Effects of Housing Condition and Cage Change on Characteristics of Sleep in Mice

Abstract

Although human subjects are widely used to study sleep and sleep disorders, animals have been invaluable in developing our understanding of the physiology of sleep and underlying mechanisms of sleep disorders. Environmental stimuli are likely to modify sleep in both animals and people, suggesting that environmental stability must be controlled carefully by both husbandry and research staff to allow collection of valid results with minimal numbers of animals. However, few studies have measured the effects of cage condition on sleep parameters in mice. Current guidelines recommend social housing and environmental enrichment for standard rodent housing. Environmental factors such as these create potential confounds in studies for which facets of sleep are outcome measures. We therefore sought to determine whether cage changes, group housing, or single housing with a shelter altered measures of sleep in C57BL/6J mice. The resulting data indicate that 1) cage changing disrupts sleep for approximately 3 h; 2) group housing is associated with shorter bouts of rapid-eye-movement sleep (REMS) and less slow-wave sleep (SWS) during the light phase and with more REMS during the dark phase; and 3) mice housed with a shelter spend less time awake and more time in SWS, with longer bouts of SWS during the dark phase. In additional, both group housing and housing with a shelter were associated with less locomotor activity than occurred in individually housed mice without a shelter. These findings provide evidence for long-held beliefs that housing conditions must be controlled carefully in studies that require assessment of sleep.

The study of sleep in animals has been invaluable in developing our understanding of sleep disorders and sleep physiology. Many external factors can influence sleep in animals and people.^{3,10,14,15,19,20,24,25,29} For example, although laboratory mice typically are considered to be a nocturnally active species, a recent study reported that mice living in a naturalistic environment are not explicitly nocturnal, exhibit feeding activity that is predominantly and sometimes exclusively diurnal, and show a negligible modulatory influence of specific genes on activity timing as compared with seasonal influences.⁵ Current recommendations for the management of research rodents advocate the use of social housing and environmental enrichment.^2,16 However, sleep studies in rodents generally are conducted in individually housed animals, particularly when animals are instrumented, such as with EEG recording electrodes. Recent data indicate that pair-housed and individually housed mice show some differences in their recuperative response to sleep loss.¹⁸ In addition, the frequency at which rodent cages are changed varies among institutions. Environmental differences of this type could modify patterns of sleep in rodents and potentially alter study outcomes. Therefore, environmental stability must be carefully controlled by both husbandry and research staff to allow collection of valid results with minimal numbers of animals. These issues apply to all research that uses animals but are particularly critical for studies of sleep.

In the current study, we sought to determine whether routine cage changes, social housing, and housing with a shelter altered measures of sleep in C57BL/6J mice. Our data indicate that 1) cage changing disrupts sleep for approximately 3 h but thereafter is not associated with significant effects on sleep; 2) group housing of mice is associated with shorter bouts of rapid-eye-movement sleep (REMS) and less slow-wave sleep (SWS) during the light phase and with more REMS during the dark phase compared with those of individually housed mice; and 3) mice housed with a shelter show trends toward a deeper plane of SWS and more state transitions during the light phase and spend significantly less time awake and more time in SWS, with longer bouts of SWS and a trend toward longer bouts of REMS, during the dark phase.

Materials and Methods

Mice and surgery.

Adult male C57BL6/J mice (n = 11; weight, 20 to 25 g; Jackson Laboratory, Bar Harbor, ME) were used in this study. Unless otherwise indicated, mice were housed individually in static microisolation caging (18 × 28 × 12 cm) with 1/8-in. corncob bedding (Bed O'Cobs, Anderson Mills, Maumee OH). Cages were maintained in temperature-controlled chambers (29 ± 1 °C; model no. 352601, Hotpack, Philadelphia, PA) on a 12:12-h light:dark cycle. All mice received rodent laboratory chow (Lab Diet 5001, PMI Nutrition International, Brentwood, MO) and drinking water ad libitum. Cage changes were performed once weekly at the same time as regularly scheduled health checks. Mice were transferred by gently picking them up by the base of the tail with gloved hands and placing them in a sterile cage with clean bedding and nesting pads. Health checks were performed daily at light onset. Sentinel testing was used to confirm that mice were free of infection with mouse hepatitis virus, mouse parvovirus, minute virus of mice, reovirus 3, pneumonia virus of mice, epizootic diarrhea of infant mice, Theiler murine encephalomyelitis virus, lymphocytic choriomeningitis virus, ectromelia virus, Sendai virus, Mycoplasma pulmonis, pinworms, and fur mites.

Telemeters were surgically implanted into all mice as previously described.^21,28 Anesthesia was induced with 4% isoflurane and maintained with1.5% to 2% isoflurane during surgery. Sterile technique was used to implant telemeters (model no. ETA-F20, Data Sciences International, St Paul, MN) into the peritoneal cavity. Transmitter biopotential leads were subcutaneously routed around the back, toward the head. Two stainless steel screws (#80 × 1/8 in.. Small Parts, Miami Lakes, FL) serving as EEG electrodes were implanted into the skull over the cranial left frontal cortex and caudal right parietal cortex. Dental acrylic (Isocryl, Lang Dental Supply, Wheeling, IL) was used to cement the screw electrodes in place and cover the exposed portion of the skull. After completion of surgery, mice received subcutaneous injections of an analgesic (0.05 mg/kg buprenorphine) and a broad-spectrum antibiotic (1,200,000 IU/kg penicillin). A topical analgesic (4% lidocaine) and a triple-antibiotic ointment (neomycin, polymixin B sulfate, and bacitracin zinc; E Fougera, Melville, NY) were applied to the surgical wound.

After surgery, mice were individually housed in environmentally controlled chambers and allowed at least 21 d for recovery from surgery. Ibuprofen (0.2 mg/mL) was included in the drinking water for 2 d after surgery.¹³ Chamber temperature was maintained at 29 ± 1 °C with a 12:12 h light:dark cycle. Mice were monitored daily, and attitude (bright, alert, responsive; quiet, alert, responsive; depressed), activity, signs of discomfort, body weight (in grams), hydration status, food and water intake, fecal output, and appearance of surgical incisions were noted.

All procedures involving the use of animals for this study were approved by the University of Washington Institutional Animal Care and Use Committee in accordance with all applicable federal regulatory policies.

Experiment 1.

After the postsurgical recovery period, mice (n = 5) were housed individually. On day 7 (that is, the day prior to cage change), EEG and body temperature were recorded beginning at light onset and continuing for 72 h. At 3 h after light onset on recording day 2, mice were placed into a sterile cage with clean bedding, and recording continued. Health checks were performed daily at 3 h after light onset.

Experiment 2.

After the postsurgical recovery period, test mice (n = 6) were housed with 2 unimplanted littermates to provide groups of 3 mice per cage. After a 7-d habituation period to this group-housed condition, EEG and body temperature were recorded for 96 h, beginning at light onset. The cage mates were then removed, and the test mice were housed individually in sterile cages with clean bedding. After a 7-d period of habituation, EEG and body temperature were recorded for 48 h. Twenty days later, a PVC pipe (diameter, 4 in.; length, 7 in.) was placed in the cage during cage change. The PVC pipe was painted black and provided a dark shelter. After 7 d of habituation to the presence of the pipe, EEG and body temperature again were recorded for 48 h. Health checks were performed daily at light onset.

Recording apparatus.

A receiver (RPC1, Data Sciences International) to detect signals from the transmitters was placed under each mouse's cage. Methods for signal preparation and determining sleep–wake behavior in mice have been previously discussed.^21,22 Briefly, signals from the transmitter were fed into an analog converter (model ART Analog-8 CM, Data Sciences International), which converted frequency signals into EEG and temperature voltages according to vendor-provided calibration coefficients specific for each transmitter. The output data from the transmitter then underwent analog-to-digital conversion (model PCI-3033E A/D Board, National Instruments, Austin, TX) with 16-bit precision at a sampling rate of 128 Hz. Motor activity was detected by using an infrared sensor (BioServ, Bonn, Germany) placed above the cages and was converted to voltage output, the magnitude of which was directly related to the magnitude of movement detected. During acquisition, EEG files were digitally filtered by using Chebyschev filters and third-order coefficients into δ (0.5 to 4.0 Hz) and θ (6.0 to 9.0 HZ) frequency bands and integrated over 1-s periods. Body temperature was recorded at a sampling rate of 1 Hz, and a single temperature reading was obtained for each 10-min interval. EEG, body temperature, and motor activity signals were stored as binary files until further processing.

Arousal states were identified according to previously determined parameters.²³ EEG signals were visually scored at 10-s epochs in conjunction with body movements to determine state-dependent changes. Custom software (ICELUS, M Opp, University of Michigan) written in LabView for Windows (National Instruments, TX) was used for sleep scoring. Raw, nonintegrated EEG signals underwent fast-Fourier transformation, which yielded power spectra between 0.5 and 40 Hz in 0.5-Hz frequency bins. Vigilance states were categorized as wakefulness, REMS, and SWS. Wakefulness was classified according to body movement and low-amplitude EEG with essentially equivalent amounts of δ and θ frequency bands. SWS was identified as a large-amplitude EEG, lack of body movement, and greater power in δ frequency bands as compared with theta bands. REMS was characterized as having a general absence of body movement, low-amplitude EEG, and higher power in θ compared with δ. Signals that were contaminated with electrical noise were identified visually and were excluded from analysis.

SWS δ power was determined for each mouse for each hour that contained a minimum of 30 epochs (5 min) of SWS. The power density values from the frequency bins encompassing the 0.5- to 4.0-Hz range for every artifact-free epoch scored as SWS were averaged. However, when a particular animal did not exhibit SWS or did not spend more than 30 epochs in SWS, values for that animal for that hour were not included in subsequent analyses. We previously have used this approach to minimize a potentially disproportionate effect of a few epochs on overall values for this parameter.¹

Statistical methods.

SPSS software (IBM, Chicago, IL) was used to analyze the data from all outcome variables. δ power values during SWS were analyzed by using one-way ANOVA due to missing values when mice spent less than 5 min in SWS. All other measures were analyzed by using repeated-measures ANOVA, comparing the singly housed state without a shelter to the group-housed or shelter-housed conditions and comparing the 2 d after cage change to the day before cage change. A P value of less than 0.05 was considered to represent a statistically significant effect, and a P value greater than 0.05 but less than 0.1 was considered to indicate a trend.

Results

Sleep–wake behavior in association with cage change.

Sleep–wake behavior was monitored in singly housed C57BL/6J mice (n = 5) before and after a normal cage change. As compared with values on the day before cage change, significant (P< 0.05) changes in sleep–wake behavior (Figure 1) and temperature (Figure 2) consistently occurred during the 3-h period after the cage change, with only sporadic significant changes during the remainder of that day (Figure 1) or on the subsequent day (data not shown). Mice spent nearly all of their time awake during the first 3 h after cage change, with long consolidated bouts of waking (Figure 1) and few state transitions (Figure 2). During that period, mice also exhibited significant (P< 0.05) increases in body temperature, which commonly develop in association with increased activity in mice. Time spent in both SWS and REMS were correspondingly low during this 3-h period. During the remainder of the light phase, mice did not show significant rebound increases in either SWS or REMS time or consolidation. However, δ power during SWS was significantly (P< 0.05) higher than baseline levels during the period immediately after cage change (Figure 2).

Figure 1.

Influence of cage change on 24-h patterns of sleep and waking. Male C57BL/6J mice (n = 5) were monitored before and after being moved from a soiled cage to a clean cage. Open circles indicate data obtained in the soiled cage, with filled circles indicating data contained for 3 h before and 21 h after transfer to a clean cage (indicated by the vertical dashed line). Data points show mean ± SEM. Data were analyzed by using repeated-measures ANOVA (*, P < 0.05; #, 0.05 < P < 0.1). The dark bar on the abscissa denotes the dark phase of the diurnal cycle.

Figure 2.

Influence of cage change on vigilance state transitions, δ power, and body temperature. Male C57BL/6J mice (n = 5) were monitored before (open circles) and after (filled circles) being moved from a soiled to a clean cage. Data points show mean ± SEM. State transitions and body temperatures were analyzed by using repeated-measures ANOVA, and δ power was analyzed using one-way ANOVA (*, P < 0.05; #, 0.05 < P < 0.1). The dark bar on the abscissa denotes the dark phase of the diurnal cycle. The vertical dashed line indicates the time of cage change.

Sleep–wake behavior in the presence of a shelter.

Mice housed individually with or without a shelter showed no significant changes in sleep measures during the 12-h light phase but did show trends (0.05 < P< 0.1) toward greater δ power during SWS and a greater number of state transitions (Table 1). During the dark phase, mice housed individually with a shelter spent significantly (P< 0.05) less time awake and more time in SWS and a trend (0.05 < P< 0.1) toward longer bouts of REMS (Table 1). These changes were distributed throughout the dark phase, with only sporadic significant differences at individual hourly time points (Figures 3 through 6). In addition, mice showed significantly (P< 0.05) less locomotor activity when housed with a shelter (Figure 6, Table 1).

Table 1.

Effects of housing condition on vigilance states and temperature in male C57BL/6J mice (n = 6 per group) during light and dark phases

		Wake			SWS			REMS
	Time (phase)	% of total time	No. of bouts	Bout length (min)	% of total time	No. of bouts	Bout length (min)	% of total time	No. of bouts	Bout length (min)	δ power during SWS	No. of state transitions	Body temperature (°C)	Locomotor activity (counts)
Singly housed,  no shelter	0100–1200 (light)	42 ± 2	90 ± 8	4.4 ± 0.8	52 ± 2	142 ± 12	2.7 ± 0.3	5.5 ± 0.6	30 ± 2	1.3 ± 0.1	1.07 ± 0.11	41 ± 4	35.8 ± 0.1	118 ± 20
Singly housed  with a shelter	0100–1200 (light)	42 ± 2	92 ± 9	4.7 ± 1.2	51 ± 2	144 ± 9	2.8 ± 0.3	5.5 ± 0.7	29 ± 4	1.2 ± 0.1	1.26 ± 0.15b	47 ± 4b	36.0 ± 0.2	48 ± 7a
Group -housed,  no shelter	0100–1200 (light)	46 ± 4	92 ± 4	6.0 ± 1.4b	48 ± 3b	144 ± 3	2.4 ± 0.2	5.9 ± 0.8	38 ± 5b	0.9 ± 0.1a	1.04 ± 0.01	45 ± 2	35.9 ± 0.2	76 ± 10a
Singly housed,  no shelter	1300–2400 (dark)	74 ± 2	52 ± 4	19.6 ± 1.8	25 ± 2	76 ± 7	2.1 ± 0.2	1.5 ± 0.2	9 ± 1	0.5 ± 0.1	1.34 ± 0.15	23 ± 3	37.4 ± 0.2	332 ± 28
Singly housed  with a shelter	1300–2400 (dark)	65 ± 2a	45 ± 4	17.6 ± 1.2	32 ± 2a	81 ± 2	2.4 ± 0.2a	2.3 ± 0.4	11 ± 2	0.7 ± 0.1 †	1.51 ± 0.18	24 ± 1	37.4 ± 0.2	171 ± 27a
Group-housed,  no shelter	1300–2400 (dark)	70 ± 1	57 ± 4	18.1 ± 3.0	26 ± 1	87 ± 4	1.7 ± 0.1	3.5 ± 0.6a	19 ± 2a	0.7 ± 0.1	1.24 ± 0.11	28 ± 1	37.3 ± 0.3	171 ± 11a

Locomotor activity values represent the sum of counts over the indicated period; for group-housed mice, summed values were divided by 3.

^aP < 0.05 compared with value for singly housed with no shelter

^b0.05 < P < 0.1 compared with value for singly housed with no shelter.

Figure 3.

Influence of housing condition on 24-h patterns of waking. Male C57BL/6J mice (n = 6) underwent group housing (filled circles, right panels), individual housing (open circles, both panels), and individual housing with a shelter (filled circles, left panels). Data were collected after 1 wk of housing under the specified condition. Data from mice housed individually without shelters are duplicated on the left and right panels to simplify visual comparison of control and experimental conditions. Data points show mean ± SEM. Data were analyzed by using repeated-measures ANOVA (*, P < 0.05; #, 0.05 < P < 0.1). The dark bar on the abscissa denotes the dark phase of the diurnal cycle.

Figure 6.

Influence of housing condition on vigilance state transitions, δ power, body temperature, and locomotor activity. Male C57BL/6J mice (n = 6) underwent group housing (filled circles, right panels), individual housing (open circles, both panels), and individual housing with a shelter (filled circles, left panels). Data were collected after 1 wk of housing under the specified condition. Data from mice housed individually without shelters are duplicated on the left and right panels to simplify visual comparison of control and experimental conditions. Data points show mean ± SEM. During group housing, values collected from the cage were divided by 3. State transitions, body temperature, and locomotor activity were analyzed by using repeated-measures ANOVA, and δ power was analyzed by using one-way ANOVA (*, P < 0.05; #, 0.05 < P < 0.1). The dark bar on the abscissa denotes the dark phase of the diurnal cycle.

Figure 4.

Influence of housing condition on 24-h patterns of SWS. Male C57BL/6J mice (n = 6) underwent group housing (filled circles, right panels), individual housing (open circles, both panels), and individual housing with a shelter (filled circles, left panels). Data were collected after 1 wk of housing under the specified condition. Data from mice housed individually without shelters are duplicated on the left and right panels to simplify visual comparison of control and experimental conditions. Data points show mean ± SEM. Data were analyzed by using repeated-measures ANOVA (*, P < 0.05; #, 0.05 < P < 0.1). The dark bar on the abscissa denotes the dark phase of the diurnal cycle.

Figure 5.

Influence of housing condition on 24-h patterns of rapid-eye-movement sleep. Male C57BL/6J mice (n = 6) underwent group housing (filled circles, right panels), individual housing (open circles, both panels), and individual housing with a shelter (filled circles, left panels). Data were collected after 1 wk of housing under the specified condition. Data from mice housed individually without shelters are duplicated on the left and right panels to simplify visual comparison of control and experimental conditions. Data were analyzed by using repeated-measures ANOVA (*, P < 0.05; #, 0.05 < P < 0.1). The dark bar on the abscissa denotes the dark phase of the diurnal cycle.

Sleep-wake behavior under the group-housed condition.

Individual mice that were housed singly or as a member of a group showed significant changes in REMS during both the 12-h light and the 12-h dark phases (Table 1). Throughout the light phase, group housed mice showed significantly shorter REMS bout lengths and trends toward a greater number of REMS bouts, less time in SWS, and longer bouts of waking (Table 1). On the other hand, throughout the dark phase, group-housed mice spent significantly more time in REMS as a result of more, as compared with longer, REMS bouts (Table 1). Mice also showed significantly less locomotor activity when group-housed (Figure 6, Table 1).

Discussion

Our data indicate that 1) cage changing disrupts sleep in mice for approximately 3 h but thereafter is not associated with significant effects on sleep; 2) compared with individually housing of mice, group housing is associated with shorter REMS bout lengths and less SWS during the light phase and with more REMS during the dark phase; and 3) compared with group-housed or mice housed individually without shelters, mice housed individually with a shelter show trends toward a deeper plane of SWS and more state transitions during the light phase and spend significantly less time awake and more time in SWS, with longer bouts of SWS and a trend toward longer bouts of REMS, during the dark phase.

A stable cage environment is crucial to the collection of valid sleep data,^3,30 and changing the cage clearly disrupts and alters the environment the mouse experiences. Because mice are an olfactory-oriented species, disruption of the olfactory environment by providing a sanitized cage and clean bedding constitutes a significant environmental perturbation that is likely to disrupt stable patterns of activity and behavior.²⁴ Recent studies in rats have found that the type of bedding affects the amount of sleep measured²⁰ and that cage color can influence circadian rhythm expression.⁸ The frequency at which mouse cages are changed, the type of bedding used, and even the manner in which mice are handled during the cage change process can influence the mice, their subsequent behavior, and the data collected, including the amount of sleep in which the mice engage.^20,24,26 For example, a study of 3 different methods of transferring mice from one cage to another (forceps transfer, gentle transfer with gloved hands, and a passive-transfer technique that did not involve active handling) found transient effects on serum corticosterone concentrations and altered open-field behaviors when mice were tested later on the same day.²⁴

Previous studies have suggested that cage changing affects the sleep of mice; however, these reports generally describe only the amount of time mice spend in sleep stages.^28,29 In our current study, we extend the observations of these previous studies to include measures of sleep quality, sleep depth, and body temperature. Our data show that sleep is disrupted and that changes in body temperature occur during the 3-h period after C57BL/6J mice are moved to a clean cage. Modest but statistically significant increases in SWS δ power occur for 3 h after this period of spontaneous arousal, suggesting that mice compensate for disrupted sleep by increasing sleep depth, without any change in sleep duration. Although both sleep duration and depth of sleep generally increase after sleep loss, a growing literature illustrates conditions under which these 2 sleep parameters may be dissociated.⁹ δ power during SWS has historically been viewed as correlating with the depth of sleep, such that spontaneous arousal is less likely to occur and induced arousal is more difficult to achieve if δ power is high.^4,11 Higher δ power in mice after a prolonged period of arousal is consistent with greater depth of sleep that develops as a homeostatic response to sleep loss.

Our data show that mice housed individually with a shelter spend significantly less time awake and more time in SWS, with longer bouts of SWS and a trend toward longer bouts of REMS and higher δ power, during the dark phase. These changes suggest that the availability of a darkened enclosure promotes sleep and sleep consolidation. In addition, housing mice with a shelter was associated with decreased locomotor activity over a 24-h period, which suggests that the mice are not sleeping more simply because they become behaviorally more active in the enriched environment. Instead, the dark shelter likely provides a location within the cage that supports sleep stability and consolidation. Although we did not include a housing condition that combined group housing with a shelter, another study showed that the presence of shelters can influence social behaviors in group-housed NIH/S mice (an aggressive strain) by preventing intracage fighting.¹⁷ Although our monitoring system does not allow us to determine the location of a mouse within the recording cage, individually housed mice appear to prefer sleeping in a tube-shelter when available.²⁷ Shelter-related changes in behavior could affect sleep in a social environment also.

Social influences on sleep have been studied previously in rodents. However, many studies involving EEG recording require rodents to be housed individually due to the need for a tether; this system has limited the abilities of researchers to study the effects of group housing on sleep. Our use of a telemetry system allows us to record from implanted animals in a social environment during an experiment. Group housing affected C57BL/6J mice differently during the light (somnolent) and dark (active) phases of the diurnal cycle. Thus, during the light phase, mice showed shorter REMS bout lengths and trends toward a greater number of REMS bouts, less time in SWS, and longer bouts of waking when group housed in comparison to individual housing. In contrast, during the dark phase, group-housed mice spent more time in REMS as a result of more, rather than longer, bouts of REMS. These findings indicate that housing with cagemates results in fragmentation of REMS (that is, shorter or more frequent bouts). In contrast, a study of rats concluded that social partnering, as compared with isolation housing, may protect stress-sensitive animals from REMS fragmentation that occurs after cue exposure in cued fear conditioning.⁶ The cited study⁶ evaluated the intervals of time that separated bouts of REMS, used these intervals to classify REMS as occurring in single (greater than 3 min) or sequential (3 min or less) episodes, and reached the conclusion that an increase in the number of sequential REMS episodes after cued fear conditioning represented REMS fragmentation.⁷ However, in the presence of a partner naïve for cued fear conditioning, stress-sensitive rats showed more sleep time during the light phase and less time in SWS during the dark phase, with the number of single REMS episodes higher during both the light and dark phases in partnered rats and the number of sequential REMS episodes higher in socially isolated rats.⁶ Whether single or sequential REMS episodes actually denote REMS consolidation or fragmentation is debatable in the absence of other information on the sleep architecture. In another study, after 6 h of sleep deprivation, male C57BL/6J mice showed the typical recuperative homeostatic response of a significant reduction in wake time and an increase in time spent in SWS and REMS, with similar but less robust effects in individually housed mice.¹⁸ δ power during SWS was higher in both groups immediately after sleep deprivation, but pair-housed mice continued to show significant elevations in δ power throughout the dark period.¹⁸ Therefore, responses to imposed sleep loss differ in pair-housed and individually housed mice, suggesting that social context influences the quantity and quality of sleep in this species.¹⁸ Our study did not examine the influence of social compared with individual housing on the response to sleep deprivation.

Maintaining environmental stability is an important consideration in designing sleep studies in rodents. Additional variables not measured in our study (for example, bedding type, presence of environmental enrichment, availability of a resting surface) may affect the behavior and consequently sleep characteristics of rodents. Although changes in sleep and EEG patterns with aging have been documented in mice, the mice we used in this study were 2 to 3 mo old, and there is little evidence that sleep of mice changes dramatically across the relatively short duration of this study. Although the possibility exists that sleep–arousal parameters could differ across an 8-wk study, such changes are reported to be subtle and are not of the same magnitude as those associated with the experimental conditions used in this study.¹²

In summary, our data indicate that factors such as cage changes, the presence of a shelter, and cagemates can all modify sleep in mice. These findings document the importance of providing a stable environment for studies of sleep using rodent models.