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COMPLEX FUNCTION OF THE CENTRAL NERVOUS SYSTEM, SLEEP AND LOCOMOTION
1Department of Psychology, Simon Fraser University, Burnaby; and 2Department of Anatomy and Cell Biology, University of British Columbia, Vancouver British Columbia, Canada V5A 1S6
Submitted 25 April 2003 ; accepted in final form 18 June 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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circadian rhythms; nonphotic zeitgeber; wheel running; sleep deprivation; immobilization stress; psychosocial stress; cortisol; electroencephalogram
40 min) phase advance shifts of their free running circadian
activity rhythms (31). Small
advance shifts could also be induced by nonsocial disturbances, such as
changing the animals' cage or litter
(31), whereas the same
stimulation in the latter half of the active period (the "subjective
night") could induce small phase delays. Much larger phase advance
shifts, in the range of 2-4 h, could be induced by confining hamsters to a
novel wheel for 3 h (36).
Animals that ran continuously, accumulating 5,000 or more wheel revolutions,
with rare exceptions shifted, whereas those that did not run, with rare
exceptions, did not shift. Phase shifts induced by other stimuli, including
triazolam injections and exposure to a 6-h dark interval in otherwise constant
light, were soon shown also to depend on the expression of wheel running
activity; shifts were blocked by physical restraint
(41,
43) and strongly attenuated by
confinement to a small nest box
(34,
35). These and other studies
demonstrated that activity or arousal during the usual sleep period can be a
potent nonphotic zeitgeber (entraining stimulus), capable of shifting or
entraining free running rhythms or altering the phase of entrainment to a
light-dark (LD) cycle (for additional references, see
29,
33). Although intense or continuous locomotor activity was implicated as a critical factor for clock resetting in response to arousal procedures, the results did not rule out the possibility that hamsters need only be kept awake for large phase shifts to occur. A role for arousal independent of continuous running was clearly established using the sleep-deprivation procedure of gentle handling (i.e., occasional light air puffs or touch when animals assumed a sleep posture). This procedure fully mimicked the large phase-advance shifting effects of continuous novelty-induced wheel running, despite minimal locomotion and intervention (3, 27). In a subset of hamsters in one study, no relation was detected between phase shifting and general cage activity (3). In aggregate, these results indicate that arousal, or sleep loss, is sufficient to reset the circadian pacemaker in Syrian hamsters and that "exercise," i.e., intense or continuous running, is not necessary.
A remaining puzzle in clarifying the behavioral state correlates of nonphotic clock resetting are reports that physical restraint in the middle of the usual rest period does not induce phase shifts in hamsters. Restraint is a well known stress stimulus and would thus be expected to arouse sleeping hamsters. If arousal without exercise is sufficient to induce phase shifts, then restraint should also be an effective zeitgeber. Yet, in the three available studies, restraint during the rest period induced no shifts and prevented shifts to triazolam injections or dark pulses (39, 41, 43). Three explanatory hypotheses will be considered. 1) The restraint procedure used in these hamster studies may not have produced a sustained arousal. 2) The stress of restraint may inhibit nonphotic phase shifting. 3) Some minimal level of locomotion may be necessary for arousal-induced phase shifts. There is no means by which to refute the first hypothesis, as behavioral or physiological measures of arousal or stress were not reported in the hamster restraint studies (39, 41, 43). There is no direct evidence to refute the second hypothesis; however, evidence consistent with the hypothesis includes observations that injections of cortisol or the glucocorticoid agonist dexamethasone do not entrain or phase shift free running rhythms in Syrian hamsters (2, 16) and that acute stressors do not induce phase shifts in another species, the rat (23, 24; but note that large phase shifts in response to activity or arousal have not yet been demonstrated in rats). Finally, there is no evidence to refute the third hypothesis. Whereas the sleep-deprivation procedure of gentle handling minimizes locomotion, the hamsters in these studies were not prevented from moving (3, 27). The reported lack of correlation between general cage activity levels and phase shifts to this procedure does not rule out the possibility that some minimal level of locomotion is necessary for clock resetting.
We report here the results of several experiments designed to assess the contributions of stress and locomotion to nonphotic phase shifting in Syrian hamsters. We first examined whether phase shifts to sleep deprivation and restraint could be enhanced by pharmacological blockade of cortisol synthesis. We then examined whether the conventional restraint procedure sustains arousal and whether increasing the stress load during restraint might induce phase shifts. We measured cortisol (Cort) to determine if sleep deprivation by gentle handling, confinement to a novel running wheel, and stress-loaded restraint are in fact differentially stressful. Finally, we compared the phase-shifting effects of three additional stress procedures in which locomotion was either prevented [confinement to a pedestal over water (37)] or permitted [resident-intruder interaction and confinement to a novel open field (17)]. The results support hypothesis 1 (the conventional restraint procedure does not sustain arousal), refute hypothesis 2 (stress does not preclude the induction of large "nonphotic" phase shifts), and are consistent with hypothesis 3 (some minimal level of forward locomotion is necessary for large phase shifts).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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30 lx:0 lx). Food and water were available
ad libitum. Wheel running activity was detected by microswitches monitored
continuously by computer. Activity data were summed and stored to disk at
10-min intervals and periodically transferred to a Macintosh computer for
analysis. Phase shift measurements. In all but one experiment, the Aschoff type II method was used for measuring phase shifts (see Ref. 32). Hamsters were maintained in LD until the day of the experimental procedure. Lights were turned off when the procedure was initiated and were kept off for 3-5 days. A computer algorithm was used to identify the onset of the main period of daily wheel running, designated circadian time (CT12), by convention (each complete circadian cycle is comprised of 24 equal "circadian hours"). The average time of activity onset for 4-5 days before the day of the experimental manipulation was compared with the time of activity onset on day 2 of constant dark (DD) after the manipulation. A within-subject design was used in most experiments, so that phase shifts to experimental procedures were expressed relative to phase shifts to the appropriate control procedures (typically lights off at the same time, with no additional manipulation; see Fig. 1, A and B). After DD, the LD cycle was reinstated for at least 5 days before additional testing.
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Phase shifts to sleep deprivation by gentle handling: effects of Cort
synthesis inhibitor. Hamsters (n = 9) were subjected to sleep
deprivation by gentle handling, as described previously
(3,
27,
38). Briefly, cage lights were
dimmed to 1 lx (red incandescent; DDred) at zeitgeber time 6 (ZT6, i.e.,
6 h before the usual time of lights off, designated ZT12 by convention). Cage
tops were removed and the hamsters were observed continuously for 3 h. Food
and water were available, but running wheels were locked. If the hamsters
attempted to adopt a sleep posture, they were stimulated by tapping on the
cage, a light air puff, or light touch. At the end of the procedure, the
wheels were unlocked and the lights were turned off for at least 3 days. All
hamsters received one 3-h sleep deprivation counterbalanced for order with one
control test (DD at ZT6, no behavioral intervention). These data were reported
previously (3) and are included
here to illustrate typical phase shift responses to sleep deprivation and
permit within-animal comparisons with the drug condition. To test whether
phase shifts induced by sleep deprivation are influenced by a cortisol
response to the procedure, the hamsters were subjected to two additional 3-h
sleep deprivations, preceded at ZT23 and ZT4.5 by injections of the
corticosteroid synthesis (11
-hydroxylase) inhibitor metyrapone (50
mg/kg, Sigma-Aldrich) or vehicle (40% polyethyleneglycol in saline). The
hamsters were reentrained to LD 14:10 for at least 5 days before each test.
Tests in four hamsters indicated that metyrapone alone did not induce phase
shifts.
Phase shift effects of restraint and stress-loaded restraint.
Hamsters (n = 12) entrained to LD 14:10 were subjected to a DD
control test and three physical restraint tests. Restraint was accomplished by
placing hamsters into polyvinylchloride tubes (15-cm long, 4-cm internal
diameter) inside a wire cage (0.5-cm2 mesh) that blocked both ends
but allowed air and waste to pass. The animals could move horizontally 6
cm, but could not turn around. Hamsters were restrained from ZT6 to ZT9 in
DDred, after which they were returned to their home cages and left undisturbed
in DD for 3 days. Hamsters reentrained to LD for at least 5 days between
tests.
During the first restraint test, behavioral state was scored every minute as active wake (eyes open, overt attempts to move), quiet wake (eyes open, motionless), and behavioral sleep (eyes closed and motionless). Behavioral observations were made using an infrared viewer.
During the second and third restraint tests, counterbalanced for order, the
hamsters were stimulated with compressed air to interrupt behavioral sleep.
During one test, a light puff of air was delivered to the face at 1-min
intervals using an aerosol dusting can (30 psi; Memorex Duster). During the
other test, a high-intensity air "blast" was applied at
interstimulus intervals of 30 s using compressed air tanks providing 110
psi through 3/8-in. tubing. Sound pressure level of the compressed air was
measured at 107 dB (Quest Technologies, model 2900 dB meter). This procedure
is designated "stress-loaded" restraint. Due to the near
continuous intervention, no attempt was made to score behavioral state.
A second group of hamsters (n = 11) was subjected to the 3-h
restraint procedure combined with continuous vestibular stimulation, produced
by horizontal rotation on a cart at 120°/s. Behavioral state was
noted but not recorded as the procedure appeared to produce sustained quiet or
active wake.
Cort responses to sleep-deprivation procedures. To determine whether the sleep-deprivation procedures of gentle handling, stress-loaded restraint, and confinement to a novel running wheel (a standard procedure for inducing large phase shifts) are differentially stressful, the effect of these procedures on Cort release was measured. Naive hamsters (n = 96) were housed in running wheel cages and entrained to LD 14:10 for at least 10 days. On the test day, lighting was reduced to DDred at ZT6, and the hamsters were subjected to either the control procedure (undisturbed in home cage), sleep deprivation by gentle handling, stress-loaded restraint, or confinement to a 33-cm diameter novel running wheel. Twenty-four hamsters were assigned to each group, and eight hamsters from each group were killed at ZT6.5, ZT7.5, and ZT9 (i.e., at 30, 90, and 180 min after the onset of the procedures). For blood collection, the animals were transferred singly to another room and decapitated (the order of death was counterbalanced across groups). Blood was collected into chilled Eppendorf tubes containing EDTA, centrifuged at 3,600 rpm for 10 min, and stored at -70°C. Total Cort (bound plus free) was measured by radioimmunoassay in plasma extracted in 95% ethanol. Tracer [3H]Cort (cat# 07-121026), unlabeled Cort for standards (cat# 07-121040), and anti-Cort (cat# 07-121015) were obtained from ICN (Costa Mesa, CA). Dextran-coated charcoal was used to absorb and precipitate free steroids after incubation. Samples were counted in Scintisafe Econo2 (Fisher Scientific, Nepean, ON, Canada). Intra- and interassay coefficients of variation were <4.3% and 5.6%, respectively.
Phase shift effects of sleep deprivation by pedestal over water. Hamsters (n = 12) were housed in wheel running cages and entrained to LD 14:10 for at least 10 days. Each hamster received three 6-h sessions on a 4.3-cm diameter pedestal set 1.5 cm above 13-cm water (23°C) in a Plexiglas cage (20 x 30 x 55 cm). The cage was enclosed by a standard stainless steel lid providing food and water ad libitum. Plastic barriers were placed in the water around the pedestal, so that if hamsters chose to swim, they could not reach the sides of the cage. These preliminary tests established that the procedure prevented sleep and that the hamsters could stay out of the water. After a few swims in the preliminary tests, all hamsters did stay out of the water. The hamsters then received, at 7-day intervals, a control test (DD for 3 days beginning at ZT6) counterbalanced for order with a 3-h (ZT6-9) sleep deprivation on the pedestal.
EEG recordings were conducted on four additional hamsters to confirm that the pedestal technique prevents sleep. Electrodes (n = 4 stainless steel jeweler screws for differential recording of midline EEG and lateral EEG and n = 2 subcutaneous wire electrodes for recording EMG from the nuchal muscle) were implanted using standard stereotaxic procedures and pentobarbital sodium (50 mg/kg) anesthesia. The pins from all six electrodes were connected to a plastic headcap bonded to the skull with dental acrylic.
After 5-7 days of recovery from surgery, the hamsters were habituated to the recording cable for 3-7 days in an electrically shielded recording chamber. The cable was left connected for 24 h before the baseline recording day. On the day after baseline, the hamsters were placed on the pedestal over water from ZT6 to ZT9, with lights lowered to DDred. EEG and electromyogram (EMG) signals were amplified (Grass model 79D, Grass Instruments), digitized (sampling rate of 250 Hz), and stored on computer using AcqKnowledge software (Biopac, Goleta, CA). Behavioral states were determined in 10-s epochs on the basis of four electrographic measures: unfiltered lateral EEG, lateral EEG filtered for delta waves (1-3 Hz), midline EEG filtered for bandwidths including theta (5-15 Hz), and EMG (high-pass filtered at 50 Hz). Each epoch was classified as whichever state was predominant, according to criteria validated previously (Ref. 4, see also 38). Wakefulness (W) was identified by low-amplitude lateral EEG, absence of delta waves, only occasional regularity in midline theta EEG, and high-amplitude EMG. This electrophysiological state corresponded to behavioral alertness with eyes open. Slow-wave sleep (SWS) was identified by high-amplitude lateral EEG, presence of delta waves, spikiness in midline theta EEG, and low-amplitude EMG. Rapid eye movement sleep (REMS) was identified by low-amplitude lateral EEG, absence of delta waves, sinusoidal midline theta rhythms, and muscle atonia. Also recognized were transitional sleep (TS; high-amplitude lateral EEG, absence of delta waves, spikiness in midline theta EEG, and low-amplitude EMG) and low-amplitude sleep (LS; low-amplitude cortical EEG, absence of delta waves, spikiness in midline theta EEG, and low-amplitude EMG).
Phase shift effects of social stress: resident-intruder paradigm. Hamsters (n = 12) were housed in running wheel cages and entrained to LD 14:10. The six lightest hamsters (123 ± 19 g) were designated as "intruders", and the six heaviest (145 ± 5 g) as "residents." At ZT6 on the test day, a 3-h resident-intruder test was conducted in DDred. Each intruder was rotated between the cages of two resident hamsters at 15-min intervals, after an initial 30-min interval. Thus each intruder interacted with two different residents and each resident with two different intruders. Sessions were videotaped using an infrared camcorder to score behavior. Running wheels were locked during the first test and unlocked during the second test. If any animal was observed to adopt a sleep posture, a light air or touch stimulus was applied. Phase shifts were quantified relative to a control DD test.
Videotapes were scored in 1-min intervals by trained observers. Behavioral variables quantified included fight, chase, flee, walk, defensive subordinate posture, offensive attack posture, nesting, number of minutes during which each subject spent some time wheel running (wheel unlocked condition), and number of experimenter interventions to prevent a sleep posture. These variables were used to determine the dominance status of the hamsters, and activity and intervention measures were correlated with the size of phase shifts.
Phase shift effects of open field stress. Hamsters (n = 12) used in the resident-intruder test were reentrained to LD for 3 wk and then maintained in DDred. During the third week in DDred, each subject was transferred at CT6 to a 1-m square open field apparatus for 3 h under the same dim lighting. The open field was made of 60-cm-high white plastic walls and floor. A 250-ml, 6-cm diameter glass beaker, with horizontal stripes of white tape, was placed in the center of the floor. An overhead infrared camera coupled to a microcomputer by an image analyzer (Chromotrack, San Diego Instruments, San Diego, CA) was used to track movements. Data were sampled every 0.055 s. The minimum displacement necessary for detection of movement was set at 4 cm. For analysis purposes, the open field was divided on the computer tracker file into an outer ring (0-15 cm from the wall), a middle ring (15-30 cm from the wall), an inner ring (30-50 cm from the wall), an object zone (15 cm in diameter centered around the glass beaker), and four corner areas (each 15 x 15 cm). Before each trial, the open field and glass beaker in it were cleaned with a solution of 70% alcohol. The trial began with the subject being placed in the open field facing the wall. The tracker recording began simultaneously with the release of the animal via a remote start switch. The trial duration was 180 min, with data summaries automatically taken each minute. Light taps on the floor or walls of the open field were used to prevent sleep postures as necessary. At the end of the trial the subject was promptly removed and returned to its home running wheel cage.
Phase shifts were measured by fitting separate regression lines to activity onsets for 10 days preceding and after the open field test. The following measures were recorded: the time spent in each of the five regions of the open field (the 3 rings, the object area, and the corner areas); the number of entries into each of the same five regions; the total distance traveled; and the number of crossings of 20 x 20-cm imaginary divisions of the open field.
Data analysis. The phase shift, cortisol, and behavioral measures were evaluated by ANOVA with Student-Newman-Keuls post hoc analyses or paired t-tests with Bonferroni corrections. Pearson product-moment correlation coefficients were calculated to examine relationships between phase shifts and behavioral variables. Means in the text are reported as ±SE.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Restraint procedures are differentially arousing but do not induce
phase shifts. All hamsters restrained for 3 h from ZT6 to ZT9, by the
conventional method (i.e., without air stimulation or other interventions),
exhibited behavioral sleep within the first hour. Mean sleep latency was 39
± 8 min and mean sleep accumulation was 120 ± 8 min. Quiet wake
occupied 9 ± 2 min and active wake 52 ± 28 min. Restraint
combined with low-intensity air stimulation appeared to reduce behavioral
sleep, whereas high-intensity compressed air applied at 30-s intervals
prevented behavioral signs of sleep.
Despite differential effects on behavioral state, phase shifts to these procedures were uniformly small (e.g., Fig. 1D), and treatment means were 20 min or less. Although there was a significant main effect of treatment (F2,22 = 5.43, P < 0.05), only restraint combined with low-intensity air stimulation differed significantly from the control condition (P < 0.05), and the magnitude of the difference was only 9 min. There were no differences among the three restraint procedures (Fig. 2). In the conventional restraint condition (no air stimulation) there was no relationship between the size of the phase shift and the amount of time in either wake (r = 0.09, P > 0.1) or behavioral sleep (r = - 0.16, P > 0.1).
Hamsters subjected to vestibular stimulation by horizontal rotation during restraint exhibited continuous behavioral arousal (eyes open), but no significant phase shift compared with the control procedure (26 ± 10 vs. 19 ± 5 min, in the restraint and control procedures, respectively; P > 0.1).
Sleep-deprivation procedures are differentially stressful: Cort measurements. There was a significant main effect of circadian time (F2,81 = 6.96, P = 0.002) and treatment (F3,81 = 24.62, P < 0.001, Fig. 3) on plasma Cort. Pairwise comparisons revealed that at the 30-min sampling point, Cort levels did not differ across procedures. At the 90-min sampling point, only the restraint procedure differed significantly from the undisturbed control condition. At the 180-min sampling point, both gentle handling and restraint were associated with significant elevations of Cort. However, in the gentle handling group, only three of eight hamsters exhibited higher Cort levels at 180 min. Hamsters confined to a running wheel for 3-h did not exhibit significant Cort elevations at any time point by comparison with the control group.
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Sleep deprivation by the pedestal over water method does not induce large phase shifts. During baseline EEG recording sessions from ZT6 to ZT9, hamsters averaged 24.8 ± 0.8% W, 42 ± 1.7% SWS, 17.4 ± 0.3% REMS, 3.6 ± 0.6% LS, and 2.2 ± 0.4% TS. During the 3-h session on the pedestal over water, the hamsters exhibited continuous waking, i.e., eyes open with low-amplitude desynchronized lateral and midline EEG and continuous high-amplitude EMG, reflecting a tonically high muscle tone required to remain upright on the pedestal (Fig. 4).
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Hamsters used for phase shift studies also exhibited continuous behavioral alertness with eyes open while on the pedestal. The hamsters did not fall into the water. A small but significant group mean phase advance shift was observed (59 ± 11 min, compared with 28 ± 4 min in the control condition; paired t = 3.63, P < 0.05; Figs. 1E and 2). However, only one shift exceeded 47 min by comparison with the control procedure, and 9 of 12 shifts were less than 30 min.
Resident-intruder interactions induce large phase shifts related to measures of activity. Dominance status in the hamster dyads was consistent with the relative size of the protagonists and the site of the encounter. Of the 24 total tests, involving 12 hamsters tested twice each, once with the wheel locked and once with the wheel open, the larger resident hamster was dominant in 21 cases, exhibiting chasing, offensive attack postures and no fleeing. One resident hamster exhibited neither chasing nor fleeing on either trial, another exhibited fleeing and no chasing on one trial, and one intruder hamster exhibited both chasing and fleeing on one trial. The remaining intruder hamsters exhibited only fleeing and defensive/submissive postures.
There was no relation between phase shifts and resident status. Compared with the DD control condition, residents shifted 62 ± 18 and 98 ± 34 min on the first and second test, respectively, whereas intruders shifted 72 ± 28 and 82 ± 43 min, respectively (all significantly different from control, P < 0.05; Fig. 2). Phase advance shifts >140 min occurred in one resident and two intruders in the first test, and in two residents and three intruders in the second test, for a total of eight large shifts in 24 total tests, involving three of six residents and four of six intruders (e.g., Fig. 1, F and G). Resorting the groups to account for the social status outliers produced virtually the same numerical outcomes. Phase shifts on the two tests were positively correlated across hamsters (r = 0.79, P < 0.005).
There was a strong positive relation between phase shifts and an aggregate measure of activity created by combining the number of episodes of chasing and fleeing (note that 10 of 12 hamsters exhibited either one or the other). This combined activity score correlated +0.59 (P < 0.05) with phase shift magnitude in test 1, and +0.48 in test 2 (P < .05). In test 2, the number of minutes during which running was observed (revolutions not recorded) was not significantly correlated with phase shifts (r = 0.31, P > 0.1).
Phase shifts were inversely related to the number of interventions required to prevent sleep postures in both test 1 (r = - 0.61, P < 0.05) and test 2 (r = - 0.56, P < 0.05). More interventions were used on residents than on intruders (12.7 vs. 8.8 and 8.3 vs. 4.7 during tests 1 and 2, respectively), but this was only a statistical trend (P > 0.1). There was a substantial group difference in the number of minutes of wheel running observed, with intruders averaging 44 ± 11 min and residents only 9 ± 10 min (independent t = 2.58, P < 0.05).
Open field exposure induces large shifts related to measures of forward locomotion. Hamsters placed into the open field at CT6 remained close to the walls during the first minute, explored the inner ring of the field within the first 2 min, and investigated the center field object within the first 3 min. All hamsters remained spontaneously active during the first 30 min, but needed at least a few interventions to interrupt sleep postures after that. Phase shifts to this open field test ranged from -23 to 177 min and averaged 74 ± 21 min. Total activity in the open field was not signifi-cantly related to the phase shifts (r = - 0.28). However, the total activity score is an integrated measure of activity related to forward locomotion, grooming, object manipulation, scratching at the corners, and other significant movements. Therefore, activity in the corners and in the outer and inner rings of the open field was quantified separately, along with the number of area crossings. Both total time and activity level in the corners were inversely related to phase shifting (r = -0.79 and -0.77, respectively, P < 0.001). Notably, most attempts to sleep occurred in the corners. By contrast, total time and activity in the center of the open field were positively correlated with shifting (r = +0.76 and 0.71, respectively, P < 0.001). The total number of crossings between 20-cm-square areas within the open field (the most direct measure of forward locomotion) correlated 0.67 with phase shifts (P < 0.005).
To further explore the apparent relation between locomotion and shifting, the hamsters were divided into shifters (>80 min, n = 6, mean = 133 ± 16 min) and nonshifters (<15 min, n = 5, mean = 4 ± 5 min). Shifters exhibited significantly more time and activity in the inner ring and significantly less in the outer ring and the corners (P < 0.002 for each comparison). Shifters exhibited 2.85-fold more line crossings than non-shifters over the entire 3 h (P < 0.005), and 4.41-fold more during the last hour of the test (P < 0.001). Although the groups did not differ in the general activity score, the shifters showed progressively more time in the inner ring of the open field while the nonshifters showed progressively more time in the corners (the "sleep attempt" zone). The two groups did not differ in either measure during the first 30 min, suggesting no group differences in "anxiety" (operationalized by thigmotaxic behavior) during initial exposure to the open field.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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60 min) in hamsters
(12,
22). These shifts were
positively correlated with a cortisol response but not with acute induction of
wheel running. However, this procedure was not effective in the mid-rest
phase, and it is unknown whether the shifts were dependent on cortisol
release, locomotor activity other than running, or some other factor. A second substantive conclusion is that stress does not prevent large phase shifts to arousal in the mid-rest period. We were led to consider this novel hypothesis by the prior evidence that restraint, a presumably arousing procedure, does not produce shifts, and by our anecdotal observations that a few hamsters that appeared very stressed by the sleep deprivation procedure of gentle handling (based on defensive body postures and vocalizations) did not exhibit phase shifts (M. C. Antle, unpublished observations). Tantalizing support for this hypothesis was provided by our first experiment, showing that a cortisol synthesis inhibitor metyrapone, at doses that block the corticosterone response to restraint in rats (8), may potentiate phase shifts to sleep deprivation by gentle handling. However, our subsequent studies appear to refute the hypothesis; large shifts were evident in socially subordinate hamsters transferred between the cages of two dominant hamsters for 3 h (a strong stressor; 17) and in hamsters confined to a novel open field for 3 h (a milder stressor). Why metyrapone was effective in the first experiment remains unclear, but it is worth noting that the mean phase shift in that condition was not greater than that exhibited by other groups subjected to sleep deprivation without metyrapone (see 3, 27).
A third substantive conclusion is that some minimal level of locomotion is necessary to induce phase shifts by arousal in the mid-rest period. This conclusion may have already been assumed based on the results of prior studies showing that restraint or confinement to a small nest box can block or attenuate phase shifts to activity-inducing stimuli (e.g., triazolam, dark pulses) and that hamsters that fail to run in novel wheels with rare exceptions fail to shift. An obvious conclusion from those results is that activity is necessary for shifts to occur, but such a conclusion oversteps the data. Our behavioral observations revealed that restrained hamsters fall asleep, and we have previously noted that hamsters that fail to run when confined to a novel wheel also fail to stay awake. If hamsters are not kept awake, then the absence of phase shifts to restraint or confinement could be due to the absence of running or the lack of arousal. Thus these studies do not permit a conclusion that locomotor activity is necessary for phase shifting. Indeed, the results of our two previous sleep deprivation studies demonstrated that continuous running activity is not necessary for phase shifting and that very large shifts can be induced despite low levels of activity (3, 27). Nonetheless, hamsters in those studies were able to move about in their cages. The results of the experiments reported here constitute the clearest evidence so far that the expression of locomotor activity is indeed necessary for phase shifts to behavioral arousal. Restrained hamsters kept aroused for 3 h did not phase shift. Hamsters confined to a small platform stayed awake for 3 h but showed only small or no phase shifts relative to the control procedure. Some hamsters subjected to psychosocial stress showed very large phase shifts, and across hamsters, shift magnitude was related to a composite activity measure (chasing and fleeing) and unrelated to social status in the dyads. Some hamsters confined to a novel open field also exhibited very large phase shifts and, again, shifting was related to indexes of forward locomotion (e.g., number of area crossings). Activity does not need to be either continuous or intense, but some minimal level appears to be necessary.
This conclusion suggests an explanation for individual differences in phase shift responses to sleep deprivation by gentle handling procedures. Some hamsters fail to shift, and this has been related to the number of interventions required to prevent behavioral sleep (3). Nonshifters appear sleepier and make more attempts to sleep. We also noted that some hamsters that appeared very stressed required few interventions yet still failed to shift. In both types of hamsters, phase shifts may be small or absent because total locomotor activity over the 3 h is low, in the one case because of sleepiness and in the other because of prolonged freezing responses. In a pilot study, we failed to see phase shifting in response to continuous rewarding lateral hypothalamic stimulation applied to hamsters in the mid-rest period; these hamsters were awake but did not have to produce an operant response to obtain stimulation, and moved very little during the procedure (M. S. Pollock, M. C. Antle, and R. Mistlberger, unpublished observations). Thus even strongly rewarding stimuli may fail to induce phase shifts in the absence of locomotion or operant responses that require motor output.
These results also have implications for elucidating the neural pathways by which nonphotic stimuli induce phase shifts. There is convergent evidence that the thalamic intergeniculate leaflet (IGL) is a gateway through which arousing stimuli shift the master circadian pacemaker located in the suprachiasmatic nucleus (SCN). The IGL contains neuropeptide Y (NPY)-releasing neurons that project to the SCN (30) and that express FOS protein in hamsters that are aroused for 3 h in the mid-rest period, either by confinement to a novel running wheel (14, 26) or by gentle handling (3). Ablation of the IGL blocks circadian phase or period changes induced by wheel running or saline injections in hamsters, rats, or mice (15, 18, 20, 21, 44). Microinjections of NPY into the SCN area induce phase shifts that mimic those induced by arousal (1), and phase shifts induced by running are blocked by microinjections of NPY-antibody (5). However, several issues remain to be resolved. The aspects of behavioral arousal coded for by IGL efferents to the SCN are unknown. Our behavioral analyses suggest that this pathway may not be activated (e.g., as assessed by FOS induction) by arousal procedures associated with little or no movement and no phase shifts. If NPY efferents are activated by such procedures, this would suggest that NPY release is necessary but not sufficient for clock resetting to arousal, and that one or more other pathways must participate. In mice, serotonin efferents also appear to be necessary (10, 20), although the evidence for this in hamsters is equivocal at best (reviewed in Ref. 29). Also remaining to be identified are the pathways that transmit information about behavioral state to the IGL. There are several candidates from among the monoamine and neuropeptide systems that regulate behavioral state (25, 42). Again, our behavioral data suggest that critical pathways may be preferentially activated during arousal accompanied by locomotion. Whether such pathways encode motor output or somatosensory or proprioceptive feedback from movement is an open question. The lack of phase shifts in restrained hamsters subjected to rotational stimulation, at a rate known to disrupt spatial memory in rodents (e.g., Ref. 11), suggests that vestibular correlates of movement are not a sufficient stimulus.
Phase advance shifts induced in hamsters by the procedures of gentle handling or confinement to a novel wheel can be detected within 1 h of the end of the procedure, using the circadian rhythm of light-induced c-Fos expression in the SCN as a cellular level phase marker (3; see also 22). In the current study, confinement to a novel wheel was not associated with a significant elevation of Cort, and sleep deprivation by gentle handling was associated with elevated Cort only in three of eight hamsters sampled at ZT9. If by this time the circadian clock were already phase advanced 2-3 h by these procedures, as inferred from the FOS data, then a significant elevation of Cort might be expected by virtue of the new circadian phase (i.e., basal Cort exhibits a significant elevation as the pacemaker approaches the beginning of the subjective night). The absence of elevated Cort, particularly in the wheel confinement group, suggests a lag in resetting of the Cort rhythm, by comparison with the circadian rhythm of light-induced FOS that is intrinsic to SCN neurons.
Manipulations of behavioral state can have potent effects on the circadian system of Syrian hamsters. There are, however, significant species differences; whereas other rodents, including nocturnal rats and mice and the diurnal European ground squirrel, can also be entrained by a daily exercise schedule, these animals do not exhibit large or reliable phase shifts in response to arousal during the mid-subjective day (9, 13, 19, 28). The Syrian hamster may be an outlier species in the responsivity of its circadian clock to behavioral manipulations in the subjective day, but only a handful of species have been examined so far. Several studies of humans have produced evidence consistent with a phase shifting effect of exercise in the subjective night or late in the subjective day (6, 7, 40). The design of these studies was informed by the early evidence that continuous wheel running was highly predictive of phase shifting in hamsters confined for 3 h to a novel wheel. However, our sleep deprivation studies demonstrate that continuous running is not required to induce maximal phase advance shifts (3, 27). The results of the present study extend this analysis by showing that the expression of locomotor activity is necessary, although even low levels appear sufficient. Perhaps casual walking, standing, or other low-intensity activities may prove to be sufficient in humans as well.
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Present address of M. C. Antle: Department of Psychology, Columbia University, New York, NY.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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