT, recovery, sleep deprivation, sustained attention, vigilance Citation: Deurveilher S, Bush JE, Rusak B, Eskes GA, Semba K. Psychomotor vigilance task performance during and following chronic sleep restriction in rats. SLEEP 2015;38(4): INTRODUCTION Chronic sleep restriction (CSR) is becoming increasingly common in many societies because of a combination of lifestyle choices and workplace demands. CSR has adverse effects on metabolism and cardiovascular function 1 and also on cognitive performance, especially sustained attention (i.e., maintenance of response readiness for an extended period of time). 2 4 Impairment in sustained attention associated with inadequate sleep jeopardizes the ability to perform operational tasks safely and efficiently, and thus can be a significant contributing factor to industrial and transportation accidents. 5 Psychomotor vigilance tasks (PVTs) have been used in humans to evaluate the effects of inadequate sleep on sustained attention. 6 8 In the most common PVT tasks, individuals are required to respond as quickly as possible with a button press to visual stimuli that are presented at irregular intervals over a period of 10 min. Restriction of time in bed to 3 6 h per night over 7 14 consecutive days in healthy, young adults resulted in cumulative, dose-dependent deficits in performance on the PVT. 6,9 11 Following such sleep restriction paradigms, full recovery of performance may require more than 2 or 3 nights of regular or extended sleep. 10,12,13 One more recent study, however, Submitted for publication April, 2014 Submitted in final revised form November, 2014 Accepted for publication November, 2014 Address correspondence to: Kazue Semba, PhD, Department of Medical Neuroscience, Dalhousie University, 5850 College Street, PO Box 15000, Halifax, Nova Scotia, B3H 4R2, Canada; Tel: (902) ; Fax: (902) ; semba@dal.ca reported that young adults who were permitted 6 h time in bed for 7 nights had no cumulative deficits on PVT performance across days of sleep restriction. 14 In addition, two recent studies reported that young adults 15 or middle-aged men 16 who were permitted 4 h time in bed per night for 5 nights had fairly stable, albeit reduced, performance on PVT over 3 5 nights of sleep restriction; recovery to baseline performance took 1 day. 15,16 Underlying reasons for this variability across studies are unknown, and deserve more investigation. Various rat models of cognitive skills have been used to investigate the mechanisms linking sleep loss and cognitive performance changes For tasks that require sustained attention, Christie et al. 21,22 developed and validated a rat version of the PVT (rpvt) that is analogous to the human PVT. In this task, rats undergoing water restriction were trained to poke their noses into a water-delivery port when they detected brief light stimuli that were presented irregularly; after 24 h of sleep deprivation, correct response latencies and the number of errors increased. 21 These findings are consistent with those obtained from people experiencing acute total sleep deprivation To the best of our knowledge, no studies have used this rpvt to investigate the effects of CSR on attention performance. We have developed and studied a rat model of CSR that takes into account the polyphasic sleep patterns of rats. 26 In this model, cycles consisting of a 3-h period of sleep deprivation (using slowly rotating wheels) followed by a 1-h period of sleep opportunity ( 3/1 CSR protocol) were imposed continuously for 4 days. This protocol reduced total daily sleep time by ~60% from baseline levels. It induced both homeostatic responses (rebound increases in sleep duration and intensity SLEEP, Vol. 38, No. 4, Vigilance Performance and Chronic Sleep Loss Deurveilher et al.

2 (see Experimental Design). The rats were 4 5 mo of age (i.e., young adults) at the end of the entire experiment. Animal handling procedures followed the guidelines of the Canadian Council on Animal Care and were approved by the Dalhousie University Committee on Laboratory Animals. Figure 1 The trial sequence of the rat psychomotor vigilance task (rpvt). In this task, rats were trained to press a lever after they detected an irregularly presented, brief light stimulus in order to obtain a water reward. Following a variable (3 7 sec) intertrial interval, a 0.5-sec light stimulus was illuminated. A lever press during the light stimulus or the following 2.5-sec response interval (i.e., a correct response) led to a water reward (20 µl). Time-out (i.e., absence of trials with house light extinguished for 10 sec) was triggered by failure to respond within 3 sec of light stimulus onset (i.e., an omission) or by a lever press occurring during the intertrial interval (i.e., a premature response). Each daily session of rpvt training/testing lasted 30 min. during sleep opportunities over the 4 days) and allostatic or adaptive responses (gradual decline in the rebound increases in sleep intensity during sleep opportunities over the 4 days, and muted post-csr sleep recovery); both of these responses were also strongly modulated by time of day. 26 In this study, we used the rpvt in conjunction with the 3/1 CSR protocol to gain insight into how CSR impairs attention performance, and how this impairment recovers following CSR. We conducted two experiments. In the first experiment, we evaluated the daily performance of rats during 100 h of the 3/1 CSR protocol followed by 96 h of recovery. Wheel-running control (WRC) animals were housed in running wheels, as were the CSR animals, but they could rotate their wheels freely at all times and were allowed to sleep ad libitum throughout the study. This control protocol took advantage of rodents natural motivation to run when they are provided with a running wheel. 27 In the second experiment, we extended the CSR from 100 to 148 h, followed by 120 h of recovery, to evaluate the stability of the effects observed using the 100-h CSR protocol. MATERIALS AND METHODS Animals Adult male Wistar rats (Charles River Canada, St. Constant, QC, Canada), weighing g (approximately 2 mo of age) on arrival, were housed in pairs under a 12 h light:12 h dark cycle (lights on at 07:00 = Zeitgeber time [ZT] 0) in a temperature-controlled (23 ± 1 C) animal colony room, with water and food available ad libitum. The rats remained housed in the animal colony room for several weeks until they were transferred to activity wheels at the completion of rpvt training Water Restriction Schedule Water restriction was used to motivate animals to perform the rpvt for water reward. 21 A previous study reported that 24 h or 5 days of total sleep deprivation affected neither the amount of water intake nor motivation to perform for water reward, 28 indicating that water restriction is well suited for motivating rats to perform the rpvt and examining the effects of CSR on their performance. Restrictions on the daily period of water access were introduced gradually starting 1 w before rpvt training, and animals remained on water restriction for the remainder of the experiment. During the first week of water restriction, daily access to water was gradually reduced from 24 h to 12, 6, 4, and 2 h, and eventually to a 1-h period from ZT4 to ZT5 by the end of the week, at which time rpvt training began. In addition to receiving water as a reward for correct responses during a daily 30-min rpvt session, animals were given free access to water for 15 min in their home cages approximately 20 min after each rpvt session. Their average daily water intake was ml, including ~4 ml of water obtained during rpvt sessions. Food was available ad libitum throughout the experiment, except during the daily 30-min rpvt sessions. Animals were weighed daily immediately before the rpvt training/ testing session throughout the experiment. Over the course of training prior to CSR, all rats continued to gain body weight. The rpvt Apparatus For rpvt training and testing, rats were placed singly in one of four clear Plexiglas operant chambers housed within individual sound-attenuating cabinets (Med Associates; St. Albans, VT). Cabinets were equipped with a ventilation fan that also provided background white noise. Each operant chamber ( cm) was equipped with an overhead house light at the top of one wall. On the opposite wall, there was a retractable lever with a stimulus light positioned above it, and, to the right of the lever, a water dispenser, which provided 20 µl of water per delivery. The front door of the cabinet had a peephole that allowed the experimenter to observe the rat during task performance. The house light, stimulus light, lever operation, and water delivery were automatically controlled and response data were collected using Schedule Manager software (Med Associates). Behavioral Training and Testing Habituation and training methods were adapted from those used by Christie et al. 21 ; the main modification was the use of a lever press as opposed to a nose poke as the instrumental response. The rpvt trial sequence is shown in Figure 1. Training and testing took place daily at the same time during the light phase (starting at ~ZT4), and each session lasted 30 min. Four rats were trained or tested at the same time. SLEEP, Vol. 38, No. 4, Vigilance Performance and Chronic Sleep Loss Deurveilher et al.

3 For rpvt training, rats were first acclimated to the operant chambers (house light on, stimulus light off, and lever retracted) for 30 min daily for 2 days, and then trained to lever-press for water on a continuous reinforcement schedule for 1 day (shaping). At the start of each training session, the house light was turned on and the lever was extended into the chamber. During subsequent training, lever presses elicited water delivery only during the 0.5 sec stimulus light and during the following 2.5 sec (3-sec reward interval). The duration of each stimulus light illumination was shortened progressively over training sessions, from 30 sec (through 15, 10, 5, 2.5, and 1 sec) to 0.5 sec. Animals were required to maintain performance criteria ( 70% correct responses and 30 omissions per session) at the current stimulus duration for at least 2 consecutive days before moving on to training at the next shorter light stimulus duration. When animals failed to respond during the reward interval (i.e., the 3-sec period following light stimulus onset), or when they pressed the lever before or after that interval (i.e., in the intertrial interval), the lever was retracted and the house light was extinguished for 10 sec (time-out, during which no reward opportunities occurred); then the house light was turned on and the next trial began starting with another intertrial interval. Time-outs were introduced starting with the 10-sec light stimulus duration and occurred at all shorter stimulus intervals. The intertrial intervals ranged from 3 7 sec in 1-sec increments on a variable time schedule. Eventually, animals were required to maintain the performance criteria listed above at the 0.5-sec stimulus light duration for 4 consecutive days, which served as a 4-day baseline period, before the CSR or WRC protocol began. Performance Measures Each rpvt testing session lasted 30 min irrespective of the total number of trials completed during that period. Because the total number of trials per session varied across CSR days (see the Results), the numbers of correct responses, omissions, and premature responses were normalized as percentages of the total number of trials, as explained below. Daily performance in the rpvt was assessed using the following behavioral measures adapted from Christie et al. 21 with some modifications: (1) Average latency of correct responses: the mean time interval between the onset of a stimulus light and a correct lever press, defined as a lever press occurring within the reward interval; (2) Percent correct responses: the number of trials with correct responses expressed as a percentage of the total number of trials; (3) Percent lapses: the number of correct responses with latencies greater than twice the animal s own average latency of correct responses during the 4-day baseline period, expressed as a percentage of the total number of correct responses; (4) Percent omissions: the number of trials with no lever presses during the reward interval expressed as a percentage of the total number of trials; (5) Percent premature responses: the number of trials with a lever press that occurred before presentation of the light stimulus (i.e., during the intertrial interval) expressed as a percentage of the total number of trials. As in the human PVT, the latency of correct responses and the percentages of lapses and omissions served as the main measures of attentional performance in the rpvt, whereas the percentages of correct and premature responses served as secondary measures of attentional performance. 21 3/1 CSR Protocol The sleep restriction protocol was implemented by maintaining rats in programmable, motorized activity wheels (11 cm in width, 36 cm in diameter; Model 80860, Lafayette Instrument, Lafayette, IN). 26 The wheels were housed inside individual experimental cabinets ( cm) which were each equipped with a fan and a light. The inside light was controlled by a timer to maintain the same 12 h light:12 h dark cycle as in the animal colony room. Food was available ad libitum from a custom-made feeder attached to the stationary side wall of the wheel. The 3/1 CSR protocol consisted of continuous cycles of 3 h of sleep deprivation imposed by slow rotation of the wheel (at approximately 2.5 m/min), followed by 1 h of sleep opportunity (locked wheels), for a total duration of either 100 h (Experiment 1; Figure 2) or 148 h (Experiment 2). When wheels were rotating during sleep deprivation periods, rats either walked slowly or rode in the wheel for a few seconds until they adjusted their postures; during these riding intervals, rats could eat, groom, or lie down. The wheels were cleaned with an antiseptic (PeroxyGuard) every other day while the rats were out of the wheels (i.e., during the rpvt sessions or during the periods of free water availability in their home cages). WRC Protocol The WRC protocol used the same wheels (and the same cabinets and room) as for the CSR protocol, except that the wheels were unlocked at all times so rats could turn them freely. This protocol resulted in daily amounts of wheel rotation that were at least as high as those imposed by the CSR protocol (see Figure S1A, supplemental material), thus controlling for the level of physical activity without imposing sleep restriction. Voluntary wheel running in rats has only small effects on daily sleep amount. 29 To monitor the amount and daily pattern of activity, wheel rotations were summed in 1-h bins using Activity Wheel Monitor software (Lafayette). The wheels were cleaned with an antiseptic (PeroxyGuard) every other day, as was done for the CSR group. Experimental Design Experiment 1: Effects of 100 h of CSR on rpvt performance Figure 2 illustrates the experimental design for rats under the 100 h CSR protocol. When rats were judged, based on their daily performance, to be likely to reach performance criteria on the rpvt in approximately 1 w, they were removed from their home cages and housed individually in locked activity wheels for 4 6 days of habituation. The wheels were housed in a room adjacent to that used for rpvt training/testing. Daily rpvt training continued during this habituation period. Rats were kept in the wheels for the remainder of the experiment, except during daily rpvt testing (~30 min) and the following 15 min of free water access in their home cages. During the habituation period, wheel motors were activated at a slow speed (~2.5 m/min) once a day during the light phase, for a period gradually increasing from 5 to 20 min, to SLEEP, Vol. 38, No. 4, Vigilance Performance and Chronic Sleep Loss Deurveilher et al.

4 Figure 2 The experimental design for Experiment 1. The design of Experiment 2 is identical except that the durations of chronic sleep restriction (CSR) and recovery periods were extended as noted later in this legend. Rats that were close to reaching performance criteria on the rat psychomotor vigilance task (rpvt) were transferred and housed individually in activity wheels (locked). Once stable daily performance occurred over at least 4 days (Baseline, B1 4 during which wheels were locked except for short adaptation intervals; see the Materials and Methods for details), the 3/1 protocol of CSR began at lights-on (Zeitgeber time or ZT0). In the 3/1 protocol, 3 h of sleep deprivation (gray; slowly rotating wheels) followed by 1 h of sleep opportunity (white; locked wheels) were cycled continuously for 100 h (148 h in Experiment 2), for a daily total of 18 h of sleep deprivation and 6 h of sleep opportunity. rpvt performance continued to be assessed on each day of CSR (SR1 5), after the first 1-h sleep opportunity in the light phase (i.e., at the same clock time as during training and baseline days, ~ZT4). Thus, rpvt testing sessions (asterisks) occurred after 4 h (SR1), 28 h (SR2), and subsequently at 24 h intervals on days SR3 5 in Experiment 1 (SR3 7 in Experiment 2). Following the 3/1 protocol, rpvt performance was assessed over 4 recovery days (5 in Experiment 2), daily at the same clock time (R1 4 or 5; locked wheels). The 12-h light (white bar) and 12-h dark (dark bar) phases are indicated at the bottom using Zeitgeber time. adapt rats to the sensations associated with wheel rotation; the wheels were otherwise locked. After each of the four rats concurrently undergoing rpvt training displayed stable daily performance for 4 6 consecutive days, the last 4 of which were considered the baseline days (B1 4), the 3/1 CSR protocol began on the following day at lights on (ZT0). CSR continued for 100 h, i.e., just over 4 days (SR1 5), and rpvt performance was tested daily starting at min after the first 1-h sleep opportunity in the light phase (i.e., at the same clock time as during the training and baseline days, ~ZT4; Figure 2). Thus, rpvt testing occurred after 4 h (SR1), 28 h (SR2), 52 h (SR3), 76 h (SR4), and 100 h (SR5) of CSR. Following CSR, wheels were permanently locked and rpvt performance was assessed daily at the same clock time over 4 recovery days (R1 4). Rats were tested at ~ZT4, immediately after a sleep opportunity (1 h), in order to reduce any short-term effect on their motivation and performance of being tested during or immediately after a 3-h period of sleep deprivation. Using this approach, the majority of the rats continued to perform on the rpvt throughout the 100 h CSR period (see the Results). Similarly, the rats assigned to the WRC group that were close to achieving performance criteria on the rpvt were transferred to unlocked wheels, which they could turn freely. The rats remained housed in the wheels for the rest of the experiment. Following a 9-day period of habituation, the rats continued to be tested on the rpvt daily at the same time of day (~ZT4) for a 13-day period, which corresponded to days B1 R4 for the CSR animals. The longer period of habituation for WRC rats (9 days versus 4 6 days for CSR rats) permitted them to increase and then maintain fairly steady daily wheel rotation levels that were at least as high as those experienced by the CSR rats (see Figure S1A). Of 26 rats trained on the rpvt, 21 (81%) met the final performance criteria; the average (± standard error of the mean [SEM]) number of daily training sessions required was 34 ± 3 (range: 23 57). All of these animals proceeded to either the CSR or WRC protocol. However, two rats in the CSR group were later removed from the experiment because of wheel software malfunctions during CSR. Another two rats in the CSR group, for unidentified reasons, did not show any responses on the second and fifth day of CSR, and were excluded from the data analyses. A fifth rat developed a urinary tract infection while on rpvt training (as did two others that did not meet criteria) before being assigned to the CSR group, and was kept on antibiotic (enrofloxacin, 0.1 mg/ml of drinking water) through the remainder of the experiment. Because this rat showed no gross behavioral abnormalities and its overall performance was within the range of the other CSR rats that did not develop an infection, it was included in the analysis. Thus, 10 rats in the CSR group and 7 in the WRC group were included in the final statistical analyses. Experiment 2: Effects of 148 h of CSR on rpvt Performance To assess the stability of performance deficits during CSR and the time required for full recovery, we extended the duration of the 3/1 protocol to 148 h followed by 120 h of recovery. Rats were trained and tested using the same water restriction paradigm as in Experiment 1; six of seven rats met the final performance criteria, requiring 43 ± 4 (range: 36 54) training sessions. One of these rats developed a urinary tract infection that appeared to have affected its performance, and was excluded from the experiment, so the final n was 5 for this group with extended CSR and recovery (CSRe). Statistical Analyses Statistical analyses were conducted with Statview 5.0 (SAS Institute Inc., Cary, NC) and IBM SPSS Statistics 20.0 (IBM Corp., Armonk, NY) software. Performance data in Experiment 1 were analyzed using two-way analyses of variance (ANOVAs) for repeated measures with a between-subjects factor of Group (CSR versus WRC) and a within-subjects factor of Day (day of a given experimental protocol). When a Group Day interaction was significant (P < 0.05), analyses of simple effects were conducted using a separate one-way repeated ANOVA for each group. Performance data in Experiment 2 were analyzed SLEEP, Vol. 38, No. 4, Vigilance Performance and Chronic Sleep Loss Deurveilher et al.

5 using one-way ANOVAs for repeated measures with a withinsubjects factor of Day. When applicable, post hoc t tests (Bonferroni-corrected for multiple comparisons) were conducted to identify differences among mean values. To reduce the number of Type I errors, multiple pairwise comparisons were run separately for baseline/csr days and baseline/recovery days. To take into account any baseline differences in performance among animals, we normalized all performance measures by expressing them relative to baseline values. Thus, in each animal, the performance value on the third day of baseline (B3) was subtracted from that on the fourth (last) day of baseline (B4) and each day of CSR (i.e., SR1 5 [Experiment 1] or SR1 7 [Experiment 2]) and recovery (i.e., R1 4 [Experiment 1] or R1 5 [Experiment 2]), and this difference in each animal was then averaged in each treatment group. We normalized performance data to B3 in order to obtain a measure of variability in baseline performance, as indexed by the difference between B3 and B4, for statistical comparison to the variability between B3 and subsequent days of CSR or Recovery. To meet the assumptions of normality and homogeneity of variances, a logarithmic or a square root transformation was used before performing parametric analyses. 30 When negative values were present in a data set, a constant was added to all the values so that the largest negative value was converted to 1 prior to applying data transformation. When required, Huynh- Feldt corrections were applied to all repeated-measures effects to correct for violations of sphericity; corrected F and P values (and original degrees of freedom) are reported. P < 0.05 was considered statistically significant. RESULTS Effects of 100 h of CSR on rpvt Performance (Experiment 1) The CSR group (n = 10) underwent 100 h of the 3/1 CSR protocol followed by 96 h of recovery with no sleep restriction (see Figure 2), whereas the WRC group (n = 7) had unlocked wheels and could sleep ad libitum throughout the study. The average daily number of wheel rotations in the WRC group (3,737 ± 727 [SEM]; Figure S1A) was higher than that imposed on the CSR group (2,454). As expected of nocturnal rodents, rats in the WRC group were more active in the daily dark phase, except for a conspicuous increase in anticipatory wheel running 1 2 h before the testing time early in the light phase (Figure S1B). Figure 3A 3E shows the five performance measures on the rpvt, as well as the total number of trials per rpvt session, in the CSR group on the last day of baseline (B4), CSR days (SR1 5) and recovery days (R1 4), and in the WRC group over the corresponding time interval. There were no significant group differences on B4 for any of the five rpvt measures (P = ; Table S1, supplemental material). Table S2 (supplemental material) lists F and P values for main effects of Group (CSR and WRC) and Day (B4 R4), and Group Day interaction, and for simple effects of Day. Average Latency of Correct Responses and Percent Correct Responses Performance deteriorated during CSR, as evidenced by generally longer average latencies of correct responses (Figure 3A) and lower percentages of correct responses (Figure 3B), in patterns that were reciprocal to each other. Neither of these measures changed across days in the WRC group during the same time period (for average latency and percent correct responses: Group Day, P = 0.01 and 0.017, respectively; CSR group: Day, P = 0.01 and 0.003, respectively; WRC group: Day, P = 0.91 and 0.55, respectively; Table S2). Specifically, in the CSR group, average latency increased sharply above baseline levels on SR2 (+213 ± 80 msec, P < 0.05 versus B4; Figure 3A), whereas the percentage of correct responses fell substantially below baseline levels on SR2 ( 26.5 ± 9.4%, P < 0.05 versus B4; Figure 3B). Subsequently, average latency largely remained above baseline levels on SR3 5, with a significant difference on SR5 (P < 0.05 versus B4), whereas the percent correct responses remained below baseline levels on SR3 5 (P < 0.05 versus B4 for each comparison); in both measures, however, there was a trend toward gradual, partial recovery (on SR5, +106 ± 39 msec latency, and 14.9 ± 3.8% correct responses). During recovery after CSR, latency returned to baseline levels on R1, whereas the percentage of correct responses was still below baseline levels on R1 ( 11.3 ± 3.5%; P < 0.05 versus B4), returning to baseline levels on R2. Compared to the WRC group, the CSR group had longer average latencies on SR2 and SR5, and fewer correct responses on SR2 5 and R1 (all P < 0.05). To further characterize the latency of correct responses in the CSR and WRC groups, we calculated the median and the value of the 10 th percentile (fastest) latencies of correct responses. 31 The median latency (Figure S2A, supplemental material) followed a pattern that was similar to that of the average latency (Figure 3A). The 10 th percentile of correct response latencies (Figure S2B) showed an increase initially on SR2 (P < 0.05 versus B4), as was the case for the average latencies. On subsequent days (SR3 5), the 10 th percentile value returned to baseline levels, whereas the average latencies remained elevated (Figure 3A) as described previously. Lapses, Omissions, and Premature Responses The deterioration in performance during CSR was also indicated by increases in the percentage of lapses (i.e., correct responses with latencies > twice the average baseline latency; Figure 3C) and omission errors (i.e., trials with no response; Figure 3D). Both measures changed across days in the CSR group but not in the WRC group (Group Day, P = and < 0.001, respectively, for lapses and omissions; CSR group: Day, P = 0.03 and < 0.001, respectively; WRC group: Day, P = 0.88 and 0.51, respectively; Table S2). In the CSR group, the percentages of both lapses and omissions increased greatly above baseline levels on SR2 (lapses: ± 5.3%; omissions: ± 10.7%, P < 0.05 for each comparison; Figure 3C and 3D, respectively). Subsequently, on SR3 5, both measures remained above baseline levels (lapses: SR5 > B4; omissions: SR3 5 > B4; P < 0.05 for each comparison), although there was a trend toward gradual, partial recovery in both measures (at SR5, lapses: +5.3 ± 2.3% and omissions: ± 4.0%, P < 0.05 versus SR2). During recovery after CSR, the percentages of lapses and omissions were at baseline levels on R1 4. Compared to the WRC group, the CSR group had more lapses on SR2 and SR5, and more omissions on SR2 5 (P < 0.05 for each comparison). SLEEP, Vol. 38, No. 4, Vigilance Performance and Chronic Sleep Loss Deurveilher et al.

6 Figure 3 Changes in performance on the rat psychomotor vigilance task (A E), total number of trials (F), and body weight (G) in Experiment 1, during 100 h of the 3/1 chronic sleep restriction (CSR) paradigm. Daily means (± standard error of the mean) of different measures are shown for the last (fourth) day of baseline (B4), CSR days (SR1 5), and recovery days (R1 4) for the CSR group (CSR, closed circles), and during time-matched intervals for the wheel-running control group (WRC, open circles). Performance on the psychomotor vigilance task was analyzed using the following measures: changes in average latency of correct responses (A), percent correct responses (B), percent lapses (C), percent omissions (D), and percent premature responses (E). Each performance measure was normalized to baseline performance on the third baseline day (B3), by subtracting the performance value on B3 from that on B4 and each day of CSR and Recovery (the data of the mean of 0 and SEM of 0 on B3 are omitted from the figure; see Materials and Methods). (F) Total number of trials per rpvt session. (G) Body weights are shown as changes in body weight expressed as percentages of the body weight on B3. CSR group, n = 10; WRC group, n = 7. * Different from B4; # different from WRC; P < 0.05 (Bonferroni-corrected post hoc comparisons). SLEEP, Vol. 38, No. 4, Vigilance Performance and Chronic Sleep Loss Deurveilher et al.

7 During trials with omissions, rats were observed to be rearing, sniffing, grooming, moving around the operant chambers, or motionless, but they were not in typical sleep postures. The percentage of premature errors (i.e., responses made before onset of the stimulus light) varied across days only in the CSR group (Group Day, P = 0.001; CSR group: Day, P = 0.01; WRC group: Day, P = 0.91; Table S2) and, in contrast to lapses and omissions, the change was only transient (Figure 3E). Thus, the percent of premature responses fell below baseline levels on SR2 ( 5.0 ± 2.3%, P < 0.05; Figure 3E), but was back to baseline levels on SR3 5, as well as on R1 4. Compared to the WRC group, the CSR group had more premature responses on R1 (P < 0.05). Total Trials As shown in Figure 3F, the total number of trials per rpvt session changed across days in the CSR group, but not in the WRC group (Group Day, ; CSR group: Day, ; WRC group: Day, P = 0.67; Table S2). For the CSR group, the total number of trials was lower (by 17 29%) on SR2 5 than on B4 (P < 0.05 for each comparison). This decrease was due to an increase in omissions (Figure 3D), which resulted in more frequent 10-sec time-outs (thus limiting the number of trials available during the 30-min session). After CSR, the total number of trials was still below baseline levels (by 12%) on R1 (P < 0.05 versus B4), but returned to baseline levels on R2. Compared to the WRC group, the CSR group had fewer trials on SR2 5, R1, and R3 (all P < 0.05). Time on Task Analyses Human performance on a PVT task typically shows worsening over the course of a test session (i.e., time on task impairment), and sleep deprivation exacerbates this effect. 23,32 To examine whether performance changed over the course of each rpvt session in our rats, we analyzed performance of the CSR group in 5-min bins across the 30-min rpvt session on B4 and SR1 5. There were no statistically significant changes in any performance measures within sessions, and no significant interactions between session bins and days (P = ). Figure S3 (supplemental material) illustrates the stability of performance (percent omissions) within sessions over days of testing. Body Weight As shown in Figure 3G, the CSR group showed a gradual loss of body weight across SR1 5, followed by a gradual gain over recovery days. The single-day weight loss (relative to the previous day) was smallest on SR2 ( 1.1 ± 0.3%) and largest on SR3 ( 2.0 ± 0.3%). The weight loss relative to body weight on B3 reached 6.5 ± 0.7% by SR5, which recovered to 4.7 ± 1.1% loss by R4. Despite the greater number of wheel rotations they produced (as mentioned previously), the WRC rats lost a much smaller percentage of body weight over the same time period ( 1.6 ± 0.5% on R4). These results are consistent with previous CSR studies in rats. 26,33 36 Summary of the Results (Experiment 1) rpvt performance was not affected acutely by 4 h of the CSR protocol, but became greatly disrupted by 28 h, as evidenced by a longer latency for correct responses, fewer correct responses, and more lapses and omissions. Thereafter, however, a trend for partial recovery of performance occurred across the remainder of the 4-day CSR period, although overall performance remained worse than at baseline. Following CSR, recovery occurred within 1 day on most performance measures, and was complete within 2 days. Voluntary wheel running over the same time period did not affect performance on the task. The partial performance recovery after initial deterioration with the 100-h CSR protocol raised the question of whether this recovery would be maintained, or whether performance would decline, should CSR be extended. This question was investigated in the next experiment. Effects of 148 h of CSR on rpvt Performance (Experiment 2) In Experiment 2, CSR was extended to 148 h, and the recovery period to 5 days (CSRe group, n = 5). Figure 4A 4F, shows rpvt measures in the CSRe group on the last day of baseline (B4), CSR days (SR1 7), and recovery days (R1 5). Table S3 (supplemental material) shows the results of the ANOVA. Consistent with the results of Experiment 1, the overall performance of the CSRe group deteriorated on SR2, with longer average latencies of correct responses (+296 ± 122 ms; Figure 4A), fewer correct responses ( 21.8 ± 11.9%; Figure 4B), more lapses (+11.9 ± 5.9%; Figure 4C) and more omissions (+29.5 ± 14.6%; Figure 4D). The increase in average latencies was accompanied by an increase in both median latencies and 10 th percentile of latencies of correct responses (Figure S2C and S2D, respectively). In contrast to Experiment 1, however, all measures returned to baseline levels on SR4 5, except for omissions which remained above baseline levels at SR5 (P < 0.05 versus B4; Figure 4A 4D), indicating faster recovery overall than in Experiment 1. During the additional 48 h (SR6 and SR7) of the CSR protocol, overall performance remained similar to that observed on SR5, with no clear signs of further deterioration or improvement in any of the rpvt measures, except for omissions that recovered to baseline levels on SR6 and SR7 (Figure 4D). Thus, all measures were fairly close to baseline levels by the end of 148 h of the CSR protocol, and post-csr recovery was complete on R1 on all measures, which was similar to the recovery time course after 100 h of CSR (Figure 4A 4F). Body Weight Consistent with the results of Experiment 1, the CSRe group showed a gradual loss of body weight across 7 CSR days ( 7.0 ± 1.4% on SR5, and 8.3 ± 1.3% on SR7) followed by a gradual gain over 5 recovery days (to 4.5 ± 1.4%; Figure 4G). The single-day weight loss (relative to the previous day) was smallest on SR2 ( 1.5 ± 0.3%) and largest on SR3 ( 2.7 ± 0.6%). The values on SR5 and R4 were not different from those in the CSR group in Experiment 1 at the same time points (t 13 < 1, not significant for both comparisons). Individual Differences rpvt performance During CSR When the performance measures were compared between the two experiments, initial impairment after 28 h of CSR was SLEEP, Vol. 38, No. 4, Vigilance Performance and Chronic Sleep Loss Deurveilher et al.

8 Figure 4 Changes in performance on the rat psychomotor vigilance task (A E), total number of trials (F), and body weight (G) in Experiment 2, during 148 h of the 3/1 chronic sleep restriction (CSR) paradigm. Daily means (± standard error of the mean) of different measures are shown for the last day of baseline (B4), CSR days (SR1 7), and recovery days (R1 5). Performance measures include: changes in average latency of correct responses (A), percent correct responses (B), percent lapses (C), percent omissions (D), and percent premature responses (E). Values represent differences from the value on B3 for each day and for each measure (see Figure 3 legend). (F) Total number of trials per rpvt session. (G) Body weights are shown as changes in body weight expressed as percentages of the body weight on B3. The dotted lines indicate the end of the first 100 h of CSR. CSRe group, n = 5. * Different from B4; P < 0.05 (Bonferroni-corrected post hoc comparisons). SLEEP, Vol. 38, No. 4, Vigilance Performance and Chronic Sleep Loss Deurveilher et al.

9 observed in both studies; thereafter, however, performance recovered partially in Experiment 1, but nearly completely in Experiment 2. We noted that the rats in both experiments showed substantial individual differences in their performance impairment, especially on the second day of CSR. To determine whether these individual differences could account for the difference in the degree of performance recovery between the two experiments, we classified our rats as Vulnerable or Resilient based on the change from baseline in their performance at SR2, as described in the next paragraphs. For each rat, we calculated the difference in performance on SR2 from that on B3, and obtained a group distribution of change values (Figure S4, supplemental material). We then used the mean performance change for all rats from B3 to B4 to establish a criterion for the degree of change that would indicate vulnerability to the CSR procedure. This criterion value for each performance measure was set at 6 standard deviations (SD) greater than the mean change in that measure from baseline day B3 to B4. Rats that showed > 6 SD change in two or more of the five performance measures at SR2 were classified as Vulnerable (n = 5 [33%], including 3 from Experiment 1 and 2 from Experiment 2). Those showing > 6 SD changes in 0 or 1 measure were classified as Resilient (n = 10 [67%], including 7 from Experiment 1 and 3 from Experiment 2; Figure S4). We pooled data from rats in both experiments to compare Resilient and Vulnerable animals. This was justified for the following reasons. First, the proportion of Resilient and Vulnerable rats was fairly similar between Experiment 1 (70% Resilient, 30% Vulnerable) and Experiment 2 (60% Resilient, 40% Vulnerable). Second, the assignment of animals to the Resilient or Vulnerable group was similar regardless of whether the threshold for vulnerability was calculated separately for each experiment, or for the two experiments combined. During rpvt training prior to the baseline period for CSR, rats in the Resilient and Vulnerable groups learned at the same rate, requiring similar numbers of sessions to reach the learning criteria (32 ± 4 sessions for Resilient and 32 ± 6 sessions for Vulnerable rats; t 13 < 1, not significant). The patterns of performance on the rpvt measures at baseline (B4) and during 100 h of CSR (SR1 5) in the Resilient and Vulnerable groups are shown in Figure 5A 5E, and Figure S5 (supplemental material). Table S4 (supplemental material) lists the results of the ANOVAs. At baseline (B4), there were no significant differences between the two groups for any of the five rpvt measures (P = ; Table S1). The Resilient group showed only small impairments in rpvt performance during CSR, and not all performance measures were affected. Specifically, in comparison with baseline levels, the Resilient animals had fewer correct responses on SR3 5, with a small peak on SR4 (Figure 5B). Average latency of correct responses was not affected (Figure 5A), although the 10 th percentile of latencies of correct responses was transiently increased on SR2 (Figure S5B). In addition, the Resilient animals had more omissions on SR2 4 compared to baseline levels (Figure 5D), with an apparent peak on SR4. The other measures, including lapses (Figure 5C) and premature responses (Figure 5E), were not affected. The Vulnerable rats were impaired compared to baseline levels on SR2 on all the performance measures except premature responses (Figure 5A 5E). They also performed poorly compared to the Resilient rats on all five performance measures on SR2, because performance on that day was the basis for grouping (Figure 5A 5E). In addition, in the Vulnerable rats, despite some recovery, correct responses and omissions remained impaired, and average latencies of correct responses and lapses tended to be impaired, at the end of 100 h of CSR (SR5) compared to baseline levels. Thus, the Vulnerable rats performed poorly relative to the Resilient rats on SR3 (Vulnerable > or < Resilient, P < 0.05 for average latency, correct responses, lapses, omissions: Figure 5A 5D). On SR4 and SR5, although the performance in the Vulnerable group tended to be impaired on many measures, the only group differences that remained statistically significant were omissions (Figure 5D) and 10 th percentile of correct response latencies (Figure S5B). Not surprisingly, based on the aforementioned results, rats that were the most affected showed the greatest recovery after SR2. For correct response latencies, lapses, and omissions, there were significant correlations, with r values > 0.9 (P = ), between the degree of impairment in Vulnerable rats on SR2 (i.e., changes from B3 to SR2) and the extent of recovery by SR5 (changes from SR2 to SR5). There were no such correlations for the Resilient rats. Body Weight The Resilient and Vulnerable groups did not differ in body weight on B4 (438 ± 19 g and 453 ± 20 g, respectively; t 13 < 1, not significant). However, compared to the Resilient group, the Vulnerable group had lost more body weight on SR3 5 (up to 9.1 ± 1.1% and 5.5 ± 0.5% on SR5 in the Vulnerable and Resilient group, respectively; Figure 5G). DISCUSSION In the current study, we assessed the effect of 100 or 148 h of the 3/1 CSR protocol 26 on vigilance performance in rats as measured by the rpvt. Performance deteriorated by 28 h after starting the CSR protocol. However, after h of CSR, performance recovered partially or nearly completely despite accumulating sleep loss. After either 100 or 148 h of CSR, all performance measures were at baseline levels on the first or second day of recovery. Post hoc analyses identified considerable differences among individuals in the magnitude and time course of performance changes in response to CSR. Initial (28 h) Deterioration of rpvt Performance rpvt performance was not affected after the first 4 h of the 3/1 CSR protocol, but was severely impaired following 28 h of the protocol, with longer latencies of correct responses, more lapses and omissions, and fewer correct responses. These performance deficits are overall more severe than those reported after 24 h of total sleep deprivation by Christie et al. 21 Specifically, the average latency in the study by Christie et al. changed from 620 ms to 706 msec, 21 whereas it increased from 610 msec to 822 msec in our Experiment 1 and from 464 msec to 760 msec in Experiment 2. Similarly, lapses (including omissions) increased from 5% to 9% of total trials in the Christie et al. study, 21 but from 10% to 44% of total trials in our Experiment 1 and from 8% to 41% in Experiment 2. In addition, correct and SLEEP, Vol. 38, No. 4, Vigilance Performance and Chronic Sleep Loss Deurveilher et al.

10 Figure 5 Changes in performance in the rat psychomotor vigilance task (rpvt) (A E), total number of trials (F), and body weight (G) in the Resilient group (n = 10, closed squares) and Vulnerable group (n = 5, open squares) from combined Experiments 1 and 2, during 100 h of chronic sleep restriction (CSR). Changes in latency of correct responses (A) and in percentages of correct responses (B), lapses (C), omissions (D), and premature responses (E) are shown for the last day of baseline (B4) and each day of CSR (SR1 5). (F) Total number of trials per rpvt session. Values represent differences from those on B3 for each day and for each measure (see Figure 3 legend). (G) Body weights are shown as changes in body weight expressed as percentages of the body weight on B3. All values represent means (± standard error of the mean). See the Results and Figure S4 for the criteria used to divide animals into the Vulnerable and Resilient groups. *Different from B4; # different from Resilient; P < 0.05 (Bonferroni post hoc comparisons). SLEEP, Vol. 38, No. 4, Vigilance Performance and Chronic Sleep Loss Deurveilher et al.

11 premature responses decreased after 28 h of CSR (current study), but no changes were reported after 24 h of sleep deprivation. 20 The time of day at which testing occurred did not contribute to these differences, because it was similar in both studies. Instead these differences may be related to differences in sleep restriction/deprivation protocols (3 h on/1 h off [current study] versus continuous 3 sec on/12 sec off 21 ); the strain of rats studied (Wistar [current study] versus Fisher-Norway 21 ); behavioral response required for the task (bar-press [current study] versus nose poke 21 ); and/or the performance criteria ( 70% correct responses and 30 omissions per session [current study] versus > 100 correct responses and < 20 omissions per session 21 ). The increases in the latency of correct responses and in the percentages of both lapses and omissions in the current study may be attributable to a failure to sustain attention to task-related stimuli as a result of sleep loss, but alternative interpretations should be considered. One possibility is that sleep loss affected perceptual or motor skills. Although these were not assessed independently, rats behavior in the operant chambers during trials ending with an omission indicated no obvious sensorimotor deficits, as rats groomed, sniffed, reared, and moved with no difficulty. The 10 th percentile (fastest) latencies of correct responses, however, became slower on SR2, suggesting that the performance deficit on SR2 could have been affected by a motor deficit. Alternatively, sleep restriction may initially have caused a severe disruption of attentional processes, so that even the fastest responses were relatively slowed. Another possibility is that sleep loss may have affected response selection, such that rats may have failed to select the conditioned lever response among other potential unconditioned responses (e.g., grooming). This could be interpreted as an increase in distractibility, which would be consistent with a failure of sustained attention. 37 Finally, it is unlikely that the experience of repeated wheel rotations during CSR contributed to the performance deficits. The WRC rats showed higher numbers of wheel rotations but showed no changes in rpvt performance. Similarly, Christie et al. 21 reported no changes in rpvt performance in an exercise group that experienced forced wheel rotation numbers matched to those of the sleep deprived group, but otherwise were allowed to sleep ad libitum. Thus, although voluntary versus forced exercise may differentially affect other behaviors (e.g., anxiety-related behaviors), 38 the experience of wheel rotations in this study is unlikely to have affected task performance. In summary, impairment in sustained attention likely contributes to the rpvt performance deficits of chronically sleep restricted rats, although nonattentional factors cannot be ruled out. Future studies in which attentional load is manipulated may help clarify the contribution of changes in sustained attention to the emergence of performance deficits in the rpvt. Recovery of rpvt Performance During CSR: Allostasis? When CSR continued beyond 28 h, overall rpvt performance did not deteriorate further, but recovery was observed in both Experiments 1 and 2, as reflected in restoration of all performance measures to baseline or near-baseline values. A number of possibilities may account for recovery. One possibility is allostatic adaptation. 39 Our previous study using electroencephalogram (EEG) and electromyogram (EMG) recordings with the same CSR protocol reported several allostatic changes in sleep regulation in response to CSR. 26 Notably, during intermittent sleep opportunities, EEG delta activity in non-rapid eye movement (NREM) sleep (a measure of sleep intensity) increased robustly after 1 day of the CSR protocol, and then gradually declined during the following 3 days of sleep restriction, while still remaining above baseline levels. This pattern is paralleled by the pattern of rpvt performance change observed in the current study using the same CSR protocol. This allostatic adaptation in rpvt performance and NREM EEG delta power may be linked to changes in adenosine tone during CSR. In mice undergoing 3 days of CSR, the gradual attenuation of homeostatic increases in NREM EEG slow wave (delta) activity was accompanied by a gradual reduction in adenosine tone. 40 Given the inhibitory effect of adenosine on rpvt performance, 22 reduction in adenosine tone might have contributed to the recovery of performance that we observed during CSR. Brain-derived neurotrophic factor may also play a role in initiating allostatic responses. 41 Another possible explanation for the recovery of rpvt performance during CSR is that rats undergoing sleep deprivation could have obtained increasing numbers of brief behavioral sleep episodes (microsleeps), as well as local cortical sleep or inactivation In our previous study using EEG and EMG recordings with the same CSR protocol, 26 however, brief NREM sleep episodes occurring during the sleep deprivation periods amounted to an average daily total of less than 25 min, and this amount increased only slightly (+10 min) from the first to second half of the 4-day CSR period. 26 These findings suggest that microsleeps are unlikely to be a significant contributor to recovery in performance that we observed during CSR; however, the contribution of a gradual increase in transient local cortical sleep episodes cannot be ruled out. A third possibility is that performance improvement resulting from practice across the CSR days could account for the apparent adaptation observed in the CSR rats. However, the control (WRC) rats, trained to the same criterion, showed no evidence of further improvement over the same time period. There was also no evidence for changes in performance strategies (e.g., higher rates of bar presses in order to obtain more correctly timed responses) in the CSR group. Another possible explanation might be increases in motivation for water reward over the course of CSR. However, this also seems unlikely, as the CSR animals decreased their overall rate of bar pressing across days of sleep restriction. In addition, a previous study reported that 5 days of total sleep deprivation (using wheel rotations on a 3 sec on/12 sec off schedule) did not affect motivation for water reward. 28 It should also be noted that the CSR animals in the current study showed a steady level of performance (i.e., no time on task effects) over the 30-min period of rpvt testing on each day, suggesting that rats maintained a similar level of motivation for water reward during testing. Performance recovery after 100 h of CSR was more complete among rats in Experiment 2 than in Experiment 1. In both studies, however, relative to the initial impairment on the second and/or third day of CSR, performance recovered fairly well by the fourth day and beyond, despite continued SLEEP, Vol. 38, No. 4, Vigilance Performance and Chronic Sleep Loss Deurveilher et al.